Application of Xinsunning preparation in preparation of medicine for preventing and treating myocardial fibrosis

Abstract

The invention provides application of a Xinsunning preparation in preparation of a medicine for preventing and treating myocardial fibrosis, and belongs to the technical field of traditional Chinese medicine application. The invention particularly relates to an application of a Xinsunning preparation in preparation of drugs for preventing and treating myocardial fibrosis by enhancing myocardial collagenous fibers.

Description

Technical Field

[0001] The present invention relates to the technical field of application of traditional Chinese medicine, and in particular to the application of traditional Chinese medicine in the preparation of medicines for preventing and treating myocardial fibrosis, and specifically to the application of Xinsuning preparation in the preparation of medicines for preventing and treating myocardial fibrosis. Background Art

[0002] Myocardial fibrosis (MF) is a reactive pathological change of the myocardium, which is induced by multiple factors such as increased load, ischemia and hypoxia, metabolic disorders, and immune disorders, and is characterized by excessive proliferation and differentiation of myocardial fibroblasts, abnormal deposition of myocardial extracellular matrix, and reconstruction of collagen network. According to epidemiological surveys, more than 800,000 people die from fibrotic diseases each year in the world, especially pulmonary and cardiac fibrosis. Western medicine believes that the pathogenesis of MF is very complex, mainly related to factors such as inflammatory cytokines and chemokines, reactive oxygen species, renin-angiotensin-aldosterone system, matrix proteins and growth factors, and thus uses angiotensin converting enzyme inhibitors (ACEI), aldosterone receptor antagonists (ARB), β-blockers, angiotensin receptor antagonists, statins and other drugs to treat it. However, because ACEI drugs can cause irritating dry cough and angioedema, many patients cannot tolerate them. The incidence of hyperkalemia is as high as 8.6% to 26% when taking ARB drugs, and arrhythmia is often the first manifestation. Blood potassium and electrocardiogram must be closely followed during medication. Beta-blockers have the risk of causing hypotension, inducing or aggravating bronchial asthma, and aggravating atrioventricular block, and sudden discontinuation of medication after long-term use often aggravates the original symptoms. Therefore, the treatment methods used in the prior art have the disadvantages of expensive treatment costs, uncertain efficacy, and many adverse reactions, which limit clinical application.

[0003] In terms of traditional Chinese medicine, modern studies have shown that traditional Chinese medicine can inhibit inflammatory response, regulate cell autophagy, proliferation and apoptosis, reduce myocardial oxidative stress and damage, inhibit fibroblast activation and collagen synthesis, and has the potential to reduce or even reverse MF by intervening in a variety of cytokines and signaling pathways. Many effective ingredients of traditional Chinese medicine, such as curcumin, quercetin, rutin, ursolic acid, berberine, and resveratrol, have the effect of inhibiting myocardial fibrosis. There are also many traditional Chinese medicine compound prescriptions, such as the ancient prescription Dahuangchong Pill, the self-prescribed Kangdaxin oral liquid, Qishen Yiqi granules, Kangxian Yixin prescription, and other effective prescriptions. However, MF is a complex pathological process, and the corresponding TCM disease names and syndrome types are also different. There are great differences in the syndrome differentiation and treatment of MF in traditional Chinese medicine. According to the different TCM syndrome differentiation or the different causes of myocardial fibrosis, different traditional Chinese medicines and their compound prescriptions need to be selected for treatment. For example, myocardial fibrosis caused by hypertension often uses the method of promoting blood circulation and removing blood stasis, and nourishing the liver and kidneys, and myocardial fibrosis caused by coronary heart disease often uses the method of promoting blood circulation and removing blood stasis, and promoting qi and relieving pain. Some scholars have also proposed to explore the pathogenesis and treatment of myocardial fibrosis in TCM from the perspective of the theory of meridians, the theory of "stasis and poison", and the theory of internal movement of collateral wind, but there is no unified treatment principle. Therefore, finding Chinese medicine for the treatment of myocardial fibrosis that conforms to the theory of TCM evidence-based treatment principles and methods has become a problem for continued research. Summary of the invention

[0004] The present invention proposes a technical scheme for treating myocardial fibrosis by using traditional Chinese medicine, specifically the use of a Xinsu Ning preparation in preparing medicines for preventing and treating myocardial fibrosis, mainly for preparing medicines for preventing and treating myocardial fibrosis.

[0005] In view of the above technical problems, the present invention adopts the following technical solutions:

[0006] The invention discloses an application of the Xinsuning preparation in preparing medicines for preventing and treating myocardial fibrosis.

[0007] It is further defined that the Xinsuning preparation is used in the preparation of a drug for preventing and treating myocardial fibrosis by enhancing myocardial collagen fibers.

[0008] It is further defined that the strengthening of myocardial collagen fibers includes reducing the number of myocardial collagen fibers, enhancing the arrangement density of myocardial collagen fibers and increasing the number of myocardial cells.

[0009] It is further defined that the Xinsuning preparation is used in the preparation of a drug for preventing and treating myocardial fibrosis by inhibiting the overexpression of myocardial collagen.

[0010] It is further defined that the myocardial collagen is type I collagen and type III collagen.

[0011] It is further defined that the Xinsuning preparation is used in the preparation of a drug for preventing or treating doxorubicin-induced myocardial fibrosis.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1. Use of the Xinsuning preparation of the present invention in the preparation of drugs for preventing and treating myocardial fibrosis, wherein the Xinsuning preparation treatment group of the present invention can significantly improve cardiac myocardial cell tissue fibrosis compared with the model group and the control group, and use of the Xinsuning preparation in the preparation of drugs for preventing and treating myocardial fibrosis by improving cardiac myocardial cell tissue fibrosis.

[0014] 2. The use of the Xinsuning preparation of the present invention in the preparation of drugs for preventing and treating myocardial fibrosis, wherein the Xinsuning preparation treatment group of the present invention can make type I collagen and type III collagen in the extracellular matrix of myocardial cells show a significant downward trend compared with the model group and the control group, with type I collagen p<0.05 and type III collagen p<0.01, which are statistically significant. Therefore, the Xinsuning preparation can effectively reduce the overexpression of myocardial collagen and prevent the occurrence of myocardial fibrosis.

[0015] 3. The Xinsuning preparation of the present invention is mainly used in the preparation of drugs for preventing and treating myocardial fibrosis induced by adriamycin and can be used as a protective drug for adriamycin-induced cardiotoxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the pathological morphology of mouse heart tissue after myocardial cell injury by Xinsuning preparation under Masson staining at a magnification of X=4;

[0017] Figure 2 This is a schematic diagram of the pathological morphology of mouse heart tissue after myocardial cell injury by Xinsuning preparation, using Masson staining, at a magnification of X=400;

[0018] Figure 3 This is a Sirius red staining image of the pathological morphology of mouse heart tissue after myocardial cell injury by Xinsuning preparation; Figure 3 Group A is the image under ordinary microscope bright field. Figure 3 Group B is the image under dark field of polarized light microscope;

[0019] Figure 4 Volcano plot of differentially expressed proteins between the model group and the Xinsuning treatment group;

[0020] Figure 5 This is the cluster diagram of differential protein expression levels between the model group and the Xinsuning treatment group;

[0021] Figure 6 The flowchart of DIA experiment is shown in Figure 1, where DIA is a targeted proteomics technology.

[0022] Figure 7 This is a basic flowchart for bioinformatics analysis.

[0023] Figure 8 This is a statistical chart of ultrasound cardiology detection in mice in a comparative experiment. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further explained below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to the implementation modes described below.

[0025] The invention proposes the use of a Xinsu Ning preparation in preparing a drug for preventing and treating myocardial fibrosis. The Xinsu Ning preparation comprises: 334 parts of coptis root, 250 parts of pinellia tuber, 250 parts of tuckahoe, 167 parts of immature bitter orange, 250 parts of radix changshanii, 42 parts of lotus seed core, 250 parts of sophora flavescens, 250 parts of artemisia annua, 167 parts of ginseng, 250 parts of ophiopogon and 167 parts of liquorice.

[0026] As a traditional Chinese medicine for antiarrhythmic treatment of phlegm-heat disturbing the heart, Xinsuning has been shown in multiple studies to have a positive effect on arrhythmia by prolonging the arrhythmia latency period of arrhythmic mice, delaying the onset of arrhythmia, shortening the duration of arrhythmia, and improving the hemorheology indexes of mice. In terms of its mechanism of action research: by conducting metabolic pathway enrichment analysis of differential metabolites, it is predicted that Xinsuning plays an antiarrhythmic role by regulating the disordered metabolic pathways; by using patch clamp technology to explore the effects of Xinsuning on the action potential and ion channels of isolated cardiac myocytes, it is shown that Xinsuning can dose-dependently prolong the action potential interval of mouse cardiomyocytes and can simultaneously regulate multiple targets, including the effects on sodium, potassium, and calcium channels; by weighted network pharmacology analysis, Xinsuning has the effects of protecting the heart from I / R injury, inhibiting cardiomyocyte apoptosis, and improving glucose and lipid metabolism.

[0027] Myocardial fibrosis is an important pathological change in the progression of cardiovascular diseases. The normal extracellular matrix (ECM) is composed of collagen, aminoglycans and proteoglycans, non-collagenous proteins, elastin, various growth factors and enzymes. Myofibroblasts secrete and synthesize collagen, mainly containing type I collagen (80%), type III collagen (11%) and a small amount of other collagens. Under normal circumstances, ECM maintains a dynamic balance, providing structure for surrounding cells and supporting cell growth and proliferation. ECM imbalance will lead to fibrosis formation, and the metabolic disorder of collagen in ECM is the main factor leading to myocardial fibrosis. Xinsuning preparations are used in the preparation of drugs for the prevention and treatment of myocardial fibrosis by improving myocardial fiber morphology. Among them, improving myocardial fiber morphology includes reducing the number of myocardial collagen fibers, enhancing the arrangement density of myocardial collagen fibers and increasing the number of myocardial cells. Xinsuning preparations are used in the preparation of drugs for the prevention and treatment of myocardial fibrosis by inhibiting the overexpression of myocardial collagen, wherein myocardial collagen is type I collagen and type III collagen.

[0028] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0029] The Xinsuning preparations of the present invention include Xinsuning capsules, Xinsuning tablets, Xinsuning pills and Xinsuning granules.

[0030] Example 1: Preparation of Xinsuning Capsules

[0031] The composition formula: 334g of Coptis chinensis, 250g of Pinellia ternata, 250g of Poria cocos, 167g of Citrus aurantium, 250g of Dichroa chinensis, 42g of Lotus seed core, 250g of Sophora flavescens, 250g of Artemisia annua, 167g of Ginseng, 250g of Ophiopogon japonicus and 167g of Licorice.

[0032] The process steps are as follows:

[0033] (1) Clean, slice and dry all raw materials for later use.

[0034] (2) Take 334g of Coptis chinensis, 167g of Citrus aurantium, 250g of Radix Dichroae, 42g of Pithmum nelumbinis and 250g of Sophora flavescens, add 60% ethanol, and reflux extract twice. For the first time, 8 times the amount of ethanol was added to the raw materials of Coptis chinensis, Citrus aurantium, Radix Dichroae, Pithmum nelumbinis and Sophora flavescens, and reflux extract was performed for 2 hours. For the second time, 6 times the amount of ethanol was added to the raw materials of Coptis chinensis, Citrus aurantium, Radix Dichroae, Pithmum nelumbinis and Sophora flavescens, and reflux extract was performed for 1.5 hours. The extracts were combined and filtered. The filtrate was decompressed at 0.04MPa and 75°C to recover ethanol, and the filtrate was further concentrated to a thick paste with a relative density of 1.36 to 1.38 at 60°C, and dried at 80°C to form a dry paste. The residue was discarded.

[0035] (3) Take 167g of ginseng, 250g of Poria cocos, and 250g of Pinellia ternata, add 70% ethanol, and reflux extract twice. The first time, add 10 times the amount of ethanol of the raw materials of ginseng, Poria cocos, and Pinellia ternata, and reflux extract for 2.5 hours. The second time, add 8 times the amount of ethanol of the raw materials of ginseng, Poria cocos, and Pinellia ternata, and reflux extract for 2 hours. Combine the extracts, filter, and collect the filtrate in another container for later use.

[0036] (4) The residue obtained in step (3) is combined with 250 g of Ophiopogon japonicus, 250 g of Artemisia annua and 167 g of Licorice, and decocted twice with water. The first time, 10 times the amount of water was added to the residue, Ophiopogon japonicus, Artemisia annua and Licorice raw materials, and the second time, 8 times the amount of water was added to the residue, Ophiopogon japonicus, Artemisia annua and Licorice raw materials. Each time, the decoction is combined, filtered, and the filtrate is concentrated to a clear paste with a relative density of 1.05 to 1.06 at 80°C, 95% ethanol is added to make the alcohol concentration reach 70%, mixed, and allowed to stand for 24 hours to precipitate, the supernatant is collected, filtered, the filtrate is combined with the filtrate collected in another device in step (3), and the ethanol is recovered under reduced pressure at 0.04 MPa and 75°C, and the mixture is further concentrated to a thick paste with a relative density of 1.36 to 1.38 at 60°C, and dried at 80°C to obtain a dry paste.

[0037] (5) Combine the two dry pastes obtained in step (2) and step (4), crush into 100-mesh fine powder, add appropriate amount of dextrin, mix well, put into capsules, and prepare 1000 capsules.

[0038] Example 2: Preparation of Xinsu Ning Tablets

[0039] The composition formula of Xinsu Ning Tablets is the same as that of Xinsu Ning Capsules, and the process steps (1) to (4) are also the same as those of Xinsu Ning Capsules, except that:

[0040] (5) The two dry pastes obtained in step (2) and step (4) are combined, crushed into a fine powder of 100 mesh, added with an appropriate amount of dextrin, granulated with 85% ethanol, dried at 60°C, granulated, added with 0.5% magnesium stearate, mixed well, pressed into 1000 tablets, and coated.

[0041] Example 3: Preparation of Xinsu Ning Pills

[0042] The composition formula of Xinsu Ning Pills is the same as that of Xinsu Ning Capsules, and the process steps (1) to (4) are also the same as those of Xinsu Ning Capsules, except that:

[0043] (5) Combine the two dry pastes obtained in step (2) and step (4), grind into fine powder of 100 mesh, add 35g-50g of refined honey and appropriate amount of water to make pills for every 100g of powder, dry and make water-honey pills; or add 80g-130g of refined honey to make small honey pills or large honey pills.

[0044] Example 4: Preparation of Xinsuning Granules

[0045] The composition formula of Xinsu Ning Granules is the same as that of Xinsu Ning Capsules, and the process steps (1) to (4) are also the same as those of Xinsu Ning Capsules, except that:

[0046] (5) The two dry pastes obtained in step (2) and step (4) are combined, and an appropriate amount of sucrose powder is added to form granules, and dried to obtain the granules.

[0047] In the preparation process, auxiliary materials used in pharmacy such as fillers, binders, disintegrants, lubricants, flavoring agents and the like may also be added to oral liquid preparations.

[0048] The Xinsu Ning capsules prepared in Example 1 were used to carry out the following experimental process.

[0049] 1. Animal model experiments

[0050] 1.1 Experimental Materials

[0051] The experimental instruments and reagents are shown in Table 1.

[0052] Table 1 Main experimental instruments

[0053]

[0054] Xinsuning capsules were purchased from Shaanxi Momede Qixuehe Pharmaceutical Co., Ltd. (approval number: National Medicine Standard Z20050131). Adriamycin, item number: D8740, specification: 500 mg, was purchased from Solebao Company.

[0055] 1.2 Experimental animals

[0056] A total of 30 male C57BL / 6 mice, weighing 20±5g, were kept in the SPF-grade standard animal breeding room of the Experimental Animal Center of Shandong University of Traditional Chinese Medicine. Breeding conditions: temperature controlled at 24±2℃; humidity controlled at 50±10%; artificial light 12h / d; ventilation rate 15 times / h, mice free drinking water and food intake. The mice used in the experiment were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2021-0011. All animals in the experiments were strictly in accordance with the "Guiding Principles for the Care and Use of Laboratory Animals" of Shandong University of Traditional Chinese Medicine (Animal Use License: SYXK (Beijing) 2021-0011). This study has been approved by the Animal Ethics Review Committee of Shandong University of Traditional Chinese Medicine (Approval Number: SDUTCM20230828004).

[0057] 1.3 Drug preparation

[0058] Xinsuning treatment group: Weigh an appropriate amount of Xinsuning capsule powder and add 10 ml of pure water to make 0.075 g kg -1 of aqueous suspension.

[0059] Control group (CON group): mice were given normal saline at 10 ml / kg / d.

[0060] Model group (DOX group) and XSN treatment group (DOX+XSN group): Modeling drug was prepared at 3 mg / kg, 15 mg of doxorubicin was dissolved in 50 ml of normal saline, and immediately divided into 5 centrifuge tubes, stored at 4°C in the dark, and can be stored for 1 month. Mice were intraperitoneally injected with modeling drug at 3 mg / kg.

[0061] 1.4 Experimental Procedure

[0062] (1) Grouping: control group (CON group), model group (DOX group), and XSN treatment group (DOX+XSN group).

[0063] (2) Myocardial fibrosis modeling: Male C57BL / 6 mice were intraperitoneally injected with doxorubicin for 5 consecutive days. The control group of mice was intraperitoneally injected with an equal amount of normal saline.

[0064] (3) Administration: Xinsuning treatment group: continuous oral administration for 14 days, with a dosage of 10 mL kg -1 , Control group: gavage with equal amount of normal saline.

[0065] 1.5: Tissue staining

[0066] Masson trichrome staining: Connective tissue contains three kinds of fibers, namely collagen fibers, elastic fibers, and reticular fibers, and collagen fibers are the most widely distributed and the most abundant type of fibers. Masson trichrome staining, also known as Masson staining, is a classic method for connective tissue staining. The trichrome usually refers to staining cell nuclei and can selectively display collagen fibers and muscle fibers. After Masson staining, muscle fibers appear red, and collagen fibers appear green (light green) or blue (aniline blue), which is used to distinguish collagen fibers from muscle fibers. Specifically, the tissue sections were stained according to the following staining steps: 1) paraffin sections were dewaxed to water and soaked in potassium dichromate overnight; 2) the sections were taken out the next day and rinsed with tap water; 3) the nuclei were stained with Weigert's hematoxylin for 5-10 minutes; 4) the sections were differentiated with hydrochloric acid ethanol differentiation solution and washed with water; 5) the sections were blued with Masson's blue solution and washed with water for 1 minute; 6) the sections were stained with acidic Ponceau red for 5-10 minutes; 7) during the above operations, the weak acid working solution was prepared in a ratio of distilled water: weak acid solution = 2:1, and the weak acid working solution Rinse for 1 minute; 8) Differentiate with 1% phosphomolybdic acid solution for 1-2 minutes; 9) Wash with the prepared weak acid working solution for 1 minute; 10) Directly immerse in aniline blue staining solution for 1-2 minutes; 11) Wash with the prepared weak acid working solution for 1 minute; 12) Rapid dehydration with 95% ethanol; 13) Dehydrate with anhydrous alcohol for 3 times, 5-10 seconds each time; 14) Transparent with xylene for 3 times, 1-2 minutes each time, and seal with neutral gum; 15) Observe under a microscope, the collagen fibers are stained blue, which represents the degree of myocardial fibrosis.

[0067] Sirius red staining: Collagen fiber is the most widely distributed and abundant fiber in connective tissue, and is widely distributed in various organs. Type I collagen fibers are mainly bones, skin, and tendons; type II collagen fibers are mainly cartilage collagen; type III collagen fibers are mainly found in embryonic tissues, adult blood vessels, and the gastrointestinal tract; and type IV collagen fibers are mainly found in the basement membrane. Sirius red and its contrast dye are both strong acid dyes that easily bind to the alkaline groups in collagen molecules and adsorb firmly. In polarized light microscopy, collagen fibers have the property of positive uniaxial birefringence light. When combined with Sirius red composite staining solution, birefringence can be enhanced, and resolution can be improved, thereby distinguishing the two types of collagen fibers. Sirius red staining shows that collagen tissue is red under an ordinary microscope (bright field) and myocardial tissue is yellow; polarized light microscope (dark field) observation shows that type I collagen fibers are yellow or red fibers, precisely arranged, showing strong birefringence; type II collagen fibers are loosely distributed in a variety of colors, showing weaker birefringence; type III collagen fibers are green fine fibers, showing weaker birefringence; type IV collagen fibers are light yellow, showing weaker birefringence. The tissue sections were stained according to the following staining steps: 1. Sampling: After completely removing the mouse heart, place the mouse heart in the direction of the ultrasonic long axis section, and put the mouse heart into the 4% paraformaldehyde prepared in advance. 2. Dehydration and embedding: After the mouse heart tissue block is fixed in 4% paraformaldehyde for 2 hours, it is placed in a dehydrator for dehydration according to the procedure in the table below. After dehydration, it is embedded in paraffin, and the embedded wax block is placed in a 4°C refrigerator for standby use. 3. Staining: 1) Take the dewaxed tissue specimen and stain it with modified Sirius red staining solution for 5 minutes; 2) Wash it with distilled water to remove the surface staining solution; 3) Dehydrate it with a series of ethanol starting from 75%, make it transparent with xylene, and seal it with neutral gum. 4) Observe it under a microscope.

[0068] 1.6 Analysis of color development results

[0069] See also Figure 1 and Figure 2 The myocardial tissue of mice in the control group (CON group) was complete and clear, and the microscopic field was purple-red, with a small amount of blue collagen tissue; compared with the control group (CON group), the myocardial tissue of mice in the model group (DOX group) was disordered, the purple-red myocardial tissue decreased, and the blue myocardial collagen fibers increased; compared with the model group (DOX group), the myocardial tissue of mice in the xinsuning treatment group (DOX+XSN group) was mainly purple-red myocardial cells, and the blue myocardial collagen fibers decreased. It was proved that the xinsuning preparation can increase the number of myocardial cells and reduce the number of myocardial collagen fibers to improve myocardial tissue fibrosis.

[0070] See also Figure 3 In group A, Sirius red staining shows collagen tissue in red and myocardial tissue in yellow under a normal microscope (bright field); see Figure 3In group B, polarized light microscopy (dark field) observation showed that type I collagen fibers were yellow or red fibers, precisely arranged, and showed strong birefringence; type II collagen fibers were loosely distributed in various colors, showing weak birefringence; type III collagen fibers were green thin fibers, showing weak birefringence; type IV collagen fibers were light yellow, showing weak birefringence. It can be seen that compared with the control group (CON group) mice, the model group (DOX group) mice heart tissue collagen fibers increased significantly, arranged disorderly, mainly yellow-red type I collagen, in addition to type I collagen fibers, also contained green type III collagen fibers, and the arrangement density of type I collagen fibers and type III collagen fibers was significantly improved; compared with the model group (DOX group), the collagen fibers in the heart speed Ning treatment group (DOX+XSN group) were significantly reduced, and the expression of type I and type III collagen in the mouse heart tissue was significantly decreased. It was demonstrated that the Xinsuning preparation could significantly reduce the expression of type I and type III collagen in cardiac tissue, thereby improving myocardial tissue fibrosis.

[0071] 2. Fibrosis proteomics test

[0072] 2.1 Experimental Procedure

[0073] See also Figure 6 , extract the total protein from the mouse heart tissue samples, take out a part for protein concentration determination and SDS-PAGE detection, take another part for trypsin hydrolysis, desalt the hydrolyzed peptides, and perform LC-MS / MS identification on the samples. According to the proteomics experiment to detect the changes in fibrosis-related proteins in mouse heart tissue, LC-MS / MS identification is divided into two main steps. The first step is to use the traditional DDA method to establish a protein spectrum library, and the second step is to use DIA technology to collect mass spectrometry data for each sample. Based on the DDA database, spectrum matching, quantitative information extraction, and subsequent statistical analysis are performed to intuitively obtain which fibrosis-related proteins are improved by Xinsuning.

[0074] 2.2 Bioinformatics analysis process

[0075] See also Figure 7 The basic process of bioinformatics analysis is: qualitative and quantitative data analysis is performed using search and analysis software, and expression level analysis and functional analysis are performed after quality assessment and preprocessing. Functional annotation analysis of identified proteins is performed using multiple common databases. GO analysis, pathway analysis, and interaction analysis are performed on the screened differential proteins. At the same time, volcano maps, expression pattern clustering heat maps, and venn analysis are performed on the data of the difference comparison group. In addition, according to the data situation, the correlation or the part of interest is studied, and key proteins and their functions or pathways are selected for subsequent key research and verification directions.

[0076] 2.3 Instruments and reagents

[0077] Table 2: Instruments and equipment

[0078]

[0079]

[0080] Table 3: Main reagents

[0081]

[0082] 2.4 Preparation of main solutions

[0083] Preparation of dithiothreitol (DTT) (25 mM) solution: weigh 192.5 mg DTT, dissolve it in 50 ml deionized water, mix well and dispense into 200 μl / tube, and store at -20°C. Do not freeze and thaw repeatedly.

[0084] Preparation of iodoacetamide solution (100 mM): Quickly weigh 95 mg of iodoacetamide powder and transfer it to a 15 ml centrifuge tube wrapped in tin foil. Mix with 3 ml of deionized water until dissolved, and make up to 5 ml. Store in a dark place.

[0085] 2.5 Protein extraction

[0086] (1) Take out the frozen mouse heart tissue sample, add liquid nitrogen, grind it thoroughly, and take out an appropriate amount into a 2 mL high-speed centrifuge tube.

[0087] (2) Add mouse heart tissue sample lysate, phosphatase inhibitors, and protease inhibitor PMSF to a final concentration of 1 mM.

[0088] (3) Grind with a cold grinder at -35°C, 60 Hz, 120 s, and repeat once.

[0089] (4) The solution was centrifuged at 12,000 rpm for 10 min at 4°C, the supernatant was collected, and the solution was centrifuged again to collect the supernatant.

[0090] (5) The supernatant is the total protein solution of the mouse heart tissue sample. The protein concentration is measured and the solution is aliquoted and stored at -80°C for future use.

[0091] 2.6 Protein concentration determination

[0092] Quantitative principle

[0093] BCA (bicinchonininc acid, polycyanoacrylate n-butyl ester) and other reagents such as copper sulfate of divalent copper ions are mixed together to form apple green, which is the BCA working reagent. Under alkaline conditions, when BCA binds to proteins, the proteins convert Cu 2+ Reduction to Cu + , a Cu + After chelating two BCA molecules, the working reagent changes from the original apple green to a purple complex. The water-soluble complex shows maximum absorbance at 562nm. The absorbance and protein concentration have a good linear relationship over a wide range. Therefore, the protein concentration can be calculated based on the absorbance value.

[0094] BCA method experimental process

[0095] (1) According to the instructions of the BCA kit, prepare the required volume of colorimetric solution at a ratio of buffer A:buffer B=50:1 (v / v).

[0096] (2) Take out a portion of the protein solution to be tested and dilute it with ultrapure water (to prevent the concentration from being too high and exceeding the working range of the standard curve).

[0097] (3) Prepare a clean 96-well plate and add the following gradient of BSA standard protein solution: 0 μL, 1 μL, 2 μL, 4 μL, 8 μL, 12 μL, 16 μL, 20 μL. Then add the corresponding volume of ultrapure water to each well to make up the volume to 20 μL.

[0098] (4) Add 2 μL of the protein solution to be tested to a 96-well plate, set up three replicate wells for each sample, and also add the volume to 20 μL.

[0099] (5) Add 200 μL of pre-prepared colorimetric solution to each well (the colorimetric solution must be prepared immediately before use) and incubate at 37°C for 30 min.

[0100] (6) Use an ELISA reader to measure the absorbance (wavelength: 562 nm).

[0101] (7) Calculate the standard curve based on the known concentration and absorbance value of the standard protein solution, substitute the absorbance value of the sample to be tested, and the protein concentration value can be calculated.

[0102] 2.7 SDS-polyacrylamide gel electrophoresis

[0103] An appropriate amount of protein was taken from each sample and separated by 12% SDS-PAGE. The separated gel was stained with Coomassie brilliant blue using eStain LG protein stainer. The stained gel was imaged using a fully automatic digital gel imaging analysis system.

[0104] 2.8 Trypsin digestion

[0105] (1) According to the measured protein concentration, take an appropriate amount of protein from each sample and dilute different groups of samples with lysis buffer to the same concentration and volume.

[0106] (2) Add DTT to the above protein solution to make the final concentration of DTT 5 mM, mix well, and incubate at 55°C for 30 min.

[0107] (3) Cool on ice until it reaches room temperature (the solution should not be too cold or too hot when felt by hand).

[0108] (4) Add the corresponding volume of iodoacetamide to make the final concentration of 10 mM, mix thoroughly, and place at room temperature away from light for 15 min.

[0109] (5) Add 6 times the volume of acetone to the above solution to precipitate the protein and place it at -20°C for more than four hours or overnight.

[0110] (6) Collect the precipitate by centrifugation at 8000 × g for 10 min at 4°C and evaporate the acetone for 2-3 min.

[0111] (7) Add 100 μL of 50 mM NH4HCO3 to dissolve the precipitate, add 1 mg / ml trypsin-TPCK at 1 / 50 of the sample mass, and digest at 37°C overnight.

[0112] (8) Phosphoric acid is added to adjust the pH to about 3 to terminate the enzymatic hydrolysis reaction.

[0113] 2.9 Peptide desalting

[0114] The peptides after enzymatic hydrolysis were treated with SOLA TM SPE 96-well plate desalting:

[0115] (1) Activation: Activate the column with 200 μL of methanol, repeat 2 times, for a total of 3 times.

[0116] (2) Equilibration: Activate the column with 200 μL of equilibration solution (0.1% formic acid, 100% water). Repeat twice, for a total of 3 times.

[0117] (3) Load sample: 50 μL-500 μL sample (total protein solution of mouse heart tissue sample), adjust the vacuum, and keep the droplet rate at 1 mL / min (about 1 drop / second).

[0118] (4) Repeat the load in step S3 once.

[0119] (5) Washing: Wash with 200 μL of 0.1% formic acid aqueous solution, repeat twice, a total of 3 times.

[0120] (6) Elution: 150 μL of 50% acetonitrile-water (containing 0.1% formic acid) was used to elute the peptides. This was repeated 2 times and 3 times in total to obtain 450 μL of eluent, which was then evaporated to dryness under vacuum.

[0121] 2.10LC-MS / MS high-resolution mass spectrometry detection

[0122] Before mass spectrometry injection, each sample (total protein solution of mouse heart tissue sample) was mixed at a volume ratio of iRT: sample to be tested = 1:20 as an internal standard. iRT standard (Biognosys, ThermoFisher) is a mixed kit containing 11 synthetic peptides that do not exist in nature. Its stability, sensitivity and retention time have been optimized, it is relatively stable, and it will not affect the sample to be tested. Therefore, it is widely used for calibration of chromatographic systems and quantitative quality control.

[0123] High pH Liquid Phase Separation:

[0124] (1) Samples: Equal amounts of peptides from all enzymatically digested samples were mixed and separated using an Agilent 1100 HPLC system in a mobile phase at pH 10.

[0125] (2) Separation conditions

[0126] Chromatographic column: Nano Chrom-C18 column (5μm, 150mm×2.1mm).

[0127] Detection wavelength: UV 210nm and 280nm.

[0128] Mobile phase A: ACN-H2O (2:98, v / v), mobile phase B: ACN-H2O (90:10, v / v) (both mobile phases were adjusted to pH 10 with ammonia water), flow rate: 250 μL / min. Gradient elution conditions: 0-10 min, 2% B; 10-10.01 min, 2-5% B; 10.01-37 min, 5-20% B; 37-48 min, 20-40% B; 48-48.01 min, 40-90% B; 48.01-58 min, 90% B; 58-58.01 min, 90-2% B; 58.01-63 min, 2% B.

[0129] Component collection: Starting from the 10th minute, the eluent was collected into centrifuge tubes 1-10 every one minute, and the fractions were collected in the order of 1→10. A total of 10 components were collected, vacuum freeze-dried and dried, and the samples were frozen and stored for mass spectrometry.

[0130] The flow rate was 300 nL / min, C 18Analytical column (25cm×75μmID,1.6μmC 18 , ionoptics), buffer A is 0.1% FA aqueous solution, buffer B is 0.1% FA in CAN, and the specific chromatographic conditions and mass spectrometry conditions are shown in Tables 4, 5, 6 and 7.

[0131] Table 4: DDA chromatography conditions

[0132]

[0133] Table 5: DDA mass spectrometry conditions

[0134]

[0135] Table 6: DIA chromatography conditions

[0136]

[0137] Table 7: DIA mass spectrometry conditions

[0138]

[0139]

[0140] 2.11 Data Analysis

[0141] The purpose of this operation is to match the spectrum output by the mass spectrometer with the theoretical spectrum generated by the fasta library, convert the machine signal into peptide and protein sequence information, and then establish a spectrum library based on sequence information, peptide retention time, fragment ion information, etc., to facilitate subsequent DIA analysis.

[0142] The LC-MS / MS raw files were imported into Spectronaut Pulsar 18.3 (Biognosys) software for library search and library construction. The main parameters are shown in Tables 8 and 9.

[0143] Table 8: DDA library construction parameters

[0144]

[0145] Carbamidomethyl (C) in Table 7 is often referred to as "Carbamidomethyl" in the fields of biochemistry and proteomics. In proteomics, it is a common chemical modification that usually introduces a methyl group on the amide group of a protein. Oxidation (M) and Acetyl (Protein N-term) both refer to specific protein modifications. Oxidation (M) usually refers to the oxidation of methionine (M) residues in proteins. This modification may cause the sulfur atom on the methionine side chain to oxidize to sulfoxide or sulfone; Acetyl (Protein N-term) describes the acetylation of the N-terminus of a protein (the amino terminus of a protein). This modification usually involves adding an acetyl group (-COCH3) to the first amino acid residue of a protein, usually an alanine (Ala) or methionine (Met) at the amino terminus.

[0146] Table 9: Key parameters for DIA data analysis

[0147]

[0148]

[0149] 2.12 Differential protein screening

[0150] Two parameters were used to evaluate the differences in protein expression between groups, namely Fold change (FC, fold change, log2 (Fold change) = experimental group mean - control group mean) and p-value obtained by t-test. In this experiment, the screening conditions for differentially expressed proteins were: Foldchange ≥ 2 or Foldchange ≤ 1 / 2 and p-value < 0.05.

[0151] The proteins regulated by XSN in fibrotic mouse heart tissue were analyzed by proteomics. In the doxorubicin-induced mouse heart tissue samples, 192 differential proteins, 102 upregulated proteins and 90 downregulated proteins were detected. In the XSN treatment group (DOX-XSN group), 181 differential proteins were detected, including 77 upregulated proteins and 104 downregulated proteins. The statistical table of differentially expressed proteins is shown in Table 10 and Figure 4 The volcano plot of differentially expressed proteins between the xinsuning treatment group (DOX-XSN group) and the model group (DOX group) was used to show the overall distribution of differentially expressed proteins.

[0152] Table 10: DDA library construction parameters

[0153]

[0154] See also Figure 5 The model group (DOX group) and the DOX-XSN treatment group (DOX-XSN group) were clustered according to protein expression levels. Red indicates highly expressed proteins, blue indicates low expressed proteins, and each row indicates the expression level of each protein in different groups. Figure 5 As can be seen from the legend in the upper right corner, the left half of the horizontal axis is group D, which represents the model group (DOX group), and the right half of the horizontal axis is group X, which represents the DOX-XSN treatment group (DOX-XSN group). The vertical axis represents the differential proteins of the two groups, and the changes in protein expression are distinguished by the red and blue in the legend. Red represents high-expression proteins, and blue represents low-expression proteins. The results of cluster analysis of the expression levels of total differential proteins revealed that the DOX-XSN preparation has a regulatory effect on the collagen family.

[0155] Collagen is the dominant extracellular protein in the myocardium. The healthy heart extracellular matrix contains about 70% type I collagen (Col1a1) and 12% type III collagen (Col3a1). The collagen family was further analyzed specifically, as shown in Table 11. Table 11 shows the expression of the collagen family in the xinsuning treatment group compared with the model group. It can be concluded that the xinsuning treatment group (DOX-XSN group) significantly downregulated type I collagen (p<0.05) and type III collagen (p<0.01), and the results were statistically significant. This result is consistent with the results of Sirius red staining polarized light imaging. It can be concluded that the xinsuning preparation prevents the occurrence of myocardial fibrosis by downregulating the expression of type I collagen and type III collagen. Q60847, Q80X19, O35206, P39061, P11087, Q01149, P28481, P02463, P08122, Q04857, Q02788 and Q8C6K9 in Table 11 are all protein identification codes.

[0156] Table 11 Differential expression of collagen family in the Xinsuning treatment group before and after administration

[0157]

[0158] 3. Comparative test

[0159] Myocardial fibrosis can lead to increased myocardial stiffness and decreased compliance, thereby affecting the contractile function of the heart and causing a decrease in ejection fraction. To further demonstrate the role of Xinsuning preparation in the preparation of drugs for the prevention and treatment of myocardial fibrosis, the following experiment was conducted.

[0160] 3.1. Formula:

[0161] Xinsuning group (DOX+XSN-Q group): Coptis chinensis 334g, Pinellia ternata 250g, Poria 250g, Citrus aurantium 167g, Dichroa 250g, Lotus seed heart 42g, Sophora flavescens 250g, Artemisia annua 250g, Ginseng 167g, Ophiopogon japonicus 250g and Licorice 167g.

[0162] Comparative group (DOX+XSN-Z group): Poria cocos and Sophora flavescens were replaced with Atractylodes macrocephala and Scutellaria baicalensis.

[0163] 3.2 Experimental Animals

[0164] Same as section 1.2.

[0165] 3.3 Drug configuration

[0166] Xinsuning group (DOX+XSN-Q group): according to the formula in 3.1, add 10 ml of pure water to make 0.075 g·kg -1 of aqueous suspension.

[0167] Comparative group (DOX+XSN-Z group): According to the formulation in 3.1, add 10 ml of pure water to prepare 0.075 g·kg -1 of aqueous suspension.

[0168] Control group (CON group): mice were given normal saline at 10 ml / kg / d.

[0169] The model group (DOX group), doxorubicin group (DOX+XSN-Q group) and control group (DOX+XSN-Z group) were all prepared with modeling drugs at 3 mg / kg. 15 mg of doxorubicin was dissolved in 50 ml of normal saline, and the mice were intraperitoneally injected with modeling drugs at 3 mg / kg.

[0170] 3.4 Experimental Process

[0171] (1) Grouping: control group (CON group), model group (DOX group), xinsuning group (DOX+XSN-Q group) and comparative group (DOX+XSN-Z group).

[0172] (2) Myocardial fibrosis modeling: Male C57BL / 6 mice were intraperitoneally injected with doxorubicin for 5 consecutive days. The control group mice were intraperitoneally injected with an equal amount of normal saline.

[0173] (4) Administration: Xinsuning group (DOX+XSN-Q group) and control group (DOX+XSN-Z group): Administered by intragastric administration for 14 consecutive days, with a dosage of 10 mL kg -1 ; Control group: gavage with equal volume of normal saline.

[0174] 3.5. Echocardiography in mice

[0175] See also Figure 8 A, compared with the model group (DOX group) and the control group (DOX+XSN-Z group), the xinsuning group (DOX+XSN-Q group) can significantly improve the ejection fraction of the mouse heart; see Figure 8 B, compared with the control group (DOX+XSN-Z group), the effect of Xinsu Ning group (DOX+XSN-Q group) was better. Figure 8 The DOX+DEX group in A is the positive group. Figure 8 A shows that compared with the model group (DOX group), the ejection fraction of the positive group (DOX+DEX group) was significantly increased, indicating that the mice were successfully modeled with doxorubicin.

[0176] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of Xinsuning preparation in the preparation of drugs for preventing and treating myocardial fibrosis.

2. The use of the Xinsuning preparation according to claim 1 in the preparation of drugs for preventing and treating myocardial fibrosis, characterized in that: The Xinsuning preparation is used in preparing medicines for preventing and treating myocardial fibrosis by strengthening myocardial collagen fibers.

3. The use of the Xinsuning preparation according to claim 2 in the preparation of drugs for preventing and treating myocardial fibrosis, characterized in that: The method of strengthening myocardial collagen fibers includes reducing the number of myocardial collagen fibers, enhancing the arrangement density of myocardial collagen fibers and increasing the number of myocardial cells.

4. The use of the Xinsuning preparation according to claim 2 in the preparation of drugs for preventing and treating myocardial fibrosis, characterized in that: The Xinsuning preparation is used in preparing drugs for preventing and treating myocardial fibrosis by inhibiting the overexpression of myocardial collagen.

5. Use of the Xinsuning preparation according to claim 4 in the preparation of drugs for preventing and treating myocardial fibrosis, characterized in that: The myocardial collagen is type I collagen and type III collagen.

6. Use of the Xinsuning preparation according to claim 1 in preparing a drug for preventing and treating myocardial fibrosis, characterized in that: The Xinsuning preparation is used in preparing a drug for preventing or treating adriamycin-induced myocardial fibrosis.

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