Application of compound wintercreeper mixture in improvement of myocardial fibrosis

Abstract

The invention discloses an application of a compound euonymus fortunei mixture (CFM) in improvement of myocardial fibrosis, and belongs to the technical field of medicines. The potential action target HSPA8 of the CFM for treating the MF is excavated for the first time by using a method of combining a cell heat transfer experiment and network pharmacology, and the binding capacity of the CFM and the HSPA8 is verified by using the cell heat transfer experiment. On the basis, the protective effect of the CFM on ISO-induced mouse myocardial fibrosis is further verified through animal experiments, and the result shows that the CFM improves MF by regulating and controlling HSPA8 and plays a myocardial protection role. In conclusion, it is found for the first time that the CFM can relieve or improve the myocardial fibrosis, the mechanism of the CFM for relieving or improving the myocardial fibrosis is further studied, the key core target HSPA8 is disclosed, and a new theoretical basis is provided for clinical treatment of the myocardial fibrosis through the CFM.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to the application of Compound Fufangteng Mixture in improving myocardial fibrosis. Background Art

[0002] Compound Fufangteng Mixture (CFM) is a proprietary Chinese medicine developed by the Pharmaceutical Factory of Guangxi University of Chinese Medicine, which is a characteristic drug in Guangxi. CFM is composed of three traditional Chinese medicines: Euonymus fortunei, Astragalus membranaceus, and Panax ginseng. The whole formula has the effects of replenishing qi and blood, strengthening the spleen and nourishing the heart, and is mainly used to treat symptoms such as qi and blood deficiency, and deficiency of both the heart and spleen. In the formula, both Panax ginseng and Astragalus membranaceus are important herbs for strengthening the healthy qi and tonifying deficiency. Among them, Panax ginseng greatly tonifies primordial qi and invigorates the spleen and benefits the lung; Astragalus membranaceus is good at replenishing qi and promoting yang, and can also replenish the qi of the spleen and lung; Euonymus fortunei functions to promote qi and activate blood circulation, relax tendons and activate collaterals. When combined with Panax ginseng and Astragalus membranaceus, they together achieve the effects of replenishing qi and blood, strengthening the spleen and nourishing the heart. Currently, there is little research on the therapeutic mechanism and molecular mechanism of CFM in improving cardiac function.

[0003] Myocardial Fibrosis (MF) is a very complex process of cardiac interstitial remodeling, mainly manifested as abnormal proliferation of cardiac fibroblasts (CFs), excessive deposition and uneven distribution of extracellular matrix (ECM). Cardiovascular disease (CVD) has become one of the most prominent health problems in the global public health field. MF is the core pathological basis of various CVDs. This pathological process can directly lead to changes in cardiac structure, affect ventricular systolic and diastolic functions, and ultimately induce heart failure. According to statistics from the World Health Organization, up to 17.9 million people die from CVDs every year, accounting for 31% of the total global deaths. Almost all CVDs are accompanied by the occurrence of MF. Due to its long course and high fatality rate, MF has become a core problem and focus of attention in the clinical prevention and treatment of heart diseases. However, there is still a lack of effective specific treatment methods and means for MF. Therefore, it is crucial to search for and develop new drugs and effective treatment strategies. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the purpose of the present invention is to provide the application of Compound Fufangteng Mixture in improving myocardial fibrosis.

[0005] In the first aspect of the present invention, there is provided the application of Compound Fufangteng Mixture in the preparation of a product for reducing the expression of HSPA8.

[0006] Furthermore, the product is a drug.

[0007] Furthermore, the HSPA8 is HSPA8 in myocardial tissue.

[0008] In the second aspect of the present invention, there is provided the use of Compound Fufangteng Mixture in the preparation of a product for reducing the contents of myocardial interstitial TGF-β1, α-SMA, COL I and COL III.

[0009] Furthermore, the product is a drug.

[0010] In the third aspect of the present invention, there is provided the use of Compound Fufangteng Mixture in the preparation of a product for reducing the expression of α-SMA around blood vessels.

[0011] Furthermore, the product is a drug.

[0012] In the fourth aspect of the present invention, there is provided the use of Compound Fufangteng Mixture in the preparation of a drug for treating or improving myocardial fibrosis.

[0013] Furthermore, the myocardial fibrosis is ISO-induced myocardial fibrosis.

[0014] In the fifth aspect of the present invention, there is provided the use of Compound Fufangteng Mixture in the preparation of a cardioprotective drug.

[0015] In the sixth aspect of the present invention, there is provided the use of Compound Fufangteng Mixture in the preparation of a drug for treating or improving left ventricular systolic dysfunction.

[0016] Furthermore, the left ventricular systolic dysfunction is ISO-induced left ventricular systolic dysfunction.

[0017] In the seventh aspect of the present invention, there is provided the use of Compound Fufangteng Mixture in the preparation of a drug for treating or improving myocardial hypertrophy.

[0018] Furthermore, the myocardial hypertrophy is ISO-induced myocardial hypertrophy.

[0019] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here.

[0020] Compared with the prior art, the present invention has the following advantages and progressiveness:

[0021] The present invention for the first time discovers the potential target HSPA8 of Compound Fufangteng Mixture (CFM) in the treatment of myocardial fibrosis (MF) by combining the Cellular thermal shift assay (CETSA) and network pharmacology, and verifies the binding ability of CFM and HSPA8 by using the cellular thermal migration experiment. On this basis, the present invention further verifies the protective effect of CFM on ISO-induced myocardial fibrosis in mice through animal experiments, and the results show that CFM improves MF by regulating HSPA8 and plays a myocardial protective role. In summary, the present invention discovers for the first time that CFM can alleviate or improve myocardial fibrosis, further studies the mechanism of CFM in alleviating or improving myocardial fibrosis, reveals its key core target HSPA8, and provides a new theoretical basis for the clinical treatment of myocardial fibrosis with CFM. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0023] Figure 1 : Coomassie brilliant blue staining result diagram of heat treatment experiment;

[0024] Figure 2 : Venn diagram of Compound Fufangteng Mixture (CFM) and myocardial fibrosis (MF) targets;

[0025] Figure 3 : PPI network diagram and secondary fragment diagram of Hspa8 peptide;

[0026] Figure 4 : GO functional enrichment analysis result diagram;

[0027] Figure 5 : KEGG pathway enrichment analysis result diagram;

[0028] Figure 6 : Cellular thermal shift assay (CETSA) result diagram and temperature curve;

[0029] Figure 7 : Effect of CFM on the body weight of isoprenaline (ISO) model mice;

[0030] Figure 8 : Comparison of cardiac ultrasound of mice in each group during 1 - 3 weeks of ISO modeling; compared with the Con group, ##P < 0.01; compared with the ISO group, **P < 0.01;

[0031] Figure 9: Effects of CFM on the cardiac index of ISO model mice; compared with the Con group, ##P<0.01; compared with the ISO group, **P<0.01;

[0032] Figure 10 : Effects of CFM on myocardial fibrosis in ISO model mice (400×); compared with the Con group, ##P<0.01; compared with the ISO group, **P<0.01, *P<0.05;

[0033] Figure 11 : Effects of CFM on the fibrosis markers TGF-β1 and COLIII in the cardiac tissue of ISO model mice; compared with the Con group, ##P<0.01; compared with the ISO group, **P<0.01, *P<0.05;

[0034] Figure 12 : Effects of CFM on the expression of α-SMA protein in the cardiac tissue of ISO model mice;

[0035] Figure 13 : Effects of CFM on the expression of HSPA8 protein in the cardiac tissue of ISO model mice; compared with the Con group, # P<0.05; compared with the ISO group, **P<0.01, *P<0.05. Detailed implementation manners

[0036] The present invention provides an application of compound Fufangteng mixture in improving myocardial fibrosis. The following is a specific description of the present invention in combination with embodiments to facilitate the further understanding of the present invention by those skilled in the art. However, the embodiments described below are only a part of the embodiments of the present invention and should not be regarded as any form of limitation to the present invention. It should be noted that the adjustments and improvements made by those of ordinary skill in the art based on the concept of the present invention should be regarded as the protection scope of the present invention. For the specific technical operation steps and operators not specified in the embodiments, they are all carried out according to the general technical conditions described in the literature in this field or the relevant product specifications.

[0037] Embodiments

[0038] 1 Experimental materials

[0039] 1.1 Experimental animals

[0040] Male C57BL / 6 mice (SPF grade, 6 - 8 weeks old, body weight 20 ± 2 g) were purchased from Beijing Huafukang Biotechnology Co., Ltd., with the license number: SCXK (Jing) 2024 - 0003. The experimental animals were housed in the SPF - level environmental barrier animal center of Tianjin University of Traditional Chinese Medicine, with the environmental temperature of 20 - 25 °C, relative humidity of 50 ± 5%, and a 12 - hour day - night cycle. All mice had free access to food and water during the experiment. The animal experiment complied with the ethical requirements and regulatory procedures of the Animal Experiment Ethics Committee of Tianjin University of Traditional Chinese Medicine (TCM - LAEC2024101z1053).

[0041] 1.2 Experimental drugs and reagents

[0042] Compound Fufangteng Mixture was provided by Guangxi Bainianle Pharmaceutical Factory; Isoprenaline (ISO) was purchased from MedChemExpress Biotechnology Co., Ltd. (product number: HY - B0468); Sacubitril / valsartan sodium tablets were purchased from Novartis Pharma AG (Beijing); Transforming growth factor - β1 (TGF - β1), Alpha smooth muscle actin (α - SMA), Collagen I (COLI), and Collagen III (COL III) were purchased from Shanghai Enzyme - linked Biotechnology Co., Ltd. (product numbers: ml002115, ml006777, ml058169, ml008755); Hspa8 antibody was purchased from Wuhan Abbexa Biotech Co., Ltd. (product number: A0415); GAPDH antibody was purchased from Wuhan Sevier Biotechnology Co., Ltd. (GB15004); Anti - rabbit IgG was purchased from Cell Signaling Technology (product number: 7074S).

[0043] 1.3 Experimental instruments

[0044] Portable VINNO 6LAB small animal ultrasound (Fiyeno Technology Co., Ltd.); R500 general - type small animal anesthesia machine (Shenzhen Reword Biotechnology Co., Ltd.); Low - temperature high - speed centrifuge (Eppendorf AG, Germany); Low - temperature high - speed tissue grinder (Sevier Biotechnology Co., Ltd.); Constant - temperature table - top shaker incubator (Shanghai Boxun Industry Co., Ltd.); INFINITE F50 microplate reader (TECAN Group Ltd., Switzerland); Amersham Imager 600 gel imaging system (General Electric Company).

[0045] 1.4 Software and databases

[0046] Cytoscape (version 3.10.1) software; Image J (version 1.8.0) software; GraphPad Prism (version 8.0.2) software; GeneCards database (https: / / www.genecards.org / ); STRING (version 12.0) platform (https: / / www.string-db.org / ); Microbioinformatics - Online Bioinformatics Analysis and Visualization Cloud Platform (http: / / www.Bioinformatics.com.cn / ).

[0047] 2 Experimental methods

[0048] 2.1 Cell culture

[0049] Rat H9c2 cardiomyocytes were purchased from Wuhan Punosai Biotechnology Co., Ltd. H9c2 cardiomyocytes were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, and placed in a cell culture incubator at 37°C and 5% CO2. Passage was carried out when the cell density reached 80%-90%.

[0050] 2.2 CETSA

[0051] H9c2 cardiomyocytes were collected, resuspended in RIPA solution containing 1 mM protease phosphatase inhibitor, lysed on ice for 30 min, centrifuged at 4°C and 13,000 rpm for 15 min, and the supernatant was taken. The supernatant was divided into four equal parts. Two of them were added with 1 mg / mL extract of Fufangteng mixture, and the other two were added with an equal volume of ultrapure water. Incubation was carried out at 37°C for 2 h. After incubation, one sample from the Fufangteng-treated group and one sample from the ultrapure water group were placed in a 55°C metal bath and heated for 3 min, cooled to room temperature for 3 min, centrifuged at 4°C and 12,000 rpm for 3 min, the supernatant was collected, 5× Loading Buffer was added, and heated in a metal bath at 100°C for 10 min to obtain protein samples for subsequent SDS-PAGE experiments.

[0052] 2.3 Coomassie brilliant blue staining

[0053] An 8% polyacrylamide gel was prepared, and the protein samples prepared above were loaded onto the gel for separation. After electrophoresis separation, the gel was placed in Coomassie brilliant blue staining solution and stained at room temperature for 1 h. After staining, it was eluted with decolorizing solution until the blue background was basically completely removed. The gel image was collected in bright field using an Amersham Imager 600 gel imaging system. The gels with differences in different groups were cut and placed in 1.5 mL EP tubes for QE proteomics detection.

[0054] 2.4 QE proteomics detection parameters

[0055] The peptide segments were separated by an UltiMate 3000 RSLCnano ultra-high performance liquid system. Mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was an aqueous solution containing 0.1% formic acid and 98% acetonitrile. The liquid phase gradient was set as follows: 5%-8% B phase from 0 to 6 minutes; 8%-30% B phase from 7 to 40 minutes; 30%-60% B phase from 41 to 45 minutes; 60%-80% B phase from 46 to 48 minutes, 80% B phase from 49 to 56 minutes, 80%-5% B phase from 56 to 58 minutes, and 5% B phase from 58 to 65 minutes. The flow rate was maintained at 400 nL / minute.

[0056] The peptide segments were analyzed by a Thermo ScientificTM Q ExactiveTM mass spectrometer. The ion source voltage was set at 1.8 kV, and both the peptide segment precursor ions and their secondary fragments were detected and analyzed using a high-resolution Orbitrap. The primary mass spectrometry scan range was set at 350 - 2000 Da, and the scan resolution was set at 70,000. The data acquisition mode used a data-dependent scanning (DDA) program, that is, after the primary scan, the top 20 peptide segment precursor ions with the highest signal intensity were sequentially selected and entered the HCD collision cell for fragmentation using 28% fragmentation energy, and secondary mass spectrometry analysis was also performed sequentially. The secondary mass spectrometry scan range was automatically selected depending on the primary precursor ion mass-to-charge ratio, and the resolution was set at 17,500. The secondary mass spectrometry data was processed and retrieved using MASCOT 2.3.0.

[0057] 2.5 Network pharmacology analysis

[0058] 2.5.1 Screening of targets related to myocardial fibrosis (MF)

[0059] The GeneCards database was used to retrieve targets related to "Myocardial fibrosis", and the targets with Relevance score > 10 were selected as potential targets for the treatment of MF.

[0060] 2.5.2 Obtaining intersection targets

[0061] The identified protein targets in the CFM treatment group and the potential targets of MF were uploaded to the microshengxin online platform to obtain the intersection of drug action targets and disease-related targets, and a Veen diagram was drawn, which were the potential targets of CFM in the treatment of MF.

[0062] 2.5.3 Construction of protein-protein interaction networks (PPI)

[0063] Intersection targets of CFM and MF were imported into the STRING database for interaction analysis. The species was set to "Rattus norvegicus," the minimum interaction threshold was set to "medium confidence," and free nodes were hidden. TSV files were downloaded and imported into Cytoscape 3.10.1 for visualization. Topological analysis was performed using the Network Analyzer plug-in, and core targets were selected based on their degree values.

[0064] 2.5.4GO and KEGG pathway enrichment analysis

[0065] The potential targets of CFM for the treatment of MF were uploaded to the Weishengxin online platform for GO and KEGG pathway enrichment analysis.

[0066] 2.6 Animal Experiment Verification

[0067] 2.6.1 Animal grouping and drug administration

[0068] After adaptive feeding, 35 mice were randomly divided into five groups based on body weight: a normal group, a model group (20 mg / kg), a low-dose Fufangteng mixture group (0.72 g / kg), a high-dose Fufangteng mixture group (1.44 g / kg), and a sacubitril / valsartan group (20 mg / kg). Fufangteng mixture was administered orally three days before modeling. Modeling was initiated by subcutaneous injection of ISO starting on the fourth day. Simultaneously, drug intervention was administered once daily for a cumulative period of three weeks. The survival status and body weight of mice in each group were recorded daily.

[0069] ISO is a β-receptor agonist that stimulates cardiac β1 receptors, thereby enhancing myocardial contractility, accelerating heart rate, and promoting cardiomyocyte hypertrophy and the occurrence and development of MF. It has been widely used in the construction of various myocardial injury models.

[0070] 2.6.2 Cardiac ultrasound examination

[0071] On the 7th, 14th, and 21st days after modeling and administration, echocardiography was performed on mice in each group to evaluate their cardiac function. One day before echocardiography, hair removal cream was used to remove the hair in the precordial area of mice in each group. After anesthesia with 1.0 - 2.0% isoflurane, the mice were fixed on the operating table, and a portable VINNO 6LAB small animal ultrasound was used to monitor left ventricular cardiac function. The ejection fraction (EF), fractional shortening (FS), left ventricular internal diameter at end-diastole (LVIDd), and left ventricular internal diameter at end-systole (LVIDs) of mice in each group were measured and calculated through the M-mode. 2.6.3 Heart weight and heart weight / tibia length

[0072] After the experiment, the hearts of mice in each group were taken out, photographed, weighed, the weights were recorded, and the tibia lengths were measured for calculating the heart weight to tibia length (HW / TL).

[0073] 2.6.4 Masson staining

[0074] The mouse heart tissues were fixed with 4% paraformaldehyde, paraffin-embedded, sectioned, dewaxed, and rehydrated. The paraffin sections of the hearts of mice in each group were stained with a Masson trichrome staining kit, sealed with neutral gum, and image acquisition was performed using a 3DHISTECH panoramic scanner. The Image J software was used to analyze the collagen deposition, and the collagen volume fraction (CVF) in the myocardial tissue was calculated. CVF (%) = positive collagen staining area / total tissue area × 100%.

[0075] 2.6.5 ELISA

[0076] An ELISA kit was used to detect the contents of TGF-β1, α-SMA, COLI, and COLIII in the mouse heart tissues. 20 mg of heart tissue was weighed for each group of mice, 180 μL of PBS was added, and the tissue was minced on ice and ground using a high-speed and low-temperature tissue grinder. After centrifugation at 5000 g for 10 min, the supernatant was taken. Detection was performed according to the instructions of the ELISA kit. The OD value was measured at a wavelength of 450 nm, a standard curve was plotted, and the concentrations of each sample were calculated.

[0077] 2.6.6 Detection of α-SMA protein expression in mouse myocardial tissue by immunofluorescence

[0078] The paraffin sections were dewaxed to water, antigen retrieval was performed using a citric acid antigen retrieval buffer, and they were shaken, washed, and soaked in PBS three times for 3 minutes each. After the sections were slightly dried by shaking, a histochemical pen was used to draw a circle around the tissue, and goat serum was added dropwise within the histochemical circle to evenly cover the tissue, and it was blocked at room temperature for 60 minutes. The blocking solution was gently shaken off, and primary antibody α-SMA was added dropwise onto the sections, and incubated overnight at 4°C. The next day, the primary antibody was recovered, and the sections were washed with PBS. After the sections were slightly dried by shaking, a fluorescence secondary antibody corresponding to the same species as the primary antibody was added dropwise within the circle in the dark to cover the tissue, and incubated at room temperature in the dark for 1 hour. The secondary antibody was recovered, and the sections were washed with PBS. After the sections were slightly dried by shaking, DAPI nuclear stain was added dropwise within the circle, and stained at room temperature for 5 minutes, and then sealed with an anti-fluorescence quenching mounting medium.

[0079] 2.6.7 Western blotting

[0080] Take 20 mg of heart tissue, add 200 μL of RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors, cut it into pieces on ice, homogenize it using a tissue homogenizer, lyse it on ice for 30 minutes, centrifuge at 13,000 rpm at 4°C for 15 minutes, take the supernatant, and perform BCA protein quantification and denaturation according to the corresponding instructions. The prepared protein samples were added to an 8% SDS-PAGE gel for separation. After electrophoresis separation, they were transferred onto a PVDF membrane, blocked with a rapid blocking solution for 30 minutes, and incubated with primary antibody Hspa8 (1:1000) overnight at 4°C. The next day, the primary antibody was recovered, and washed with TBST five times for 3 minutes each; HRP-labeled secondary antibody (1:5000) was added, incubated at room temperature for 1 hour, and washed with TBST. Developed with ECL chemiluminescence solution, images were acquired using an Amersham Imager 600 gel imaging system, and the gray values of the bands in each group were measured using ImageJ software.

[0081] 2.6.8 Statistical analysis

[0082] Statistical analysis was performed using GraphPad Prism 8.0.2 software. Experimental data were all expressed as mean ± standard error (Mean ± SEM). When comparing multiple groups, if they conformed to a normal distribution, one-way ANOVA was used; if they did not conform to a normal distribution, a non-parametric independent samples test was used. P < 0.05 was considered statistically significant.

[0083] 3 Experimental results

[0084] 3.1 Coomassie brilliant blue staining

[0085] The target protein and the drug molecule can form a tightly bound complex through intermolecular forces. Compared with the pure protein, this protein-small molecule complex can be more stable during heat treatment. The results of the heat treatment experiment are as Figure 1As shown, after incubating H9c2 cardiomyocytes with 1 mg / mL CFM, there is a protein band in each of the molecular weight ranges of 35 - 55 KDa and 70 - 100 KDa, which is darker in color and has a higher protein content compared to the untreated group, suggesting that there may be target proteins interacting with CFM at these two sites.

[0086] We used proteomics to qualitatively and quantitatively analyze the proteins in these two protein bands. The results showed that a total of 208 proteins were identified in CFM - treated H9c2 cardiomyocytes.

[0087] Conclusion: The results of the heat treatment experiment showed that in CFM - treated H9c2 cardiomyocytes after heating, compared with the untreated group, there was a protein band with a darker color and a relatively higher protein content in each of the molecular weight ranges of 35 - 55 KDa and 70 - 100 KDa. It is speculated that there may be target proteins interacting with CFM at these two sites. Next, we identified the proteins in these two protein bands by proteomics. A total of 208 proteins were identified, indicating that CFM may form a tightly - bound complex with these proteins through interaction forces, thereby improving the thermal stability of the target proteins.

[0088] 3.2 Network pharmacology analysis

[0089] 3.2.1 Screening of MF - related targets

[0090] A total of 5042 MF - related targets were retrieved through the GeneCards database, and among them, 514 targets had a Score > 10.

[0091] 3.2.2 Results of obtaining intersection targets

[0092] The MF targets obtained from the GeneCards database were mapped with the potential targets of CFM identified by QE. A total of 19 potential targets for CFM in the treatment of MF were obtained, as shown in Figure 2 .

[0093] 3.2.3 Construction of PPI network diagram

[0094] The 19 intersection targets for CFM in the treatment of MF were imported into the STRING database for PPI network analysis. The analysis results (TSV format) were downloaded and imported into the Cytoscape 3.10.1 software for visualization. The Network Analyzer plugin was used for topological analysis. This network included 11 nodes and 24 edges. The top 2 targets with the highest Degree value were Hspa8 and Hspa4. Based on proteomic detection and protein function analysis, it is speculated that Hspa8 may play a crucial role in the process of CFM treating MF, as shown in Table 1; the specific band of Hspa8, query8468, was identified, as shown in Figure 3 ; the specific band of Hspa8, query8468, was identified, as shown inFigure 3 。

[0095] Table 1 Identification information of hspa8 and Hspa4 protein spectra

[0096]

[0097] 3.2.4 GO and KEGG pathway enrichment analysis

[0098] The online platform of Weishengxin was used to perform GO functional enrichment analysis and KEGG pathway enrichment analysis on 19 intersection targets of CFM in the treatment of MF, with P < 0.05 as the screening condition. A total of 775 entries were obtained from the GO functional enrichment analysis, including 636 biological processes (BP), 80 cell components (CC), and 59 molecular functions (MF). The top 10 entries were selected for visualization, as shown in Figure 4 。BP is mainly related to positive regulation of cell-substrate adhesion, cell matrix adhesion, positive regulation of fibroblast migration, etc.; CC is mainly related to membrane region, collagen-containing extracellular matrix, extracellular matrix, etc.; MF is mainly related to collagen binding, fibronectin binding, extracellular matrix protein binding, etc. The KEGG pathway enrichment analysis is mainly related to ECM-receptor interaction, regulation of actin cytoskeleton, cell adhesion molecules, etc., as shown in Figure 5 。

[0099] 3.3 Cell thermal migration experiment

[0100] As Figure 6 shown, compared with the H2O treatment group, 1 mg / mL CFM can improve the thermal stability of HSPA8 protein in H9c2 cardiomyocytes in the range of 40-55 °C, indicating that under heating conditions, CFM plays a role in stabilizing the structure of HSPA8 protein by binding to HSPA8 protein.

[0101] Conclusion: The results of network pharmacology analysis showed that there were 514 MF-related targets, and 19 intersection targets between CFM and MF. PPI network analysis showed that HSPA8 might be the core target for CFM to treat MF. The results of GO functional enrichment analysis showed that BP was mainly related to positive regulation of cell-substrate adhesion, cell matrix adhesion, positive regulation of fibroblast migration, etc.; CC was mainly related to membrane region, collagen-containing extracellular matrix, extracellular matrix, etc.; MF was mainly related to collagen binding, fibronectin binding, extracellular matrix protein binding, etc. The KEGG pathway enrichment analysis was mainly related to ECM-receptor interaction, regulation of actin cytoskeleton, cell adhesion molecules, etc. In addition, the results of thermal shift assay showed that CFM could improve the thermal stability of HSPA8 protein. HSPA8 is a constitutively expressed homologous protein belonging to the HSP70 family and is the main housekeeping protein of this family. It is lowly expressed or not expressed under normal conditions. When cells are subjected to physiological and environmental stresses, the HSP-encoding genes are activated for expression, and it participates in processes such as correct folding, translocation, and degradation of intracellular proteins. Existing studies have found through proteomic analysis that compared with normal CFs, Hsp70 is significantly up-regulated in keloid fibroblasts, indicating that the overexpression of Hsp70 may be involved in the pathogenesis of keloids. It has been found that the overexpressed Hsp70 in keloid fibroblasts may cause excessive collagen accumulation, leading to the formation of keloids. In addition, it has also been found that Hsp27, Hsp47, and Hsp70 are significantly overexpressed in keloid tissues. Therefore, CFM may regulate HSPA8 to improve MF.

[0102] 3.4 Results of animal experiments

[0103] 3.4.1 Effects of CFM on the living status of ISO mice

[0104] During the ISO modeling process, mice in the ISO group, CFM-L group, CFM-H group, and Sac-Val group all showed listlessness, slow movement, reduced food and water intake, and a large amount of fluid secretion in the mouth. With the extension of the drug intervention time, the above symptoms were all improved, and the body weights of mice in each group showed an upward trend, as shown in Figure 7 .

[0105] 3.4.2 Effects of CFM on the cardiac function of ISO mice

[0106] Echocardiogram results showed that compared with the Con group, in the ISO group, the EF and FS of mice significantly decreased from the 1st week after modeling (P < 0.01), the LVIDs significantly increased (P < 0.01), and from the 3rd week after modeling, the LVIDd significantly increased (P < 0.01); compared with the ISO group, in the CFM-L group, CFM-H group and Sac-Val group, the EF and FS significantly increased from the 1st week after modeling (P < 0.01), the LVIDs significantly decreased (P < 0.01), and from the 3rd week after modeling, the LVIDd significantly decreased (P < 0.01), indicating that CFM can improve ISO-induced cardiac dysfunction and play a myocardial protective role, see Figure 8 。

[0107] 3.4.3 Effects of CFM on the cardiac index of ISO mice

[0108] HW / TL is the most commonly used index of cardiac hypertrophy. Compared with the Con group, the heart weight (HW) and HW / TL of mice in the ISO group significantly increased (P < 0.01); compared with the ISO group, the HW and HW / TL of mice in the CFM-L group, CFM-H group and Sac-Val group significantly decreased (P < 0.01), indicating that CFM can significantly improve ISO-induced cardiac hypertrophy, see Figure 9 。

[0109] 3.4.4 Effects of CFM on myocardial fibrosis in ISO mice

[0110] Results of Masson staining showed that compared with the Con group, the myocardial fibers in the ISO group were disordered and fractured, and the collagen deposition in the myocardial interstitium significantly increased, with a significant increase in CVF (P < 0.01); compared with the ISO group, after treatment with CFM and Sac-Val, the disorder and fracture of myocardial fibers were significantly improved, the collagen deposition in the myocardial interstitium significantly decreased, and the CVF significantly decreased (P < 0.05, P < 0.01), see Figure 10 。

[0111] 3.4.5 Effects of CFM on fibrosis markers TGF-β1, α-SMA, COLI and COLIII in the cardiac tissue of ISO mice

[0112] Compared with the Con group, the contents of fibrosis markers TGF-β1, α-SMA, COLI, and COLIII in the heart tissues of ISO group mice were significantly increased (P < 0.01); compared with ISO group mice, the contents of fibrosis markers TGF-β1, α-SMA, COLI, and COLIII in the heart tissues of CFM-L group and CFM-H group mice were significantly decreased (P < 0.01, P < 0.05), the contents of fibrosis markers α-SMA and COLI in the heart tissues of Sac-Val group mice were significantly decreased (P < 0.01), and the contents of TGF-β1 and COLIII showed a decreasing trend, indicating that CFM can improve ISO-induced myocardial fibrosis, see Figure 11 .

[0113] 3.4.6 Effect of CFM on the expression of α-SMA protein in the heart tissues of ISO mice

[0114] Compared with the Con group, the expression of α-SMA around blood vessels in ISO group mice was significantly increased; compared with the ISO group, the expression of α-SMA around blood vessels in CFM-L group, CFM-H group, and Sac-Val group mice was significantly decreased, see Figure 12 .

[0115] 3.4.7 Effect of CFM on the expression of HSPA8 protein in the heart tissues of ISO mice

[0116] Compared with the Con group, the protein expression level of HSPA8 in the heart tissues of ISO group mice was significantly increased (P < 0.05); compared with the ISO group, after treatment with CFM-L group, the protein expression level of HSPA8 decreased, but there was no statistical difference. The protein expression levels of HSPA8 in the heart tissues of CFM-H group and Sac-Val group mice were significantly decreased (P < 0.05, P < 0.01), see Figure 13 .

[0117] Conclusion: The present invention uses intraperitoneal injection of ISO to establish a mouse MF model, evaluates whether CFM has an improvement effect on MF at the animal level, and explores whether its mechanism is related to the regulation of HSPA8. The results of animal experiments show that the body weights of mice in each group show an overall upward trend; compared with the Con group, the EF and FS of mice in the ISO group are significantly decreased, and the LVIDd and LVIDs are significantly increased, indicating that ISO can cause left ventricular systolic dysfunction; the hearts of mice in the model group become larger, and the HW and HW / TL are significantly increased, indicating that ISO can cause myocardial hypertrophy; the results of Masson staining show that there are a large number of collagen fiber deposits in the myocardial interstitium and around blood vessels in mice in the ISO group, and the contents of TGF-β1, α-SMA, COL I and COL III in the myocardial interstitium are significantly increased, and the expression of α-SMA around blood vessels is significantly increased; the protein expression of HSPA8 in myocardial tissue is significantly up-regulated, indicating that ISO can induce the occurrence and development of MF. After CFM intervention, the EF and FS of mice are significantly increased, and the LVIDd and LVIDs are significantly decreased, suggesting that CFM can effectively improve the left ventricular systolic dysfunction caused by ISO; the hearts of mice in the CFM group gradually return to normal size, and the HW and HW / TL are significantly decreased, suggesting that CFM can reduce ISO-induced myocardial hypertrophy; CFM can improve the degree of MF, reduce the contents of TGF-β1, α-SMA, COL I and COL III in the myocardial interstitium, reduce the expression of α-SMA around blood vessels, and down-regulate the protein expression of HSPA8 in myocardial tissue, suggesting that CFM can inhibit ISO-induced MF to a certain extent. The above results indicate that CFM can reduce ISO-induced MF in mice, and its mechanism of action may be related to the regulation of HSPA8.

[0118] The above are only the preferred embodiments of the present invention and do not impose any limitations on the present invention. For those skilled in the art of this technology, any modifications and changes made to the above embodiments according to the technical essence of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. Use of compound Fufangteng mixture in the preparation of a product for reducing the expression of HSPA8.

2. Use of compound Fufangteng mixture in the preparation of a product for reducing the contents of myocardial interstitial TGF-β1, α-SMA, COL I and COL III.

3. Use of compound Fufangteng mixture in the preparation of a product for reducing the expression of α-SMA around blood vessels.

4. Use of compound Fufangteng mixture in the preparation of a drug for treating or improving myocardial fibrosis.

5. Use of compound Fufangteng mixture in the preparation of a myocardial protective drug.

6. Use of compound Fufangteng mixture in the preparation of a drug for treating or improving left ventricular systolic dysfunction.

7. Use of compound Fufangteng mixture in the preparation of a drug for treating or improving myocardial hypertrophy.

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