Effect Of Quercetin On Cyclosporine-induced Epithelial-mesenchymal Transition And TGF-β1/smads Signaling Molecules In Renal Tubular Epithelial Cells

Objective:To evaluate the effect of quercetin on cyclosporine-inducedepithelial-mesenchymal transition(EMT) and TGF-β1/Smads signaling molecules in renaltubular epithelial cells.Methods:1.Normal rat kidney tubular epithelial cells（NRK-52E）was culture in vivo,anddivide into nomal control group and different drugs(CsA,TGF-β1blockers and quercetin)treatment groups. Multiplication-inhibiting of cells treated by different drugs in the differentconcentrations was evaluated by CCK8assay.2. The culturing cells was divided into6groups:normal control group(A group,medium containing only fetal bovine serum), cyclosporine Agroup (B group, medium containing CsA), quercetin group(C group, medium containingquercetin), blocks group (D group, pretreatment of medium containing TGF-β1blockersSB431542for24h, plus CsA posttreatment), quercetin intervention group1(E group,pretreatment of medium containing CsA for24h, plus quercetin posttreatment), quercetinintervention group2(F group, pretreatment of medium containing CsA for24h, plusSB431542and quercetin posttreatment). Cells of aforesaid groups was cultured for24hours.The cells gene and protein levels of fibroblast specific protein (FSP-1), α-smooth muscleactin(α-SMA),TGF-β1,Smad2and Smad7in groups were evaluated with real-timequantitative reverse transcription polymerase chain reaction (RT-PCR),immunohistochemistry or Western-blot method.Results:1.The cells viability was obviously decreased in pretreatment with CsA, butsignificantly increased in posttreatment with different concentration of SB431542andquercetin for24h(P<0.05). Their optimal concentrations were10μmol/L and40μmol/L,respectively. The cells viability was higher in posttreatment of quercetin than SB431542withoptimal concentrations(P<0.05).2.FSP-1: FSP-1immumostaining was mainly detected in endochylema. Noexpressional difference of FSP-1mRNA and protein was found between C and A groups (P>0.05). FSP-1mRNA and protein expression in B,E and F groups were obviously increasedas compared with A group (P<0.05),especially higher in B than E, F groups; The level ofFSP-1mRNA and protein expression in D group was obviously lower than B,Eand F groups,but was higher than that in A,C groups (P<0.05); The expression of FSP-1mRNA and proteinwas higher in F than E groups(P<0.05).3.α-SMA: α-SMA immumostaining was mainly detected in endochylema. Noexpressional difference of α-SMA mRNA and protein was found between C and A groups(P>0.05). α-SMA mRNA and protein expression in B,E and F groups were obviouslyincreased as compared with A group (P<0.05),especially higher in B than E, F groups; Thelevel of α-SMA mRNA and protein expression in D group was obviously lower than B,Eand Fgroups, but was higher than that in A,C groups (P<0.05); The level of α-SMA mRNA in Fgroup was not statistical differences as compared with E group,but protein expression washigher in F than E group (P<0.05).4. TGF-β1: TGF-β1immumostaining was mainly detected in endochylema. Noexpressional difference of TGF-β1mRNA and protein was found between C and A groups(P>0.05). TGF-β1mRNA and protein expression in B,E and F groups were obviouslyincreased as compared with A group (P<0.05),especially higher in B than E, F groups; Thelevel of TGF-β1mRNA and protein in D group was not expressional differences as comparedwith C group (P>0.05), but obviously lower than that in B,Eand F groups (P<0.05); TGF-β1protein expression in F group was not statistical differences as compared with E group(P>0.05),but the level of TGF-β1mRNA was higher in F than E group (P<0.05).5.Smad2: Smad2immumostaining was mainly detected in endochylema. Noexpressional difference of Smad2mRNA and protein was found between C and A groups(P>0.05). Smad2mRNA and protein expression in B,E and F groups were obviouslyincreased as compared with A group (P<0.05),especially higher in B than E,F groups; Thelevel of Smad2mRNA and protein in D group was not expressional differences as comparedwith C group (P>0.05), but obviously lower than that in B,Eand F groups (P<0.05); Smad2protein expression in F group was not statistical differences as compared with E group (P>0.05),but the level of Smad2mRNA was higher in F than E group (P<0.05).6. Smad7: Smad7immumostaining was mainly detected in endochylema. Noexpressional difference of Smad7mRNA and protein was found between C and A groups.Smad7mRNA and protein expression in B,D groups were obviously decreased as comparedwith A group (P<0.05), especially higher in B than D group; The level of Smad7mRNA andprotein expression in D group was lower than A,C groups(P<0.05);Smad7protein expressionin E, F groups was higher than A,B,C and D groups (P<0.05);The level of Smad7mRNA washigher in E,F than B groups,but lower than that in C,Dgroups (P<0.05); The expression ofS m a d7m R N A a n d p r o t e i n w a s h i g h e r i n E t h a n F g r o u p (P <0.05).Conclusions:1.Quercetin can effectively inhibit renal tubular EMT stimulated by CsA, andthis effect may be, at least in party, through regulating expression of TGF-β1/Smads signalingmolecules.2. TGF-β1/Smads signaling pathway is one of the most important mechanismsin renal tubular EMT stimulated by CsA.