CN112704681B

CN112704681B - Composition, traditional Chinese medicine composition containing composition and application of traditional Chinese medicine composition

Info

Publication number: CN112704681B
Application number: CN202011496578.2A
Authority: CN
Inventors: 冯宇新; 朱彦; 高秀梅; 张俊华; 王涛; 王跃飞; 张晗; 贺爽; 王欣桐; 彭圳
Original assignee: Tianjin University of Traditional Chinese Medicine
Current assignee: Tianjin University of Traditional Chinese Medicine
Priority date: 2020-12-17
Filing date: 2020-12-17
Publication date: 2022-04-15
Anticipated expiration: 2040-12-17
Also published as: CN112704681A

Abstract

One or more embodiments of the specification provide a composition, a traditional Chinese medicine composition containing the composition and an application of the composition, the composition takes the existing traditional Chinese medicine composition on the market as a research object, the effective key components are screened and recombined, the limitations of complex components, difficult quality control guarantee, clinical safety hidden danger and the like brought by the traditional Chinese medicine injection at present are broken through, a new composition of compounds for effectively preventing and treating bacterial sepsis is obtained, a new core compound composition is provided for developing safer and more effective anti-systemic infection medicines, and the material and theoretical basis for developing new anti-infection medicines are laid.

Description

Composition, traditional Chinese medicine composition containing composition and application of traditional Chinese medicine composition

Technical Field

One or more embodiments of the present disclosure relate to the technical field of traditional Chinese medicine, and more particularly, to a composition, a traditional Chinese medicine composition containing the composition, and applications of the composition in preparation of drugs for treating or preventing bacterial sepsis.

Background

Sepsis, known as the "most typical disease of the 21 st century," has far received little attention as to the threat to human health. Given that current clinical trials (over 100) for single-target sepsis therapy worldwide all end up in failure, the world health organization announces that measures will be taken to preferentially improve the prevention, diagnosis and treatment of sepsis, a human killer, on day 26, month 5, 2017.

Sepsis is a complex disease that is one of the leading causes of human death worldwide. It has high lethality and expensive treatment cost. Currently there is a lack of effective therapies for sepsis. Sepsis (sepsis) is a critical response of the body to systemic infection. The sepsis has high incidence, serious illness and high death rate and is divided into two stages of sepsis and septic shock. There are over 1800 million cases of severe sepsis worldwide per year and escalate at a rate of 1.5% to 8.0% per year. About 14,000 people die each day worldwide from sepsis and its complications. According to foreign epidemiological investigation, the mortality rate of sepsis exceeds that of myocardial infarction, and becomes a main cause of death of non-cardiac patients in intensive care units. In recent years, despite significant advances in anti-infective therapy and organ function support technologies, sepsis has still suffered from a mortality rate of up to 30% to 70%.

Sepsis treatment costs a lot, seriously affects the quality of life of human beings, and poses a great threat to human health. Thus, in 2001 the european severe society, the american severe society, and the international Sepsis forum for Sepsis "rescue Sepsis battle" (SSC) was initiated, in 2002 a number of organizations in europe and america initiated and signed the "barcelona declaration" together, and in 2003 a guideline for Sepsis therapy, i.e., the SSC guideline, was formulated based on evidence-based medical evidence for Sepsis research and continuously updated to improve the therapy for Sepsis, reduce the mortality of Sepsis, and revised again in 2008. With the insight into sepsis, the concept of sepsis has also been updated. Sepsis 3.0 emphasizes that sepsis is a critical response induced by systemic infection.

Sepsis develops in response to immune dysregulation of the host to infection, and systemic organ failure is a serious consequence of sepsis and is closely associated with an inflammatory factor storm. When sepsis progresses to septic shock, the cardiovascular system undergoes severe disorders well known to intensive care physicians; and a circulating series of proinflammatory cytokines directly act on cardiac myocytes and the vascular system, thereby causing cardiac dysfunction.

Clinically, the bacterial sepsis and the fungal sepsis are proved, wherein pathogenic bacteria of the bacterial sepsis comprise gram-negative bacteria (such as pseudomonas aeruginosa, escherichia coli, klebsiella pneumoniae, enterobacter cloacae and the like) and gram-positive bacteria (such as coagulase-negative staphylococcus, staphylococcus aureus and the like); wherein, the pathogenic bacteria of the fungal sepsis are mainly candida. In a retrospective analysis case, 349 strains of pathogenic bacteria were co-isolated from 277 positive sepsis patients, wherein 54.44% of 190 strains of gram-negative bacteria were selected, and the main pathogenic bacteria were pseudomonas aeruginosa, escherichia coli, klebsiella pneumoniae, enterobacter cloacae, acinetobacter baumannii and serratia marcescens; the gram-positive strain 115 accounts for 32.95 percent, and takes coagulase-negative staphylococcus and staphylococcus aureus as the first 2 sites; candida strain 44, accounting for 12.61%. Therefore, the incidence of fungal sepsis is significantly lower than that of bacterial sepsis, or bacterial sepsis is more common in clinical settings.

Sepsis therapy includes respiratory maintenance, oxygen supply assurance, intravenous injection, antibiotics, and the like. A large body of evidence suggests that a single treatment modality does not achieve good results, and therefore early-stage cluster therapy is advocated in the treatment of sepsis, with effective measures taken in order as soon as possible.

Sepsis caused by bacterial infection can cause aggregation of neutrophils, and the antibacterial effect is achieved by releasing antibacterial peptide and NETs (neutrophil extracellular bactericidal network) and the like, however, some components in the NETs, such as histone, can stimulate platelet aggregation to form thrombus, and can directly activate thrombin and coagulation factors to cause Disseminated Intravascular Coagulation (DIC). DIC is one of the major complications of bacterial sepsis and is also one of the important manifestations of bacterial sepsis. DIC may lead to organ damage by inadequate blood supply to the organ, while persistent inflammation caused by the release of large amounts of cytokines by immune cells may also lead to coagulation and organ damage. DIC is the most severe form of coagulation abnormality, whose development is closely associated with endothelial injury, leading to multiple organ dysfunction and hypotension (septic shock). Anticoagulation is one of means for treating DIC, and two anticoagulants, heparin and recombinant human activator protein C (RhAPC), are widely used clinically, but have large side effects; or the antibiotics such as dobutamine injection, levosimendan, aspirin, clopidogrel, ticagrelor and the like are adopted, and the antibiotics can improve the survival result of early sepsis through timely and positive initial supportive care, but easily cause the drug resistance of the body, and are not beneficial to the long-term treatment of bacterial sepsis or DIC.

Sepsis has the characteristics of multiple factors, multiple targets, complex mechanism and the like, the conventional single-target treatment is difficult to take effect, and the existing treatment method usually treats the symptoms and the root causes. The compound traditional Chinese medicine has the advantage of multi-target system intervention, and has preliminarily obtained the curative effect of preventing and treating sepsis and septic shock. Therefore, more and more researchers turn research and development to the field of traditional Chinese medicine and obtain certain progress. Previous researches show that the traditional Chinese medicine can relieve endothelial injury, inhibit blood coagulation activation, reduce inflammatory reaction and has positive treatment effects on bacterial sepsis and disseminated blood coagulation. However, the traditional Chinese medicine still needs to be combined with therapies such as antibiotics for treating sepsis at present, and no traditional Chinese medicine for treating sepsis alone obtains the approval of CFDA.

A Chinese medicinal preparation, XBJ, is composed of Carthami flos extract, radix Paeoniae Rubra extract, rhizoma Ligustici Chuanxiong extract, Saviae Miltiorrhizae radix extract, and radix Angelicae sinensis extract, and is approved for CFDA for treating sepsis in combination with antibiotic. However, the ingredients of the traditional Chinese medicine are complex, and more than 100 compounds are contained in the traditional Chinese medicine. Therefore, the potential safety hazard of clinic is possibly brought, and the clinic use of a plurality of traditional Chinese medicine injections is limited. The adverse reaction of the traditional Chinese medicine injection is concerned by the society. According to a clinical study of the Unionidae medical college in 2019, the incidence rate of adverse reactions of Xuebijing and other medicines is over 5 percent. The quality control is a difficult problem faced by the traditional Chinese medicine preparation, and because the raw material medicinal materials are subject to the differences of climate, producing area, extraction process and the like, the content difference of effective compound components of the traditional Chinese medicines in different batches is obvious, and the problems of overproof heavy metal content of the traditional Chinese medicines and the like caused by environmental pollution exist, and the problems become potential safety hazards of clinical medication. In addition, the clinical effectiveness of the traditional Chinese medicine for treating sepsis still needs to be deeply researched, and a large-scale clinical research is needed to clarify whether the traditional Chinese medicine can effectively reduce the 28-day mortality of patients with sepsis. Too low a concentration of the active ingredient is one of the bottlenecks that limit the efficacy of the composition. Therefore, the development of a new drug formulation for sepsis and sepsis-induced cardiac dysfunction with a definite material basis can meet the clinical needs of improving the safety and effectiveness of the drug.

Disclosure of Invention

In view of the above, one or more embodiments of the present disclosure are directed to a composition and its application in preparing a drug for treating or preventing bacterial sepsis, so as to solve the problem of the treatment defect of bacterial sepsis caused by traditional Chinese medicine and western medicine in the prior art.

In view of the above, one or more embodiments of the present disclosure provide a composition, which includes the following raw materials in parts by weight: the weight ratio of paeoniflorin to hydroxysafflor yellow A is 1:10-10: 1. A composition comprising paeoniflorin and hydroxysafflor yellow A is named C0127-2.

Optionally, the weight ratio of paeoniflorin to hydroxysafflor yellow A is 1:4-4: 1.

The weight ratio of the paeoniflorin to the hydroxysafflor yellow A is 1:4-2: 1.

Optionally, the weight ratio of paeoniflorin to hydroxysafflor yellow A is 1:10, 1:4, 1:2, 1:1, 2:1, 4:1 or 10: 1.

Based on the same inventive concept, one or more embodiments of the invention also disclose the application of the composition in preparing a medicament for preventing or treating bacterial sepsis or organ dysfunction diseases caused by bacterial infection.

Optionally, the specific method of the application is as follows: dissolving the composition C0127-2 in PBS for injection, filtering, and sterilizing to obtain injection, wherein the concentration range of paeoniflorin in the injection is 0.5-30mg/ml, and the concentration range of hydroxysafflor yellow A is 0.5-30 mg/ml.

Optionally, the concentration of paeoniflorin in the injection is in the range of 1-4mg/ml, and the concentration of hydroxysafflor yellow A is in the range of 0.1-2 mg/ml.

Based on the same inventive concept, one or more embodiments of the invention also disclose a Chinese medicinal composition, which comprises a Chinese medicament containing paeoniflorin and a Chinese medicament containing hydroxysafflor yellow A, wherein the weight ratio of the paeoniflorin to the hydroxysafflor yellow A in the Chinese medicinal composition is 1:10-10: 1.

Optionally, the paeoniflorin-containing traditional Chinese medicine comprises at least one of radix paeoniae rubra or radix paeoniae alba; and/or the traditional Chinese medicine containing hydroxysafflor yellow A comprises at least one of safflower and hawthorn.

Optionally, the weight ratio of paeoniflorin to hydroxysafflor yellow A in the Chinese medicinal composition is 1:4-2: 1.

Based on the same inventive concept, one or more embodiments of the invention also disclose application of the traditional Chinese medicine composition in preparing a medicine for preventing or treating bacterial sepsis or organ dysfunction diseases caused by bacterial infection.

It should be noted that, the components of the extracts of most traditional Chinese medicine injection are complex, and compared with the monomeric medicine, the quality control faces a great challenge, and the extracts are difficult to meet the international standard, and become one of the barriers to internationalization of traditional Chinese medicine. The effectiveness of the existing sepsis therapy needs to be improved urgently, and the clinical safety and the effectiveness of the traditional Chinese medicine need to be improved.

Hydroxy safflower yellow A (hydroxysafflor yellow A) is a compound with a single chalcone glycoside structure, has a structural formula shown in a formula (I), is the most effective water-soluble part with pharmacological effects of safflower, can inhibit platelet activation factor-induced platelet aggregation and release under pressure stimulation of bacteria and viruses and the like, can competitively inhibit the combination of the platelet activation factor and a platelet receptor, is an effective component of the safflower yellow for promoting blood circulation and removing blood stasis, and can effectively relieve myocardial ischemia reperfusion injury through resisting oxidative stress injury, resisting apoptosis, protecting mitochondria, expanding blood vessels and the like.

Paeoniflorin belongs to terpenoid, the structural formula of the paeoniflorin is shown in a formula (II), the paeoniflorin is the most effective water-soluble part with the pharmacological effect of red paeony root, and the paeoniflorin can obviously reduce the levels of inflammatory factors such as interleukin-1 (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and the like in nucleus pulposus cells induced by Lipopolysaccharide (LPS), obviously inhibit the expression of chemotactic factor mRNA of human dermal microvascular endothelial cells (HMEC-1) induced by the TNF-alpha and reduce the secretion of the chemotactic factor in culture supernatant; and the paeoniflorin is found to inhibit the nuclear transcription factor-kB (NF-kB) from being transported to the nucleus, which indicates that the paeoniflorin has the anti-inflammatory capacity for resisting the TNF-alpha induced chemokine generation and leukocyte migration.

In one or more embodiments of the invention, C0127-2 formed by matching paeoniflorin and hydroxysafflor yellow A has various pharmacological actions of regulating and controlling inflammatory response, improving blood coagulation function, protecting endothelial cells, improving microcirculation, resisting oxidative stress, regulating immunologic function and the like, overcomes the trouble of antibiotic resistance of the organism on sepsis treatment, simplifies the components of the prescription, is beneficial to improving the safety and quality control of the medicine, and can be used as a new basic prescription for preparing medicines for preventing or treating bacterial sepsis while ensuring the efficacy.

From the above, it can be seen that one or more embodiments of the present specification provide a composition, a Chinese medicinal composition containing the composition, and an application of the composition in preparing a medicament for preventing or treating bacterial sepsis or organ dysfunction diseases caused by bacterial infection, the composition takes the existing Chinese medicinal composition as a research object, breaks through the limitations of complex components, difficult quality control guarantee, clinical safety hidden dangers and the like caused by the complex components and the difficult quality control of the existing traditional Chinese medicinal injection, obtains a compound combination for effectively preventing and treating bacterial sepsis, provides a new medicament combination for developing safer and more effective anti-infective medicaments, lays a material and theoretical basis, and has a hint on developing a new generation of medicaments for resisting systemic infection. Based on its unique organ protection, anticoagulation and immunoregulation functions, C0127-2 is expected to become a core component of a new generation for treating systemic infections including sepsis and new coronavirus infections.

Drawings

In order to more clearly illustrate one or more embodiments or prior art solutions of the present specification, the drawings that are needed in the description of the embodiments or prior art will be briefly described below, and it is obvious that the drawings in the following description are only one or more embodiments of the present specification, and that other drawings may be obtained by those skilled in the art without inventive effort from these drawings.

FIG. 1 is a graph showing the survival curves and changes in body temperature of various groups of sepsis mice in the examples; wherein, fig. 1A is a schematic diagram of the mouse pre-and post-operative intervention procedure; FIG. 1B is the effect of XBJ (positive control), C0127-2 on survival of septic mice within 72 hours; FIG. 1C is a graph of the effect of XBJ, C0127 and C0127-2 on anal body temperature of septic mice; in contrast to the sham group,^#P<0.05，^##P<0.01，^###P<0.001, compared with CLP group^*P<0.05，^**P<0.01。

FIG. 2 is the heart ultrasonic image and its data analysis of each group of sepsis mice in the example; wherein FIG. 2A is a representative image of a left ventricular echocardiogram, through which different sets of cardiac performance are examined; FIG. 2B is left ventricular inner diameter diastole (LVID; D), FIG. 2C is left ventricular inner diameter systole (LVID; s)%, FIG. 2D is left ventricular posterior wall diastole (LVPW; D), FIG. 2E is left ventricular posterior wall systole (LVPW; s), FIG. 2F is ejection fraction (EF%), FIG. 2G is fractional shortening (FS%), measured using type M; results are expressed as mean SEM (n-7-10); in contrast to the sham group,^#P<0.05，^##P<0.01，^###P<0.001, compared with CLP group^*P<0.05，^**P<0.01。

FIG. 3 shows the expression of genes associated with cardiac tissue damage in each of the sepsis mice groups in the examples; wherein, fig. 3A shows that the levels of different groups of amino-terminal pro-brain natriuretic peptide (NT-pro BNP) are measured by Elisa method, and N is 4/group; real-time PCR detection of different groups of cardiac tissue type B Natriuretic Peptides (BNP) (FIG. 3B), TLR4 (FIG. 3C), HMGB1 (FIG. 3D), periglobulin (FIG. 3C)E) GRP78 (fig. 3F) mRNA expression, N-4-5/group; in contrast to the sham group,^#P<0.05，^##P<0.01，^###P<0.001, compared with CLP group^*P<0.05，^**P<0.01.

FIG. 4 shows Rho2 (Ca) of each H9C2 cell group in examples²⁺Fluorescent probe) and DCFH (active oxygen fluorescent probe); wherein, FIG. 4A shows mitochondrial Ca in different administration treatments²⁺A horizontal representative image; FIG. 4B is a representative image of ROS levels in different dosing treatments; FIG. 4C shows Ca in different administration treatments²⁺Quantification of the level; FIG. 4D is a quantification of fluorescence values of ROS production in different dosing treatments; compared with the blank control group, the composition of the composition,^#P<0.05，^##P<0.01，^###P<0.001, compared to LPS-stimulated group^*P<0.05，^**P<0.01，^***P<0.001; the experiment was independently repeated 3 times; wherein control represents blank control.

FIG. 5 is a graph of the effect of XBJ, C0127-2 on mRNA expression of proinflammatory cytokines and cytokine receptors in heart tissue following systemic infection in example; wherein, FIG. 5A is a heatmap of gene expression in the cytokine-cytokine receptor affected by XBJ, C0127, and C0127-2; FIG. 5B, FIG. 5C, FIG. 5D are the expression of the proinflammatory cytokines IL6, IL1a and IL1B, respectively, detected in heart tissue of different groups of mice; fig. 5E, fig. 5F, fig. 5G are analysis results respectively verifying the expression of pro-inflammatory cytokines IL6, IL1 α and IL1b in heart tissues of different groups of mice by real-time PCR experiments; n is 4-5/group; # P<0.05, compared to the sham group,^#P<0.05，^##P<0.01，^###P<0.001, compared with CLP group^*P<0.05，^**P<0.01。

FIG. 6 is the effect of C0127 and C0127-2 on tumor necrosis factor TNF signaling pathway mRNA expression in heart tissue of sepsis mice in example; wherein, FIG. 6A is a heat map of gene expression affected by C0127 and C0127-2 in TNF signal in RNA-seq analysis; fig. 6B, 6C, 6D, 6E are results of analysis of the expression of BIRC3, CEBPB, FOS, CXCL5, respectively, validated RNA-seq for real-time PCR experiments; n is 4/group; all real-time PThe CR experiment was repeated twice; in contrast to the sham group,^#P<0.05，^##P<0.01，^###P<0.001, compared with CLP group^*P<0.05，^**P<0.01。

FIG. 7 is a graph of the effect of C0127 and C0127-2 on gene expression of NF-kB signaling pathway in heart tissue of CLP mice in example; wherein, FIG. 7A is the RNA sequencing results of gene expression affected by XBJ, C0127 and C0127-2 in NF- κ B signaling, and is embodied in a heatmap; FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F, FIG. 7G are the results of the real-time PCR experiments to verify the expression of RNA-seq. CD14, TNFAIP3, PTGS2, CXCL2, CXCL12, ICAM1, respectively; all real-time PCR experiments were performed twice; n is 4/group; in contrast to the sham group,^#P<0.05，^##P<0.01，^###P<0.001, compared with CLP group^*P<0.05，^**P<0.01。

FIG. 8 is a graph of a mouse survival experiment in combination with antibiotics; in contrast to the sham group,^###P<0.001, compared with CLP group^*P<0.05。

FIG. 9 is a graph of the effect of different dosing on the survival of septic mice; in contrast to the sham group,^###P<0.001, compared with CLP group^*P<0.05，^**P<0.01，***P<0.001, ns means no significant difference; c0127-2 Medium dose compared to Low dose group^*P<0.05。

Detailed Description

For the purpose of promoting a better understanding of the objects, aspects and advantages of the present disclosure, reference is made to the following detailed description taken in conjunction with the accompanying drawings.

It is to be noted that unless otherwise defined, technical or scientific terms used in one or more embodiments of the present specification should have the ordinary meaning as understood by those of ordinary skill in the art to which this disclosure belongs.

As described in the background art, the existing traditional Chinese medicine cannot be used for treating clinical sepsis independently, and has the problems of complex components, undefined active ingredients, unknown action mechanism and the like, the quality control of the compound traditional Chinese medicine is difficult and serious, the quality control needs to be perfect, the fluctuation of main active ingredients in different batches of traditional Chinese medicines is large, certain differences are more than one time, even the effective ingredients cannot be detected, and the effectiveness and the mechanism of the traditional Chinese medicine for treating sepsis need to be deeply researched.

Therefore, the Chinese patent CN 108743600A uses paeoniflorin (the most effective water-soluble part of the pharmacological efficacy of red paeony root), hydroxysafflor yellow A (the most effective water-soluble part of the pharmacological efficacy of safflower), ferulic acid (the most effective water-soluble part of the pharmacological efficacy of szechuan lovage rhizome) and protocatechualdehyde (the most effective water-soluble part of the pharmacological efficacy of salvia miltiorrhiza) as a formula (named C0127) to research the treatment effect of the fungal sepsis; the pathogenic mechanisms of fungal sepsis and bacterial sepsis are different, and the component of the C0127 formula is mainly screened and recombined, and the treatment effect of the component serving as a traditional Chinese medicine preparation on the bacterial sepsis is researched.

The Xuebijing involved in the experimental method disclosed by the present disclosure is produced by Tianjin Hongyi pharmaceutical industry Co.

As an alternative example, the bacterial sepsis mouse model was established as follows: performing Cecal Ligation and Perforation (CLP) for molding, specifically, anesthetizing mouse with tribromoethanol, removing abdominal hair, cutting an incision smaller than 1cm below abdomen, exposing cecal, and ligating ileocecal flap at distance base 1/3 with 2-0 line. The cecum was perforated with an 18G needle (1) and a small amount of stool was expressed. And (3) recovering the caecum in situ after operation, suturing the wound by 4-0 lines, and putting the mouse on an electric blanket for recovery after operation, wherein the mouse subjected to the CLP operation is referred to as the CLP mouse.

As an optional embodiment, the composition of the present disclosure includes the following raw materials in parts by weight: the weight ratio of paeoniflorin to hydroxysafflor yellow A is 1:10-10: 1;

dissolving paeoniflorin and hydroxysafflor yellow A in PBS for injection, filtering, and sterilizing to obtain injection, wherein the concentration of paeoniflorin in the injection is 0.5-30mg/ml, and the concentration of hydroxysafflor yellow A is 0.5-30 mg/ml.

The weight ratio of each component in the C0127-2 is screened in the table 1, the concentration range of paeoniflorin in the C0127-2 injection is 2mg/ml, the concentration range of the hydroxysafflor yellow A is 0.5mg/ml, and the table 1 shows that when the weight ratio of the paeoniflorin to the hydroxysafflor yellow A is 4:1, the survival and the cardiac function of the mice with bacterial sepsis can be effectively improved.

TABLE 1

Effective in improving survival of mice with sepsis

No significant effect on the survival/cardiac function of septic mice

It should be noted that, unless otherwise specified, the weight ratio of paeoniflorin, hydroxysafflor yellow a, ferulic acid and protocatechuic aldehyde in C0127 in the following examples is 200: 50: 2:1, the concentrations of paeoniflorin, hydroxysafflor yellow A, ferulic acid and protocatechuic aldehyde in the C0127 injection are respectively 2mg/ml, 0.5mg/ml, 0.02mg/ml and 0.01 mg/ml; the weight ratio of hydroxysafflor yellow A to paeoniflorin in C0127-2 is 4:1, and the concentrations of hydroxysafflor yellow A and paeoniflorin in C0127-2 injection are 2mg/ml and 0.5mg/ml respectively.

As an alternative example, 50 ICR mice aged 8 weeks were randomly divided into a Sham control group, a CLP group, a Xuebijing group, a C0127 group and a C0127-2 group, each of which comprises 10 mice, wherein the Sham control group is a normal mouse, the CLP group is a CLP mouse, the Xuebijing group is a positive control group, the CLP mice were treated with Xuebijing injection (dosage is 9ml/kg), the C0127 group is treated with the C0127 injection (dosage is 9ml/kg), and the C0127-2 group is treated with the C0127-2 injection (dosage is 9 ml/kg). The drug treatment of mice in the CLP model group, the Xuebijing group, the C0127 group and the C0127-2 group is started 3 days before modeling, after the mice are weighed, the Xuebijing, the C0127 and the C0127-2 tail veins are respectively injected into the mice with the dosage of 9ml/kg, and the PBS is injected into the Sham group and the CLP group for 2 times each day. After the molding is completed, the corresponding medication is continued to be given 2 times a day. The survival and temperature changes of the mice were observed and the simplified procedure is shown in FIG. 1A.

The experimental results showed that within 48 hours after surgery, as shown with reference to figure 1B, all CLP mice died and all mice in the Sham control group survived, indicating that the model was successfully established; in addition, fig. 1B also shows that the survival rate of mice in the C0127-2 group and the C0127 group is lower than that of XBJ group, which may be caused by the lower concentration of the composition in the C0127-2 injection and the C0127 injection, and the volume ratio of the liquid traditional Chinese medicine extract to the solvent (such as PBS or normal saline) in the clinically applied XBJ injection is 1:1, i.e., the Xuebijing is diluted by 1:1, and if the dosage of Xuebijing is increased, the toxic and side effects may be more brought, but each component of the present disclosure is in powder form, which is favorable for storage, and has a high solubility in normal saline or PBS, and the therapeutic effect can be improved by increasing the concentration of the composition in the C0127-2 injection and the C0127 injection, as shown in fig. 9. The Xuebijing group, the C0127 group and the C0127-2 group can obviously reduce the fever of CLP mice (see figure 1C).

As an alternative example, the mouse echocardiography experiment was performed 24 hours post-operatively, and the heart left ventricular function and coronary flow were non-invasively assessed using an ultra-high resolution small animal ultrasound Vevo 2100 imaging system (Visual sonic, Toronto, Canada) and a 30MHz transducer. Mice were anesthetized prior to imaging (2% isopentane with 0.5L/min 100% O₂Mixed), the depilatory cream removed the hair from the chest and the anesthetized mice were laid on a hot pad with ecg leads to maintain body temperature. The nose cone connected to the anesthesia system remained at a stable level throughout the procedure (1.0% -1.5% isoflurane mixed with 0.5L/min 100% O₂). The level of anesthesia was adjusted to reach a target heart rate (bpm) of 450 + -50 beats/min. Four paws were fixed on the ECG electrode with electrode gel. During the test, the probe was gently placed on the chest of the mouse to determine the location of the left ventricle, and three cardiac cycles of each mouse were measured and averaged. Data analysis was performed after the experiment was completed using software provided by the ultrasound system.

FIG. 2A is a left ventricular echocardiogram of mice in each group, showing that the CLP group has impaired cardiac function, decreased ejection fraction (EF%), decreased fraction (FS%), left ventricular posterior wall diastolic period (LVPW; d), and left ventricular posterior wall systolic period (LVPW; s) while increasing left ventricular inner diameter diastolic period (LVID; d), and left ventricular inner diameter systolic period (LVID; s) compared to the Sham control group. The Xuebijing group, C0127 group and C0127-2 group significantly improved the LVID of CLP mice; d. an LVID; s, LVPW; d. LVPW; s, EF%, FS% (see FIGS. 2B, 2C, 2D, 2E, 2F, 2G, respectively).

As an alternative example, the Real-time PCR experiment is as follows: 24 hours after CLP surgery, mouse hearts were removed and stored in liquid nitrogen, which was then added to a pre-cooled mortar. 1ml of lysis buffer was added to each tissue sample, separated with chloroform, precipitated with isopropanol, washed with 75% ethanol, and dissolved in ultrapure water. RNA concentration, purity and integrity were determined using the Qubit RNA kit in the Qubit 2.0 fluorometer and the NanoPhotometer spectrophotometer, respectively. Immediately after RNA extraction, total RNA was reverse transcribed into cDNA according to the instructions of the transcript first strand cDNA synthesis kit. Real-time PCR was then performed in 25ul reaction. The sample was held at 95 ℃ for 30s, annealed at 60 ℃ for 30s, and extended at 72 ℃ for 40 s.

The experimental results showed that C0127s (including C0127 group and C0127-2 group) reduced NT-ProBNP (N-terminal-pro hormone type B natriuretic peptide) in peripheral blood (see fig. 3A) and reduced mRNA expression of BNP (type B natriuretic peptide) in heart tissue of CLP mice (see fig. 3B). Furthermore, in groups C0127 and C0127-2, expression of TLR4 gene and its downstream effector HMGB1 gene, periostin and GRP78 (effector of endoplasmic reticulum stress) was moderately reduced (see fig. 3C, fig. 3D, 3E, fig. 3F).

As an alternative embodiment, the cell assay detection method: H9C2 cells were seeded at 10000 cells/well on a 96-well blackboard in DMEM medium containing 10% fetal bovine serum at 37 deg.C and 5% CO₂After 24h incubation under the conditions of (1ug/ml), LPS (1ug/ml) stimulation was performed, and incubation was performed with diluted Xuebijing injection (1: 20 and 1:100 dilution of Xuebijing injection, respectively), diluted C0127 injection (1: 20 and 1:100 dilution of C0127 injection, respectively), and diluted C0127-2 injection (1: 20 and 1:100 dilution of C0127-2 injection, respectively). After 12 hours DCFH-DA and RHOD2 dyes were added, incubated at 37 ℃ for 30 minutes, PBS washed 3 times, images were taken and analyzed as described using the Perkinelmer high content imaging System, the stronger the fluorescence intensity in the images representingCa²⁺The more severe the overload and ROS (reactive oxygen species) generation.

The results of the experiments showed that C0127 and C0127-2 reversed Ca in mitochondria at the cellular level²⁺C0127-2 has some advantage over XBJ in reducing ROS expression in H9C2 cells by overloading (see fig. 4A and 4C) and reducing ROS production in LPS (bacterial lipopolysaccharide) stimulated H9C2 cells (see fig. 4B and 4D). This indicates that XBJ, C0127 and C0127-2 can protect the cellular function of cardiomyocytes.

As an alternative example, the experimental method of the transcriptome sequencing technology is as follows: mouse hearts were collected 24 hours after CLP surgery (Sham control group, CLP group, hematobizin group, C0127-2 group). The blood in the chamber was washed clean with PBS and the heart was immediately placed in liquid nitrogen. These hearts were then subjected to high throughput sequencing on the Illumina sequencing platform (4 cases per group).

The experimental results show that in the heart transcriptome results of the sepsis mice, different genes (namely CLP vs sham, CLP vs XBJ, CLP vs C0127 and CLP vs C0127-2) after the treatment of sham, Xuebijing and C0127-2 are obtained by taking the CLP model group as a control group, and 952 different genes are obtained by taking the difference multiple of more than 2 times as a standard. In this example, KEGG (Kyoto Encyclopedia of Genes and genomics Kyoto Encyclopedia of genome) analysis was performed on the differential Genes of the CLP vs XBJ group alone (see Table 2), and the results showed that the three inflammatory signaling pathways of cytokine-cytokine receptor interaction signaling pathway, TNF signaling pathway, and NF-kb signaling pathway ranked 5 positions first in the top 10 signaling pathway. In this example, the 952 common differential genes were analyzed by KEGG, and the three inflammatory signal pathways, i.e., cytokine-cytokine receptor interaction signal pathway, TNF signal pathway, and NF-kb signal pathway, were ranked 6-top (see Table 3). It was thus revealed that C0127 and C0127-2 were achieved by regulating cytokine-cytokine receptor interaction signaling pathway, TNF signaling pathway and NF-kb signaling pathway.

In this example, the differential genes included in the three inflammatory signaling pathways were subjected to thermographic analysis and PCR verification, specifically shown in fig. 5, fig. 6, and fig. 7.

5A, 6A, 7A are heat map analyses performed on the differential genes contained in the three inflammatory signaling pathways, with heat values between 0 and 1.5 indicating a tendency for the differential genes to be up-regulated compared to the other groups in the CLP group, and heat values between 0 and 1.5 indicating a tendency for the differential genes to be down-regulated compared to the other groups in the CLP group.

Selecting genes on the upstream, the middle and the downstream of a cytokine-cytokine receptor interaction signal path for PCR verification, wherein the difference reading values of IL-6, IL1a and IL-1B transcriptomes are respectively shown in figures 5B, 5C and 5D, and the PCR verification results of IL-6, IL-1a and IL-1B are respectively shown in figures 5E, 5F and 5G, and the results show that the CLP group can enable the expression levels of IL-6, IL-1a and IL-1B to be up-regulated, and the Xuebijing group, the C0127 group and the C0127-2 group can obviously down-regulate the expressions of IL-6, IL-1a and IL-1B.

Selecting the upstream, middle and downstream genes of the TNF signal channel for PCR verification, wherein the PCR verification results of BIRC3, CEBPB, FOS and CXCL5 are shown in fig. 6B, 6C, 6D and 6E respectively, and the results show that the expression levels of the CLP groups BIRC3, CEBPB, FOS and CXCL5 are up-regulated; the Xuebijing group, the C0127 group and the C0127-2 group can obviously down-regulate the expression of BIRC3, CEBPB, FOS and CXCL 5.

Selecting upstream, middle and downstream genes of an NF-KB signal channel for PCR verification, and performing PCR verification on CD14, TNFAIP3, PTGS2, CXCL2, CXCL12 and ICAM1 respectively on a figure 7B, a figure 7C, a figure 7D, a figure 7E, a figure 7F and a figure 7G to show that the expression levels of CD14, TNFAIP3, PTGS2, CXCL2 and ICAM1 in a CLP group are up-regulated, so that the expression of CXCL12 is reduced; the Xuebijing group, the C0127 group and the C0127-2 group can obviously reduce the expression of CD14, TNFAIP3, PTGS2, CXCL2 and ICAM1 and enhance the expression of CXCL 12.

The PCR result is consistent with the variation trend of the transcriptome difference gene heat map analysis, so that the authenticity of the transcriptome result is verified.

TABLE 2

TABLE 3

In addition to the thermographic analysis and PCR verification of the differential genes contained in the three inflammatory signaling pathways, the present example also performed the transcription control analysis of two control groups, CLP vs C0127-2 and CLP vs XBJ, on the target genes ALK, TLR5 and Stat3 reported in the literature as having therapeutic effects on bacterial sepsis, and the results are shown in table 4.

As can be seen from Table 4, Xuebijing has no obvious regulation effect on the expression of ALK, TLR5 and Stat3 genes, and C0127-2 has significant effect on the expression of these genes, for example, the expression of ALK gene and Stat3 gene in CLP group mice is higher than that in C0127-2 group mice, but the high expression of ALK gene and Stat3 gene can aggravate bacterial sepsis; the expression of TLR5 in C0127-2 group mice is higher than that of CLP group mice, and the expression of TLR5 gene can relieve the damage of sepsis; while Xuebijing has no obvious influence on the expression of the three genes. The reason for this is probably that the Xuebijing cannot regulate the expression of some key genes affecting the progress of bacterial sepsis due to the complex components of Xuebijing, which is not beneficial to the prevention and treatment of bacterial sepsis. Therefore, compared with Xuebijing, C0127-2 has the advantage of preventing and treating bacterial sepsis. This result confirms the results in the subsequent examples, for example, the combination of C0127-2 and antibiotic significantly improves the survival of sepsis mice, and is significantly superior to the effect of Xuebijing and antibiotic (see fig. 8 in particular); as another example, the medium dose of C0127-2 given one day in advance mentioned in the examples which follow is substantially equivalent to the therapeutic effect of Xuebijing given three days in advance on bacterial sepsis (see in particular FIG. 9).

TABLE 4

+: indicating that the expression of the gene is higher in CLP group mice than in C0127-2 group mice, i.e., C0127-2 down-regulates gene expression;

-: indicating that the expression of the gene is lower in CLP group mice than in C0127-2 group mice, i.e., C0127-2 up-regulates gene expression;

ns: indicating no significant difference.

As an alternative embodiment, normal mice are taken as a sham group, CLP mice are randomly divided into a CLP group, a C0127-2+ imipenem cilastatin group, an XBJ + imipenem cilastatin group and an imipenem cilastatin group, wherein each group comprises 10 mice, wherein the C0127-2+ imipenem cilastatin group is formed by respectively injecting 9ml/kg (twice per day) of C0127-2 injection and 250 micrograms/day of antibiotic imipenem cilastatin to the CLP mice; the XBJ + imipenem cilastatin group is 9ml/kg of Xuebijin injection (injected twice per day) and 250 micrograms/day of antibiotic imipenem cilastatin which are respectively injected into CLP mice; the imipenem cilastatin group is that CLP mice are injected with 250 micrograms/piece/day of antibiotic imipenem cilastatin; before and after the operation, the combined treatment of the Xuebijing and the antibiotics and the combined treatment of the C0127s and the antibiotics are carried out.

The survival chart of mice (see figure 8) shows that the combination of Xuebijing and C0127-2 with antibiotics can improve the survival rate of sepsis mice more than the antibiotics alone. And the combination of C0127-2 and antibiotics is superior to the combination of Xuebijing and antibiotics. This result indicates that C0127-2 is suitable for use in combination with antibiotics to prevent bacterial sepsis.

As an alternative example, the mice were injected intravenously at the tail vein with a maximum volume of 250ul, and according to literature reports that the maximum dose of XBJ for mice was 18ml/kg per day, the test mice weighed about 25g, and were injected twice per day at a dose of 9ml/kg, with about 225ul per injection. The low-dose C0127-2 can not improve the survival rate of the sepsis mouse by injecting the mouse through tail vein one day ahead (the concentration of paeoniflorin is 2mg/ml, the concentration of hydroxysafflor yellow A is 0.5mg/ml, the injection dose is 9ml/kg, and the injection is performed twice every day); the low-dose C0127-2 three days ahead tail intravenous injection mouse (C0127-2 low-dose three days ahead administration group) can obviously improve the survival rate of sepsis mice, but has certain disadvantage compared with the XBJ group. After the administration dose of the C0127-2 is increased (the dose in the C0127-2 is advanced one day in the administration group, the paeoniflorin concentration is 4mg/ml, the hydroxysafflor yellow A concentration is 1mg/ml, the injection dose is 9ml/kg, and the injection is performed twice every day), the survival rate of the sepsis mice is obviously increased compared with the dose group with lower survival rate, and the administration effect is basically equivalent to that of Xuebijing three days in advance, and the specific figure is shown in FIG. 9. The result shows that the survival rate of the mice can be effectively improved by using higher concentration paeoniflorin and hydroxysafflor yellow A, and the traditional Chinese medicine composition has no toxic or side effect on the mice.

It is intended that the one or more embodiments of the present specification embrace all such alternatives, modifications and variations as fall within the broad scope of the appended claims. Therefore, any omissions, modifications, substitutions, improvements, and the like that may be made without departing from the spirit and principles of one or more embodiments of the present disclosure are intended to be included within the scope of the present disclosure.

Claims

1. The composition is applied to the preparation of medicines for preventing or treating bacterial sepsis or heart dysfunction diseases caused by bacterial sepsis, and comprises the following raw materials in parts by weight, wherein the weight ratio of paeoniflorin to hydroxysafflor yellow A is 1:4-4: 1.

2. The application of claim 1, wherein the specific method of the application is as follows: dissolving the composition of claim 1 in PBS for injection or physiological saline, filtering, sterilizing to obtain injection, wherein the concentration of paeoniflorin in the injection is 0.5-30mg/ml, and the concentration of hydroxysafflor yellow A is 0.5-30 mg/ml.

3. The use of claim 1, wherein the concentration of paeoniflorin in the injection is in the range of 1-4mg/ml, and the concentration of hydroxysafflor yellow A is in the range of 0.1-2 mg/ml.

4. The use of claim 1, wherein said paeoniflorin is derived from at least one of red peony or white peony; and/or

The hydroxysafflor yellow A is derived from at least one of Carthami flos and fructus crataegi.