Zhou Zhou, Zhen Wang , Yang Liu, Yong Wang, Chuan-Hua Yang?

1. Shandong University of Traditional Chinese Medicine, Jinan 250000, China

2. Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250000, China

Keywords:Bioinformatics Myocardial fibrosis Traditional Chinese traditional Pathological mechanism

ABSTRACT Objectives:Bioinformatics was applied to screen the key genes of Myocardial fibrosis, explore its pathogenesis and predict the potential traditional Chinese medicines for the treatment of Myocardial fibrosis. Methods: Based on raw data of gene chip GSE59437 from gene expression database (GEO), myocardial tissue samples from 3 control mice and 3 mice treated with angiotensin II-induced myocardial fibrosis were included.Using R language processing data and screening of gene express significant differences (DEG), use a database of DAVID and the R language finish Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment for differences gene, using the STRING database structure protein protein interactions (PPI) networks, using Cytoscape software visualization and use the MCODE plug-in screening key function modules in the network.Coremine Medical database was used to map the key genes, construct the gene-Chinese medicine network, and screen the traditional Chinese medicines for the treatment of myocardial fibrosis.Results: 208 DEGs were screened, 94 of which were up-regulated and 114 were downregulated.DEGs is mainly involved in a variety of biological processes such as extracellular matrix remodeling, collagen fiber deposition and lipid metabolism disorders. KEGG pathway enrichment involves Platelet activation,Oxytocin signaling pathway,Insulin secretion,ECMreceptor interaction,GnRH signaling pathway,TNF signaling pathway and other signaling pathways. Key modules of PPI network including: CTGF,TIMP1,SPP1,SERPINE1,COL3A1,POSTN and FOS. The potential traditional Chinese medicines for the treatment of myocardial fibrosis are Astragalus membranaceus (Fisch.), Lepidium apetalum Willd and Salvia miltiorrhiza Bge. Conclusion: Myocardial fibrosis is a complex pathological process, and the genes related to the imbalance of extracellular matrix synthesis and degradation and excessive deposition of collagen fibers play an important role in this process. This study provides a scientific reference for further exploring the pathogenesis of myocardial fibrosis, looking for therapeutic targets and potential therapeutic traditional Chinese medicines.

1. Introduction

Myocardial fibrosis is the pathological basis of many heart diseases. When the heart is damaged or the heart is overloaded,a variety of substances cause fibroblasts to activate and transdifferentiate to myofibroblasts. Myocardial fibrosis plays a core role in the process of myocardial remodeling[1]. Severe myocardial fibrosis can induce myocardial hypertrophy, relatively insufficient blood supply, increased ventricular wall stiffness, restricted ventricular activity, and subsequent cardiac pumping dysfunction,leading to heart failure. Some people even suggested that if 20% of the "risk myocardium" is saved, heart failure can be avoided [2]. "Cardiovascular Health and Disease Report of China 2019" shows[3]: The current estimated value of heart failure patients in China is 8.9 million. From January 2012 to September 2015, among 13 687 patients with heart failure in 132 hospitals across the country selected in the China-HF study, the case fatality rate of heart failure patients in hospital was 4.1%. Therefore, the treatment of heart failure is still the focus of current cardiovascular disease research,and improving myocardial fibrosis is a key link in the treatment of heart failure. Although modern medicine has made some progress in the treatment of heart failure, the pathogenesis of myocardial fibrosis and other factors has not yet been fully clarified. Improving myocardial fibrosis is still a difficult problem to overcome, and the curative effect of existing treatment programs is limited. Traditional Chinese medicine has the advantages of multiple components,multiple targets, and multiple pathways. It has significant effects on heart failure. It can significantly improve patients' clinical symptoms,improve heart function, and reverse the development of myocardial fibrosis, but its pharmacology and molecular mechanisms are lacking elaborate [4-6]. Therefore, it is necessary to deeply explore the molecular mechanism of Chinese medicine in the treatment of myocardial fibrosis, find potential therapeutic targets, and provide new scientific reference for the treatment of heart failure by Chinese medicine.

Bioinformatics is the science of storing, retrieving and analyzing biological information using computers. Medical research can use bioinformatics to mine and analyze databases to find potential therapeutic targets from differences in functional gene expression.Combining the multi-target macroscopic view of bioinformatics with the micro-molecular mechanism to explore the molecular mechanism of the treatment of diseases and predict potential Chinese medicines is not only in line with the concept of TCM diagnosis and treatment that emphasizes the whole, but also conducive to the modernization of TCM research and the development of TCM And step into the world.

2. Materials and methods

2.1 Data set acquisition

Use "heart failure", "myocardial remodeling", "myocardial fibrosis", etc. as search terms to retrieve the relevant gene chip data set in the GEO database[7], and download the original file of the chip with the chip number GSE59437 after screening. This chip comes from the remodeling of Capital Medical University of China The key laboratory of related cardiovascular diseases contains the sequencing data of 3 control mice and mouse cardiac fibrosis tissue samples constructed with angiotensin II intervention for 7 days. The sequencing platform is: Affymetrix GeneChip Mouse Genome 430 2.0 Array , Uploaded to GEO database by Li H et al[8].

2.2 Data preprocessing

The gene expression chip is processed based on the relevant R package in the R language Bioconductor library (http://www.bioconductor.org/). Use the affyPLM package to perform fitting regression calculation on the data set, and draw the relative logarithmic expression (RLE) box plot and the relative standard deviation (NUSE) box plot. Use the affy package to obtain degradation data and draw the RNA degradation map, evaluate the RNA 5'～3' degradation slope, and eliminate unreliable samples with severe degradation. After quality control, use the affy package to use robust mulitichip average (RMA) to preprocess the control group and model group data and merge the two sets of data to convert the probe level data into gene expression data, based on the sequencing platform The annotation file is re-annotated, the probe name is converted to the gene ID, the nearest neighbor method (k-Nearest Neighbor, KNN) is used to supplement the missing values of the original data, and the expression value at the gene level is read.

2.3 Screening of differentially expressed genes

Using the limma package of the R language, the Bayesian method is used for multiple inspection corrections, and the difference analysis of the standardized chip expression profile is performed.The threshold is set to: |log2FC|>1, P<0.05. FC (fold change)represents the fold difference between the two groups of gene expression values. Use R language for visualization.

2.4 Enrichment Analysis of Gene Ontology Annotation and Kyoto Encyclopedia of Genes and Genomes

Import genes homologous to humans in DEGs into the DAVID database[9] (https://david.ncifcrf.gov/), and set the protein source species to "Homo sapiens" for GO biological process annotation,with a P value <0.05 as the threshold Filter annotations and visualize the top 10 annotations with the most significant enrichment. Based on the KEGG database[10] ,differential genes were enriched in pathways, and the signal pathways with significant enrichment were obtained with P value<0.05 as the threshold, and the first ten pathways were visualized.

2.5 Protein-Protein Interaction (PPI) network construction and screening of core gene modules

Import DEGs homologous to humans into the STRING database[11](https://string-db.org/), set the protein source species to "Homo sapiens", and set the lowest interaction threshold to "medium confidence (>0.4)", hide For non-interacting proteins, keep the default settings for other parameters, and perform protein interaction analysis to obtain interaction data. Use Cytoscape 3.8.0 software to realize the visualization of PPI network, use the molecular complex detection[12] (MCODE) plug-in to filter the protein interaction modules in the PPI network with strong correlation, and set the parameters as follows: degree cutoff=2, node score limit ( node score cutoff)=0.2, K-core=2, and maximum depth (Max.Depth)=100.

2.6 Key targets predict potential Chinese medicine

Import the selected core genes into the Coremine Medical database(https://coremine.com/medical/), use P value<0.05 as the threshold to obtain core gene related Chinese medicines, use Cytoscape to visualize, and use degree ≥ 3 as the threshold to obtain Chinese medicine with multiple targets, based on the theoretical knowledge of Chinese medicine and the research progress of modern Chinese medicine pharmacology, analysis and screening of predictive Chinese medicine.

2.7 Verification of expression differences of key targets

Obtain the other three samples contained in the chip GSE59437 from the GEO database: "GSM1436903", "GSM1436904", and"GSM1436905". The samples are fibrotic myocardial tissues of mice that have been treated with angiotensin II for 3 days. The same method is used to compare the data. Perform differential expression analysis to verify whether the key targets screened are also significantly differentially expressed in this test group.

3.Result

3.1Chip data

After screening, 6 samples in the data set with the chip number GSE59437 (Table 1) were included in the study. The researchers of this chip implanted osmotic pumps in C57BL/6 male mice aged 8 to 10 weeks. Angiotensin II was injected into Ringer's solution(physiological saline solution containing 0.01 mmol/L acetic acid)at a rate of 1500 mg/kg/min to construct a tissue fibrosis model.After 7 days of intervention, the total RNA in myocardial tissue was extracted using TRIzol method (3 samples), and based on the Affymetrix gene chip mouse genome 430 2.0 array sequencing platform to sequence the total RNA of myocardial tissues in the three model groups and three control groups.

Table 1 Information of sample

3.2Quality control and preprocessing of chip data

The REL box plot shows that the overall expression level of each sample is basically the same (the ratio is close to 1, and the logarithmic value is close to 0), as shown in Figure 1A. The NUSE box plot shows that the standard deviation of gene expression in all samples is basically the same (the relative standard deviation is close to 1), as shown in Figure 1B. The RNA degradation curve graph shows that the 5'end of each sample is lower than the 3'end,and the slope of the degradation curve is smaller, indicating that the sample is less degraded, as shown in Figure 1C. It can be seen that the quality of this chip is good and meets the research standards.The RMA method was used to preprocess the data, and the probe sequence was converted into gene ID, and the KNN method was used to supplement the missing values. A total of 20744 gene expression data were obtained.

Figure1 Quality analysis of chip

3.3Differential gene analysis

Comparing the model group data with the control group data, using the limma package for difference analysis, a total of 208 differential genes meeting the threshold (|log2FC|>1, P＜0.05) were obtained,of which 94 were up-regulated genes and 114 were down-regulated genes , Use the R language ggplot package to visualize the data and draw a volcano map (Figure 2). According to the differential gene expression, draw a violin chart of the total expression of each group of differential genes (Figure 3A). Perform cluster analysis according to the heterogeneous level of differential gene expression and draw a circular heat map (Figure 3B). The violin chart indicates that the model group and the control group are significantly different in terms of differential gene expression, while the expression levels within the group tend to be the same. The circular heat map indicates that there are significant differences in the expression of different clustering modules between the model group and the control group,and the modules in the group are basically the same, suggesting that myocardial fibrosis is the result of the differential expression of these differential genes.

Figure2 Volcano plot of gene expression

Figure3 Analysis of differential gene expression

3.4 GO enrichment analysis

Import all DEGs into the DAVID database and identify 116 genes homologous to humans (Table 2). Use the DAVID online database to annotate the GO biological process (BP) of the genes that are homologous to humans among the differential genes, and obtain the enrichment results, and visualize the top ten enrichment with significant (P value <0.05) (Figure 4) The size of the bubble reflects the number of enriched genes, the larger the enrichment, the more the number of genes; the2 color of the bubble represents the significance of enrichment, and the deeper the color, the more significant the enrichment. It can be seen from the figure that differential genes are mainly enriched in extracellular fibril organization, extracellular matrix organization, muscle filament sliding, cell-matrix adhesion,collagen fibril organization, negative regulation of lipid metabolic process, negative regulation of cholesterol transport, negative regulation of very-low -Density lipoprotein particle clearance,muscle contraction, chylomicron remnant clearance and other biological processes. It is suggested that these differential genes are closely related to extracellular matrix remodeling, collagen fiber deposition and lipid metabolism disorders.

Table 2 Differentially expressed genes homologous to human

Figure4 Enrichment analysis of GO biological processes

3.5 KEGG pathway enrichment analysis

The pathway enrichment analysis of DEGs homologous to humans revealed a total of 16 pathways with significant enrichment (P value<0.05). The first ten pathways were visualized using R language(Figure 5). It can be seen that these differential genes are mainly enriched in Platelet activation, Oxytocin signaling pathway, Insulin secretion, ECM-receptor interaction, GnRH signaling pathway,TNF signaling pathway, etc., suggesting that myocardial fibrosis is a complex pathological process involving multiple pathways.

Figure5 Enrichment analysis of KEGG pathway

3.6PPI network construction and core gene screening

The STRING database was used to construct a PPI network of homologous differential genes, and the lowest interaction threshold was set to 0.4 to hide independent proteins without interaction.Import the data into Cytoscape for visualization. The resulting network has 64 nodes and 98 edges. The nodes represent differential gene proteins. The higher the degree of protein, the darker the color.The edges represent the interaction between protein and protein.The higher the combined score, the thicker the connected edges(Figure 6A). Use the MCODE plug-in to filter the modules in the PPI network (Figure 6B). The largest module is composed of 7 nodes and 19 edges. The MCODE score is 6.33. This module is the core module of the PPI network. It is suggested that CTGF, TIMP1,SPP1, SERPINE1, COL3A1, POSTN, FOS and other proteins are important nodes in the PPI network.

Figure 6 Network of PPI

3.7Prediction and screening of potential Chinese medicines for the treatment of myocardial fibrosis

The selected core genes were imported into the Coremine Medical database, and the relevant Chinese medicines of the core genes were obtained with the P value<0.05 as the threshold. Cytoscape software was used for visualization (Figure 7). A total of 127 Chinese medicines were obtained, and the Chinese medicines with the degree value among the top five flavors were obtained. The degree values of Salvia miltiorrhiza Bge.(Dan shen,DS), fruit of Leonurus japonicus Houtt.(Chong wei zi,CWZ), Astragalus(Huang qi,HQ), Caulis Aristolochiae Manshuriensis (Guan mu tong,GMT), and Lepidium apetalum Willd.(Ting li zi,TLZ) were 4, 4, 4, 3, and 3 respectively,which could be included in the analysis. Based on the theoretical analysis of traditional Chinese medicine: DS, HQ and TLZ have the effects of promoting blood circulation, removing blood stasis,replenishing qi and promoting yang, and promoting water and swelling. They are often used in clinical Chinese medicine for heart failure, asthma, chest pain and other myocardial remodeling related diseases The treatment has the value of treating myocardial fibrosis[13, 14].

Figure 7 Prediction of target related traditional Chinese medicine

3.8 Verification of expression differences of key targets

Using the same method as described above, after checking the quality of the chip (Figure 8A), the difference in gene expression between the verification group and the control group was analyzed and visualized (Figure 8B). It can be seen from the figure that the data quality of the verification group is reliable. The difference analysis obtained a total of 254 significantly differentially expressed genes. The expression of the 7 key targets screened in 2.6 of this article was searched in the data. The results showed that the 7 key targets were in the verification process. There are significant differences in expression in the groups (marked in the volcano map),confirming the reliability of the key target screening in the previous article.

4. Discussion

The previous research of the research team members found that myocardial fibrosis is a complex pathological process involving multiple regulatory pathways such as endocrine, autocrine,and paracrine. Among them, the renin-angiotensin-aldosterone system (RAS), endothelial-to-mesenchymal transition (Endo-MT)and myocardial fibroblast proliferation are important causes of myocardial fibrosis[6, 15]. At present, the research on myocardial fibrosis is mostly limited to a certain pathway or target, and the results often can not fully explain the mechanism of myocardial fibrosis, so it is difficult to make a major breakthrough in the treatment. High throughput sequencing technology has the advantages of comprehensive coverage, accurate measurement and convenient screening. It is an efficient and multi-level research method to explore the pathogenesis of diseases based on gene expression differences.

Angiotensin Ⅱ is the main effect hormone of the renin-angiotensin system (RAS). It has been confirmed that angiotensin Ⅱ plays an important role in the process of myocardial fibrosis, cardiac hypertrophy, and heart failure. Pathological hypertrophy and myocardial interstitial fibrosis lead to increased ventricular wall stiffness, which damages the heart’s systolic and diastolic function.It is an important risk factor for a variety of experimental models and patients with heart failure, and studies have shown that antagonizing angiotensin II can reduce Reversing the development of myocardial fibrosis to a certain extent is a valuable treatment strategy for myocardial fibrosis. Therefore, this study selected a gene chip including angiotensin Ⅱ-induced myocardial fibrosis mouse model to conduct research [16, 17].

4.1 Extracellular matrix remodeling and collagen fiber deposition are the core links of myocardial fibrosis

The chip GSE59437 was preprocessed and analyzed to obtain 208 differentially expressed genes, of which 116 genes were homologous to humans. GO enrichment indicates that homologous differential genes are mainly related to extracellular fibrous tissue, extracellular matrix tissue, myofilament sliding, cell matrix adhesion, collagen fibril tissue, negative regulation of lipid metabolism, negative regulation of cholesterol transport, and extremely low The negative regulation of density lipoprotein particle clearance, muscle contraction, and chyle micro-residual gaps are related to biological processes. Previous studies have shown that myocardial fibrosis is a pathological process characterized by excessive deposition of extracellular matrix proteins in the myocardium. The disordered synthesis of extracellular matrix proteins leads to structural changes in the heart and affects the excitation-contraction coupling of myocardial cells. Process, followed by heart failure characterized by systolic or diastolic dysfunction[18]. The extracellular matrix of myocardium is mainly composed of type I and type III fibrillar collagen and a small amount of type IV, V, VI collagen and other proteins (fibronectin, laminin, elastin, etc.), of which about 85%of I Type III fibrillar collagen is the thick collagen responsible for imparting tensile strength to the myocardium, and type III fibril collagen, which accounts for about 11%, is responsible for the network elasticity of the matrix[19]. The relative balance of the components of the extracellular matrix and the dynamic balance of the synthesis and degradation of the extracellular matrix are essential to maintain the integrity of the myocardial structure. After acute myocardial infarction, type I and type III fibrillar collagen are synthesized in large quantities, leading to myocardial fibrosis Intensify, induce the occurrence of heart failure. Studies have shown that the increase of type III collagen propeptide (PⅢNP) in the pre-hospitalization of myocardial infarction patients (a marker in the process of type III fibril collagen synthesis, indicating a large amount of fibril collagen synthesis) is the heart of the follow-up period. Predictors of death or heart failure[20]. In addition, other biological processes are mostly involved in lipid metabolism. The study found that gerbils fed high-fat for 16 weeks showed obvious body obesity and hyperlipidemia, but there was no significant change in blood sugar levels. Pathological anatomy showed high-fat The heart of gerbils with hyperemia has structural disorders, including cardiomyocyte hypertrophy, lipid deposition, interstitial and vascular fibrosis, and an increase in the number of infiltrating neutrophils,suggesting that myocardial metabolism caused by lipid metabolism disorders also promotes the heart The development of muscle fibrosis[21]. Therefore, reversing extracellular matrix remodeling,collagen fiber deposition, and lipid metabolism disorders are important links in the treatment of myocardial fibrosis.

4.2 The occurrence of myocardial fibrosis is the result of the cooperative participation of multiple pathways

The enrichment of KEGG pathway showed that the pathways of significant enrichment of differential genes were: platelet activation, oxytocin signaling pathway, ECM receptor interaction,tumor necrosis factor-αIt suggests that myocardial fibrosis involves endocrine regulation, extracellular matrix remodeling,inflammation and other mechanisms. Platelet activation is essential for the process of primary hemostasis, but it should be noted that platelet activation also plays an important role in promoting inflammation, which can promote the development of hypertension mediated myocardial fibrosis and heart failure[22]. Liu GZ et al.Found that platelets from hypertensive WT mice constructed with angiotensin II were intravenously injected into WT mice without treatment with angiotensin II in the same nest, and inflammatory factors (Mac-2, TGF) in heart tissue of WT mice after receiving platelets were found- β 1) The expression of collagen I and TGF- β was increased- β The results of tissue sections showed that the degree of myocardial fibrosis of WT mice after receiving platelets was higher than that of other groups, suggesting that activated platelets can promote the occurrence of myocardial fibrosis and inflammation[23].Previous studies have confirmed that oxytocin can reduce blood pressure, negative cardiac force, regulate parasympathetic nerve, dilate blood vessels, protect the heart and improve the healing process[24].However, recent studies have found that transgenic mice that specifically overexpress oxytocin in the heart have heart failure 3 months after birth, and have cardiac dilation, decreased systolic function, myocardial fibrosis and other phenomena, suggesting that oxytocin has two-sided effects on the myocardium. Therefore, oxytocin should be used cautiously in the prevention and protection of cardiovascular diseases, and further study the mechanism of protecting or aggravating myocardial damage[25]. Integrins are heterodimeric cell adhesion molecules that connect extracellular matrix and cytoskeleton. They are the main components of extracellular matrix (ECM) receptors and play an important role in the regulation of proliferation, differentiation,apoptosis and cell migration[26]. Studies have shown that the expression of integrin increases in the healing stage of myocardial infarction, but decreases in the process of myocardial remodeling β Compared with the model control group, the cardiac function of integrin knockout mice was significantly decreased, and the levels of myocardial fibrosis and apoptosis were higher than those of the model control group, suggesting that integrin plays an important role in the process of anti myocardial fiber and myocardial protection after myocardial infarction[27]. Tumor necrosis factor- α(TNF and its soluble TNF receptors (TNFRs) are independent predictors of mortality in patients with heart failure. TNF signaling is mainly realized through TNFR1 and TNFR2 cells. Studies have shown that compared with normal wild-type (WT) mice, the 4-week survival rate of TNFR1 and TNFR2 knockout WT mice is significantly improved, However, TNFR1 knockout mice showed significant improvement in myocardial fibrosis and systolic dysfunction, while these pathological phenomena were promoted in TNFR2 knockout mice, suggesting that the specific role of TNF receptor in heart failure should be considered when formulating therapeutic anti TNF strategies[28]. TNF in circulating blood of patients with heart failureα And a variety of inflammatory factors are at a high level, these factors are secreted by cardiac fibroblasts, and through paracrine effect on cardiomyocytes, leading to ventricular remodeling and heart failure. In addition, studies have shown that in the cardiac fibroblast culture medium alone, TNF-αIn the co culture medium of cardiac fibroblasts and cardiac myocytes, the level of TNF was lower- α These findings indicate that the communication between cardiac myocytes and cardiac fibroblasts and paracrine function play an important role in the development of myocardial fibrosis[29].

4.3 Myocardial fibrosis is related to the expression of multiple targets

According to the PPI network construction and core gene screening,the PPI network contains seven genes, CTGF, TIMP1, SPP1,serpine1, COL3A1, postn and Fos, which are located in the core of the network and may be the key module of myocardial fibrosis.The genes in this module are mainly involved in extracellular matrix synthesis and fibroblast proliferation and transdifferentiation[30,31]. Connective tissue growth factor (CTGF) is a kind of vasoactive substance with extensive biological effects, which can promote angiogenesis, cell chemotaxis, fibroblast proliferation and collagen synthesis[32]. Chen l et al. Found that CTGF expression in myocardial tissue of rats with myocardial fibrosis was significantly up regulated, and the level of myocardial fibrosis was inhibited after down regulating the expression of CTGF [33]. Interestingly,endothelial cells have poor ability of Endo MT to produce collagen in myocardial injury, but they secrete a large amount of CTGF in the process of Endo MT, which stimulates cardiac fibroblasts to transdifferentiate into cardiac fibroblasts with stronger ability of collagen synthesis and promotes the development of myocardial fibrosis[34],it is suggested that the regulation of cardiac endothelial paracrine on fibroblasts may be an important driving factor of myocardial fibrosis. Matrix metalloproteinase (MMP) can degrade extracellular matrix and make it in dynamic balance, but TIMP1 can inhibit the effect of MMP, and TIMP1 is significantly upregulated in the development of myocardial fibrosis[35]. Collagen III (COL3A1) is an important component of extracellular matrix.Clinical studies have shown that the ratio of type III procollagen to type I procollagen (a marker of collagen III and collagen I synthesis)is related to left ventricular remodeling one month after myocardial infarction[36]. Previous studies by members of the research group showed that the intervention of traditional Chinese medicine in the treatment of heart failure rat model induced by myocardial infarction can significantly improve the cardiac function of rats, inhibit the occurrence and development of myocardial fibrosis, and the mRNA expression of CTGF, TIMP1, COL3A1 and COL1A1 in rat heart tissue were significantly down regulated, suggesting that traditional Chinese medicine intervenes the expression of these genes and plays an anti myocardial fibrosis role, It also proves the rationality of this study[6, 37].

4.4 The results of validation group showed that the screening of core targets was reliable

Through the gene differential expression analysis between the validation group and the control group, it was found that the above core targets were significantly expressed in the validation group.According to the volcanic map (Fig. 8B), all the seven core targets were up-regulated. The previous paper was to clarify that these genes played a role in promoting fibrosis in the development of myocardial fibrosis, so the validation results were consistent with the existing conclusions, The reliability of the research results is verified.

4.5 DS , HQ , TLZ has the value of anti myocardial fibrosis

Drug prediction and screening to obtain the most relevant three traditional Chinese medicine with myocardial fibrosis: DS, HQ,TlZ. The three drugs are based on promoting blood circulation by removing blood stasis, air supply and invigorating splenic yang, and inducing diuresis to alleviate edema to treat myocardial fibrosis.

There is no name of "myocardial fibrosis" in Chinese medicine.According to the clinical symptoms of patients with myocardial fibrosis, they can be classified into "heart failure", " gasp syndrome"and " chest stuffiness and pains". According to the theory of traditional Chinese medicine, the pathogenesis of this disease can be summarized as "deficiency in origin and excess in superficislity",in which deficiency of origin is mostly Qi deficiency and yang deficiency, and excess of superficislity belongs to phlegm and blood stasis. Qi deficiency is unable to promote the blood in the pulse,blood stasis is not smooth; Blood stasis stops in the heart, which blocks the movement of Qi and blood and damages the heart qi.Therefore, the treatment should be based on " supplementing qi and restoring yang, invigorate blood circulation and promoting diuresis"[38]. Later scholars of traditional Chinese medicine believed that promoting the elimination of body fluid through diuresis and defecation was the principle of treating the disease[39]. DS is bitter and slightly cold. It is recorded in Sheng Nong's herbal classic that it has the function of "mainly treating heart and lower abdomen...Breaking disease, removing obstruction, stopping vexation and filling Qi",when it is used in myocardial fibrosis, it can remove the blood stasis caused by the excess and can be combined with HQ,which can replenish the Qi of deficiency of origin, can treat both the symptoms and the root causes, and it has been widely used in the treatment of myocardial fibrosis. Modern pharmacological studies show that Salvianic acid can inhibit the expression of collagen in myocardial tissue of SD rats with myocardial infarction and improve myocardial fibrosis[40].HQ and TLZ are commonly used in the treatment of heart failure. HQ can Reinforcing Qi and elevate Yang, and stimulate Heart Qi of failure. It is just the so-called "Qi is the marshal of blood". If the heart Qi is abundant, the blood stasis can be dispersed, and the whole blood can be transported.TLZ has the function of diuresis and detumescence, dispersing the water remaining in the chest, and also has the function of relieving asthma, the combination of the two coincides with the strategy of"strengthening heart" and "diuresis" adopted by western medicine.Previous studies have confirmed that Luqi formula, including HQ and TLZ, has a clear curative effect in the treatment of heart failure.It can inhibit RAS system, reverse myocardial fibrosis, improve heart function, and down regulate the mRNA expression of CTGF, TIMP1,COL3A1 and COL1A1[6, 15, 37, 41, 42] . In addition, astragaloside and quercetin-3-O-b-D-glucose-7-O-b-D-gentiobiosiden is the main component of HQ and TLZ respectively. Pharmacological studies have confirmed that astragaloside IV has function of anti myocardial fibrosis[43].Extraction of TLZ can reduce the total collagen content of rat ventricles caused by abdominal aortic bands and inhibit myocardial remodeling[44]. Therefore, the three traditional Chinese medicines of DS, HQ and TLZ have rich pharmacological research and theoretical basis of traditional Chinese medicine in the treatment of myocardial fibrosis.

In conclusion, Myocardial fibrosis is a multi process, multi-channel,multi-target coordinated regulation of pathological process, involving extracellular matrix synthesis, collagen synthesis, inflammation and other biological pathways. Through a variety of cell secretion,extracellular matrix regulation related pathways, CTGF, TIMP1,SPP1, serpine1, COL3A1, postn, Fos and other targets are regulated.This study is based on bioinformatics to explore its mechanism,clarify the related functions and pathways of different genes involved in its pathogenesis, and verify the results show that the key targets screened are reliable. This paper also predicted the potential traditional Chinese medicine related to the treatment of myocardial fibrosis, and combined with the previous research of the research group, proved that DS, HQ and TLZ have theoretical and practical support in the treatment of myocardial fibrosis, so we should pay attention to the use of these three traditional Chinese medicine in the process of clinical treatment of myocardial fibrosis. At the same time,we found that the occurrence of myocardial fibrosis may involve the autocrine of cardiac fibroblasts or the paracrine regulation of other cardiac cells, but the specific mechanism has not been clarified.The research group will carry out the next step research based on this scientific problem to clarify its mechanism, and provide more research basis for the in-depth research and treatment of myocardial fibrosis.

Author's contribution

Zhou Zhou: completed the project implementation, result analysis and thesis writing; Wang Zhen, Liu Yang: finished the literature collation, scheme inspection and result analysis; Wang Yong: completed the examination and revision of the thesis; Yang ChuanHua: completed the project design, research selection and final audit.