首页|Curcumin Interferes with TGF-β 1-lnduced Fibrosis in NRK-49F Cells by Reversing ADAMTS18 Gene Methylation
Curcumin Interferes with TGF-β 1-lnduced Fibrosis in NRK-49F Cells by Reversing ADAMTS18 Gene Methylation
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Objective:To explore the molecular mechanism by which curcumin affects renal interstitial fibrosis(RIF)progression by regulating ADAM metallopeptidase with thrombospondin type 1 motif 18(ADAMTS18)methylation.Methods:NRK-49F cells RIF model were induced with transforming growth factor β1(TGF-β1).Effects of different concentrations of curcumin(0,10,20,and 30 µmol/L)on cell proliferation,cell cycle,cell apoptosis as well as cyclin D1 expression were analyzed by cell counting kit-8,flow cytometry and Western blot,respectively.ADAMTS18 methylation levels were determined by methylation-specific polymerase chain reaction.ADAMTS18,fibronectin(FN),type Ⅰ collagen(Col-Ⅰ)and alpha-smooth muscle actin(α-SMA)mRNA and protein expressions were analyzed by real-time PCR(RT-PCR)and Western blot,respectively.Meanwhile,cells were treated with 50 mmol/L 5-aza-2'-deoxycytidine(5-aza-dC,demethylation agent)for 72 h.Effect of curcumin on extracellular matrix(ECM)deposition was evaluated by immunochemical staining and Western blot.NRK-49F cells were transfected with ADAMTS18 small interfering RNA and grouped into a normal control,ADAMTS18-knock-out(KO),and ADAMTS18-KO+30 µmol/L curcumin groups,and whether curcumin can reverse the effect of ADAMTS18 knockdown on RIF was evaluated.Results:Compared with the control group,TGF-β 1 significantly inhibited the proliferation of NRK-49F cells,blocked the G1/G0 phase,promoted cell apoptosis and inhibited cyclin D1 expression(P＜0.01).Among the different concentrations of curcumin,30 μmol/L curcumin significantly reversed these processes(P＜0.01).Immunochemical staining and Western blot results showed that curcumin significantly inhibited the deposition of FN,Col-Ⅰ and α-SMA(P＜0.01).Curcumin and 5-zaz-dC had synergistic effects,promoting ADAMTS18 expression,removing ADAMTS18 methylation,and reducing ECM deposition.ADAMTS18 knockdown promoted ECM accumulation,and curcumin reversed this process(P＜0.01).Conclusion:TGF-β 1 induced fibrosis in NRK-49F cells.Curcumin promoted ADAMTS18 expression,reduced ECM accumulation,and alleviated RIF progression by inhibiting ADAMTS18 methylation.
Department of Urology,Peking University First Hospital and Institute of Urology,Peking University,National Urological Cancer Center,Beijing(100034),China
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