Twinfilin-1过表达缓解镉诱导肾细胞重金属中毒及凋亡

Study of Twinfilin-1 overexpression relieving heavy metal poisoning and apoptosis of cadmium-induced renal cells

目的探究细胞骨架调控蛋白Twinfilin-1过表达对镉诱导肾细胞重金属中毒及凋亡的影响。方法体外培养猪肾细胞LLC-PK1,随机分为对照组、模型组、模型+pc DNA空载体组、模型+Twinfilin-1过表达组,以8μmol/L镉处理细胞24 h建立重金属中毒模型。采用实时荧光PCR技术和蛋白印迹实验检测Twinfilin-1的表达,CCK8实验检测细胞增殖,流式细胞术检测细胞凋亡和线粒体膜电位的变化,蛋白印迹检测凋亡相关蛋白[B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸天冬氨酸特异性蛋白酶(Caspase3、9)]水平。结果与对照组比较,模型组Twinfilin-1 mRNA及蛋白水平、细胞增殖能力、线粒体膜电位降低,细胞凋亡率、Bax/Bcl-2、cleaved cas9/cas9及cleaved cas3/cas3水平增加(P<0.05);与模型组比较,模型+Twinfilin-1过表达组Twinfilin-1 mRNA及蛋白水平、细胞增殖能力、线粒体膜电位增加,细胞凋亡率、Bax/Bcl-2、cleaved cas9/cas9及cleaved cas3/cas3水平降低(P<0.05),而模型组+pc DNA空载体组无明显变化(P>0.05)。结论Twinfilin-1过表达可能通过下调凋亡关键因子Bax/Bcl-2水平,抑制Caspase3、9的活化,减少细胞凋亡,维持正常线粒体膜电位,从而保护镉诱导肾细胞重金属中毒。

Objective To explore the effects of cytoskeleton regulatory protein Twinfilin-1 overexpression on heavy metal poisoning and apoptosis of cadmium-induced renal cells.Methods Porcine renal cells LLC-PK1 were cultured in vitro and randomly divided into control group,model group,model+pc DNA empty vector group and model+Twinfilin-1 overexpression group.Cells were treated with 8μmol/L of cadmium for 24 h to establish the model of heavy metal poisoning.The expression of Twinfilin-1 was detected by real-time fluorescent PCR and Western blot.The cells proliferation was detected by CCK8.The apoptosis and changes in mitochondrial membrane potentials(MMP)were detected by flow cytometry.The expressions of apoptosis-related proteins[B cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),cysteine aspartate specific proteases(Caspase3,9)]were detected by Western blot.Results Compared with control group,expression of Twinfilin-1 mRNA and protein,ability of cells proliferation,MMP decrease,apoptosis rate,Bax/Bcl-2,cleaved cas9/cas9 and cleaved cas3/cas3 levels were increased in model group(P<0.05).Compared with model group,expression of Twinfilin-1 mRNA and protein,ability of cells proliferation,MMP increase,apoptosis rate,Bax/Bcl-2,cleaved cas9/cas9 and cleaved cas3/cas3 levels were decreased in model+Twinfilin-1 overexpression group(P<0.05),but there was no significant change in model+pc DNA empty vector group(P>0.05).Conclusion Twinfilin-1 overexpression may inhibit the activation of Caspase3 and 9,reduce apoptosis and maintain normal MMP by reducing level of key apoptosis factor Bax/Bcl-2,thereby protecting from heavy metal poisoning of cadmium-induced renal cells.