CN114668757A - Application of pseudolaric acid B in preparing medicine for preventing and/or treating malignant tumor - Google Patents
Application of pseudolaric acid B in preparing medicine for preventing and/or treating malignant tumor Download PDFInfo
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Abstract
An application of pseudolaric acid in preparing a medicine for preventing and/or treating malignant tumor relates to the field of new medical application. The invention discloses a new application of pseudolaric acid B in preparing a medicine for preventing and/or treating malignant tumor; the malignant tumor includes: brain glioma, liver cancer, breast cancer and ovarian cancer; the concentration of the pseudolaric acid is 12.5-200 umol/L. Experiments prove that the pseudolaric acid B can effectively kill various malignant tumor cells such as human glioma cells, human hepatoma cells, human breast cancer cells, human ovarian cancer cells and the like in a concentration-dependent manner, and has a certain dose-effect relationship; the pseudolaric acid has low toxicity to normal human cells, can activate autophagy flow in human glioma cells, and kills the human glioma cells by activating autophagy flow in the human glioma cells. The invention develops the new application of the pseudolaric acid and provides a new treatment scheme for treating various malignant tumor cells.
Description
Technical Field
The invention relates to the technical field of new medical application, in particular to application of pseudolaric acid B in preparing a medicine for preventing and/or treating malignant tumors.
Background
Cancer, medically commonly referred to as malignant tumor, is a complex and serious disease that seriously threatens human health, and is the second leading cause of human death next to cardiovascular and cerebrovascular diseases worldwide.
Human brain glioma is the most common primary malignant tumor in craniums, can be seen in all age groups, is frequently found in adults, has no obvious difference in male and female morbidity, and has the characteristics of high disability rate, high recurrence rate and high mortality rate. The treatment of human brain glioma mainly adopts surgical excision of tumor, and combines with postoperative radiotherapy and chemotherapy. However, WHO graded grade III and IV gliomas patients have a mean post-operative survival of no more than 1 year. At present, the treatment effect of Temozolomide (Temozolomide), a first-line chemotherapeutic drug adopted in the treatment of human brain glioma, on high-grade brain glioma (WHO is classified into grades III-IV) is not ideal all the time, and compared with the average survival of patients with high-grade brain glioma treated by surgical operation, the average survival of the patients can be prolonged by 3-5 months only by combining Temozolomide treatment after the operation. Therefore, there is a lack of effective drugs for chemotherapy of human brain gliomas.
Liver cancer is divided into primary and secondary types, refers to malignant tumors derived from liver cells and hepatobiliary cells, and is one of common malignant tumors in China. The common symptoms of early liver cancer are not specific, while the symptoms of middle and late liver cancer are more, and the common clinical manifestations are as follows: pain in the liver area, abdominal distension, anorexia, asthenia, emaciation, progressive hepatomegaly or epigastric mass; some patients have low fever, jaundice, diarrhea, upper gastrointestinal hemorrhage, etc. The current methods for treating liver cancer mainly comprise: operation treatment, blood vessel interventional embolization, ablation, radiotherapy, targeted treatment, traditional Chinese medicine treatment and the like. Therefore, there is no effective drug for treating liver cancer.
Breast cancer is a malignant tumor occurring in mammary epithelium or ductal epithelium, the cause of which is not completely clear, and may be related to family history, breast cancer-related genes, reproductive factors, sex hormones, nutrition, diet, environmental factors, and the like. Most of early-stage breast cancers have no obvious symptoms, are difficult to find, are mostly breast painless masses, and have the symptoms of retraction of nipples, breast skin 'dimple disease' or cellulitis, axillary lymphadenectasis and the like at the late stage. The treatment method of the breast cancer mainly comprises the following steps: surgical resection, chemotherapy, radiation therapy, endocrine therapy, targeted therapy. Wherein, the operation treatment is suitable for the patient whose tumor can be completely removed, and whether other treatment means are combined or not is considered according to the pathological stage, the pathological type and the molecular typing; targeted therapy: the gene immunohistochemical method detects more than 23 + HER, or the FISH method detects the HER2 amplification positive person; radiotherapy: irradiating the local and lymph drainage area of the mammary gland by using high-energy X rays; endocrine treatment: tamoxifen, letrozole, are commonly used. Therefore, there is a lack of effective drugs for the treatment of breast cancer.
Ovarian cancer is one of the common malignant tumors of female reproductive organs, and refers to a malignant tumor disease occurring in the ovary, and the incidence rate is second to that of cervical cancer and uterine corpus cancer. Ovarian cancer is the most common of epithelial cancers, and is followed by malignant germ cell tumors, wherein the death rate of ovarian epithelial cancers accounts for the first part of various gynecological tumors, and can cause serious threat to the life of women. Ovarian cancer is asymptomatic in the early stage, digestive tract symptoms such as lower abdominal discomfort, abdominal distension and appetite decrease can appear in the late stage, and the treatment mode mainly comprises the following steps: surgical resection, drug therapy and radiation therapy have a poor overall prognosis. Therefore, there is a lack of effective drugs for the treatment of ovarian cancer.
Pseudolaric acid (PseudolaricAcid C) is a natural small molecule compound extracted from dried root bark or near-root bark of medicinal plant Lysimachia christinae Hance, is white crystal, is insoluble in water, and is easily soluble in organic solvent such as DMSO. The molecular formula is as follows: c21H26O7(ii) a Molecular weight: 390; CAS number: 82601-41-0. Pseudolaric acid (PseudolaricAcid C) has the advantages of easily-accessible raw materials and low cost. Cortex pseudolaricis can be used for treating localized neurodermatitis. However, the use and mechanism of action of the natural small molecule compound pseudolaric acid extracted from pseudolarix is unknown, especially for the malignant tumorThe prevention and/or treatment aspects are not reported.
Disclosure of Invention
The invention aims to provide a new application of pseudolaric acid in preparing a medicine for preventing and/or treating malignant tumors.
The technical scheme adopted by the invention for solving the technical problem is as follows:
the pseudolaric acid B is applied to preparing the medicine for preventing and/or treating malignant tumors.
As a preferred embodiment, the malignant tumor comprises: brain glioma, liver cancer, breast cancer and ovarian cancer.
In a preferred embodiment, the concentration of the pseudolaric acid B is 12.5 to 200 umol/L.
In a more preferred embodiment, the concentration of the pseudolaric acid B is 25 to 50 umol/L.
As the most preferred embodiment, the concentration of the pseudolaric acid is 25 umol/L.
The invention has the beneficial effects that:
experiments prove that the pseudolaric acid B can effectively kill various malignant tumor cells such as human brain glioma cells, human liver cancer cells, human breast cancer cells, human ovarian cancer cells and the like in a concentration-dependent manner, and has a certain dose-effect relationship. Meanwhile, experiments prove that the pseudolaric acid has low toxicity to normal cells of human, can activate autophagy flow in the human glioma cells, and can kill the human glioma cells by activating the autophagy flow in the human glioma cells.
The invention discovers that the pseudolaric acid can prevent and/or treat various malignant tumor cells such as human brain glioma cells, human liver cancer cells, human breast cancer cells, human ovarian cancer cells and the like, develops new application of the pseudolaric acid, and provides a new therapy and a new treatment scheme for treating various malignant tumor cells such as human brain glioma cells, human liver cancer cells, human breast cancer cells, human ovarian cancer cells and the like.
Drawings
FIG. 1 is a graph showing the effect of pseudolaric acid B on the inhibition of proliferation of human brain glioma cell U251 in example 1.
FIG. 2 is a graph showing the effect of pseudolaric acid B on the inhibition of proliferation of human glioma cells U87 in example 1.
FIG. 3 is a graph showing the effect of pseudolaric acid B on the inhibition of proliferation of human glioma cell U118 in example 1.
FIG. 4 is a graph showing the toxic effect of pseudolaric acid B on HUVEC cells in umbilical vein epithelial cells in example 2.
FIG. 5 is the regulation effect of pseudolaric acid B in human brain glioma cell U87 on autophagy-related proteins in example 3.
FIG. 6 is a graph showing the activation of autophagy flux in human brain glioma cells U87 by pseudolaric acid B in example 3.
FIG. 7 is a graph of the inhibition of pseudolaric acid induced human brain glioma cell death by 3MA and bafilomycin A1 in example 3.
FIG. 8 is a graph of the effect of 3MA and bafilomycin A1 on protein induction by pseudolaric acid B in example 3.
FIG. 9 shows the effect of Hibiscus pseudolaricis propionic acid on inhibiting the proliferation of human hepatoma cells in example 4.
FIG. 10 is a graph showing the effect of pseudolaric acid B on the inhibition of proliferation of breast cancer cells in example 5.
FIG. 11 is a graph showing the effect of pseudolaric acid B on the inhibition of human ovarian cancer cell proliferation in example 6.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Material sources are as follows:
the pseudolaric acid B is obtained from Shanghai leaf Biotech limited and is prepared into required concentration with DMSO solution when in use.
Human glioma cells U251, U87, and U118, were purchased from Gickab Biotech, China.
Human umbilical vein epithelial cells HUVEC, purchased from Gyka Biotechnology, China.
Human hepatoma cell Hep3B, breast cancer cell MB-231 and human ovarian cancer cell A2780 were purchased from Chigkat Biotech, China.
Example 1 proliferation inhibitory Effect of Pseudolaric acid Hibisci on human brain glioma cells
Inoculating human brain glioma cells U251, U87 and U118 into DMEM culture medium in a 96-well plate respectively, incubating overnight with carbon dioxide, incubating with pseudolaric acid (12.5umol/L, 25umol/L, 50umol/L, 100umol/L and 200umol/L) at different concentrations for 48 hours, and detecting cell proliferation capacity by using an MTT method, wherein the cell survival rates of U251 are 96.67%, 91.97%, 68.75%, 43.59% and 34.05% respectively as shown in FIG. 1 and Table 1; as shown in fig. 2 and table 2, U87 cell survival rates were 97.19%, 91.73%, 69.27%, 46.51% and 31.94%, respectively; as shown in fig. 3 and table 3, U118 cell viability was 98.36%, 93.04%, 67.57%, 44.42% and 34.60%, respectively.
As can be seen from the test results, the survival rate of the brain glioma cells is correspondingly reduced along with the increase of the concentration of the pseudolaric acid. Thus, it was confirmed that pseudolaric acid can kill brain glioma cells in a concentration-dependent manner.
TABLE 1 proliferation inhibitory effect of pseudolaric acid B on human brain glioma cell U251 (. about.. p <0.01)
TABLE 2 proliferation inhibitory Effect of pseudolaric acid on human brain glioma cells U87 (. about.. p <0.01)
TABLE 3 proliferation inhibitory effect of pseudolaric acid B on human brain glioma cell U118 (p < 0.05;. p <0.01)
Example 2 toxic Effect of Pseudolarix acid on Normal cells
In order to detect the toxic effect of pseudolaric acid B on normal cells, HUVEC of human umbilical vein epithelial cells was inoculated into DMEM medium in a 96-well plate, incubated overnight with carbon dioxide, incubated with pseudolaric acid B (12.5umol/L, 25umol/L, 50umol/L, 100umol/L, 200umol/L) at different concentrations for 48 hours, and then the cell proliferation capacity was detected by MTT method, and the survival rates of HUVEC cells were 100%, 99.15%, 95.84%, 89.80% and 82.42%, respectively, as shown in FIG. 4 and Table 4.
According to the detection result, compared with the survival rates of the human brain glioma cells U251, U87 and U118, the pseudolaric acid with the concentration of 25umol/L and 50umol/L has less toxicity to the human umbilical vein epithelial cell HUVEC, and can effectively kill the human brain glioma cells.
TABLE 4 proliferation inhibition of HUVEC by pseudolaric acid B on human umbilical vein epithelial cells
Example 3 mechanism of action of Pseudolaric acid B on killing human brain glioma cells
(1) Activation effect of pseudolaric acid on autophagic flow in human brain glioma cells
In order to clarify the killing action mechanism of pseudolaric acid on human brain glioma cells, 50umol/L of pseudolaric acid and human brain glioma cells U87 are incubated for 6, 12, 24 and 48 hours, the expression condition of autophagy-related proteins is detected by a Westernblotting method after protein extraction, and the result is shown in figure 5, the pseudolaric acid can up-regulate the expression of protein ATG5 and protein LC3B II in a time-dependent manner, and simultaneously correspondingly inhibits the expression of autophagy substrate protein P62. The red-green tandem fluorescent LC3B protein plasmid is transfected into a human brain glioma cell U87 by adopting lentivirus, and whether autophagy is induced by pseudolaric acid B is further verified. As shown in FIG. 6, when 50umol/L of pseudolaric acid B was reacted for 48 hours, U87 cells showed red, green and yellow spots, and the red fluorescence spot was significantly increased, as compared with the control group. Thus, it was demonstrated that pseudolaric acid activates autophagic flow in human brain glioma cells.
(2) Pseudolaric acid B kills human glioma cells by activating autophagy
To illustrate the effect of autophagy in killing human brain glioma cells by pseudolaric acid, in the experiment, an autophagy-initiating inhibitor 3MA (concentration of 5mmol/L) and an autophagy flux inhibitor bafilomycin A1 (concentration of 0.2umol/L) are incubated with human brain glioma cells U87 for 1h, 50umol/L pseudolaric acid is added, and the death of the cells is detected by an LDH release method after 48h of action, so that the 3MA and the bafilomycin A1 can effectively inhibit the death of the human brain glioma cells induced by the pseudolaric acid as shown in FIG. 7. After the protein is extracted, a Westernblotting method is adopted for analysis, and the result is shown in figure 8, 3MA effectively inhibits the increase of protein LC3B II and the decrease of autophagy substrate protein P62 induced by pseudolaric acid; the bafilomycin A1 further increases the level of protein LC3B II while inhibiting the decrease of autophagy substrate protein P62. Thus, it was demonstrated that pseudolaric acid B kills human brain glioma cells by activating autophagy.
Example 4 proliferation inhibitory Effect of Pseudolaric acid on human hepatoma cells
In order to verify that the pseudolaric acid can kill other malignant tumor cells such as human liver cancer cells, in the experiment, the human liver cancer cells Hep3B are inoculated into a DMEM culture medium in a 96-well plate, a carbon dioxide incubator is used for overnight incubation for 48 hours with pseudolaric acid (12.5umol/L, 25umol/L, 50umol/L, 100umol/L and 200umol/L) with different concentrations, and then the cell proliferation capacity is detected by adopting an MTT method. As shown in fig. 9 and table 5, the survival rates of the human liver cancer cell Hep3B were respectively reduced to 98.37%, 94.82%, 81.62%, 56.02% and 40.10%. Thus, pseudolaric acid can kill human hepatoma cells in a concentration-dependent manner.
TABLE 5 proliferation inhibitory effect of pseudolaric acid B on human hepatoma cell Heb3B (. about.p <0.01)
Example 5 proliferation inhibitory Effect of Pseudolaric acid on Breast cancer cells
In order to verify that the pseudolaric acid can kill other malignant tumor cells such as human breast cancer cells, in the experiment, the human breast cancer cells MB-231 are inoculated into a DMEM medium in a 96-well plate, carbon dioxide is incubated overnight, and the pseudolaric acid with different concentrations (12.5umol/L, 25umol/L, 50umol/L, 100umol/L and 200umol/L) is incubated together for 48 hours, and then the cell proliferation capacity is detected by adopting an MTT method. As shown in FIG. 10 and Table 6, the survival rate of human breast cancer cell MB-231 was reduced to: 94.02%, 88.34%, 75.90%, 58.83%, 43.87%. Thus, pseudolaric acid can kill human breast cancer cells in a concentration-dependent manner.
TABLE 6 proliferation inhibitory effect of pseudolaric acid B on human breast cancer cells MB-231 (. about.. p <0.01)
Example 6 proliferation inhibition of human ovarian cancer cells by Pseudolaric acid B
In order to verify that the pseudolaric acid can kill other malignant tumor cells such as human ovarian cancer cells, in the experiment, human ovarian cancer cells A2780 are inoculated into a DMEM culture medium in a 96-well plate, a carbon dioxide incubator is used for overnight incubation for 48 hours with pseudolaric acid (12.5umol/L, 25umol/L, 50umol/L, 100umol/L and 200umol/L) with different concentrations, and then the cell proliferation capacity is detected by adopting an MTT method. As shown in FIG. 11 and Table 7, the survival rate of human ovarian cancer cell A2780 decreased to: 93.78%, 87.34%, 71.82%, 55.64% and 41.39%. Thus, pseudolaric acid can kill human ovarian cancer cells in a concentration-dependent manner.
TABLE 7 proliferation inhibitory effect of pseudolaric acid B on human ovarian cancer cells A2780 (. about.: p <0.01)
The invention discloses an application of pseudolaric acid B in preparing a medicine for preventing and/or treating malignant tumor, which can be realized by appropriately improving process parameters by taking the contents of the pseudolaric acid B as reference by the technical personnel in the field. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations and modifications, as appropriate, may be made to the products and methods described herein to implement and use the techniques of the invention without departing from the spirit and scope of the invention.
Claims (5)
1. Application of pseudolaric acid B in preparing medicine for preventing and/or treating malignant tumor is disclosed.
2. The use of claim 1, wherein the malignancy comprises: brain glioma, liver cancer, breast cancer and ovarian cancer.
3. The use according to claim 1, wherein the concentration of pseudolaric acid is 12.5-200 umol/L.
4. The use according to claim 3, wherein the concentration of pseudolaric acid is 25 to 50 umol/L.
5. The use according to claim 4, wherein the concentration of pseudolaric acid is 25 umol/L.
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