Involvement Of The JAK/STAT/Klf4 Signaling Pathway Against Renal Proteinuria Effects Of Ling Zhu Tu Si Zi Pill

Objective:1.This study aimed to explore the core targets and signaling pathways of Ling Zhu Tu Si Zi Pill(LZTSTP)against renal proteinuria through network pharmacology.2.To observe the protective effect of isorhamnetin,the main active compounds of LZTSTP,on adriamycin-induced podocyte injury and to verify the effect of JAK/STAT/Klf4 pathway in vitro and in vivo.Methods:1.TCMSP was used to obtain the compounds and targets of LZTSZP.The targets of renal proteinuria were collected from OMIM and Gene Cards databases.Subsequently,built compound-target-disease network with the help of Cytoscape.String database were employed for constructing protein interaction network.GO and KEGG enrichment analysis were performed by DAVID database.With the study of Network Pharmacology,the compounds of LZTSZP and the targets of renal proteinuria were found,While the potential pathways were identified.2.CCK8 assay was used to detect cell viability to MPC-5.Levels of TNF-α and MCP-1 were measured using ELISA kits.HE,Masson and PAS staining were employed to observe the morphology of kidney.Klf4 and Nephrin m RNA expression was detected by RT-PCR;Western-Blot was applied to detect the protein expression of pJAK2,pSTAT3,pNF-κB,MCP-1,TGF-β,Nephrin,WT1,F-actin and Klf4.Results: Based on network pharmacology,138 active compounds with the effect of improving renal proteinuria were obtained in LZTSZP.There were 58 significantly enriched pathways and 93 biological processes,mainly including NF-κB pathway,JAK/STAT pathway,TNF pathway,redox and nuclear receptor activity.In vitro,Isorhamnetin improved the adriamycin-induced injury of MPC-5.In vivo,Isorhamnetin reduced adriamycin-induced the 24-hour urine protein content,raised the content of Alb and improved the pathological structure of kidney.Increased the m RNA expression of Klf4 and nephrin.Moreover,Isorhamnetin were able to not only inhibit TNF-α,MCP-1expression but also down-regulated the expressions of pJAK,pSTAT3,pNF-κB,MCP-1,TGF-β and up-regulated Nephrin,WT1,F-actin and Klf4 expressions.Conclusion:.1.Isorhamnetin is the core ingredient of LZTSZP which can effectively improve renal proteinuria.2.Isorhamnetin,can protect podocytes from adriamycin-induced injury by modulating the JAK/STAT/Klf4 pathway.Isorhamnetin could improve podocyte injury and is worthy of further development and application.