The Anti-Renal Fibrosis Mechanism Of Shenkang Injection Was Researched Based On Network Pharmacology And BMP-7/Smads Signaling Pathway

Objective:The purpose of this study was to explore the mechanism of Shenkang injection(SKI)against renal fibrosis through network pharmacological techniques,and to verify the predicted mechanism through cell experiments.Methods:1.From traditional Chinese medicine extract the SKI(TCMSP)database system pharmacology contains rhubarb,safflower,salvia miltiorrhiza and astragalus membranaceus 4 Chinese herb,the active ingredient and potential targets,then with the help of a database search renal fibrosis associated with disease related targets,both targets after the integration of overlapping genes,draw the protein-protein interaction(PPI)network diagram,And construct a complete system of network regulation diagram,and use Cytoscape and Clusterprofiler to carry out GO and KEGG function enrichment analysis of the obtained key targets.2.The effect of SKI on TGF-β1-induced mesenchymal transdifferentiation of human renal tubular epithelial cells(HK-2)was studied by network pharmacological analysis based on BMP-7/Smads signaling pathway.The specific operation steps are as follows:(1)HK-2 cells were randomly divided into blank control group,model group(TGF-β1 10 ng/mL induction for 48 h),SKI low-concentration group(TGF-β110 ng/mL+1:640 SKI intervention for 48 h),and SKI medium-concentration group(TGF-β1 10 ng/mL)+1:160 SKI intervention for 48 h),SKI high concentration group(TGF-β1 10 ng/ml +1:10 SKI intervention for 48 h)5 groups;(2)CCK-8 assay was used to evaluate the effects of SKI concentrations on TGF-β1-induced HK-2 cell proliferation at 12 h,24 h and 48 h.(3)The morphological changes of HK-2 cells were observed with inverted microscope to understand the situation of cell transdifferentiation;(4)The localization and expression of p-Smad1/5 complex were detected by immunofluorescence assay;(5)The m RNA and protein expressions of BMP-7,Smad1,Smad5,Smad6,Fn,Vimentin and α-SMA were detected by RT-PCR and Western Blotting.Results:1.A total of 75 active compounds and 99 target genes were collected,involving92 signaling pathways.Functional enrichment analysis suggested that the anti-renal fibrosis of SKI may be produced through the regulation of multiple signaling pathways,such as TGF-β/Smads and p38 MAPK signaling pathways.2.Different concentrations of SKI(1:640,1:160,1:10)had certain inhibitory effects on TGF-β1-induced HK-2 cell proliferation after 24 h.Compared with blank control group,HK-2 cells in TGF-β1-induced model group showed typical fusiform changes.After TGF-β1-treated HK-2 cells were treated with SKI at different concentrations,the results showed that SKI could down-regulate the expression of Vimentin and α-SMA protein in a concentration dependent manner,reduce FN secretion,and up-regulate the expression of BMP-7,Smad1,Smad5,p-Smad1/5,and Smad6 protein in a certain degree.Conclusion:1.Through a variety of network pharmacological analysis methods,the association between drugs and disease targets and the information of related pathways were intuitively predicted and understood.It was preliminarily speculated that the BMP-7/Smads signaling pathway might be an important pathway of Shenkang injection against renal fibrosis.2.Shenkang injection can inhibit the mesenchymal transdifferentiation of HK-2cells induced by TGF-β1 by regulating the BMP-7/Smads signaling pathway,thus exerting the anti-renal fibrosis effect.