The Effects And Mechanism Of Poricoic Acid A On Renal Tubulointerstitial Fibrosis

Poria cocos(Fuling in Chinese)is the dried sclerotia of Poria cocos(Schw.)Wolf and commonly used to treat edema and insomnia.The surface layer of P.cocos(Fulingpi in Chinese)has similar effects to Fuling but exhibits stronger effects against edema than Fuling.Tetracyclic triterpenoids and polysaccharides are both major and active chemical components of Fuling,while tetracyclic triterpenoids are both major and active chemical components of Fulingpi.Wulingsan that is a classical Chinese compound prescription and contains Fuling are commonly used to treat chronic kidney disease(CKD)clinically.However their active chemical constituents and mechanisms remain unclear,which hinders further clinical applicatioon and durg development.Tubulointerstitial fibrosis is the common characteristic of CKD and the suppression of tubulointerstitial fibrosis is beneficial for progressive CKD treatment.Here,we isolated and identified poricoic acid A(PAA)that is the major components of Fuling and Fulingpi and investigated its effects and mechanisms of PAA for treating tubulointerstitial fibrosis.Additionally,we identified biomarkers that involved in CKD progression and investigated the intervention effects of wulingsan on these biomarkers.Based on the biomarkers,we identified the therapeutic target for CKD treatment and investigated the effects of PAA on the therapeutic target.Objective:1.The present study investigates the role of PAA in oxidative stress and inflammation,fibroblast activation,epithelial-mesenchymal transition(EMT)and extracellular matrix(ECM)accumulation during fibrogenesis,which will demonstrate the effects and mechanisms of PAA against CKD and tubulointerstitial fibrosis.2.We identify a panel of biomarkers that relate to clinical CKD progression and further investigate the intervention effects of wulingsan on these biomarkers,which will elucidate the biochemical mechanisms of wulingsan for treating CKD.3.We identify the major mediator involves in CKD progression and explore the PAA effects on the mediator that could be identified as the therapeutic target for PAA to inhibit tubulointerstitial fibrosis,which will provide experimental data and theoretical basis for research and development of drug against tubulointerstitial fibrosis Methods:After pulverized,Fulingpi was extracted by ethanol and ethyl acetate and isolated by MCI and reversed-phase chromatographic separation,and then PAA was obtained.We investigate the effects and mechanisms of PAA against tubulointerstitial fibrosis in several animal models including 5/6 nephrectomy(Nx),renal ischemia-reperfusion injury(IRI),unilateral ureteral obstruction(UUO)and adenine induced CKD models,and in vitro experiments including hypoxia/reoxygenation(H/R)-induced NRK-52 E and HK-2 cells,TGF-β1-induced HK-2,NRK-52 E and NRK-49 F cells by using PCR,western blotting,immunofluorescent,immunohistochemical staining,co-immunoprecipitation,luciferase reporter,chromatin immunoprecipitation methods.By using the combined approach of metabolomics and bioinformatics,the biomarkers of CKD progression were identified and the intervention effects of wulingsan on biomarkers was assessed.Results:1.The isolation and structural identification of PAA.PAA was isolated from Fulingpi and the chemical structure of PAA was confirmed by using nuclear magnetic resonance and mass spectrometry.2.PAA suppressed fibroblast activation through enhancing AMPK activity and further selectively inhibiting Smad3 phosphorylation.PAA promoted AMPK activity and further suppressed fibroblast activation and ECM remodelling induced by Nx and UUO.PAA inhibited tubulointerstitial fibrosis through selectively suppressing Smad3 phosphorylation via blocking the interaction of Smad3 with TGFβRI and SARA,which indicated that PAA attenuated tubulointerstitial fibrosis through activating AMPK.PAA hardly affected the protein expression of p-Smad2,Smad2,Smad4 and Smad7.3.PAA inhibited oxidative stress and inflammation through regulating Gas6/Axl-NF-κB/Nrf2 signaling pathway.PAA,melatonin and their combined intervetnion significantly reduced the increased levels of creatinine and urea,attenuated epithelium cell injury,alleviated tubulointerstitial fibrosis and ameliorated AKI-to-CKD transition induced by renal IRI.PAA and melatonin could modulate Gas6/Axl signaling pathway in different stages,and further inhibit IκB/NF-κB signaling pathway activation and Keap1/Nrf2 signaling pathway inactivation.4.PAA inhibited EMT and ECM accumulation through suppressing TGF-β/Smad,Wnt/β-catenin signaling pathways and their interaction.PAA selectively inhibited Smad3 phosphorylation but hardly affected other Smad proteins and Smad-independent pathway in kidney tissues of renal IRI-induced rats,while melatonin not only inhibited the phosphorylation of Smad2 and Smad3,enhanced Smad7 expression,and also inhibited the phosphorylation of ERK,p38 and PI3 K.Smad3 was required for PAA against tubulointerstitial fibrosis.PAA and melatonin also suppressed Wnt/β-catenin signaling pathway and profibrotic downstream gene expression.In addition,melatonin inhibited the interaction of Smad2 and Smad3 with β-catenin,while PAA only inhibited the interaction of Smad3 with β-catenin.5.The five biomarkers including 5-MTP distinguished progressive CKD and assessed the effects of wulingsan.Based on metabolomics and bioinformatic analysis of large-scale clinical serum samples,five metabolites including 5-MTP were identified as biomarkers of progressive CKD.Serum 5-MTP level gradually decreased during CKD progression.Further studies revealed the high sensitivity and specificity of five biomarkers.The five biomarkers also could evaluate the clinical effects of wulingsan,which illuminated partial pathogenesis of CKD and therapeutic mechanisms of wulingsan in the biochemical level.6.TPH-1 had the potential as the therapeutic target for CKD treatment.Both 5-MTP and its regulatory enzyme TPH-1 ameliorated renal function and tubulointerstitial fibrosis.TPH-1 could be developed as therapeutic target for CKD treatment,while PAA attenuated tubulointerstitial fibrosis through regulating TPH-1 expression.5-MTP treatment suppressed tubulointerstitial fibrosis through modulating IκB/NF-κB and Keap1/Nrf2 signaling pathways.TPH-1 is the key enzyme of 5-MTP biosynthesis,and TPH-1 expression gradually reduced during CKD progression.TPH-1 overexpression enhanced 5-MTP level,and inhibited tubulointerstitial fibrosis by regulating IκB/NF-κB,Keap1/Nrf2 and Wnt/β-catenin signaling pathways.TPH-1 deficiency aggravated inflammation and fibrosis while TPH-1 overexpression inhibited inflammation and fibrosis.PAA could inhibit tubulointerstitial fibrosis by enhancing TPH-1 expression.Conclusion:The present study investigated the molecular and biochemical mechanisms of PAA for treating CKD and tubulointerstitial fibrosis,and we found that PAA could inhibit oxidative stress and inflammation,fibroblast activation,EMT and ECM accumulation during fibrogenesis through modulating Gas6/Axl,IκB/NF-κB,Keap1/Nrf2,AMPK,TGF-β/Smad and Wnt/β-catenin signaling pathways.TPH-1 had the potential to be the therapeutic target for treating CKD and tubulointerstitial fibrosis,and PAA inhibited tubulointerstitial fibrosis through promoting TPH-1 expression.The present study illuminated the molecular and biochemical mechanisms of PAA against CKD and tubulointerstitial fibrosis,which not only provides theoretical basis and experimental data for drug research and development of PAA,and also reveals the active chemical components and mechanisms of Fuling,Fulingpi and Wulingsan that provide evidence for their clinical application.