肾茶黄酮对急性肾衰中肾小管上皮细胞保护作用的研究

Study on the Protective effect of Flavone of Clerodendranthus Spicatus on Renal Tubular Epithelial Cells in Acute Renal Failure

目的探究肾茶黄酮对急性肾衰中肾小管上皮细胞保护作用。方法甘油制备大鼠急性肾衰模型,检测成功后提取肾小管上皮细胞,分为模型组、(100、200、300、400)μg·mL^-1肾茶黄酮组,其中(100、200、300、400)μg·mL^-1肾茶黄酮组分别添加对应剂量肾茶黄酮,对照组未经处理的正常肾小管上皮细胞,对照组和模型组正常培养。分别在培养(0、3、6、12、24、36)h Cell Counting Kit-8(CCK-8)法检测细胞增殖能力;各组细胞处理24h流式细胞术检测细胞凋亡能力,超氧化物歧化酶(SOD)活性检测试剂盒、丙二醛(MDA)检测试剂盒分别检测SOD活性、MDA水平,蛋白免疫印迹(WB)检测细胞中半胱氨酸天冬氨酸蛋白酶(caspase)3、caspase9、B淋巴细胞瘤-2基因(BCL2)、Bcl-2相关X蛋白(BAX)、转化生长因子-β1(TGF-β1)、Sma和Mad相关蛋白3(Smad3)蛋白水平。结果与对照组相比,模型组在细胞培养(3、6、12、24、36)h OD450降低(P<0.05);与模型组相比,(200、300、400)μg·mL^-1肾茶黄酮组在细胞培养6h OD450升高(P<0.05);(300、400)μg·mL^-1肾茶黄酮组在细胞培养(12、24、36)h OD450升高(P<0.05)。各组细胞处理24h,与对照组相比,模型组SOD活性、细胞凋亡率,caspase3、caspase9、BAX、TGF-β1、Smad3蛋白水平升高(P<0.05),MDA活性、BCL2蛋白水平降低(P<0.05),随着剂量的升高,各组呈剂量依赖性变化。与模型组相比,(100、200、300、400)μg·mL^-1肾茶黄酮组细胞凋亡率,SOD活性,BCL2、TGF-β1、Smad3蛋白水平降低(P<0.05),MDA活性、caspase3、caspase9、BAX蛋白水平升高(P<0.05),呈剂量依赖效应。结论肾茶黄酮可促进急性肾衰中肾小管上皮细胞增殖、抑制凋亡,抑制氧化应激,抑制促凋亡蛋白的表达从而减缓急性肾衰肾小管上皮细胞的损伤。

Objective To explore the protective effect of flavone of clerodendranthus spicatus on renal tubular epithelial cells in acute renal failure.Methods The rat model of acute renal failure was prepared by glycerol.After successful detection,divided into model group,(100,200,300,400)μg/ml flavone of clerodendranthus spicatus group,(100,200,300,400)μg/ml flavone of clerodendranthus spicatus group were added with corresponding doses of theaflavones,the control group was treated normal renal tubular epithelial cells were cultured normally in the control and model groups.Cell proliferation ability was detected at(0,3,6,12,24,36)h Cell Counting Kit-8(CCK-8)method;cells in each group were treated for 24 h by flow cytometry to detect apoptosis and superoxidation Physical dismutase(SOD)activity detection kit and malondialdehyde(MDA)detection kit to detect SOD activity and MDA level;protein Western blot(WB)detection of caspase 3,caspase 9,B lymphocyte tumor-2 gene(BCL2),Bcl-2 related X protein(BAX),transforming growth factor-β1(TGF-β1),Sma and Mad related protein 3(Smad3)protein levels.Results Compared with the control group,the OD450 decreased in the cell culture(3,6,12,24,36)h in the model group(P<0.05);compared with the model group,(200,300,400)μg in the cell culture 6 h the OD450 of the flavone of clerodendranthus spicatus group increased(P<0.05);at(12,24,36)h in cell culture,the OD450 of the flavone of clerodendranthus spicatus group(300,400)increased(P<0.05).Cells in each group were treated for 24 hours,Compared with the control group,SOD activity,apoptosis rate,caspase3,caspase9,BAX,TGF-β1,and Smad3 protein levels were increased in the model group(P<0.05),MDA.The activity and BCL2 protein levels were decreased(P<0.05),and each group showed a dose-dependent change with increasing dose.Compared with model group,the apoptosis rate,SOD activity,BCL2,TGF-β1 and Smad3 protein levels of(100,200,300,400)μg/mL flavone of clerodendranthus spicatus group were decreased(P<0.05).MDA activity,caspase3,caspase9,and BAX protein levels were elevated(P<0.05)in a dose-dependent manner.Conclusions Flavone of clerodendranthus spicatus can promote the proliferation,inhibit the apoptosis,inhibit the oxidative stress,inhibit the expression of apoptotic protein,and thus slow down the damage of renal tubular epithelial cells in acute renal failure.