Inhibitory Effect Of Stevioside On Mouse Renal Fibrosis And Its Potential Mechanisms

Objective:To investigate the inhibitory effect of stevioside on mouse renal fibrosis and its potential mechanisms.Methods:Male KM mice were randomly divided into sham operation group,unilateral ureteral obstruction(UUO)model group,stevioside 50 and 100 mg/kg treatment group,and positive drug spironolactone 20 mg/kg group.The drug-treated mice were intragastrically administered with stevioside or spironolactone 0.2 ml/10 g body weight daily,and the mice in the sham operation and UUO model groups were intragastrically treated with an equivalent volume of double-distilled water.After 14 days of administration,kidney and blood were collected,and biochemical indicators were measured,including serum creatinine(SCr),urea nitrogen(BUN),renal hydroxyproline(Hyp),and renal glutathione peroxidase(GSH-Px).In addition,HE staining and VG staining were used to observe the pathological changes of renal tissues.The Western blot was used to determine the expression levels of renal α-SMA,collagen Ⅰ,and collagen Ⅲproteins for evaluating the effect of stevioside on renal fibrosis,and determine the expressions of renal PPARγ,NF-κB p65,TGF-β1,STAT3,p-STAT3,Smad2/3,p-Smad2/3,Smad4 and Smad7 proteins for exploring the potential mechanisms of stevioside action.In vitro,the LPS-induced rat renal tubular epithelial cells(NRK-52E)and TGF-β1-stimulated rat renal fibroblasts(NRK-49F)were used to further observe the effects of stevioside on epithelial-mesenchymal transition(EMT)and fibroblast proliferation and differentiation.For LPS-induced NRK-52E cell model,the expressions of E-cadherin,Vimentin,α-SMA,PPARy,NF-κB p65,STAT3,p-STAT3,TGF-β1,Smad2/3 and p-Smad2/3 proteins were detected by Western blot method.To confirm the activation of PPARy by stevioside,GW9662,a PPARy inhibitor,was used to pre-treat NRK-52E cells for 2 h and to observe whether the inhibitory effect of stevioside on EMT was affected.For TGF-β1-stimulated NRK-49F cell model,the expressions of α-SMA,collagen Ⅰ,collagenⅢ,STAT3,p-STAT3,Smad2/3,and p-Smad2/3 proteins were detected by Western blot method.Results:In vivo,the results showed that after continuous treatment of stevioside 50 and 100 mg/kg for 14 days,the levels of BUN and renal Hyp were decreased,the level of renal GSH-Px was increased,and the renal tubular atrophy,interstitial fibrosis,and collagen area ratio were also decreased,especially in stevioside 100 mg/kg group(P<0.01).Western blot results showed that stevioside could reduce the expressions of renal α-SMA,collagen Ⅰ,collagen Ⅲ,NF-κB p65,TGF-β1,STAT3,p-STAT3,Smad2/3,and p-Smad2/3 proteins(P<0.05 or P<0.01),increase the expressions of PPARy and Smad7 proteins(P<0.05 or P<0.01).In vitro,the results showed that in LPS-induced NRK-52E cells,stevioside treatment could significantly up-regulate the E-cadherin and PPARy protein expressions,and down-regulate the Vimentin,α-SMA,NF-βB p65,p-STAT3,TGF-β1,Smad2/3,and p-Smad2/3 protein expressions(P<0.05 or P<0.01).After pretrement of LPS-induced NRK-52E cells with the PPARγ inhibitor GW9662 for 2 h，these effects of stevioside were reduced or disappeared(P<0.05 or P<0.01).In TGF-β1-stimulated NRK-49F cells,stevioside treatment could significantly reduce the expressions of α-SMA,collagen Ⅰ,collagen Ⅲ,p-STAT3,Smad2/3,and p-Smad2/3 proteins(P<0.05 or P<0.01).Conclusion:Stevioside possessed an inhibitory effect on mouse renal fibrosis,and its mechanisms might be associated with the PPARy activation and antioxidative effect,which might subsequently inhibit the NF-κB-mediated STAT3 and TGF-β1 expressions and block the Smad signaling pathway.And,stevioside could directly inhibit the phosphorylations of STAT3 and Smad2/3,which might also be one of its anti-fibrotic mechanisms.