预先给予白藜芦醇苷对TGF-β1诱导生长小鼠肾足细胞株MPC5增殖的影响及其机制

Effect of pretreatment of polydatin on cell proliferation of renal podocytes MPC5 cells of mice induced by TGF-β1

目的观察预先给予白藜芦醇苷对TGF-β1诱导生长小鼠肾足细胞株MPC5增殖的影响,并探讨其可能作用机制.方法将取MPC5细胞分为A、B、C、D组四组,A、B、C组分别加入100μmol/L白藜芦醇苷预处理24 h.预处理后,A组细胞加入100μmol/L白藜芦醇苷培养2 h,再加入10 ng/mL TGF-β1培养24 h;B组加入30μmol/L的miR-21抑制剂培养6 h,再加入100μmol/L白藜芦醇苷培养2 h,再加入10 ng/mL TGF-β1培养24 h;C组加入10 ng/mLTGF-β1培养24 h;D组为空白对照,加入普通培养基.培养结束时,MTT法观察各组细胞增殖情况(光密度OD值表示增殖能力),分光光度计测算各组细胞氧化应激指标超氧化物歧化酶(SOD)、丙二醛(MDA)含量,2′,7′-二氯二氢荧光素二乙酸酯负载法测算活性氧簇(ROS)含量,Western blotting法检测各组miR-21及NOD样受体热蛋白结构域3(NLRP3)、caspase-1、白细胞介素1β(IL-1β)蛋白.结果A、B、C、D组细胞OD值分别为14.73±1.60、12.70±1.13、19.31±1.79、12.62±1.26,与D组比较,C组细胞OD值升高(P<0.05);与C组比较,A组细胞OD值降低(P<0.05);与B组比较A组细胞OD值升高(P<0.05).培养24 h时与D组比较,C组细胞MDA、ROS、miR-21、NLRP3、caspase-1及IL-1β相对表达量均升高,SOD相对表达量降低(P均<0.05);与C组比较,A组细胞MDA、ROS、miR-21、NLRP3、caspase-1及IL-1β相对表达量均降低,SOD相对表达量升高(P均<0.05);与B组比较,A组细胞MDA、ROS、miR-21、NLRP3、caspase-1及IL-1β相对表达量升高,SOD相对表达量降低(P均<0.05).结论白藜芦醇苷可抑制TGF-β1诱导的MPC5细胞增殖.其可能作用机制为调控miR-21相关信号通路,增加SOD含量,降低胞内MDA、ROS含量,抑制细胞NLRP3、caspase-1及IL-1β蛋白表达.

Objective To observe the effect of polydatin on the proliferation of renal cell line MPC5 induced by TGFp1 in mice,and to explore its possible mechanism.Methods MPC5 cells were divided into four groups:groups A,B,C,and D.The cells in the groups A,B and C were pretreated with polydatin 100μmol/L for 24 h.After pretreatment,cells in the group A were cultured with polydatin for 2 h,and two hours later,10 ng/mL TGF-β1]were added in cells for 24 h.In the group B,30μmol/L of the inhibitor of miR-21 was added,and six hours later,100μmol/L polydatin were added,for 2 h,followed by addition of TGF-β1]for culturing 24 h.In the group C,10 ng/mL TGF-β1]were added for culturing 24 h.However,the group D was regarded as the blank control group,and it was added the ordinary culture medium.At the end of the training,the cell proliferation[optical density(OD value]was determined by MTT,the cell oxidative stress indicators superoxide dismutase(SOD)and malondialdehyde(MDA)content was detected by spectrophotometer,the reactive oxygen species(ROS)content was detected by 2′,7′-dichloro dihydrogen fluorescein diacetate ester load method,and the miR-21 and Nod-like receptor pyrin containing 3(NLRP3),caspase-1,interleukin 10(IL-10)protein were determined by Western blotting.Results The OD values in the groups A,B,C and D were 14.73±1.60,12.70±1.13,19.31±1.79 and 12.62±1.26,respectively.Compared with group D,the OD value increased in the group C(P<0.05);compared with group C,the OD value decreased in the group A(P<0.05);compared with group B,the OD value of the group A increased(P<0.05).At 24 h after culture,compared with group D,the relative expression levels of MDA,ROS,miR-21,NLRP3,caspase-1 and IL-10 in the group C all increased,while the relative expression level of SOD decreased(all P<0.05).Compared with the group C,the relative expression levels of MDA,ROS,miR-21,NLRP3,caspase-1 and IL-10 in the group A all decreased,while the relative expression level of SOD increased(all P<0.05).Compared with the group B,the relative expression levels of MDA,ROS,miR-21,NLRP3,caspase-1 and IL-10 in the group A increased,while the relative expression of SOD decreased(all P<0.05).Conclusions Polydatin can inhibit the proliferation of MPC5 cells induced by TGF-β1].The possible mechanism is that polydatin regulates miR-21-related signaling pathways,further increases SOD content,reduces intracellular MDA and ROS content,and inhibits the expression of NLRP3,caspase-1 and IL-10 proteins.