发酵红参总皂苷对高糖诱导大鼠肾小管上皮细胞间充质转化的抑制作用及其机制

Inhibitory effect of fermented red ginseng total saponins high glucose-induced renal tubular cell epithelial-mesenchymal transition and its mechanism

目的:探讨发酵红参总皂苷(FRGTS)对高糖诱导下肾小管上皮细胞发生上皮细胞-间充质转化(EMT)的抑制作用,并阐明其作用机制。方法:将大鼠肾小管上皮NRK-52E细胞分为正常对照组(5.5 mmol·L^(-1)D-葡萄糖)、高糖组(30.0 mmol·L^(-1)D-葡萄糖)、沉默信息调节因子1(SIRT1)抑制剂EX527组(30.0 mmol·L^(-1)D-葡萄糖+10.0μmol·L^(-1).EX527)、 FRGTS组(30.0 mmol·L^(-1)D-葡萄糖+25 mg·L^(-1)FRGTS)和EX527+FRGTS组(30.0 mmol·L^(-1)D-葡萄糖+10μmol·L^(-1)EX527+25 mg·L^(-1)FRGTS)。采用免疫荧光法检测各组细胞中E-钙黏蛋白(E-cadherin)和α-平滑肌肌动蛋白(α-SMA)蛋白表达水平,实时荧光定量PCR (RT-qPCR)法检测各组细胞中E-cadherin、α-SMA和SIRT1 mRNA表达水平,ELISA法检测培养上清液中Ⅰ型胶原(ColⅠ)水平,Western blotting法检测各组细胞中SIRT1、转化生长因子β1 (TGF-β1)和Smad3蛋白表达水平。结结果果:高糖培养48 h后,与正常对照组比较,高糖组NRK-52E细胞中E-cadherin蛋白表达水平和mRNA表达水平降低(P<0.01),α-SMA蛋白表达水平和mRNA表达水平升高(P<0.01),NRK-52E细胞上清液中ColⅠ水平升高(P<0.01),SIRT1 mRNA和蛋白表达水平均明显降低(P<0.01),TGF-β1和Smad3蛋白表达水平升高(P<0.01);与高糖组比较,FRGTS组NRK-52E细胞中E-cadherin mRNA表达水平升高(P<0.01),α-SMA mRNA表达水平降低(P<0.05),NRK-52E细胞上清液中ColⅠ水平降低(P<0.05), SIRT1 mRNA和蛋白表达水平均升高(P<0.05或P<0.01),TGF-β1和Smad3蛋白表达水平降低(P<0.05或P<0.01);与FRGTS组比较,EX527+FRGTS组NRK-52E细胞中E-cadherin mRNA表达水平明显降低(P<0.01),α-SMA mRNA表达水平明显升高(P<0.05), NRK-52E细胞上清液中ColⅠ水平升高(P<0.05), SIRT1 mRNA和蛋白表达水平明显降低(P<0.01), TGF-β 1和Smad3蛋白表达水平明显升高(P<0.01)。结结论论:FRGTS可通过上调SIRT1表达,进而抑制TGF-β1/Smad信号通路,有效抑制肾小管上皮细胞发生EMT。

Objective:To investigate the inhibitory effect of fermented red ginseng total saponins(FRGTS)on the epithelial-mesenchymal transition(EMT)of renal tubular epithelial cells induced by high glucose,and to clarify its mechanisms. Methods:The rat renal tubular epithelial cells NRK-52E were divided into normal control group(5. 5 mmol·L^(-1)D-glucose),high glucose group(30. 0 mmol·L^(-1)D-glucose),silent information regulator 1(SIRT1)inhibitor EX527 group(30. 0 mmol·L^(-1)D-glucose+10 μmol·L^(-1)EX527),FRGTS group(30. 0 mmol·L^(-1)D-glucose+25 mg·L^(-1)FRGTS)and EX527+FRGTS group(30. 0 mmol·L^(-1)Immunofluorescence method was used to detect the expression levels of E-cadherin and α-smooth muscle actin(α-SMA) protein in the cells in various groups. Real-time fluorescence quantitative PCR(RTqPCR) method was used to detect the expression levels of E-cadherin,α-SMA and SIRT1 mRNA,ELISA method was used to detect the levels of collagen type Ⅰ (Col Ⅰ) in culture supernatant,and Western blotting method was used to detect the expression levels of SIRT1,transforming growth factor-β1(TGF-β1)and Smad3 proteins in the cells in various groups. Results:After 48 h of high glucose culture,compared with normal control group,the expression levels of E-cadherin mRNA and protein in the NRK-52E cells in high glucose group were decreased(P<0. 01),the expression levels of α-SMA mRNA and protein were increased(P<0. 01),the level of Col Ⅰ in culture supernatant was increased(P<0. 01),the expression levels of SIRT1 mRNA and protein were significantly decreased(P<0. 01),and the expression levels of TGF-β1 and Smad3 proteins were increased(P<0. 01). Compared with high glucose group,the expression level of E-cadherin mRNA in the NRK-52E cells in FRGTS group was increased(P<0. 01),the expression level of α-SMA mRNA was decreased(P<0. 05),the level of Col Ⅰ in culture supernatant was decreased(P<0. 05),the expression levels of SIRT1 mRNA and protein were increased(P<0. 05 or P<0. 01),and the expression levels of TGF-β1 and Smad3 proteins were decreased(P<0. 05 or P<0. 01). Compared with FRGTS group,the expression level of E-cadherin mRNA in the cells in EX527+FRGTS group was significantly decreased(P<0. 01),the expression level of α-SMA mRNA was increased(P<0. 05),the level of Col Ⅰ in culture supernatant was increased(P<0. 05),the expression levels of SIRT1 mRNA and protein were significantly deceased(P<0. 01),and the expression levels of TGF-β1 and Smad3 were significantly increased(P<0. 01). Conclusion:FRGTS can upregulate the expression of SIRT1 and then inhibits TGF-β1/Smad signaling pathway,which effectively inhibits the EMT of renal tubular epithelial cells.