CN108403941B - Traditional Chinese medicine composition for treating pancreatic cancer and preparation method and application thereof - Google Patents

Traditional Chinese medicine composition for treating pancreatic cancer and preparation method and application thereof Download PDF

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CN108403941B
CN108403941B CN201810462816.4A CN201810462816A CN108403941B CN 108403941 B CN108403941 B CN 108403941B CN 201810462816 A CN201810462816 A CN 201810462816A CN 108403941 B CN108403941 B CN 108403941B
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于海涛
费洪新
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Qiqihar Medical University
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Abstract

The invention discloses a traditional Chinese medicine composition for treating pancreatic cancer, and a preparation method and application thereof. The traditional Chinese medicine composition is prepared from the following raw materials: 20-25 parts of Chinese yam, 4-8 parts of rhizoma atractylodis, 15-20 parts of Chinese date, 10-20 parts of white hyacinth bean, 5-8 parts of pawpaw, 5-8 parts of morinda officinalis, 5-8 parts of rhizoma arisaematis, 10-15 parts of snakegourd fruit, 10-15 parts of winter jasmine flower, 15-25 parts of microcos paniculata, 5-12 parts of fructus akebiae, 2-7 parts of edible tulip, 10-15 parts of bauhinia championii, 1-5 parts of lobular lotus, 2-7 parts of acorus calamus and 5-15 parts of herba aristolochiae. The Chinese medicinal composition has effects of invigorating spleen, promoting diuresis, eliminating phlegm, clearing heat away, removing blood stasis, promoting blood circulation, warming stomach, regulating qi-flowing, etc. Animal experiments show that the traditional Chinese medicine composition with high and medium doses can obviously improve the tumor inhibition rate, the spleen index, the cell cycle and the apoptosis of a nude mouse transplanted tumor model of pancreatic cancer, block the expression of key molecules of a Hedgehog signal path and has no side effect. The traditional Chinese medicine composition has wide application prospect in the aspect of treating pancreatic cancer.

Description

Traditional Chinese medicine composition for treating pancreatic cancer and preparation method and application thereof
Technical Field
The invention relates to a traditional Chinese medicine composition, a preparation method and application thereof, in particular to a traditional Chinese medicine composition for treating pancreatic cancer, a preparation method and application thereof. The invention belongs to the technical field of medicines.
Background
Pancreatic Cancer (Pancreatic Cancer) refers to a malignant tumor of the digestive system that occurs in the Pancreatic exocrine region of the body. The incidence, prevalence and mortality of pancreatic cancer in the whole World have been on the rise in recent years, wherein the incidence of pancreatic cancer in rural areas is higher than that in urban areas, and the annual diagnosis rate and mortality of pancreatic cancer in countries exceed those in developed countries in North America (Lin QJ, Yang F, Jin C, et al. Current status and progress of pancreatic cancer in China [ J ]. World J Gastroenterol, 2015, 21 (26): 7988-.
Pancreatic cancer is mainly unconscious in onset and rarely has specific symptoms and signs, which can cause difficult early diagnosis and early treatment of pancreatic cancer, most patients have metastasis when pancreatic cancer is diagnosed and are easy to spread to liver, peritoneum, lung and local lymph nodes, and the treatment of compatible medicines for surgical treatment is the best scheme for treating pancreatic cancer, but the operative mortality and postoperative recurrence rate of pancreatic cancer are relatively high, and the 5-year survival rate is relatively low. Therefore, new strategies for treating pancreatic cancer are urgently needed.
In recent years, many studies have shown that the Hedgehog signaling pathway is closely related to the occurrence and development of pancreatic cancer. In embryonic development, the Hedgehog signal pathway is highly conserved, and can participate in the regulation of the development of various tissues and organs of an organism. The Hedgehog Signaling Pathway includes ligands (Shh), membrane receptors (Ptch and Smo), nuclear transcription factors (family G1 i), regulatory factors, etc. (Qin S, Sun D, Li H, et al. the Effect of SHH-Gli Signaling Pathway on the synthetic fiber promotion in Rheumatoid Arthritis [ J ]. Inflammation, 2016, 39 (2): 503-. In general, Hedgehog Signaling pathway expression is not usually detected in normal organisms, and it has been demonstrated that various malignant tumors are closely associated With Hedgehog Signaling pathway abnormal high expression (Skoda AM, Simovic D, Karin V, et al. the role of the Hedgehog Signaling pathway in Cancer: A complex revisive revisiview [ J ]. Bosn J Basic Med Sci, 2018, 18 (1): 8-20.), for example, Pancreatic Cancer (Gu J, Saiyin H, Fu D, et al. Stroma A Double Ed Sword in pancreative Cancer: A Lesson Targeting Stroma in pancreative Cancer: A Lesson Targeting prostate Cancer in pancreative Cancer, prostate Cancer, ovarian Cancer, prostate Cancer, etc.). It has been proved that activation of the Hedgehog signaling pathway occurs in most pancreatic cancer patients, which will change the biological characteristics of pancreatic cancer, and especially high expression of the Hedgehog signaling pathways Shh, Ptch, Smo and Gli in pancreas can be detected in early stage of pancreatic cancer patients, and thus the Hedgehog signaling pathway plays an important role in pancreatic cancer.
Three ligands, Shh, Ihh and Dhh, exist in the Hedgehog signaling pathway, wherein the ligand Shh is studied more. Ligand Shh is a highly conserved secretory signal protein in the Hedgehog signaling pathway, which is also a key protein that activates the Hedgehog signaling pathway and plays a role in intercellular regulation, and functions similarly to cholesterol transferase, has autocatalytic processing ability, and can also release Ptch from inhibiting Smo and activate Smo (Du Z, Zhou F, Jia Z, et al. the Hedgehog/Gli-1 signaling pathway involved in the inhibition of the inbibitorey of the innovative alcohol on human color cancer HCT116 cells [ J ]. Iran J Basic Med Sci, 2016, 19 (11): 1171-. Ptch is a membrane protein receptor, and can be classified into Ptch1 and Ptch2, and Ptch1 and Ptch2 both have Shh-binding and Smo-inhibiting effects. Ptch is a key protein of the Hedgehog signaling pathway, the up-regulation of Ptch expression is a key factor for activation of the Hedgehog signaling pathway, mutant Ptch loses the inhibitory effect on Smo, Smo activates downstream Gli, causing activation of Hedgehog signaling pathway target genes (Wang LW, Lin H, Lu Y, et al. Smo is a proto-oncogene, which is a key member of the Hedgehog signaling pathway. In the absence of Shh under normal conditions, Smo activity can be inhibited by Ptch, and Smo controls the activation state of the downstream transcription factor Gli. Gli mainly comprises three molecules Gli1, Gli2 and Gli3, and Gli1, Gli2 and Gli3 are particularly similar in structure. Gli1, Gli2 and Gli3 all have 5 zinc fingers in DNA domains, and can be combined with a specific sequence of a downstream target gene (GAC CAC CCA) of a Hedgehog signal path to play a regulating role. Among them, Gli1 has a potent transcription activator effect, Gli2 exists mainly in the form of a transcription activator, and Gli3 mainly plays a transcription repressor effect.
The data show that aberrant activation of the Hedgehog signaling pathway in pancreatic cancer can be accompanied by overexpression of Gli1 and its function in binding to specific sequences of downstream target genes (GAC CAC CCA) (Gan H, Liu H, Zhang H, et al SHh-Gli1 signaling pathway cells and by mediating bacterial IAP repeat-linking 3(BIRC3) gene in pancreatic cancer cells [ J ]. Tumour Biol, 2016, 37 (7): 9943-9950.) by the formation of a negative feedback regulatory loop that induces Ptch transcription of the Hedgehog signaling pathway and activates the target genes, leading to loss of control of the Hedgehog signaling pathway. The Gli target gene is proved to be involved in the growth, apoptosis, metastasis, neovascularization and epithelial-mesenchymal transition of tumor cells, for example, Gli can induce cyclin D1 expression to promote the proliferation of pancreatic cancer cells, direct activation can cause epithelial tissue to be transcribed to mesenchymal transition factors and enhance the infiltration of pancreatic cancer cells, direct induction of the expression of anti-apoptosis factors such as Bcl-2 and the like and the expression of pro-apoptosis factors such as Bax and the like can inhibit the apoptosis of the pancreatic cancer cells, and finally the activation of a key protein Ptch of a Hedgehog signal path is directly regulated to promote the proliferation of the pancreatic cancer cells. Therefore, the effective blockage of the Hedgehog signaling pathway can inhibit the growth, invasion and metastasis of pancreatic cancer cells, which is an active strategy for treating pancreatic cancer.
At present, many drugs, such as 5-fluorouracil (Wang WB, Yang Y, Zhao YP, et al, recipient students of 5-fluorogenic resistance in pancreatic cancer [ J ]. World J Gastroenterol, 2014, 20 (42): 15682. acid 15690.), antioxidants, anti-apoptotic drugs, resveratrol (Xu Q, Zong L, Chen X, et al, Reservarol in the prostate of the pancreatic cancer Sci, 2015, 8 (1): 10-19.), cisplatin, etc., can treat pancreatic cancer at home and abroad, but many drugs have side effects to a certain extent, cause harm to the body of pancreatic cancer patients, and thus have no side effects or side effects, and very effective drugs for treating pancreatic cancer are particularly important.
The theory of traditional Chinese medicine considers that the spleen governs transportation and transformation and can play the basic functions of transportation, digestion, absorption and the like. The spleen governs transportation and transformation and can be divided into transportation and transformation of food and water, and the spleen can promote the viscera, meridians and other tissues to obtain sufficient nutrition through transportation and transformation functions. The spleen has vigorous transportation and transformation functions, so that the generation of pathological products such as water dampness, phlegm retention, damp obstruction, blood stasis, heat evil and the like can be prevented. Once dysfunction of spleen in the body causes spleen deficiency, wood stagnation is caused by spleen deficiency, dampness is generated by spleen deficiency, transportation and transformation are abnormal due to spleen deficiency, heat is transformed from exterior and interior stagnation, spleen deficiency and dampness obstruction are formed to induce pancreatic cancer, and therefore, the pancreatic cancer needs to invigorate spleen and promote diuresis. In addition, spleen deficiency in the body damages the spleen, and is accompanied by the combination of internal and external damp-heat pathogens, heat toxin is stored internally, and the heat toxin cannot be removed for a long time, and the heat toxin is gradually changed into lumps, so that phlegm, heat, stasis and blood clots are accumulated in the spleen, and cancer and tumor can be formed, and therefore, the pancreatic cancer needs to dispel phlegm, clear heat, resolve phlegm and promote blood circulation. In addition, spleen deficiency of the body causes disorder of transportation and transformation of food and water, obstruction of collaterals, disorder of qi movement of liver and gallbladder, liver and gallbladder obstruction of qi movement, disorder of liver and gallbladder flow, deficiency of healthy qi, exogenous dampness and heat, and diet disorder, and finally forms cancer tumor, so that the pancreatic cancer needs to regulate qi and dredge collaterals. Therefore, the disease pathogenesis analysis based on the theory of the traditional Chinese medicine shows that the pancreatic cancer needs to invigorate the spleen and promote diuresis, eliminate phlegm and clear heat, eliminate phlegm and promote blood circulation, regulate qi and dredge collaterals.
The traditional Chinese medicine has the positive characteristics of stronger synergistic effect, smaller side effect, more drug action targets, integral regulation and the like, has unique positive advantages in the aspects of delaying the pancreatic cancer process, enhancing the immune function, improving the oxidation resistance, weakening the inflammatory response, protecting normal cells of a body and the like, and brings hope for clinically treating pancreatic cancer (Caonida, daojia, ChenBin, and the like, the influence of the traditional Chinese medicine compound WCA-P and the formula thereof on subcutaneous inoculation of a naked mouse with a human pancreatic cancer BxPC-3 cell suspension [ J ]. Western traditional Chinese medicine, 2017-10 of (1)). Therefore, it is very important to find a traditional Chinese medicine composition with good curative effect and no side effect for treating pancreatic cancer.
Disclosure of Invention
The invention aims to provide a traditional Chinese medicine composition for treating pancreatic cancer and a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical means:
the invention selects 16 Chinese medicaments such as Chinese yam, rhizoma atractylodis, Chinese date, white hyacinth bean, pawpaw, morinda officinalis, rhizoma arisaematis, snakegourd fruit, winter jasmine, microcos paniculata, fructus akebiae, edible tulip, Japanese bauhinia stem, lobular lotus, acorus calamus and herba aristolochiae to form the Chinese medicinal composition for treating pancreatic cancer. In order to prove the effect of the traditional Chinese medicine composition on treating pancreatic cancer, the invention carries out the following tests:
experiment shows the growth of the Chinese medicine composition on pancreatic cancer BxPC-3 cell strain. The results show that the inhibition effect of the traditional Chinese medicine composition on the pancreatic cancer BxPC-3 cell line is gradually enhanced along with the increase of the concentration of the traditional Chinese medicine composition and the prolongation of the intervention time of the traditional Chinese medicine composition, which indicates that the pharmacological effect of the traditional Chinese medicine composition on the pancreatic cancer BxPC-3 cell line has a dose-effect relationship. After the active dry prognosis of the traditional Chinese medicine composition, the high and medium doses of the traditional Chinese medicine composition have an inhibiting effect on the growth of pancreatic cancer BxPC-3 cell strains; meanwhile, compared with the 5-fluorouracil group, the live cell count of the pancreatic cancer BxPC-3 cell line at the time points of the high and medium dose groups is not obviously changed (P is more than 0.05), while the live cell count of the pancreatic cancer BxPC-3 cell line at the time points of the low dose groups of the traditional Chinese medicine composition is obviously increased (P is more than 0.05), which indicates that the high and medium dose of the traditional Chinese medicine composition and the 5-fluorouracil have equivalent inhibition effect on the pancreatic cancer BxPC-3 cell line, and the low dose of the traditional Chinese medicine composition has poor effect on treating the pancreatic cancer BxPC-3 cell line. The combination shows that the traditional Chinese medicine composition can inhibit the growth of pancreatic cancer BxPC-3 cell strains at high and medium doses, and the inhibition effect is the same as that of 5-fluorouracil.
The experiment determines the influence of the traditional Chinese medicine composition on the motor ability level of the pancreatic cancer BxPC-3 cell strain. The results show that after the treatment of the traditional Chinese medicine composition and the 5-fluorouracil, the high dose of the traditional Chinese medicine composition, the medium dose of the traditional Chinese medicine composition and the 5-fluorouracil have strong inhibition effect on the motility of the pancreatic cancer BxPC-3 cell strain, and the influence of the traditional Chinese medicine composition on the motility of the pancreatic cancer BxPC-3 cell strain is more obvious along with the increase of the dose; meanwhile, the traditional Chinese medicine composition has poor influence on the motor capacity of the pancreatic cancer BxPC-3 cell strain at low dose. The combination shows that the Chinese medicinal composition can inhibit the motor ability of the pancreatic cancer BxPC-3 cell strain at high and medium doses, and the positive intervention effect is obvious.
In the research, a pancreatic cancer BxPC-3 cell strain is selected to construct a pancreatic cancer BxPC-3 nude mouse transplantation tumor model mouse. Compared with a model group, the high-dose Chinese medicinal composition, the medium-dose Chinese medicinal composition, the low-dose Chinese medicinal composition and the 5-fluorouracil have obvious inhibition effect on the transplanted tumor of a pancreatic cancer nude mouse (P is less than 0.05), wherein the high-dose Chinese medicinal composition group has a good tumor inhibition effect. The tumor inhibition rate of each experimental group is more than 30%, and according to the anti-tumor effectiveness standard of Chinese herbal medicines and the related standard regulation of modern tumor treatment pharmacology (the tumor inhibition rate is more than 30%), the high dose of the traditional Chinese medicine composition, the medium dose of the traditional Chinese medicine composition and the low dose of the traditional Chinese medicine composition are preliminarily considered to have the inhibition effect on the pancreatic cancer BxPC-3 transplanted tumor. In general, the spleen produces mainly B cells, and the immune function of the body can be evaluated by measuring the spleen, which is measured by an electronic balance to measure the spleen index of mice. The experimental results show that the spleen indexes of the mice in the high-dose group, the medium-dose group and the low-dose group of the traditional Chinese medicine composition are higher, which indicates that the spleen of the mice in the high-dose group, the medium-dose group and the low-dose group of the traditional Chinese medicine composition mainly generates more B cells, and prompts that the humoral immunity level of the mice in the high-dose group, the medium-dose group and the low-dose group of the traditional Chinese medicine composition is higher and the immune function of an organism is stronger; the spleen index of the mice in the 5-fluorouracil group is reduced, which indicates that the humoral immunity level of the mice in the 5-fluorouracil group is reduced, and indicates that the immune function of an organism is poor and the side effect is large. The combination shows that the high dosage of the traditional Chinese medicine composition, the medium dosage of the traditional Chinese medicine composition and the low dosage of the traditional Chinese medicine composition have anti-tumor effect and small side effect.
Flow cytometry analysis shows that cell cycles of pancreatic cancer BxPC-3 transplantable tumors are obviously changed in comparison of a traditional Chinese medicine composition high-dose group, a traditional Chinese medicine composition low-dose group and a 5-fluorouracil group with a model group, wherein the pancreatic cancer BxPC-3 transplantable tumors in the traditional Chinese medicine composition high-dose group and the traditional Chinese medicine composition medium-dose group obviously increase in the G1 phase and obviously decrease in the G2 phase (P is less than 0.05), which indicates that the cell cycles of the pancreatic cancer BxPC-3 transplantable tumors are shortened in the G2 phase and prolonged in the G1 phase after the action of the traditional Chinese medicine composition high and medium doses, and indicates that pancreatic cancer cells are blocked in the G1 phase. Meanwhile, compared with a high-dose group, a low-dose group and a 5-fluorouracil group of the traditional Chinese medicine composition and a model group, the apoptosis rate of the pancreatic cancer BxPC-3 transplanted tumor cells is obviously increased (P is less than 0.05). Comprehensively shows that the high-dose traditional Chinese medicine composition and the medium-dose traditional Chinese medicine composition play an anti-tumor role by blocking the cell cycle of the pancreatic cancer BxPC-3 transplanted tumor in the G1 stage and reducing the apoptosis rate.
The data show that the Hedgehog signaling pathway is highly conserved, Hedgehog expression is usually undetectable in normal humans, while the pancreatic cancer Hedgehog signaling pathway is highly expressed, and is closely related to the abnormal expression of the Hedgehog signaling pathways Shh, Ptch1, Smo (Dosch JS, Pasca di Magliano M, Simone DM. pancreatic cancer and Hedgehog pathway signaling: new energies [ J ]. pancreatologyo, 2010, 10 (3): 151-) 157.). It has been demonstrated that malignant cells are capable of secreting Shh, which acts as a secretory signal molecule on themselves and on surrounding cells. In the early stage of pancreatic cancer, a Hedgehog signaling pathway is activated to cause malignant transformation of pancreatic ductal epithelium, Ptch1 is used as a membrane protein receptor on the Hedgehog signaling pathway, is a recipient and a transmitter of the Hedgehog signaling pathway, and relieves the inhibition effect of Ptch1 on Smo, so that Smo transmits signals into cells, activates a downstream transcription factor Gli family and regulates the expression of related target genes. After activation, Gli1 enters into nucleus from cytoplasm, so that expression of genes downstream of Glil activation is up-regulated, thereby inducing pancreatic cancer. From the in vivo perspective, the research adopts immunohistochemistry, Real-time PCR and Western blot methods to discuss whether the anti-pancreatic cancer effect of the traditional Chinese medicine composition acts through the signal channel. Immunohistochemistry results show that Hedgehog signaling pathways Shh, Ptch1, Smo localize to cytoplasm and/or cell membrane, and Gli1 localize to cytoplasm and/or nucleus. High expressions of a Hedgehog signal channel Shh, Ptch1 and Smo are detected in a pancreatic cancer BxPC-3 transplantation tumor model, and after the dry prognosis of the traditional Chinese medicine composition, compared with a model group, the high-dose group of the traditional Chinese medicine composition, the medium-dose group of the traditional Chinese medicine composition and the 5-fluorouracil group pancreatic cancer BxPC-3 transplantation tumor model Hedgehog signal channel Shh, Ptch1 and Smo are all low-expressed (P <0.05), while the low-dose group of the traditional Chinese medicine composition can lower the expressions of the pancreatic cancer BxPC-3 transplantation tumor model Hedgehog signal channels Shh, Ptch1 and Smo, so that the high-dose of the traditional Chinese medicine composition and the medium-dose of the traditional Chinese medicine composition can lower the expressions of the pancreatic cancer BxPC-3 transplantation tumor model Hedgehog signal channels Shh, Ptch1 and Smo.
The research adopts Real-time PCR and Western blot to quantitatively detect the activation level of the Hedgehog signal path more accurately. The results show that model group pancreatic cancer BxPC-3 transplantable tumor model Hedgehog signaling pathway Shh, Ptch1, Smo, Gli1 are highly expressed. When Shh is abnormally highly expressed, Shh and Ptch1 are combined to release the inhibition of Smo, zinc finger greening factors are promoted to be separated from Sufu, Gli in cytoplasm and Cos2 form a complex and are separated from microtubules, Gli enters into cell nucleus to activate target gene transcription to cause pancreatic cancer cell proliferation, and the situation that the Shh high expression of a Hedgehog signal channel of a pancreatic cancer BxPC-3 transplantation tumor model is one of inducers for activating the Hedgehog channel is shown. After treatment by the traditional Chinese medicine composition and 5-fluorouracil, the intervention of a high-dose group of the traditional Chinese medicine composition, a medium-dose group of the traditional Chinese medicine composition and a 5-fluorouracil group promotes the expression of a pancreatic cancer BxPC-3 transplantation tumor model Hedgehog signal channel Shh, Ptch1, Smo and Gli1 to be down-regulated in each group, so that the activation of the Hedgehog signal channel is inhibited. However, the traditional Chinese medicine composition has weak intervention effect on the pancreatic cancer BxPC-3 transplantation tumor model Hedgehog signal pathways Shh, Ptch1, Smo and Gli 1. Comprehensively shows that the traditional Chinese medicine composition has high dosage, the dosage in the traditional Chinese medicine composition can reduce the expression of the Hedgehog signal paths Shh, Ptch1, Smo and Gli1 of the pancreatic cancer BxPC-3 transplantation tumor model, and the Hedgehog signal paths Shh, Ptch, Smo and Gli1 play an important role in the pancreatic cancer development process.
In order to effectively evaluate the side effects of the traditional Chinese medicine composition, the experiment also determines the change levels of brain, heart, liver, kidney and lung indexes of a pancreatic cancer BxPC-3 transplantation tumor model mouse and the change levels of the mouse body weight at different time points. The experimental result shows that compared with the model group, the indexes of the brain, the heart, the liver, the kidney and the lung of the traditional Chinese medicine composition high-dose group, the traditional Chinese medicine composition medium-dose group, the traditional Chinese medicine composition low-dose group and the 5-fluorouracil group pancreatic cancer BxPC-3 transplantation tumor model mouse are not obviously changed (P is more than 0.05); the results show that the high, medium and low dosages of the traditional Chinese medicine composition have no influence on the important organs of the mice model of the pancreatic cancer BxPC-3 transplantation tumor. Meanwhile, in the administration process of each group of mice, the weight of the mice of the pancreatic cancer BxPC-3 transplantation tumor model is in an increasing trend, which shows that the high, medium and low doses of the traditional Chinese medicine composition have no influence on the weight of the mice of the pancreatic cancer BxPC-3 transplantation tumor model. Compared with the model group, the traditional Chinese medicine composition has the advantages that the change of the body weight of 0d, 7d, 14d and 28d mice with pancreatic cancer BxPC-3 transplantable tumors is not obvious (P is more than 0.05) in the high, medium and low dose groups; statistical analysis also shows that the high, medium and low doses of the traditional Chinese medicine composition have no influence on the body weight of mice model with the pancreatic cancer BxPC-3 transplantation tumor. Finally, the Chinese medicinal composition for treating the pancreatic cancer BxPC-3 transplanted tumor model mouse does not change the important organ index and the weight of the pancreatic cancer BxPC-3 transplanted tumor model mouse while treating the pancreatic cancer BxPC-3 transplanted tumor model mouse, so that the Chinese medicinal composition has no side effect, and the Chinese medicinal composition can be used for treating the pancreatic cancer BxPC-3 transplanted tumor model mouse at high and medium doses with confidence.
The experiments show that the traditional Chinese medicine composition with high and medium doses (43.48g/kg and 21.74g/kg) can obviously improve the tumor suppression rate, the spleen index, the cell cycle and the apoptosis of a nude mice transplanted tumor model of pancreatic cancer, can also improve the expression of key molecules Shh, Ptch1, Smo and Gli1 of a Hedgehog signal pathway of the nude mice transplanted tumor model of pancreatic cancer, and has no influence on the brain index, the heart index, the liver index, the kidney index, the lung index and the body weight of the nude mice transplanted tumor model of pancreatic cancer.
On the basis of the research, the invention provides a traditional Chinese medicine composition for treating pancreatic cancer, which is prepared from the following raw materials in parts by weight: 20-25 parts of Chinese yam, 4-8 parts of rhizoma atractylodis, 15-20 parts of Chinese date, 10-20 parts of white hyacinth bean, 5-8 parts of pawpaw, 5-8 parts of morinda officinalis, 5-8 parts of rhizoma arisaematis, 10-15 parts of snakegourd fruit, 10-15 parts of winter jasmine flower, 15-25 parts of microcos paniculata, 5-12 parts of fructus akebiae, 2-7 parts of edible tulip, 10-15 parts of bauhinia championii, 1-5 parts of lobular lotus, 2-7 parts of acorus calamus and 5-15 parts of herba aristolochiae.
Wherein, the yam has mild nature and sweet taste, belongs to spleen channel, stomach channel and kidney channel, and has the efficacies of strengthening spleen, promoting fluid production, nourishing stomach, benefiting lung, tonifying kidney and arresting seminal emission; the rhizoma atractylodis is warm in nature, pungent and bitter in taste, enters spleen channel, stomach channel and liver channel, and has the effects of strengthening spleen, dispelling wind, eliminating dampness, dispelling cold, improving eyesight and resolving stagnation; the Chinese dates are warm in nature and sweet in taste, enter spleen channels and stomach channels, and have the effects of tonifying spleen and qi, soothing nerves and nourishing blood and relieving drug properties; the white hyacinth beans are neutral in nature, sweet and bland in taste, enter spleen channels and stomach channels, and have the effects of promoting diuresis and reducing edema, strengthening spleen and inducing diuresis, and regulating stomach and relieving summer heat; the pawpaw is warm in nature, sour and astringent in taste, enters liver channel, spleen channel and stomach channel, and has the effects of promoting diuresis, harmonizing stomach, soothing liver, relieving pain, inducing diuresis and reducing swelling; morinda officinalis is slightly warm in nature, pungent and sweet in flavor, enters liver and kidney meridians, and has the effects of promoting diuresis, tonifying kidney, dispelling wind and strengthening tendons; rhizoma arisaematis, warm in nature, bitter and pungent in taste, enters liver channel, lung channel and spleen channel, and has the effects of eliminating phlegm and swelling, dispelling pathogenic wind and removing dampness, and arresting convulsion and resolving masses; the snakegourd fruit is cold in nature, sweet and bitter in taste, enters lung channel, stomach channel and large intestine channel, and has the effects of eliminating phlegm and clearing heat, dissipating stagnation and relieving chest stuffiness, moistening dryness and lubricating intestines, and inducing diuresis to alleviate edema; the winter jasmine flower is mild in nature, sweet and astringent in taste, enters kidney channels and bladder channels, and has the effects of clearing away heat and toxic materials, inducing diuresis to reduce edema, promoting blood circulation and relieving pain; the paniculate microcos leaves are neutral in nature and sour in taste, enter spleen channels, liver channels and stomach channels, and have the effects of clearing heat, resolving stagnation, promoting digestion and removing dampness; fructus akebiae, cold in nature, bitter in taste, enters stomach channel, liver channel, gallbladder channel and bladder channel, and has effects of removing blood stasis, dispersing stagnated liver qi, regulating qi-flowing, promoting blood circulation, promoting urination and killing parasite; the edible tulip is cold in nature, sweet in taste and slightly pungent, enters liver and spleen channels, and has the effects of removing blood stasis, eliminating phlegm, clearing heat and removing toxicity; bauhinia championii is neutral in nature, bitter and pungent in taste, enters spleen channels, liver channels and stomach channels, and has the effects of promoting blood circulation, dispelling wind, eliminating dampness, relieving pain, promoting qi circulation and removing blood stasis; the fructus podophylli is warm in nature and bitter in taste, enters stomach channel, liver channel and lung channel, and has the effects of promoting blood circulation, removing dampness, dispelling wind, relieving cough, removing toxicity and relieving pain; the calamus has warm nature, bitter and pungent taste, enters stomach channel and heart channel, and has the effects of warming stomach, relieving pain, tonifying yang and diminishing inflammation; herba Aristolochiae, warm in nature, bitter in taste, entering spleen meridian and liver meridian, has effects of regulating qi-flowing, relieving pain, promoting blood circulation and promoting diuresis.
Preferably, the traditional Chinese medicine composition consists of the following raw materials in parts by weight: 22.5 parts of Chinese yam, 6 parts of rhizoma atractylodis, 17 parts of Chinese date, 15 parts of white hyacinth bean, 6.75 parts of pawpaw, 6.75 parts of morinda officinalis, 6.5 parts of rhizoma arisaematis, 12 parts of snakegourd fruit, 12.5 parts of winter jasmine flower, 19.5 parts of microcos paniculata, 9 parts of fructus akebiae, 4.5 parts of edible tulip, 12 parts of bauhinia championii, 3.75 parts of lobular lotus, 4.5 parts of acorus calamus and 9 parts of henbane.
Furthermore, the invention also provides application of the traditional Chinese medicine composition in preparing a medicine for treating pancreatic cancer.
Wherein, preferably, auxiliary materials required by preparation forming are added into the traditional Chinese medicine composition, and various clinically suitable medicinal preparations are prepared according to a conventional method of medicinal preparations.
Wherein, the preferred medicinal preparation is water decoction, capsule, pill, granule, powder, tablet or oral liquid.
Furthermore, the invention also provides a method for preparing the traditional Chinese medicine composition, which comprises the following steps:
(1) weighing the raw materials in parts by weight;
(2) mixing the raw materials weighed in the step (1), adding distilled water with the weight 8-15 times of the total weight of the raw materials, soaking for 1.5-3h, decocting for 1.5-3h, and filtering to obtain a first traditional Chinese medicine composition filtrate and residues;
(3) adding distilled water with the same volume as that of the residue of the traditional Chinese medicine composition obtained in the step (2) into the residue of the traditional Chinese medicine composition obtained in the step (2), soaking the residue of the traditional Chinese medicine composition for 1.5-3h, decocting the residue of the traditional Chinese medicine composition for 1.5-3h, and filtering to obtain a second filtrate of the traditional Chinese medicine composition;
(4) mixing the first and second Chinese medicinal composition filtrates, and mixing to obtain decoction of the Chinese medicinal composition.
In the method, preferably, the obtained decoction is placed in an evaporation container, and the evaporation container is placed in a laboratory electric heating constant temperature air blast drying oven for drying at 72 ℃ for 6d, 8 times per day, and 3 hours at intervals each time to obtain dry extract of the traditional Chinese medicine composition; pulverizing the obtained dry extract of Chinese medicinal composition with conventional pulverizer, filtering the pulverized material with 100 mesh 304 stainless steel sieve for 2 times, and 400 mesh 304 stainless steel sieve for 2 times to obtain dry powder of Chinese medicinal composition, subpackaging, and storing in refrigerator at-80 deg.C.
In the method, preferably, the first filtration is performed by sequentially filtering 6 layers of white gauze and 17 layers of white gauze, and the second filtration is performed by sequentially filtering 8 layers of white gauze and 19 layers of white gauze.
Compared with the prior art, the invention has the beneficial effects that:
the inventor selects Chinese yam, rhizoma atractylodis, Chinese date, white hyacinth bean, papaya, morinda officinalis, arisaema cum bile, trichosanthes kirilowii Maxim, winter jasmine, microcos paniculata, fructus akebiae, rhizoma anemones altaicae, lobular chinensis, acorus calamus and herba aristolochiae to form the traditional Chinese medicine composition for treating pancreatic cancer according to the theoretical basis of traditional Chinese medicine and by combining the disease pathogenesis of pancreatic cancer, wherein the Chinese yam, the rhizoma atractylodis, the Chinese date, the white hyacinth bean, the papaya and the morinda officinalis can play the effects of tonifying spleen and removing dampness, the arisaema cum bile, the trichosanthes kirilowii Maxim, the winter jasmine and the paniculate microcos stem can play the effects of eliminating phlegm and clearing heat, the akebia paniculata, the bulbus appendiculata, the caulis piperis and. Comprehensively shows that the traditional Chinese medicine composition has the effects of strengthening spleen, promoting diuresis, eliminating phlegm, clearing heat, removing blood stasis, activating blood circulation, warming stomach, regulating qi and the like. The invention provides a new and effective technical means for treating pancreatic cancer.
Drawings
FIG. 1 shows the effect of the Chinese medicinal composition on the growth of BxPC-3 cell line of pancreatic cancer;
note: comparison with model groups:*P<0.05，#P＞0.05；
FIG. 2 is a graph showing the effect of a Chinese medicinal composition on the scratch distance of a pancreatic cancer BxPC-3 cell line;
note: comparison with model groups:*P<0.05，#P＞0.05；
FIG. 3 shows the effect of the Chinese medicinal composition on tumor mass of pancreatic cancer BxPC-3 nude mouse transplantation tumor model mouse;
note: comparison with model groups:*P<0.05，#P＞0.05；
FIG. 4 shows the effect of the Chinese medicinal composition on spleen index of nude mouse transplanted tumor model mouse with pancreatic cancer BxPC-3;
note: comparison with model groups:*P<0.05，#P＞0.05；
FIG. 5 shows the effect of the Chinese medicinal composition on the cell cycle of a pancreatic cancer BxPC-3 nude mouse transplantation tumor model;
note: comparison with model groups:*P<0.05，#P＞0.05；
FIG. 6 shows the effect of the Chinese medicinal composition on the apoptosis rate of pancreatic cancer BxPC-3 nude mouse transplantation tumor model cells;
note: comparison with model groups:*P<0.05；
FIG. 7 shows the effect of the Chinese medicinal composition on the expression of key molecules Shh, Ptch1, Smo and Gli1 of a pancreatic cancer BxPC-3 nude mouse transplantation tumor model Hedgehog signal pathway;
note: comparison with model groups:*P<0.05，#P＞0.05；
FIG. 8 shows the effect of the Chinese medicinal composition on the relative expression of Shh, Ptch1, Smo and Gli1 proteins, which are key molecules of a pancreatic cancer BxPC-3 nude mouse transplantation tumor model Hedgehog signal pathway;
note: comparison with model groups:*P<0.05，#P＞0.05；
FIG. 9 shows the effect of the Chinese medicinal composition on the Shh, Ptch1, Smo and Gli1 genes, which are key molecules of a Hedgehog signal pathway of a pancreatic cancer BxPC-3 nude mouse transplantation tumor model;
note: comparison with model groups:*P<0.05，#P＞0.05；
FIG. 10 shows the effect of the Chinese medicinal composition on the vital organ index of a pancreatic cancer BxPC-3 nude mouse transplantation tumor model;
note: comparison with model groups:*p is more than 0.05; in comparison with the high-dose group,#P＞0.05；
FIG. 11 shows the effect of the Chinese medicinal composition on the body weight of nude mouse transplanted tumor model mice with pancreatic cancer BxPC-3.
Note: comparison with model groups:*p is more than 0.05; in comparison with the high-dose group,#P＞0.05。
Detailed Description
The invention will be further described with reference to specific examples, the advantages and features of which will become clearer from the following description, which are given for illustrating the invention and do not set any limit to the scope of protection of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
EXAMPLE 1 preparation of the Chinese medicinal composition
The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 22.5 parts of Chinese yam, 6 parts of rhizoma atractylodis, 17 parts of Chinese date, 15 parts of white hyacinth bean, 6.75 parts of pawpaw, 6.75 parts of morinda officinalis, 6.5 parts of rhizoma arisaematis, 12 parts of snakegourd fruit, 12.5 parts of winter jasmine flower, 19.5 parts of microcos paniculata, 9 parts of fructus akebiae, 4.5 parts of edible tulip, 12 parts of bauhinia championii, 3.75 parts of lobular lotus, 4.5 parts of acorus calamus and 9 parts of henbane.
EXAMPLE 2 preparation of the Chinese medicinal composition
The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 20 parts of Chinese yam, 4 parts of rhizoma atractylodis, 15 parts of Chinese date, 12 parts of white hyacinth bean, 8 parts of pawpaw, 5 parts of morinda officinalis, 6 parts of rhizoma arisaematis, 14 parts of snakegourd fruit, 10 parts of winter jasmine flower, 25 parts of microcos paniculata, 10 parts of fructus akebiae, 2 parts of edible tulip, 15 parts of bauhinia stem, 5 parts of lobular lotus, 2 parts of acorus calamus and 10 parts of henbane.
EXAMPLE 3 preparation of the Chinese medicinal composition
The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 25 parts of Chinese yam, 8 parts of rhizoma atractylodis, 20 parts of Chinese date, 20 parts of white hyacinth bean, 5 parts of pawpaw, 8 parts of morinda officinalis, 7 parts of rhizoma arisaematis, 10 parts of snakegourd fruit, 15 parts of winter jasmine flower, 15 parts of microcos paniculata, 5 parts of fructus akebiae, 5 parts of edible tulip, 10 parts of bauhinia stem, 2 parts of lobular lotus, 7 parts of calamus and 5 parts of henry.
EXAMPLE 4 preparation of the Chinese medicinal composition (Dry powder)
(1) Weighing the following raw materials in parts by weight:
22.5g of Chinese yam, 6g of rhizoma atractylodis, 17g of Chinese date, 15g of white hyacinth bean, 6.75g of pawpaw, 6.75g of morinda officinalis, 6.5g of rhizoma arisaematis, 12g of snakegourd fruit, 12.5g of winter jasmine, 19.5g of microcos paniculata, 9g of fructus akebiae, 4.5g of edible tulip, 12g of bauhinia stem, 3.75g of lobular nelumbinis, 4.5g of acorus calamus and 9g of henbane, and 16 raw medicinal materials in total are prepared, wherein the weight of each raw medicinal material of the Chinese medicinal composition is 167.25g, and 4 times of the weight of the Chinese medicinal composition (669 g of the raw medicinal materials) is.
(2) Putting 669g of the traditional Chinese medicine composition into a 15000mL middle-sized round-bottomed flask for experiment, adding 7500mL of double distilled water, soaking 669g of the traditional Chinese medicine composition into 7500mL of double distilled water for 2.5h, then decocting the traditional Chinese medicine composition for 2.5h by using 7500mL of double distilled water, and filtering through 6 layers of white medical gauze and 17 layers of white medical gauze respectively to obtain a first traditional Chinese medicine composition filtrate and residue of 669g of the traditional Chinese medicine composition raw materials.
(3) Putting traditional Chinese medicine composition residues obtained after filtering a first traditional Chinese medicine composition of 669g of raw traditional Chinese medicine composition into a 15000mL middle-sized round-bottom flask for experiments, adding 7500mL of double distilled water, soaking the traditional Chinese medicine composition residues obtained after filtering the first traditional Chinese medicine composition for 2.5h, decocting for 2.5h, and filtering through 8 layers of white medical gauze and 19 layers of white medical gauze respectively to obtain a second traditional Chinese medicine composition filtrate.
(4) Mixing the first Chinese medicinal composition filtrate and the second Chinese medicinal composition filtrate;
(5) after the two are mixed uniformly, all filtrate is placed in an evaporation container, the evaporation container is placed in a laboratory electric heating constant temperature air blast drying oven (Tester apparatus Co., Ltd., Tianjin) for drying for 6d (the temperature is 72 ℃), the evaporation condition of the liquid in the drying oven is checked for 8 times per day (3 h intervals each time), 201.32g of traditional Chinese medicine composition dry paste which is equivalent to 669g of the traditional Chinese medicine composition raw medicinal material is finally obtained, the cream yield of the traditional Chinese medicine composition which is equivalent to 669g of the traditional Chinese medicine composition is (the traditional Chinese medicine composition dry paste (g)/the traditional Chinese medicine composition raw medicinal material (g)) x 100%, and (201.32g/669g) x 100% is obtained by calculation according to a formula and is 30.09%, and the cream yield of the traditional Chinese medicine composition which is equivalent to 669g of the traditional Chinese medicine composition is 30.09%.
(6) Thoroughly crushing the dry paste of the traditional Chinese medicine composition obtained in the step (5) by using a conventional crusher, filtering the crushed material for 2 times by using a 304 stainless steel screen mesh (100 meshes), and filtering the crushed material for 2 times by using a 304 stainless steel screen mesh (400 meshes), so as to obtain 195.27g of dry powder of the traditional Chinese medicine composition which is equivalent to 669g of the raw medicinal material of the traditional Chinese medicine composition, wherein the powder yield of the traditional Chinese medicine composition which is equivalent to 669g of the raw medicinal material is [ dry powder (g) of the traditional Chinese medicine composition)/raw medicinal material (g) of the traditional Chinese medicine composition ] x 100%, and the powder yield of the traditional Chinese medicine composition which is equivalent to 669g of the raw medicinal material of the traditional Chinese medicine composition is 29.19% by calculating (195.27g/669 g.
The weight of raw medicinal materials of the traditional Chinese medicine composition is 669g, 201.32g of dry traditional Chinese medicine composition paste and 195.27g of dry traditional Chinese medicine composition powder are finally obtained, namely 6.05g of loss of the dry traditional Chinese medicine composition powder after crushing by a crusher and filtering by a screen, and the loss rate of the traditional Chinese medicine composition is [ (201.32g-195.27g)/201.32g ] x 100% to 3.01% by calculation, so that the loss rate of the traditional Chinese medicine composition is 3.01% when the weight of the raw medicinal materials of the traditional Chinese medicine composition is 669 g.
Finally, 669g of raw medicinal materials of the traditional Chinese medicine composition 4 times is decocted by double distilled water to obtain 195.27g of dry powder of the traditional Chinese medicine composition, namely 1g of the dry powder of the traditional Chinese medicine composition is equivalent to 3.43g of the raw medicinal materials of the traditional Chinese medicine composition. 195.27g of the dry powder of the traditional Chinese medicine composition is subpackaged by 20 g/bag, and finally 15.27g of the dry powder of the traditional Chinese medicine composition is packaged by 1 bag, and 10 bags are counted, and the weight of the dry powder of the traditional Chinese medicine composition in each bag is marked and stored in a refrigerator at-80 ℃ for standby.
Example 5 application of the Chinese medicinal composition in the treatment of pancreatic cancer
1 Material
1.1 animals
70 male BALB/c nude mice for 4-6 weeks for experiments, the body mass (18 +/-2) g, purchased from Splendid Wushuliang, anatomical textroom of Wushuliang, university of Harbin medical university, the basic medical college, Shanghai Si lake animal experiment Limited, the permission number of animals for experiments is SCXK (Shanghai) 2012-0002, and all animals for experiments are bred and administrated by gavage of Lilong Sujongyuan of safety evaluation center of Heilongjiang traditional Chinese medicine university under the conventional aseptic condition (SPF level) at the appropriate temperature and humidity of the medicine safety evaluation center of Heilongjiang traditional Chinese medicine university.
1.2 drugs
Example 4 the resulting dry powder was prepared. In addition, the positive control drug 5-fluorouracil of the present invention was purchased from Sigma, Inc. of America by Zhang Yingbo auxiliary professor of the Qizihaar medical college.
1.3 reagents
RPMI-1640 medium, trypsin, was purchased from HyClone; fixing powder (CW0062), high-sensitivity chemiluminescence detection kit (CW0049A), anti-GAPDH (CW0100A) were purchased from Bio-technologies, Inc., of the century, Kangji; the BxPC-3 human pancreatic cancer cell strain is purchased from the cell resource center of Shanghai Life science research institute of Chinese academy of sciences; medical X-ray film (034402311) was purchased from Xiamen Keke medical devices, Inc.; RNeasy Mini kit was purchased from QIAGEN; Anti-Shh (2207), Anti-Gli1(3538) from INT, USA; Anti-Ptch1(ab39266), Anti-Smo (ab72130) were purchased from abcam, USA; SDS (L5750), Tween20(P1379) was purchased from Sigma; PrimeScript RT kit was purchased from TaKaRa Biotechnology, Inc., Dalian, China; protein extraction reagent A (P0028-1) was purchased from Biyuntian; other domestic analytical purities are provided by professor yingbo of the ziqihaar medical college.
1.4 instruments
The electric heating constant temperature air blast drying oven is purchased from Tester instruments ltd of Tianjin; the HMIAS high-definition color medical image-text analysis system is purchased from Wuhan thousand-screen image technology limited company; the PLZOZ-S electronic balance was purchased from Mettler-Torledo instruments, Inc.; SIMF124 ice maker is available from SANYO corporation, japan; tissue slide machines are available from Leica, germany; TGL-16G desk centrifuge was purchased from Shanghai' an pavilion scientific Instrument factory; tissue hydroextractor available from Leica, germany; flow cytometry was purchased from BD, usa; the protein marking electrophoresis apparatus is purchased from six instrument factories in Beijing, China; CO 22Incubators were purchased from Thermo corporation, usa; the clean bench is purchased from Harr instruments manufacturing company, Beijing Dong, China; the laboratory animal gas exchanger was purchased from Suzhou instruments and Equipment works; real-time quantitative PCR detectors were purchased from Burley, USA, and the like.
2 method
2.1 setting of dosage of Chinese medicinal composition
The traditional Chinese medicine composition comprises 16 raw medicinal materials, namely 22.5g of Chinese yam, 6g of rhizoma atractylodis, 17g of Chinese date, 15g of white hyacinth bean, 6.75g of pawpaw, 6.75g of morinda officinalis, 6.5g of rhizoma arisaematis, 12g of snakegourd fruit, 12.5g of winter jasmine flower, 19.5g of microcos paniculata, 9g of fructus akebiae, 4.5g of Indian iphigenia bulb, 12g of bauhinia championii, 3.75g of lobular lotus, 4.5g of acorus calamus and 9g of henryvine, wherein the weight of each raw medicinal material of the traditional Chinese medicine composition is 167.25 g/adult standard weight. 167.25 g/adult standard weight is the conventional dosage identified by Zhou Zhong Zhi Guang professor of Xue Longjiang university of traditional Chinese medicine and the Liming subsidiary major ren physician of the endocrinology department of the first hospital affiliated to Xue Longjiang university of traditional Chinese medicine. The 167.25 g/adult standard body weight is calculated according to the ratio of the surface area of the mouse to the body (0.0026: 1), and the dosage of the traditional Chinese medicine composition is (167.25g × 0.0026)/0.02kg is 21.74g/kg, thus the dosage of the traditional Chinese medicine composition is 21.74 g/kg. According to the traditional Chinese medicine composition, the high dose is 2 times of the dose in the traditional Chinese medicine composition, and the low dose is 1/2 times of the dose in the traditional Chinese medicine composition, so that the experiment sets that the high dose of the traditional Chinese medicine composition is (21.74g/kg) × 2 ═ 43.48g/kg, and the low dose of the traditional Chinese medicine composition is (21.74g/kg)/2 ═ 10.87 g/kg. Finally, the invention respectively sets the high, medium and low dosages of the traditional Chinese medicine composition to be 43.48g/kg, 21.74g/kg and 10.87 g/kg.
2.2 setting of Positive drug dose
The conventional dose of the positive drug 5-fluorouracil is 150-300 mg, the calculation is carried out according to the ratio of the body surface area of a mouse to the body surface area of a human being (0.0026: 1), the minimum value of the conventional dose of the positive drug 5-fluorouracil is (150 × 0.0026 × 50) mg/kg ═ 19.5mg/kg and the maximum value of the conventional dose of the positive drug 5-fluorouracil is (300 × 0.0026 × 50) mg/kg ═ 39mg/kg, after the conventional dose of the positive drug is converted into unit g, the minimum value of the conventional dose of the positive drug 5-fluorouracil is 0.0195g/kg and the maximum value of the conventional dose of the positive drug 5-fluorouracil is 0.039g/kg, the evaluation of the chief physician in emitsuki professor the peri of the university of Heilongjiang medicine and the Limingming assistant in the first hospital of the department of Heilo.
2.3 cell culture
The experiment takes out a freezing tube filled with pancreatic cancer BxPC-3 cell strain (provided by Shanghai Life science research institute of China academy of sciences) and puts the tube in water with the temperature of 37 ℃ for rapid melting, uses a TGL-16G desktop centrifuge (Shanghai Tingting scientific Instrument factory) for centrifugation at 2000rpm/10min to discard freezing solution, adds 10 times of precooled RPMI1640 for complete culture solution for washing 1 time, inoculates the tube in a culture bottle, puts the tube in 5% CO2CO at 37 ℃ and saturated humidity2Culturing in incubator (Thermo corporation, USA) and changing the culture solution every other day. After the pancreatic cancer BxPC-3 cell strain is cultured to form a monolayer and the pancreatic cancer BxPC-3 cell strain is in a logarithmic growth phase, the pancreatic cancer BxPC-3 cell strain is digested by 0.25% trypsin (containing EDTA), passaged and collected.
2.4 measurement of cell growth
Can be worn according to trypan blueCells that are not stained by trypan blue, i.e. viable cells, can be distinguished by penetrating the cell membrane of dead or necrotic cells and binding DNA features. The experiment adopts trypan blue staining to detect the growth of the pancreatic cancer BxPC-3 cell strain, identify living cells and evaluate the initial effect of the medicament on the pancreatic cancer BxPC-3 cell strain. Experiment pancreatic cancer BxPC-3 cell strain single cell suspension in logarithmic growth phase is taken, trypsinized and washed for 3 times by phosphate buffer solution to prepare pancreatic cancer BxPC-3 cell strain, and the concentration of the cells is adjusted to be 1 multiplied by 104Selecting 300 μ L of each cell/mL, inoculating in 24-well culture plate, adding different drugs, and adding 5% CO237 ℃ sterile CO2Incubators (Thermo corporation, usa) culture 1, 2, 3, 4, 5, 6, 7 d. Taking single cell suspension every d, and according to the ratio of the single cell suspension to trypan blue of 9: 1, adding 4% trypan blue for mixing, counting within 3min, collecting data for statistical analysis, and evaluating the growth condition of the pancreatic cancer BxPC-3 cell strain.
2.5 measurement of cell motility
In the experiment, a pancreatic cancer BxPC-3 cell strain in logarithmic phase is taken, digested by trypsin and washed for 3 times by phosphate buffer solution to prepare a pancreatic cancer BxPC-3 cell strain single-cell suspension, and the concentration of the cells is adjusted to be 1 multiplied by 105Individual cells/mL. The black pen is arranged at the back of the 6-hole culture plate, transverse lines are drawn at intervals of 0.8cm, and at least 3 lines pass through each hole. Pancreatic cancer BxPC-3 cell line was added to each well at approximately 1X 105cells/mL, after 1 day of incubation, were perpendicular to the back of the 6-well plate and scored using crosshatching. Cells were washed 3 times/well with phosphate buffer, and pancreatic cancer BxPC-3 cell lines were removed in 5% CO237 ℃ sterile CO2Incubators (Thermo corporation, usa). The control group was added with normal complete medium and the other groups were added with different drugs and cultured for 2 d. Cell samples are taken at 0h, 24h and 48h, scratches are photographed, the distance between the scratches of the pancreatic cancer BxPC-3 cell line is measured, and the motor capacity of the pancreatic cancer BxPC-3 cell line can be evaluated by using the healing degree of the cells of the pancreatic cancer BxPC-3 cell line.
Calculating the healing degree of the cells: degree of cell healing (value) ═ l- (48h scratch distance/0 h scratch distance).
2.6 modeling
Reference is made to the article published by the Zhou Zhong Xue Guang professor of the university of medicine in Heilongjiang (Zhou ZG, Zhang CY, Fei HX, et al. Phenolic alkaloids from Menispermum dauricum inhibitions BxPC-3 cosmetic cancer cells by blocking of Hedgehog signalling pathway [ J]Pharmacogn Mag, 2015, 11 (44): 690 and 697.) establishing a pancreatic cancer BxPC-3 transplantation tumor model. Pancreatic cancer BxPC-3 cell lines were collected in this experiment and the concentration was adjusted to 5X 10 in RPMI1640 complete medium6pieces/mL of cell suspension are ready for use. Each mouse is 0.2mL (about 5X 10 cells)6Individually) inoculating the rat to the right axillary part of a nude mouse to establish a pancreatic cancer BxPC-3 transplantation tumor model. Meanwhile, a blank group (only physiological saline is injected) is set to be subcutaneous in the right axilla of the nude mice, and the model of the pancreatic cancer BxPC-3 transplantation tumor is further verified. When pancreatic cancer BxPC-3 cell line (5X 10)6One cell per mL) of the tumor cells are inoculated to 7-10 d of nude mice, and no flat or round tumor cells appear under the right axilla of the nude mice in the blank group, which indicates that the pancreatic cancer BxPC-3 transplantation tumor model is successfully established.
2.7 animal groups
The experiment determines 50 pancreatic cancer BxPC-3 transplanted tumor models, and the 50 pancreatic cancer BxPC-3 transplanted tumor model mice are randomly divided into five groups of 10 mice/group by adopting a random digital table, wherein the five groups comprise a model group, a traditional Chinese medicine composition high-dose group (short for a high-dose group), a traditional Chinese medicine composition medium-dose group (short for a medium-dose group), a traditional Chinese medicine composition low-dose group (short for a low-dose group) and a 5-fluorouracil group. In addition, 10 nude mice with the same background and the same age need to be selected as a control group in the experiment.
2.8 animal dosing
Model group: inoculation of pancreatic cancer BxPC-3 cell line 5X 106Per mL, perfusing equal volume of normal saline through the oral cavity of a mouse; high dose group of Chinese medicinal composition: inoculation of pancreatic cancer BxPC-3 cell line 5X 106The amount per mL is 43.48g/kg of the traditional Chinese medicine composition by intrastomatic administration through the oral cavity of a mouse; the traditional Chinese medicine composition comprises the following dosage groups: inoculation of pancreatic cancer BxPC-3 cell line 5X 106The amount per mL is 21.74g/kg of the traditional Chinese medicine composition by intrastomatic gavage of the oral cavity of a mouse; the low-dose group of the traditional Chinese medicine composition comprises: inoculation of pancreatic cancer BxPC-3 cell line 5X 106Small root/mL, small diameterThe traditional Chinese medicine composition for intragastric administration in the oral cavity of the mouse is 10.87 g/kg; 5-fluorouracil group: inoculation of pancreatic cancer BxPC-3 cell line 5X 106And each mouse is coated with positive 5-fluorouracil 0.03g/kg through oral cavity. The experiment is continuously administrated for 28 days by oral gavage of mice.
2.9 determination of tumor inhibition Rate and spleen index
After each group of pancreatic cancer BxPC-3 transplantation tumor model mice are subjected to drug intervention for 28 days, the mice are weighed by a PLZOZ-S electronic balance (Mettler-Torledo instruments, Inc.) on the next day, the pancreatic cancer BxPC-3 transplantation tumor model mice are killed by neck removal, sterilized by alcohol, the tumor bodies and the spleens of the pancreatic cancer BxPC-3 transplantation tumor model mice are completely peeled off by sterile operation on a super clean operation table (Beijing Toyobo instruments, Inc. in China), the mice are washed by normal saline, and the mice are dried by sucking through filter paper, and the tumor and the spleen weight of the pancreatic cancer BxPC-3 transplantation tumor model mice are weighed.
Calculating the tumor inhibition rate: tumor inhibition rate (%) (tumor mass of model group-tumor mass of experimental group)/average tumor mass of model group × 100%;
calculating the spleen index: spleen index (mg/g) ═ spleen mass (mg)/mouse body weight (g).
2.10 determination of the cell cycle
After the experiment is finished, tumor tissue blocks of various groups of pancreatic cancer BxPC-3 transplantation tumor model mice are cut, the tumor bodies are completely stripped in an ultra-clean operating platform (Beijing Toyobo Harr instruments manufacturing Co., Ltd.) through aseptic operation, the fiber envelopes on the surfaces of the tumors are removed, the tumor bodies are placed on an ice tray, the ice tray is cut into pieces and ground, the tumor body single cell suspension of the pancreatic cancer BxPC-3 transplantation tumor model mice is prepared, and the cell concentration is adjusted to be 2 multiplied by 106and/mL. Cells were seeded in 6-well plates and treated with the traditional Chinese medicine composition, 5-fluorouracil or normal saline for 24 h. And fixed with 70% ethanol overnight at-20 ℃ and washed. Adding 1.6mL (1mg/mL) of RNase into 1mL of suspension, incubating in a water bath at 37 ℃ for 30min, centrifuging at 1500rpm/min for 5min, adding 0.4mL of phosphate buffer into the precipitate, and shaking to mix uniformly. The tumor cell cycle of mice, a model of BxPC-3 transplanted tumor, was analyzed by staining with propidium iodide (100. mu.g/mL; Sigma-Aldrich) for 10min and detecting with a flow cytometer (BD Co., USA).
2.11 determination of apoptosis
After the experiment, tumor tissue blocks of mice as pancreatic cancer BxPC-3 transplantation tumor model were cut out on an ultra-clean operating platform (Beijing Toyobo Harr instruments manufacturing Co., Ltd., China), single cell suspension of pancreatic cancer BxPC-3 transplantation tumor was prepared, and the cell concentration was adjusted to 2X 106and/mL, inoculating the cells in a 6-well plate, treating with the traditional Chinese medicine composition, 5-fluorouracil or normal saline for 24h, adding a fluorescent staining solution, incubating for 30min, and keeping out of the sun and vibrating occasionally. The mixture was purified according to the manufacturer's protocol using Annexin-V: FITC apoptosis detection kit I determines the tumor cell apoptosis level of pancreatic cancer BxPC-3 transplanted tumor model mice.
2.12 immunohistochemical detection of protein expression
After the experiment is finished, tumor tissues of a pancreatic cancer BxPC-3 transplantation tumor model mouse are obtained on an ultra-clean operating platform (China Beijing Dong Union Harr apparatus manufacturing Co., Ltd.), 10% formalin is fixed for 4H, gradient ethanol is dehydrated, dimethylbenzene is transparent for 30min, soft wax is soaked for 1H at 45 ℃, hard wax is soaked for 1H at 55 ℃, an embedding device is used for embedding, a microtome is used for cutting the tissues into sections with the thickness of 4 mu m, poly-L-lysine glass slides are fished out, sheets are baked for 12H at 60 ℃, dimethylbenzene is dewaxed to water, and 3% H2O2Incubating at room temperature for 10min, washing with distilled water, heating and repairing by a microwave oven for 5min, cooling at room temperature, incubating at the serum room temperature for 20min, discarding the serum, dropwise adding a primary antibody according to a reagent specification, incubating at 37 ℃ for 2h, washing with PBS for 5min, adding a secondary antibody, incubating at 37 ℃ for 30min, washing with PBS for 3min, developing with DAB for 10min, washing with running water, counterstaining for 5min, dehydrating, transparentizing, sealing, observing the expression levels of key molecules Shh, Ptch1, Smo and Gli1 of tumor Hedgehog signal pathway of a pancreatic cancer BxPC-3 transplantation tumor model mouse under a mirror, and analyzing the OD values of the key molecules Shh, Ptch1, Smo and Pti 1 of the Hedgehog signal pathway by adopting an HMIAS high-definition color medical graphic and graphic system (Wuhan Qian Qianzan imaging technology, Co., Ltd.).
2.13 Western blot protein expression detection
After the experiment is finished, taking tumor tissues of a pancreatic cancer BxPC-3 transplanted tumor model mouse, homogenizing on ice, centrifuging at 4 ℃, centrifuging at 12000rpm for 10min, preserving a protein sample at-20 ℃ in a refrigerator, quantifying the protein by adopting a BCA method, calculating the protein concentration on a standard curve, performing denaturation in boiling water for 5min, subpackaging at-80 ℃ for preservation, preparing SDS polyacrylamide gel, performing electrophoresis at 75V for 1h, balancing after electrophoresis for 20min, performing electrotransformation at 75V for 2h, and sealing at 4 ℃ overnight. Adding a primary antibody: GAPDH (1: 3000), Shh (1: 800), Ptch (1: 1000), Smo (1: 2000), Gli1 (1: 2000), incubated at 37 ℃ for 2 h. Removing primary antibody and washing. Secondary antibody was added and incubated at 37 ℃ for 1 h. Removing the secondary antibody and washing. The relative expression quantity of key molecules Shh, Ptch1, Smo and Gli1 of a pancreatic cancer BxPC-3 transplantation tumor model mouse Hedgehog signal channel is analyzed by an HMIAS high-definition color medical graphic system (Wuhan thousand-screen image technology, Inc.) after exposure, development, fixation and rinsing with clear water in a dark room.
2.14 RT-PCR detection of Gene expression
After the experiment is finished, tumor tissues of pancreatic cancer BxPC-3 transplantation tumor model mice are taken to extract mRNA according to the instructions of a Trizol kit. The primer is synthesized and designed by Shanghai biological engineering company, wherein GAPDH: upstream 5'-AGA AGG CTG GGG CTC ATT TG-3', downstream 5'-AGG GGC CAT CCA CAG TCT TC-3'; shh: upstream 5'-GCT CGG TGA AAG CAG AGA ACT-3', downstream 5'-CCA GGA AAG TGA GGA AGT CG-3'; smo: upstream 5'-CTG GTG TGG TTT GGT TTG TG-3', downstream 5'-CAG GTG GAA GTA GGA GGT CTT G-3'; ptch 1: upstream 5'-CTC CTT TGC GGT GGA CAA-3', downstream 5'-CCT CAG CCT TAT TCA GCA TTT C-3'; gli 1: upstream 5'-ATC CTT ACC TCC CAA CCT CTG T-3', downstream 5'-CCA ACT TCT GGC TCT TCC TGT A-3'. Preparing a PCR reaction system according to the procedure of the kit specification, and measuring by using a real-time quantitative PCR detector (Berle company, USA)
Figure BDA0001661270740000181
Method data processing experimental data and analysis of relative mRNA expression of key molecules Shh, Ptch1, Smo and Gli1 of tumor Hedgehog signaling pathway of pancreatic cancer BxPC-3 transplanted tumor model mice.
2.15 measurement of body weight and organ index
After the detection of each group of pancreatic cancer BxPC-3 transplantation tumor model mice is finished, the weight of the pancreatic cancer BxPC-3 transplantation tumor model mice is weighed by using a PLZOZ-S electronic balance (Mettler-Torledo instruments, Ltd.), and the weight data change of the pancreatic cancer BxPC-3 transplantation tumor model mice in the administration process is counted and analyzed. After the experiment, the mice were picked up, decapitated, sacrificed, dissected on an ultraclean operating table (beijing donghong haar instruments manufacturing ltd, china), organs such as brain, heart, lung, kidney, and liver of the pancreatic cancer BxPC-3 transplantation tumor model mice were obtained, washed, and water was drained, the organ mass was weighed using a PLZOZ-S electronic balance (mettler-toledo instruments ltd), and the organ index of the pancreatic cancer BxPC-3 transplantation tumor model mice was calculated according to the organ index calculation formula.
Calculating the formula: organ index (mg/g) is organ mass (mg)/mouse body weight (g).
2.16 statistical analysis
SPSS19.0 software is adopted to analyze data, experimental data are expressed by x +/-s, measured data are subjected to normality test analysis, single-factor variance analysis data are adopted for comparison among multiple groups, P <0.05 shows that the difference has statistical significance, and P >0.05 shows that the difference does not have statistical significance.
3 results
3.1 Effect of Chinese medicinal composition on growth of pancreatic cancer BxPC-3 cell line
The results show that compared with the model group, the viable cell count of the pancreatic cancer BxPC-3 cell line is obviously reduced (P is less than 0.05) at the time points of 3, 4, 5, 6 and 7d in the high-dose group, the medium-dose group and the 5-fluorouracil group of the traditional Chinese medicine composition, while the viable cell count of the pancreatic cancer BxPC-3 cell line is not obviously changed (P is more than 0.05) at the time points of 3, 4, 5, 6 and 7d in the low-dose group of the traditional Chinese medicine composition. (see Table 1, FIG. 1).
TABLE 1 Effect of Chinese medicinal composition on growth of pancreatic cancer BxPC-3 cell line: (
Figure BDA0001661270740000191
n＝6，1×104)
Figure BDA0001661270740000192
Note: comparison with model groups:*P<0.05，#P＞0.05
3.2 Effect of Chinese medicinal composition on pancreatic cancer BxPC-3 cell line motility
The results show that compared with the model group, the 48h scratch distance of the pancreatic cancer BxPC-3 cell strain in the high-dose group, the medium-dose group and the 5-fluorouracil group of the traditional Chinese medicine composition is obviously increased (P is less than 0.05), the cell healing degree is obviously reduced (P is less than 0.05), and the 48h scratch distance and the cell healing degree of the pancreatic cancer BxPC-3 cell strain in the low-dose group of the traditional Chinese medicine composition are not obviously changed (P is more than 0.05). (see Table 2, FIG. 2).
TABLE 2 Effect of Chinese medicinal composition on pancreatic cancer BxPC-3 cell line motility: (
Figure BDA0001661270740000193
n＝9)
Figure BDA0001661270740000194
Note: comparison with model groups:*P<0.05，#P＞0.05
3.3 Effect of the Chinese medicinal composition on tumor inhibition rate of pancreatic cancer BxPC-3 nude mouse transplantation tumor model
Results show that compared with a model group, the tumor quality of a pancreatic cancer BxPC-3 nude mouse transplantation tumor model mouse is obviously reduced in a high-dose group of the traditional Chinese medicine composition, a medium-dose group of the traditional Chinese medicine composition, a 5-fluorouracil group and a low-dose group of the traditional Chinese medicine composition (P is less than 0.05). The tumor inhibition rates of the traditional Chinese medicine composition high-dose group, the traditional Chinese medicine composition medium-dose group, the traditional Chinese medicine composition low-dose group and the positive drug 5-fluorouracil group are respectively (47.8372 +/-6.7827)%, (36.8352 +/-8.4470)%, (32.2523 +/-5.3657)%, and (54.7148 +/-7.1300)%. Therefore, the traditional Chinese medicine composition high-dose group, the traditional Chinese medicine composition medium-dose group, the traditional Chinese medicine composition low-dose group and the 5-fluorouracil group all have an inhibiting effect on pancreatic cancer BxPC-3 nude mouse transplantation tumor model mice, and the blank group does not generate pancreatic cancer BxPC-3 nude mouse transplantation tumors. The tumor inhibition effect of the traditional Chinese medicine composition high-dose group and 5-fluorouracil group pancreatic cancer BxPC-3 nude mouse transplantation tumor model mouse is good. (see Table 3, FIG. 3).
TABLE 3 influence of Chinese medicinal composition on tumor mass and tumor inhibition rate of pancreatic cancer BxPC-3 nude mouse transplantation tumor model mice ((
Figure BDA0001661270740000201
n＝10)
Figure BDA0001661270740000202
Note: comparison with model groups:*P<0.05
3.4 Effect of Chinese medicinal composition on spleen index of pancreatic cancer BxPC-3 nude mouse transplantation tumor model
The result shows that compared with the model group, the spleen index of the 5-fluorouracil group pancreatic cancer BxPC-3 nude mouse transplantation tumor model mouse is obviously reduced (P <0.05), while the spleen index of the traditional Chinese medicine composition high-dose group, the traditional Chinese medicine composition medium-dose group and the traditional Chinese medicine composition low-dose group pancreatic cancer BxPC-3 nude mouse transplantation tumor model mouse is not statistically different (P > 0.05). (see Table 4, FIG. 4).
TABLE 4 influence of Chinese medicinal composition on spleen index of nude mouse transplanted tumor model with pancreatic cancer BxPC-3: (
Figure BDA0001661270740000203
n＝8)
Figure BDA0001661270740000204
Note: comparison with model groups:*P<0.05，#P＞0.05
3.5 Effect of Chinese medicinal composition on pancreatic cancer BxPC-3 nude mouse transplantation tumor model cell cycle
The results show that compared with the model group, the G1 phase of the traditional Chinese medicine composition high-dose group, the G1 phase of the traditional Chinese medicine composition medium-dose group and the 5-fluorouracil pancreatic cancer BxPC-3 nude mouse transplantation tumor model mouse is obviously increased (P is less than 0.05), the G2 phase is obviously reduced (P is less than 0.05), and the G1 phase of the traditional Chinese medicine composition low-dose group pancreatic cancer BxPC-3 nude mouse transplantation tumor model mouse is not obviously changed (P is more than 0.05); compared with the model group, the S phase of the pancreatic cancer BxPC-3 nude mouse transplantation tumor model mouse in the traditional Chinese medicine composition high-dose group, the traditional Chinese medicine composition medium-dose group and the 5-fluorouracil group is obviously reduced (P is less than 0.05), and the S phase of the pancreatic cancer BxPC-3 nude mouse transplantation tumor model mouse in the traditional Chinese medicine composition low-dose group is not obviously changed (P is more than 0.05); compared with the model group, the pancreatic cancer BxPC-3 nude mouse transplantation tumor model mouse with the traditional Chinese medicine composition in the high-dose group, the traditional Chinese medicine composition in the medium-dose group and the 5-fluorouracil group has the obviously reduced G2 phase (P is less than 0.05), while the pancreatic cancer BxPC-3 nude mouse transplantation tumor model mouse with the traditional Chinese medicine composition in the low-dose group has no obvious change in the G2 phase (P is more than 0.05). (see Table 5, FIG. 5).
TABLE 5 Effect of Chinese medicinal composition on pancreatic cancer BxPC-3 nude mouse transplantation tumor model cell cycle
Figure BDA0001661270740000211
n＝6)
Figure BDA0001661270740000212
Note: comparison with model groups:*P<0.05，#P＞0.05
3.6 Effect of the Chinese medicinal composition on apoptosis rate of pancreatic cancer BxPC-3 nude mouse transplantation tumor model cells
Results show that the apoptosis rates of pancreatic cancer BxPC-3 nude mouse transplantation tumor model mice in the model group, the traditional Chinese medicine composition high-dose group, the traditional Chinese medicine composition medium-dose group, the traditional Chinese medicine composition low-dose group and the 5-fluorouracil group are respectively (5.2933 +/-0.9249)%, (7.5717 +/-0.6930)%, (8.1983 +/-0.7523)%, (8.1667 +/-0.7392)%, and (8.0067 +/-1.0206)%. Compared with the model group, the apoptosis rates of pancreatic cancer BxPC-3 nude mouse transplanted tumor model cells in the high-dose group, the medium-dose group, the 5-fluorouracil group and the low-dose group of the traditional Chinese medicine composition are all obviously reduced (P is less than 0.05). (see Table 6, FIG. 6).
TABLE 6 influence of Chinese medicinal composition on apoptosis rate of pancreatic cancer BxPC-3 nude mouse transplanted tumor model cell
Figure BDA0001661270740000213
Figure BDA0001661270740000214
Figure BDA0001661270740000221
Note: comparison with model groups:*P<0.05
3.7 Effect of Chinese medicinal composition on pancreatic cancer BxPC-3 nude mouse transplantation tumor model Hedgehog signal pathway key molecules Shh, Ptch1, Smo and Gli1 protein expression
Results show that compared with a model group, the traditional Chinese medicine composition high-dose group, the traditional Chinese medicine composition medium-dose group and the 5-fluorouracil group pancreatic cancer BxPC-3 nude mouse transplanted tumor model Hedgehog signal pathway key molecules Shh, Ptch1, Smo and Gli1 protein expression OD values are obviously reduced (P is less than 0.05), while the traditional Chinese medicine composition low-dose group pancreatic cancer BxPC-3 nude mouse transplanted tumor model Hedgehog signal pathway key molecules Shh, Ptch1, Smo and Gli1 protein expression OD values are not obviously changed (P is more than 0.05). (see Table 7, FIG. 7).
TABLE 7 influence of Chinese medicinal composition on the expression of key molecules Shh, Ptch1, Smo and Gli1 of Hedgehog signaling pathway model of pancreatic cancer BxPC-3 nude mouse transplantation tumor (
Figure BDA0001661270740000222
n is 8, OD value)
Figure BDA0001661270740000223
Note: comparison with model groups:*P<0.05，#P＞0.05
3.8 influence of the Chinese medicinal composition on relative expression of Shh, Ptch1, Smo and Gli1 proteins of key molecules of a pancreatic cancer BxPC-3 nude mouse transplantation tumor model Hedgehog signal pathway
The results show that compared with the model group, the relative expression amounts of the key molecules Shh, Ptch1, Smo and Gli1 of the Hedgehog signal path of the traditional Chinese medicine composition high-dose group, the traditional Chinese medicine composition medium-dose group and the 5-fluorouracil group pancreatic cancer BxPC-3 nude mouse transplanted tumor model are obviously reduced (P is less than 0.05), while the relative expression amounts of the key molecules Shh, Ptch1, Smo and Gli1 of the Hedgehog signal path of the traditional Chinese medicine composition low-dose group pancreatic cancer BxPC-3 nude mouse transplanted tumor model are not obviously changed (P is more than 0.05). (see Table 8, FIG. 8).
TABLE 8 influence of Chinese medicinal composition on relative expression of Shh, Ptch1, Smo and Gli1 proteins, which are key molecules of Hedgehog signal pathway of pancreatic cancer BxPC-3 nude mouse transplantation tumor model (
Figure BDA0001661270740000224
n＝7)
Figure BDA0001661270740000225
Figure BDA0001661270740000231
Note: comparison with model groups:*P<0.05，#P＞0.05
3.9 influence of the Chinese medicinal composition on the Shh, Ptch1, Smo and Gli1 genes of key molecules of a pancreatic cancer BxPC-3 nude mouse transplantation tumor model Hedgehog signal pathway
Results show that compared with a model group, the expression levels of the key molecules Shh, Ptch1, Smo and Gli1mRNA of the Hedgehog signal pathway of the traditional Chinese medicine composition high-dose group pancreatic cancer BxPC-3 nude mouse transplanted tumor model are obviously reduced (P is less than 0.05), while the expression levels of the key molecules Shh, Ptch1, Smo and Gli1mRNA of the Hedgehog signal pathway of the traditional Chinese medicine composition low-dose group pancreatic cancer BxPC-3 nude mouse transplanted tumor model are not obviously changed (P is more than 0.05). (see Table 9, FIG. 9).
TABLE 9 influence of Chinese medicinal composition on the Shh, Ptch1, Smo and Gli1 genes, which are key molecules of Hedgehog signal pathway of pancreatic cancer BxPC-3 nude mouse transplantation tumor model ((
Figure BDA0001661270740000232
n＝7，2-△△Ct)
Figure BDA0001661270740000233
Note: comparison with model groups:*P<0.05，#P＞0.05
3.10 Effect of the Chinese medicinal composition on the important organ index of pancreatic cancer BxPC-3 nude mouse transplantation tumor model
The results show that compared with the model group, the index change of the brain, heart, liver, kidney and lung of the traditional Chinese medicine composition high-dose group, the traditional Chinese medicine composition medium-dose group, the traditional Chinese medicine composition low-dose group and the 5-fluorouracil pancreatic cancer BxPC-3 nude mouse transplantation tumor model is not obvious (P is more than 0.05), and the results show that the high, medium and low doses and the 5-fluorouracil of the traditional Chinese medicine composition have no influence on the important organs of the pancreatic cancer BxPC-3 nude mouse transplantation tumor model; compared with the traditional Chinese medicine composition high-dose group, the indexes of brain, heart, liver, kidney and lung of the BxPC-3 nude mouse transplanted tumor model of pancreatic cancer in the traditional Chinese medicine composition medium-dose group, the traditional Chinese medicine composition low-dose group and the 5-fluorouracil group are not obviously changed (P is more than 0.05), which shows that even if the dosage of the traditional Chinese medicine composition is increased, the traditional Chinese medicine composition has no influence on the indexes of brain, heart, liver, kidney and lung of the BxPC-3 nude mouse transplanted tumor model of pancreatic cancer. (see Table 10, Table 11, FIG. 10).
TABLE 10 influence of Chinese medicinal composition on brain index, heart index and liver index of nude mouse transplanted tumor model of pancreatic cancer BxPC-3: (
Figure BDA0001661270740000241
n＝8)
Figure BDA0001661270740000242
Note: comparison with model groups:*p is more than 0.05; in comparison with the high-dose group,#P＞0.05
TABLE 11 influence of Chinese medicinal composition on renal index and pulmonary index of nude mouse transplanted tumor model of pancreatic cancer BxPC-3: (
Figure BDA0001661270740000243
n＝8)
Figure BDA0001661270740000244
Note: comparison with model groups:*p is more than 0.05; in comparison with the high-dose group,#P＞0.05
3.11 Effect of Chinese medicinal composition on pancreatic cancer BxPC-3 nude mouse transplantation tumor model weight
The results show that in the administration process of each group of pancreatic cancer BxPC-3 nude mouse transplantation tumor model mice, the weight of the pancreatic cancer BxPC-3 nude mouse transplantation tumor model mice shows an ascending trend, which indicates that the high, medium and low doses of the traditional Chinese medicine composition have no influence on the weight of the pancreatic cancer BxPC-3 nude mouse transplantation tumor model mice. Compared with the model group, the weight of the pancreatic cancer BxPC-3 nude mouse transplantation tumor model mice in the traditional Chinese medicine composition high-dose group, the traditional Chinese medicine composition medium-dose group, the traditional Chinese medicine composition low-dose group and the 5-fluorouracil group is not obviously changed (P is more than 0.05), further the condition that the high, medium and low doses of the traditional Chinese medicine composition have no influence on the weight of the pancreatic cancer BxPC-3 nude mouse transplantation tumor model mice is further explained, and the condition that the high, medium and low doses of the traditional Chinese medicine composition and the 5-fluorouracil have no influence on the weight of the pancreatic cancer BxPC-3 nude mouse transplantation tumor model mice is further explained; compared with the traditional Chinese medicine composition high-dose group, the weight of mice of the pancreatic cancer BxPC-3 nude mouse transplanted tumor model in the traditional Chinese medicine composition medium-dose group, the traditional Chinese medicine composition low-dose group and the 5-fluorouracil group is not obviously changed (P is more than 0.05), which indicates that the traditional Chinese medicine composition has no influence on the weight of mice of the pancreatic cancer BxPC-3 nude mouse transplanted tumor model even if the dosage of the traditional Chinese medicine composition is increased. (see Table 12, FIG. 11).
TABLE 12 Effect of Chinese medicinal composition on body weight of nude mice transplanted tumor model with pancreatic cancer BxPC-3: (
Figure BDA0001661270740000251
n＝8)
Figure BDA0001661270740000252
Note: comparison with model groups:*p is more than 0.05; in comparison with the high-dose group,#P＞0.05。

Claims (8)

1. the traditional Chinese medicine composition for treating pancreatic cancer is characterized by comprising the following raw materials in parts by weight: 20-25 parts of Chinese yam, 4-8 parts of rhizoma atractylodis, 15-20 parts of Chinese date, 10-20 parts of white hyacinth bean, 5-8 parts of pawpaw, 5-8 parts of morinda officinalis, 5-8 parts of rhizoma arisaematis, 10-15 parts of snakegourd fruit, 10-15 parts of winter jasmine flower, 15-25 parts of microcos paniculata, 5-12 parts of fructus akebiae, 2-7 parts of edible tulip, 10-15 parts of bauhinia championii, 1-5 parts of lobular lotus, 2-7 parts of acorus calamus and 5-15 parts of herba aristolochiae.
2. The traditional Chinese medicine composition of claim 1, wherein the traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 22.5 parts of Chinese yam, 6 parts of rhizoma atractylodis, 17 parts of Chinese date, 15 parts of white hyacinth bean, 6.75 parts of pawpaw, 6.75 parts of morinda officinalis, 6.5 parts of rhizoma arisaematis, 12 parts of snakegourd fruit, 12.5 parts of winter jasmine flower, 19.5 parts of microcos paniculata, 9 parts of fructus akebiae, 4.5 parts of edible tulip, 12 parts of bauhinia championii, 3.75 parts of lobular lotus, 4.5 parts of acorus calamus and 9 parts of henbane.
3. Use of the Chinese medicinal composition of claim 1 or 2 in the preparation of a medicament for the treatment of pancreatic cancer.
4. The use of claim 3, wherein the Chinese medicinal composition of claim 1 or 2 is added with auxiliary materials required by preparation molding, and prepared into various clinically suitable medicinal preparations according to conventional methods for preparing medicinal preparations.
5. The use of claim 4, wherein the pharmaceutical formulation is a decoction, capsule, pill, granule, powder, tablet or oral liquid.
6. A method of preparing the traditional Chinese medicine composition of claim 1 or 2, comprising the steps of:
(1) weighing the raw materials according to the parts by weight of the raw materials in the claim 1 or 2;
(2) mixing the raw materials weighed in the step (1), adding distilled water with the weight 8-15 times of the total weight of the raw materials, soaking for 1.5-3h, decocting for 1.5-3h, and filtering to obtain a first traditional Chinese medicine composition filtrate and residues;
(3) adding distilled water with the same volume as that of the residue of the traditional Chinese medicine composition obtained in the step (2) into the residue of the traditional Chinese medicine composition obtained in the step (2), soaking the residue of the traditional Chinese medicine composition for 1.5-3h, decocting the residue of the traditional Chinese medicine composition for 1.5-3h, and filtering to obtain a second filtrate of the traditional Chinese medicine composition;
(4) mixing the first and second Chinese medicinal composition filtrates, and mixing to obtain decoction of the Chinese medicinal composition.
7. The method of claim 6, further comprising placing the obtained decoction in an evaporation container, and placing the evaporation container in a laboratory electric heating constant temperature air drying oven for drying at 72 deg.C for 6d, 8 times per day at 3h intervals to obtain dry extract of Chinese medicinal composition; pulverizing the obtained dry extract of Chinese medicinal composition with conventional pulverizer, filtering the pulverized material with 100 mesh 304 stainless steel sieve for 2 times, and 400 mesh 304 stainless steel sieve for 2 times to obtain dry powder of Chinese medicinal composition, subpackaging, and storing in refrigerator at-80 deg.C.
8. The method of claim 6, wherein the first filtration is performed with 6 layers of white gauze followed by 17 layers of white gauze, and the second filtration is performed with 8 layers of white gauze followed by 19 layers of white gauze.
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