Department of Urology, Peking University First Hospital and Institute of Urology, Peking University, National Urological Cancer Center, Beijing, 100034, China.
Chin J Integr Med. 2024 Jul;30(7):600-607. doi: 10.1007/s11655-023-3564-9. Epub 2023 Sep 27.
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.
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.
Compared with the control group, TGF-β 1 significantly inhibited the proliferation of NRK-49F cells, blocked the G/G 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).
TGF-β 1-induced fibrosis in NRK-49F cells. Curcumin promoted ADAMTS18 expression, reduced ECM accumulation, and alleviated RIF progression by inhibiting ADAMTS18 methylation.
通过调节 ADAM 金属肽酶与血小板反应蛋白 1 型基序 18（ADAMTS18）甲基化，探讨姜黄素影响肾间质纤维化（RIF）进展的分子机制。
用转化生长因子-β 1（TGF-β 1）诱导 NRK-49F 细胞 RIF 模型。分别采用细胞计数试剂盒-8、流式细胞术和 Western blot 法分析不同浓度（0、10、20 和 30 μmol/L）姜黄素对细胞增殖、细胞周期、细胞凋亡以及周期蛋白 D1 表达的影响。采用甲基化特异性聚合酶链反应（PCR）检测 ADAMTS18 甲基化水平。采用实时 PCR（RT-PCR）和 Western blot 法分别检测 ADAMTS18、纤连蛋白（FN）、I 型胶原（Col- I）和α-平滑肌肌动蛋白（α-SMA）mRNA 和蛋白表达。同时，用 50 mmol/L 5-氮杂-2'-脱氧胞苷（5-aza-dC，去甲基化剂）处理细胞 72 h。通过免疫化学染色和 Western blot 法评价姜黄素对细胞外基质（ECM）沉积的影响。用 ADAMTS18 小干扰 RNA 转染 NRK-49F 细胞，并分为正常对照组、ADAMTS18 敲除（KO）组和 ADAMTS18-KO+30 μmol/L 姜黄素组，评价姜黄素是否能逆转 ADAMTS18 敲低对 RIF 的作用。
与对照组相比，TGF-β 1 显著抑制 NRK-49F 细胞增殖，阻滞 G1/G0 期，促进细胞凋亡，抑制周期蛋白 D1 表达（P<0.01）。在不同浓度的姜黄素中，30 μmol/L 姜黄素能显著逆转这些过程（P<0.01）。免疫化学染色和 Western blot 结果表明，姜黄素显著抑制 FN、Col- I 和α-SMA 沉积（P<0.01）。姜黄素和 5-aza-dC 具有协同作用，促进 ADAMTS18 表达，去除 ADAMTS18 甲基化，减少 ECM 沉积。ADAMTS18 敲低促进 ECM 积累，姜黄素逆转了这一过程（P<0.01）。
TGF-β 1 诱导 NRK-49F 细胞纤维化。姜黄素通过抑制 ADAMTS18 甲基化促进 ADAMTS18 表达，减少 ECM 堆积，减轻 RIF 进展。
