Study On The Mechanism Of Hongjingtian Injection Inhibiting The Abnormal Migration And Phenotype Transformation Of TGF-β1 Induced Proximal Renal Tubular Cells Based On Network Pharmacology

Background: The study was designed to explore the mechanism of HJT inhibit the abnormal migration and phenotypic transformation of TGF-β1induced proximal renal tubular cells based on network pharmacology and in vitro cell experiment,and provide new ideas and approaches for the clinical application of HJT in the treatment of TIF.Methods:1.The network pharmacology method was used to analyze the active components,pathways and targets of HJT interfering with TGF-β1-induced HK-2 cells.The active ingredients of HJT obtained from the literature were screened using the Traditional Chinese Medicine Systems Pharmacology database,and the targets of the active ingredients were screened using the Chemmapper,SEA,and Swiss target prediction databases.Subsequently,the "HJT active ingredient-target" network was established by using Cytoscape3.7.2 software.In addition,TIF-related chips were searched from the GEO database to obtain TIF disease-related targets.Visualize differential genes with volcano plot.Then,the genes regulated by HJT active compounds and the differential genes of TIF were intersected by the Jvenn tool to obtain cross-target points.Input the obtained gene targets into the STRING online platform to establish a protein-protein interaction(PPI)network,and signaling pathways and key targets were identified by Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis.Finally,vitro cell experiments were performed.2.Taking HK-2 cells as the research object,the toxicity of HJT to cells was detected by CCK-8 method and the appropriate concentration was screened.Real-time fluorescence quantitative PCR was used to detect the effects of TGF-β1 and HJT on the expression of key genes and to determine the experimental concentration of TGF-β1.The effect of HJT on the expression of key gene SPHK1 was further verified by Western Blot method.The migration of HK-2 cells was observed by wound healing assay and transwell assay,and the expression of Vimentin and Cytokeratin-18 in HK-2 cells was detected by immunofluorescence experiment.Results:1.The results of network pharmacology analysis showed that there were36 active compounds in HJT,among which p-coumarinic acid and ethyl gallate were the core components.The active ingredients of HJT contained a total of 1044 predicted targets,and the overlapping gene functions of79 differential genes with TIF were obtained by constructing a Venn diagram.By constructing the PPI network,a total of 25 core genes were screened.KEGG pathway analysis found that in the treatment of TIF by HJT,the metabolic pathway is the most important pathway,and GAD1,SPHK1,P4HA2,AKR1B1 and PTGES among the 25 core genes are involved in the metabolic pathway.2.In the cell model experiments induced in vitro,we verified that TGF-β1 would have corresponding effects on the expression of AKR1B1,GAD1,P4HA2,SPHK1 and PTGES in HK-2 cells,and HJT can significantly restore TGF-β1-induced SPHK1 expression in HK-2 cells.The results of wound healing assay and transwell assay showed that HJT inhibited the abnormal migration of HK-2 cells induced by TGF-β1.The results of immunofluorescence experiments showed that TGF-β1 up-regulated the expression of Vimentin and down-regulated the expression of Cytokeratin-18.In contrast,the expressions of Vimentin and Cytokeratin-18 were reversed after HJT intervention.Conclusions: This study comprehensively illuminated the active compounds,potential targets,and molecular mechanism of HJT against TIF.HJT treatment of TIF may reverse EMT caused by TGF-β1 by targeting SPHK1.