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Salvia miltiorrhiza Bunge (S. miltiorrhiza) is an important Traditional Chinese herbal Medicine (TCM) used to treat cardio-cerebrovascular diseases. Based on the pharmacodynamic substance of S. miltiorrhiza , the aim of present study was to investigate the underlying mechanism of S. miltiorrhiza against cardiac fibrosis (CF) through a systematic network pharmacology approach, molecular docking and dynamics simulation as well as experimental investigation in vitro. A systematic pharmacological analysis was conducted using the Traditional Chinese Medicine Pharmacology (TCMSP) database to screen the effective chemical components of S. miltiorrhiza , then the corresponding potential target genes of the compounds were obtained by the Swiss Target Prediction and TCMSP databases. Meanwhile, GeneCards, DisGeNET, OMIM, and TTD disease databases were used to screen CF targets, and a protein-protein interaction (PPI) network of drug-disease targets was constructed on S. miltiorrhiza /CF targets by Search Tool for the Retrieval of Interacting Genes/Proteins (STING) database. After that, the component-disease-target network was constructed by software Cytoscape 3.7. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were performed for the intersection targets between drug and disease. The relationship between active ingredient of S. miltiorrhiza and disease targets of CF was assessed via molecular docking and molecular dynamics simulation. Subsequently, the underlying mechanism of the hub compound on CF was experimentally investigated in vitro. 206 corresponding targets to effective chemical components from S. miltiorrhiza were determined, and among them, there were 82 targets that overlapped with targets of CF. Further, through PPI analysis, AKT1 and GSK3β were the hub targets, and which were both enriched in the PI3K/AKT signaling pathway, it was the sub-pathways of the lipid and atherosclerosis pathway. Subsequently, compound-disease-genes-pathways diagram is constructed, apigenin (APi) was a top ingredients and AKT1 (51) and GSK3β (22) were the hub genes according to the degree value. The results of molecular docking and dynamics simulation showed that APi has strong affinities with AKT and GSK3β. The results of cell experiments showed that APi inhibited cells viability, proliferation, proteins expression of α-SMA and collagen I/III, phosphorylation of AKT1 and GSK3β in MCFs induced by TGFβ1. Through a systematic network pharmacology approach, molecular docking and dynamics simulation, and confirmed by in vitro cell experiments, these results indicated that APi interacts with AKT and GSK3β to disrupt the phosphorylation of AKT and GSK3β, thereby inhibiting the proliferation and differentiation of MCFs induced by TGFβ1, which providing new insights into the pharmacological mechanism of S. miltiorrhiza in the treatment of CF. [Display omitted] [ABSTRACT FROM AUTHOR]

The abnormal proliferation and differentiation of cardiac fibroblasts (CFs) are universally regarded as the key process for the progressive development of cardiac fibrosis following various cardiovascular diseases. Huoxin Pill (Concentrated pill, HXP) is a Chinese herbal formula for treating coronary heart disease. However, the cellular and molecular mechanisms of HXP in the treatment of myocardial fibrosis are still unclear. To investigate the effects of HXP on CFs transdifferentiation and collagen synthesis under isoproterenol (ISO) conditions, as well as the potential mechanism of action. In vivo , we established a rat model of cardiac fibrosis induced by ISO, and administered with low or high dose of HXP (10 mg/kg/day or 30 mg/kg/day). The level of α-SMA was detected by immunohistochemistry examination, and combined with RNA-sequencing analysis to determine the protective effect of HXP on myocardial fibrosis rats. In vitro , by culturing primary rat CFs, we examined the effects of HXP on the proliferation and transdifferentiation of CFs using CCK8, scratch wound healing and immunofluorescence assays. Western blot was used to determine protein expression. The findings revealed that HXP protects against ISO-induced cardiac fibrosis and CFs transdifferentiation in rats. RNA-sequencing and pathway analyses demonstrated 238 or 295 differentially expressed genes (DEGs) and multiple enriched signal pathways, including transforming growth factor-beta (TGF-β) receptor signaling activates Smads, downregulation of TGF-β receptor signaling, signaling by TGF-β receptor complex, and collagen formation under treatment with low or high-dose of HXP. Moreover, HXP also markedly inhibited ISO-induced primary rat CFs proliferation, transdifferentiation, collagen synthesis and the upregulation of TGF-β1 and phosphorylated Smad2/3 protein expression. HXP suppresses ISO-induced CFs transdifferentiation and collagen synthesis, and it may exert these effects in part by inhibiting the activation of the TGF-β/Smads pathway. This may be a new therapeutic tool for cardiac fibrosis. [Display omitted] [ABSTRACT FROM AUTHOR]

Cardiac fibrosis is a common characteristic of many cardiac diseases. Our previous results showed that TRPM7 channel played an important role in the fibrosis process. MicroRNA-135a was reported to get involved in the fibrotic process. Astragalus membranaceus (Fisch.) Bunge was widely used in Chinese traditional medicine and showed cardiac protective effects in previous researches. Astragaloside IV(ASG), which is regarded as the most important ingredient of Astragalus, has been showed the effect of cardiac protection via various mechanisms, while no data suggested its action related to miRNAs regulation. The objective of this article is to investigate the inhibition effect of ASG on cardiac fibrosis through the miR-135a-TRPM7-TGF-β/Smads pathway. We extracted the active components from herb according to the paper and measured the content of ASG from the mixture via HPLC. The inhibition potency of cardiac hypertrophy between total extract of Astragalus and ASG was compared. SD rats were treated with ISO (5 mg/kg/day) subcutaneously (s.c.) for 14 days, ASG (10 mg/kg/d) and Astragalus extract (AE) (4.35 g/kg/d, which contained about ASG 10 mg) were given p.o. from the 6th day of the modeling. Cardiac fibroblasts (CFs) of neonatal rats were incubated with ISO (10 μM) and treated with ASG (10 μM) simultaneously for 24 h. The results showed that both AE and ASG treatment reduced the TRPM7 expression from the gene level and inhibited cardiac fibrosis. ASG group showed similar potency as the AE mixture. ASG treatment significantly decreased the current, mRNA and protein expression of TRPM7 which was one of targets of miR-135a. The activation of TGF-β/Smads pathway was suppressed and the expression of α-SMA and Collagen I were also decreased obviously. In addition, our results showed that there was a positive feedback between the activation of TGF-β/Smads pathway and the elevation of TRPM7, both of which could promote the development of myocardial fibrosis. AE had the effect of cardiac fibrosis inhibition and decreased the mRNA expression of TRPM7. ASG, as one of the effective ingredients of AE, showed the same potency when given the same dose. ASG inhibited cardiac fibrosis by targeting the miR-135a-TRPM7-TGF-β/Smads pathway. Image 1 [ABSTRACT FROM AUTHOR]