Curcumin Interferes with TGF- β 1-Induced Fibrosis in NRK-49F Cells by Reversing ADAMTS18 Gene Methylation
Chinese Journal of Integrative Medicine(2023)
摘要
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 β 1 (TGF- β 1). 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 G 1 /G 0 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.
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关键词
fibrosis
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