LIU Li-ying, HUANG Ju-kai, LI Gao-biao, ZHANG Li, YANG Xiao-hui?

1. Beijing University of Chinese Medicine Dongzhimen Hospital, Beijing 100700, China

2. Xiyuan Hospital of China Academy of Chinese Medical Science,Beijing 100091,China

3. Department of Scientific Research, Oriental Hospital, Beijing University of Chinese Medicine, Beijing 100078, China

Keywords:Astragalus Prunella Diabetic cardiomyopathy Network pharmacology Molecular docking

ABSTRACT Objective: To study the main chemical components and mechanism of Astragalus and Prunella vulgaris in the treatment of diabetes cardiomyopathy (DCM) based on network pharmacology and in vitro experiments. Methods: The main active components and prediction targets of Astragalus membranaceus and Prunella vulgaris herbal pairs were obtained by TCM Pharmacology database and analysis platform (TCMSP), and the disease genes were retrieved by genecards, OMIM, PharmGKB and drugbank databases. The disease and drug prediction targets were intersected to screen out common potential therapeutic targets.Cytoscape3.7.2 software was used to construct "drug component disease target" interaction network diagram; The PPI network of protein-protein interaction was constructed by using string database. R software was used to analyze the function enrichment of GO and KEGG for drug disease common targets, and autodock Vina 1.1.2 for molecular docking. Finally,the specific mechanism of Astragalus and Prunella vulgaris medicated serum on high glucose stimulated cardiomyocytes was verified in vitro. H9c2 cardiomyocytes were divided into five groups: normal group: low glucose (5.5 mmol/L) culture group, model group: high glucose(33 mmol/L) culture group, 5% serum group: high glucose+5% Astragalus membranaceus Prunella vulgaris herb serum culture group, 10% serum group: high glucose+10% Astragalus membranaceus Prunella vulgaris herb serum culture group, 15% serum group: Hg high glucose+15% Astragalus membranaceus Prunella vulgaris herb serum culture group. MTT assay was used to detect the cell survival rate, and Western blot was used to detect the effect of Astragalus and Prunella vulgaris medicated serum on the expression of AKT1, p-AKT1,MAPK14 and p-MAPK14 proteins. Results: In this study, 31 active components of Astragalus and Prunella vulgaris were screened, involving 157 targets of diabetes cardiomyopathy and 178 related signal pathways. The results of network analysis showed that Astragalus and Prunella vulgaris herbs may play a role in the treatment of DCM by acting on key targets such as AKT1,FOS, MAPK1, MAPK8, MAPK14, Jun and key pathways such as PI3K-AKT. Molecular docking showed that Astragalus membranaceus and Prunella vulgaris medicine had good binding between the active components luteolin, quercetin, pistil isoflavone, kaempferol and key targets such as AKT1, MAPK14, MAPK1, FOS, mapk8 and Jun, and the Vina score of luteolin and AKT1 was the lowest. The results in vitro showed that Astragalus and Prunella vulgaris medicated serum significantly improved the inhibition of H9c2 cardiomyocyte proliferation induced by high glucose, and increased the phosphorylation levels of AKT1 and MAPK14 proteins to play a role in the treatment of DCM. Conclusion: Astragalus and Prunella vulgaris have the characteristics of multi-target and multi-channel in the treatment of DCM. Its mechanism may be related to the regulation of the protein expression of p-AKT1 and p-MAPK14. These findings provide a new idea and basis for further experimental study on the mechanism of Astragalus and Prunella vulgaris in the treatment of diabetes cardiomyopathy.

1. Introduction

Diabetic cardiomyopathy (diabetic cardiomyopathy, DCM) is closely related to diabetes mellitus (diabetes mellitus, DM), which can be caused by persistent increase of blood glucose and abnormal changes of heart function and structure caused by insulin resistance.A large number of studies have shown that deaths caused by DCM account for about 65%～80% of deaths caused by DM complications[1]. With the continuous increase in the number of DM patients around the world, the incidence of DCM continues to rise, and the prevention and treatment measures of DCM can be implemented effectively [2].

The western medicine treatment of DCM mainly includes dilating blood vessels and anti-inflammation, improving abnormal metabolism and so on, with many side effects and poor therapeutic effect, while traditional Chinese medicine can effectively reduce insulin resistance, regulate glucose and lipid metabolism, alleviate oxidative stress, inhibit abnormal apoptosis of cardiomyocytes and reduce myocardial fibrosis. Professor Yang Xiao-hui is one of the academic inheritors of Professor Lu Ren-he, a master of Chinese medicine. He inherits the pathogenesis concept of DM complication"micro-syndrome" put forward by Lu Shi-ji, and integrates many years' rich experience in diagnosis and treatment of DM, and analyzes and establishes the core idea of "heart-collateral microsyndrome" of DCM. It is considered that the deficiency of heart qi is the preliminary pathological change on the basis of the imbalance of qi and blood yin and yang, and then there are pathological changes such as qi stagnation and blood stasis, long-term disease entering collaterals and obstruction of heart collaterals. First gather in Qi, gather and disperse impermanently, become invisible "Qi",accumulate in blood for a long time, accumulate but not disperse,block in collaterals, and form tangible "Qi", which leads to the formation of heart-collaterals. The key factor for the formation of "Qi deficiency" is the deficiency of vital qi, and the collaterals of blood stasis in the heart is its pathological basis, just like the complete Book of Jingyue: "there is no accumulation of strong people, but there are virtual people". Yang Shi established Yixiao prescription according to the above discussion and clinical practice. Astragalus and Prunella vulgaris are the main drugs of this prescription, making good use of Astragalus to "replenish qi and nourish the heart",Prunella vulgaris to remove blood stasis and accumulate well. The combination of Astragalus and Prunella vulgaris can supplement but not stagnate, remove blood stasis without injuring, and delay the progress and aggravation of the disease. Some studies have shown that for STZ-induced DCM rats, Astragalus-summer quat herbal medicine can not only significantly improve the disorder of glucose and lipid metabolism, reduce the degree of myocardial necrosis,inhibit fibroblast infiltration, delay cardiomyocyte hypertrophy and slow down the thickening of myocardial microvascular basement membrane, but also improve cardiac autonomic nerve function and enhance the activities of aerobic and anaerobic metabolic enzymes in myocardial tissue [3-6]. However, the chemical composition of the drug pair is complex, and the target and mechanism of the treatment of DCM are not completely clear.

In this study, bioinformatics combined with network pharmacology,several data analysis software and databases were used to analyze and integrate the corresponding action target and chemical composition of traditional Chinese medicine, and the molecular network system of "component-disease-target" was constructed. its action mechanism is described systematically. Its research method combines the characteristics of multi-components, multi-targets and multi-pathways of traditional Chinese medicine, which is consistent with the medical concept of holistic and systematic view of traditional Chinese medicine [7,8]. The research group used the"component-target-disease" theory of network pharmacology to fully explore the target and mechanism of Astragalus-Prunella vulgaris in the treatment of DCM. Molecular docking study was used to further explore the mechanism of Radix Astragali-Prunella vulgaris herbs in the treatment of DCM, supplemented by in vitro experiments, to provide scientific basis for its clinical application in DCM.

2. Materials and methods

2.1 Screening of active components of Astragalus and Prunella

All the chemical constituents of Astragalus and Prunella vulgaris were searched by TCMSP (http://lsp.nwu.edu.cn/tcmsp.php).According to the screening conditions of oral bioavailability OB

30% and drug-like DL 0.18, the related active components of Radix Astragali and Prunella vulgaris were screened[9].

2.2 Search for targets of diabetic cardiomyopathy

Using "diabetic cardiomyopathy (DCM)" and "Diabetic heart disease" as search keywords, we searched GeneCards, OMIM,PharmGkb and DrugBank databases to obtain disease targets related to diabetic cardiomyopathy.

2.3 Drug target-disease-related gene acquisition and intersection

The drug target gene and disease gene were read by PERL software, the gene name was extracted, the Venn map was drawn,and the intersection gene of drug and disease was obtained. To find the corresponding relationship between drug components and target genes.

2.4 Construction of "component-disease-target" network

The "component-disease-target" interaction network diagram was constructed by using Cytoscape 3.8.0 [10] software and JAVA software, and the main active components of Astragalus-Prunella vulgaris pairs were analyzed by Network Analyzer function.

2.5 Establishment of protein-protein interaction (PPI)network

Upload the common target to STRING database (https://www.string-db.org/) to build a protein-protein interaction network. The protein type is set to Homo sapiens, the confidence level is set to"highest confidence level (0.900)", and the isolated proteins in the network are hidden to obtain the PPI network. Input the PPI network data into the Cytoscape 3.7.2 software to build the network and network topology analysis operation, and filter the key targets in the network. Core target screening parameters: nodal centrality (degree centrality, DC), medium centrality (betweenness centrality, BC),compact centrality (closeness centrality, CC). Screening conditions:the DC, BC and CC values of intersection genes were calculated by R software online package, and the genes larger than the median value of each group were retained. The R script was run twice to screen the key targets.

2.6 GO and KEGG analysis

David6.8 database (https://david.ncifcrf.gov/) was used to analyze the functional enrichment of gene ontology (GO) and KEGG pathway enrichment, respectively, and the enrichment results of GO and KEGG were obtained. GO analysis included biological process (BP), cellular composition (CC) and molecular function(MF). Clusters Profiler R software package was used to analyze the potential targets of Astragalus and Prunella vulgaris on DCM by GO and KEGG. The statistical significance threshold was set to P＜0.05.

2.7 Verification of component-target molecular docking

The 3D structure of key targets was downloaded from PDB (http://www.rcsb.org/). The core components were processed into mol2 format files by Chembio Office software, and the operations such as hydrogenation, dehydration and ligand separation were carried out.Autodock Vina 1.1.2 software was used to dock the key targets with the core components [11,12]. The binding energy between each key target and the core component is obtained. the lower the negative binding energy is, the better the compound can combine with the target and produce a stable conformation. The conformation with the best affinity was taken as the final docking conformation, and the results were visualized by PyMOL2.3 software.

2.8 In vitro experiment of H9c2 cardiomyocytes

2.8.1 Animal

Six adult SPF grade SD male rats, weighing 200～250 g, were purchased from Sbeifer (Beijing) Biotechnology Co.Ltd. [SCXK(Beijing) 2019-0010]. Animal feeding environment: SPF animal room of Dongzhimen Hospital of Beijing University of traditional Chinese Medicine, room temperature 24℃, relative humidity(60±5)%, 12 hours alternating day and night, free to drink and eat.

2.8.2 Serum containing Astragalus and PrunellaThe rats were treated with Astragalus and Prunella vulgaris solution(6.0 g/kg) once a day for 7 days. On the 7th day, blood was taken from the orbit after intragastric administration for 1 hour. 3 000 r/min centrifuged 10 min, then separated the serum, put the serum into a 56℃water bath to inactivate 30 min, filtered and sterilized by 0.22 mm filter, and preserved at-20℃. This experiment meets the requirements of animal ethics in Dongzhimen Hospital of Beijing University of traditional Chinese Medicine, and the number of animal experimental ethics examination and approval is 2021205.

2.8.3 Cell culture and grouping

Rat H9c2 cardiomyocytes, provided by Wanxian Biotechnology Company. The cells were cultured in low-sugar DMEM medium(containing 5.5 mmol/L glucose) containing 10% fetal bovine serum and 1% penicillin-streptomycin and cultured in 5% CO2incubator at 37℃. When the fusion degree reached 70%～80%, the cells were digested with trypsin containing 0.25% ethylenediamine tetraacetic acid (EDTA) and subcultured. Finally, the cells in logarithmic growth phase were taken for follow-up experiments.

The cells were divided into 5 groups: ① normal group: low glucose(5.5 mmol/L) culture; ② model group: high glucose (33 mmol/L)culture; ③ 5% serum group: high glucose+5% Astragalus-Prunella serum culture; ④ 10%serum group: high glucose+10% Astragalus-Prunella serum culture; ⑤ 15% serum group: HG high sugar+15%Astragalus-Prunella serum culture.

2.8.4 Detection of cardiomyocyte survival rate in H9c2 by MTT method

The cells in logarithmic growth phase were selected and resuspended after trypsin digestion, and the cells were counted. About 2 000 cells were added to each well of 96-well plate. The cells of each group were treated according to the experimental requirements.After the cells adhered to the wall for 48 hours, 100 μL of medium was changed in each well, and 10 μL MTT solution was added. The cells were incubated in the cell incubator for 4 hours. After that,Then 150 μL DMSO was added to each hole, continue to incubate 15 min in the cell incubator. Until it was found that the blue-purple crystals were all dissolved under the microscope, the absorbance was measured at the 490 nm wavelength of the enzyme labeling instrument.

2.8.5 Detection of related protein expression by Western blot

The proteins of H9c2 cardiomyocytes in each group were extracted according to the instructions of RIPA cleavage buffer (Shanghai Beyotime Biotechnology Co., Ltd., Shanghai China), and quantified by BCA protein detection kit (Thermo Fisher Technology Co.Ltd., Shanghai China). The protein (40 μg/swimming lane) was separated by SDS-PAGE and transferred to PVDF membrane. Seal with 5% skim milk in TBST buffer at room temperature for 2 hours.The membrane was incubated with AKT1, p-AKT1, MAPK14,p-MAPK14 and β-actin at 4 ℃ overnight and washed with TBST for 10 minutes each time. Then add the second antibody and incubate the membrane for 1 hour. Wash the membrane with TBST 3 times for 10 minutes each time. Enhanced chemiluminescence system and ImagePro-Plus were used to image and quantify the protein bands.

2.9 Statistical processing

Statistical analysis using SPSS26.0 statistical software measurement data are expressed by mean±standard deviation (±s) , Comparison of measurement data between groups using single factor ANOVA test, If the variance is uniform, the LSD method is used, and if the variance is uneven, the Dunnett T3 method is used. The difference was statistically significant (P＜0.05).

3. Results

3.1 Acquisition of potential active components and targets of Astragalus and Prunella

The results of TCMSP database showed that there were 20 active components in Astragalus and 11 active components in Prunella vulgaris. The main active components are shown in Table 1.Isoflavone, the active ingredient of Radix Astragali, molecular number MOL000398, has a high OB value (109.99%). A total of 839 drug targets were obtained. after eliminating repeated targets,210 drug targets were obtained.

Tab 1 Main active components of Astragalus-prunella

3.2 Screening of disease targets

3 632 related genes were obtained based on GeneCards, 304 related genes were screened by OMIM, 76 related genes were obtained based on PharmGkb, 94 related genes were obtained based on DrugBank. A total of 3 739 disease-related genes were obtained after weight removal, as shown in Figure 1. Using Wayne software, 3 739 disease-related targets were intersected with 210 targets predicted by the active components of Astragalus and Prunella vulgaris, and 157 intersection targets were obtained, which may be the potential key targets of Astragalus and Prunella vulgaris herbs in the treatment of DCM, as shown in Figure 2.

Fig 1 Intersection target diagram of disease database

Fig 2 Astragalus-prunella and disease targets venn

3.3 Construction of component-disease-target network

The interaction network between Astragalus and DCM was constructed by using Cytoscape3.7.2 software. Figure 3 consists of 178 nodes and 530 edges. The main core index of the network node is the degree centrality parameter. The larger the value is, the closer the node is to the network center [13]. The four components with the highest Degree value in Astragalus and Prunella vulgaris were luteolin, quercetin, Calycosin and kaempferol.

3.4 Construction of PPI Network

Fig 3 Astragalus-prunella active ingredient-DCM intersection targets network

A total of 132 nodes and 564 lines are included in the PPI network,which indicates that the 157 targets produce a total of 564 interaction links, as shown in Figure 4. According to the screening criteria, the specific process of screening core targets is shown in Figure 5. A total of 13 core targets were obtained: JUN, TP53, MYC, MAPK1,AKT1, ESR1, FOS, MAPK14, CCND1, MAPK8, CDKN1A,RELA, RB1, as shown in Table 2. These targets may be the core targets of Radix Astragali-Prunella vulgaris herbs in the treatment of DCM.

Fig 4 Target protein interaction map of Astragalus and Prunella vulgaris in the treatment of diabetic myocardial injury

Tab 2 Core target results

3.5 GO enrichment analysis and KEGG enrichment analysis

A total of 157 disease-drug common targets were analyzed by R software, and the GO results were shown in Figure 6. The set of overlapping genes was enriched into 2 576 BP pathways, of which 553 pathways with more than 10 genes were mainly in response to oxidative stress and cells to chemical stress, and 67 pathways were enriched to CC expression pathways, of which 23 pathways with more than 10 genes were mainly related to membrane rafts,membrane microregions and membrane regions. In the process of enrichment of intersecting genes, 23 pathways with more than 10 genes were enriched, which were mainly related to DNA binding transcription factor, RNA polymerase Ⅱ specific DNA binding transcription factor and ubiquitin-like protein ligase.

169 KEGG pathways were obtained after running on 157 common targets by R software. The functional enrichment KEGG bubble diagram of the first 30 is shown in figure 7. The Abscissa in the picture shows the number of enrichment, and the ordinate represents the smaller the P, the redder the color, and the larger the P, the bluer the color. The results of KEGG pathway enrichment analysis showed that the common targets were mainly concentrated in PI3K-AKT signal pathway, MAPK signal pathway, AGE-RAGE signal pathway,TNF signal pathway, IL-17 signal pathway, apoptosis, cell aging and other signal pathways.

Fig 5 Construction of Core Network Diagram of protein interaction

Fig 6 Results of GO enrichment Analysis of Astragalus and Prunella in the treatment of DCM

3.6 Molecular docking analysis

The core active components luteolin, quercetin, Calycosin and kaempferol were docked with the key target AKT1, FOS, MAPK1,MAPK8, JUN, MYC, MAPK14, RELA. It is generally considered that the binding energy is lower than -4.25 kcal·mol-1, indicating that the receptor has the corresponding binding ability to the ligand,which is lower than -5.0 kcal·mol-1, indicating that it has better binding activity, and less than -7.0 kcal·mol-1has strong binding activity [14]. Docking 4 active components with 8 target proteins respectively, The results showed that luteolin, quercetin, Calycosin and kaempferol had strong binding ability to AKT1, MAPK14,MAPK1, FOS, MAPK8, JUN, RELA, as shown in Table 3,The PyMOL software is used to visualize the docking results between the components with strong binding activity (binding energy -7 kcal·mol-1) and the target, as shown in Figure 8.

3.7 Experimental study of Astragalus-prunella containing serum on the intervention of H9c2 Cardiacmyocytes in vitro

3.7.1 Effect of Astragalus-prunella containing serum on the survival rate of normal H9c2 Cardiacmyocytes

Effect of Radix Astragali-Prunella vulgaris herbs on the survival rate of normal rat H9c2 cardiomyocytes in the experiment, different concentrations of Astragalus-Prunella vulgaris herbs on serum incubated 12 h/24 h/48 h cells, the cell survival rate was detected by MTT method. The effect of Astragalus-Prunella vulgaris herbs on serum on H9c2 cardiomyocytes cultured in normal medium showed that, compared with the normal group, when the serum level of Astragalus-Prunella vulgaris herbs was lower than 17.5%, The survival rate of cells was not significantly inhibited, and Astragalus-Prunella vulgaris herbs could promote the growth of normal cultured H9c2 cardiomyocytes when the serum was less than 15%, as shown in Table 4. Therefore, Radix Astragali-Prunella vulgaris serum will be selected to intervene the cells cultured with high glucose with in the range of 5%～15%, and the survival rate and modeling time of 12 h/24 h/48 h cells will be intervened according to different concentrations of drug-containing serum. In the followup experiment, each concentration of drug-containing serum will be used to intervene for 24 hours.

Fig 7 KEGG enrichment bubble map of Astragalus-prunella in the treatment of DCM

Tab 3 Astragalus-prunella main active components are docked with the target proteins molecule Required binding energy (kcal/mol)

3.7.2 Effect of Astragalus-prunella containing Serum on Survival rate of H9c2 Cardiacmyocytes induced by high glucose

Fig 8 Molecular docking pattern diagram

The effect of Astragalus-prunella on the survival rate of H9c2 cardiomyocytes induced by high glucose showed that compared with the normal group, the cell survival rate of the model group was significantly lower than that of the normal group (P＜0.001).Compared with the model group, the cell survival rate of 15% serum group and 10% serum group was significantly higher than that of the model group, and showed a certain concentration dependence, as shown in Table 5.

Tab 4 Effect of Astragalus-prunella containing serum on the survival rate of normal cardiomyocytes (n=3,±s)

Tab 4 Effect of Astragalus-prunella containing serum on the survival rate of normal cardiomyocytes (n=3,±s)

Time Group Survival rate% F P normal group 97.67±2.082 12 h 5% serum group 108.67±1.528 10% serum group 108.00±2.000 15% serum group 108.67±1.528 17.5% serum group 90.33±2.517 20% serum group 59.00±4.583 166.600 ＜0.001 normal group 96.67±3.055 24 h 5% serum group 107.00±3.000 10% serum group 108.33±2.082 15% serum group 107.33±3.055 17.5% serum group 80.67±2.082 20% serum group 45.67±2.517 254.577 ＜0.001 normal group 94.33±3.786 48 h 5% serum group 103.33±3.055 10% serum group 104.67±4.163 15% serum group 106.00±3.606 17.5% serum group 76.67±3.215 20% serum group 39.00±3.606 158.452 ＜0.001

Tab 5 Effect of Astragalus-prunella containing serum on the survival rate of H9c2 cardiomyocytes induced by high glucose(n=3, ±s )

Tab 5 Effect of Astragalus-prunella containing serum on the survival rate of H9c2 cardiomyocytes induced by high glucose(n=3, ±s )

Group Survival rate% F P normal group 97.67±3.215 model group 70.33±3.512 5% serum group 81.00±4.583 10% serum group 84.67±4.163 15% serum group 91.33±3.215 22.699 ＜0.001

3.7.3 Effect of Astragalus-prunella containing serum on the expression of key gene proteins

The results of WB showed that there was no significant change in the expression of PI3K and MAPK14 protein in each group.Compared with the normal group, the ratio of p-AKT1/AKT1 and p-MAPK14/MAPK14 in the model group decreased significantly(P＜0.001), while the ratio of p-AKT1/AKT1 and p-MAPK14/MAPK14 in the 15% serum group increased significantly compared with the model group (Figure 9). The results showed that Astragalus membranaceus-Prunella vulgaris herbs could increase the phosphorylated expression of AKT1 and MAPK14 proteins in H9c2 cardiomyocytes induced by high glucose.

Fig 9 Expression of AKT1, p-AKT1, MAPK14, and p-MAPK14 proteins in various groups of H9c2 cardiomyocytes

Tab 6 Expression of AKT1, p-AKT1, MAPK14 and p-MAPK14 proteins in H9c2 cardiomyocytes in each group(n=3, ±s)

Tab 6 Expression of AKT1, p-AKT1, MAPK14 and p-MAPK14 proteins in H9c2 cardiomyocytes in each group(n=3, ±s)

Index Group Expression quantity F P N 0.8600±0.03000 p-AKT1/ AKT1 M 0.5933±0.02082 15% group 0.8267±0.03055 10% group 0.7967±0.02517 5% group 0.6500±0.03000 53.873 ＜0.001 N 0.9533±0.03055 p-MAPK14/ MAPK14 M 0.4967±0.02517 15% group 0.8167±0.04509 10% group 0.5567±0.03055 5% group 0.4300±0.04583 113.296 ＜0.001

4. Discussion

DCM belongs to the category of "eliminating thirst and heart disease" in traditional Chinese medicine, with qi deficiency and blood stasis as its basic pathogenesis. The results show that as one of the most frequently used single drugs in the treatment of DM and its complications, Astragalus membranaceus occupies the primary position in the treatment of cardiovascular complications of DM [15]. The results of pharmacological experiments confirmed that Astragalus membranaceus can reduce blood sugar and regulate glucose metabolism [16,17]. Quercetin is the main active component of Radix Astragali and Prunella vulgaris, and it is a typical natural flavonoid. Some studies have shown that quercetin can inhibit oxidative stress, reduce apoptosis, reduce inflammatory reaction, and inhibit myocardial ischemia-reperfusion injury[18].Some studies have confirmed that quercetin plays an important role in reducing the production of inflammatory factors by regulating HMGB1-TLR4-NF-κB signal pathway[19]. Luteolin is the main active ingredient of Prunella. Some studies have shown that luteolin can reduce IR by interfering with PI3K/AKT signal pathway, which can improve cardiac function and protect heart[20]. Some studies have confirmed,through the model of H9c2 cardiomyocyte apoptosis induced by H2O2, the experimental group was treated with luteolin[21]. The results showed that the expression of AKT and anti-apoptotic mediator Bcl-2 in the experimental group was lower than that in the control group, while the expression of pro-apoptotic proteins Bax, Caspase-8, Cleaved-Caspase-3 and p53 increased. Luteolin can reduce ERS-induced mitochondrial damage and down-regulate the expression of p53 protein, thus protecting cardiomyocytes[22]. Luteolin can reduce the entry of Ca2+into cells, promote the excretion of Ca2+, increase the activity of Ca2+-Mg2+-ATPase, and reduce the concentration of CaM and MCU[23]. Luteolin can regulate the balance of Ca2+in cardiomyocytes, which may be the main mechanism of its inhibition of cardiomyocyte apoptosis.

This study shows that the key target proteins of Astragalus-Prunella vulgaris herbs in the treatment of DCM are AKT1, MAPK14,MAPK1, FOS, MAPK8, JUN, MYC and RELA, respectively. The above target genes are involved in the regulation of cell proliferation,apoptosis and inflammation. As one of the AKT kinases, serine/ threonine protein kinase 1 (RAC-alphaserine/threonineproteinkinase1, AKT1) can regulate cell proliferation, survival,metabolism and angiogenesis, induce glucose transport, regulate glucose uptake, and regulate diabetes and complications [24]. AKT plays an important role in regulating insulin-dependent metabolic response [25]. After activating AKT, it can regulate glucose uptake and lipid metabolism. Overexpression of AKT can increase blood glucose and induce insulin resistance [26-28]. Mitogen-activated protein kinase 14 (Mitogen-activatedproteinkinase14, MAPK14), as the main component of MAP kinase signal transduction pathway,regulates apoptosis and plays an important role in the cellular cascade induced by extracellular stimulation. Some scholars have confirmed that MAPK plays an important role in the regulation of myocardial fibrosis [29-30]. Another part of scholars has found that in skeletal muscle cells, MAPK pathway is involved not only in the expression of collagen IV, but also in the expression of MMP-9 in matrix metalloproteinases family, leading to cardiac remodeling and myocardial fibrosis [31]. Studies have confirmed that [32], in DCM, the expression of FOS, which can induce apoptosis and promote differentiation, decreased. Some studies have found that the expression of FOS has temporal characteristics in the occurrence and development of DCM. It is well known that JUN is a protooncogene involved in the process of cell proliferation and apoptosis.Studies have shown that the expression of JUN can be significantly increased when apoptosis is induced by inducers[33]. In DCM,tumor necrosis factor-ɑ(TNF-ɑ) can activate MAPK, up-regulate the expression of bax, down-regulate the expression of bcl-2, and then induce apoptosis, while TNF-ɑ can increase the expression of FOS and MYC, which leads to myocardial interstitial fibrosis.

The analysis of GO enrichment results showed that the molecular function of Astragalus-Prunella vulgaris herbs in treating DCM was closely related to DNA binding transcription factor, RNA polymeraseⅡ specific DNA binding transcription factor, ubiquitin-like protein ligase and so on. According to the analysis of the enrichment results of KEGG pathway, the effects of Astragalus on Prunella vulgaris are mainly related to PI3K/AKT signal pathway, MAPK signal pathway,AGE-RAGE signal pathway, TNF signal pathway, apoptosis, cell senescence, IL-17 signal pathway and so on. Foreign scholars have shown through animal experiments [34] that insulin affects glucose and lipid metabolism and contractile function of cardiomyocytes,mainly through MAPK pathway and PI3K signal pathway. PI3K/AKT signaling pathway plays an important role in regulating cell growth, proliferation, survival, transcription and protein synthesis [35]. Phosphatidylinositol kinase is an important kinase of phosphatidylinositol and inositol. Activation of PI3K can further promote the activation of AKT. Phosphorylation of activated AKT can activate or inhibit its downstream target proteins, and inhibit cell apoptosis by regulating apoptosis-related proteins Bax, BAD, Bcl-2,caspase-9 and so on through PI3K/AKT signal pathway[36].Some studies have shown[37] that Astragalus can reduce myocardial injury and improve cardiac function in DCM rats by activating MAPK signal pathway. RAGE is closely related to the occurrence of DM and its complications [38]. The combination of RAGE and AGE can form AGE-RAGE system, which can activate NF-κB, oxidative stress,Janus kinase and other signal pathways, thus promote the production of related cytokines and growth factors, and then affect the process of diabetic complications. Some studies have shown that the severity of DM complications is positively correlated with the protein level of AGE [39]. TNF-ɑ causes inflammatory cells to gather, adhere and dilate microvessels, which further aggravates the inflammatory response [40-41]. Some studies have shown that [42] by knocking out IL-17, the up-regulation of AK081284 induced by high glucose is eliminated, thus reducing the production of TGFβ1 and collagen in cardiac fibroblasts (CFs), thus achieving the effect of treating DCM.The results of network analysis in this study show that Astragalus-Prunella herbs may act on AKT1, MAPK14, MAPK1, MAPK8 and other key targets, activate PI3K/AKT, MAPK and other key signal pathways to play the role of anti-myocardial fibrosis. The results of molecular docking showed that Astragalus membranaceus-Prunella had good binding ability of luteolin with AKT1, stamens isoflavones and MAPK14. The cell experiment in vitro showed that Astragalus-Prunella could significantly improve the inhibitory effect of serum on the proliferation of H9c2 cardiomyocytes induced by high glucose, and Astragalus-Prunella could increase the phosphorylated expression of AKT1 and MAPK14 proteins in H9c2 cardiomyocytes injured by high glucose.

In summary, by studying the multiple network components between Astragalus-Prunella and DCM disease, this study identified the potential active components and key action targets of Astragalus-Prunella, as well as the main signal pathways related to them, which provided new ideas and insights for further exploration of its action mechanism.

Author's contribution:

Liu Li-ying conceived the study, completed online pharmacological analysis, and participated in experimental design, implementation and data statistics; Li Gao-biao is in charge of cell experimental operation and experimental data analysis; Yang Xiao-hui contributed to the censorship and supervision of writing; Zhang Li and Huang Ju-kai participated in the revision and guidance of the paper. All the authors designed the experiment, wrote the manuscript and revised the paper. All the authors read and approved the final version of the manuscript.

All authors declare that there is no conflict of interest.