The Mechanism Of Poricoic Aicd ZI On Renal Fibrosis Through Inhibiting Matrix Metalloproteinase-13 To Ameliorate Epithelialmesenchymal Transition

Background:Chronic kidney disease(CKD)is a clinical syndrome with continuous changes in kidney structure,function or both.At present,CKD has constituted a significant danger to healthcare in worldwide.Renal fibrosis is an important outcome in the process of CKD.For a long time,epithelial-mesenchymal transition(EMT)has been regarded as a key mechanism of renal fibrosis.Moreover,matrix metalloproteinases(MMPs)can stimulate the development of renal fibrosis by mediating EMT process in renal tubular epithelial cells.The surface layer of Poria cocos(Schw.)Wolf(SLPC)is a damp-clearing Chinese medicine,whose main chemical component is tetracyclic triterpenoids,and has anti-renal fibrosis pharmacological activity.This study revealed the mechanism of PZI on renal fibrosis through inhibiting MMP-13 to ameliorate EMT.Objective:To explore the effect and mechanism of the active ingredients,Poricoic acid ZI(PZI),Poricoic acid ZJ(PZJ),Poricoic acid ZK(PZK)and Poricoic acid ZL(PZL),that were isolated from SLPC,on renal fibrosis by inhibiting the expression of MMP-7,MMP-9 and MMP-13 to mediate EMT process,and to offer reference theory and experimental basis to take reasonable advantage of SLPC and the research of anti-renal fibrosis drugs.Methods:1.We used molecular docking simulation to analyze the interaction between tetracyclic triterpenoids extracted and separated from ethyl acetate fraction of SLPC with MMP-7,MMP-9 and MMP-13,and preliminarily screen the active ingredients against renal fibrosis in SLPC.2.We established TGF-β1-induced HK-2,NRK-52 E and NRK-49 F cell models to investigate the inhibitory effects of PZI,PZJ,PZK and PZL on the expression of MMP-7,MMP-9,MMP-13 and EMT-related proteins by Western blotting analysis,cell immunofluorescence staining analysis,si RNA interference and MMP-13 activity assay,and to explore the effect and mechanism of the four new poricoic acids on renal fibrosis.3.We established a unilateral ureteral obstruction(UUO)mice model.The ameliorative effects of PZI on renal fibrosis in UUO mice were evaluated through certain pathological tissue staining methods,including HE staining,Masson’s trichrome staining,and immunohistochemical staining.The selective inhibitory effect of PZI on the expression of MMP-13 and EMT-related proteins was further investigated via Western blotting analysis,MMP-13 activity assay and MMP-13 sh RNA.Results:1.Molecular docking results demonstrated that PG and PZD could bind to the active sites of MMP-7,MMP-9,MMP-13,and PZI,PZJ,PZK,PZL could bind to the active sites of MMP-9,MMP-13.Among them,four new poricoic acids had better affinity with MMP-13.2.In TGF-β1-induced HK-2,NRK-52 E and NRK-49 F cells,PZI,PZJ,PZK,and PZL could significantly down-regulate the expression of MMP-7,MMP-9 and MMP-13,and affect the expression of EMT-related proteins,such as down-regulate Collagen I,Collagen III,Fibronectin,α-SMA,Vimentin and up-regulate E-Cadherin,among which PZI had the strongest inhibitory effect on MMP-13 and EMT-related proteins.In MMP-13 si RNA transfection experiment,the inhibition of PZI was partially weakened,indicating that PZI can improve renal fibrosis by selectively inhibiting the expression of MMP-13 to regulate EMT process.3.In UUO mice model,PZI can significantly reduce the pathological changes of renal tissue fibrosis and selectively inhibit the expression of MMP-13 and EMT-related proteins,that is associated with the in vitro cell experiment results.Conclusion:This study revealed that four new poricoic acids,PZI,PZJ,PZK,and PZL,can regulate EMT process through inhibiting the expression of MMP-7,MMP-9 and MMP-13 to ameliorate renal fibrosis.It further revealed the selective inhibitory effect of PZI to MMP-13,which is a pharmic candidate for renal fibrosis treatment.