摘要目的 观察氧化低密度脂蛋白(ox-LDL)对小鼠肾小球足细胞RhoA激酶1(ROCK1)表达和活性的影响,并初步探讨ROCK1在ox-LDL诱导的足细胞损伤中的作用.方法 体外培养小鼠条件永生性足细胞,以ox-LDL 20 μg/ml刺激足细胞24h,采用Western印迹法检测正常对照组、ox-LDL刺激组、ROCK1 siRNA+ox-LDL组以及ROCK1过表达(wtROCK1)+ox-LDL组磷酸化肌球蛋白磷酸化亚单位(p-MYPT)、nephrin、LC3-Ⅱ、p62、磷酸化(p)-ULK1(ULK1是一种丝/苏氨酸氨酸激酶)的表达水平;Dil-ox-LDL与足细胞共孵育4h,荧光显微镜观察各组足细胞脂质的含量情况.结果 与对照组相比,ox-LDL刺激增加足细胞ROCK活性,同时伴有nephrin、LC3-Ⅱ表达下降(P＜0.05),p62、p-ULK1表达上升(P＜0.05),细胞内胆固醇含量增加(P＜0.05).增加ROCK1表达能进一步增加ox-LDL诱导的p-MYPT水平,并进一步降低nephrin、LC3-Ⅱ的表达,增加p62、p-ULK1的表达(P＜0.05)和细胞内胆固醇含量(P＜0.05);与之相反,抑制ROCK1表达能抑制ox-LDL诱导的p-MYPT水平上调,同时上调nephrin、LC3-Ⅱ的表达(P＜0.05),下调p62、p-ULK1的表达(P＜0.05),降低细胞内胆固醇含量(P＜0.05).结论 ROCK1介导的脂质自噬障碍是ox-LDL诱导足细胞损伤的重要途径.

abstractsObjective To explore the role of ROCK1 in oxidized low-density lipoprotein (ox-LDL) induced podocyte injury and its possible mechanism.Methods The conditionally immortalized mouse podocyte cells were cultured in vitro and exposed to 20 μg/ml ox-LDL for 24 h.Western blotting was used to analyze the expression level of p-MYPT,nephrin,LC3-Ⅱ,p62,p-ULK1 in groups of control,ox-LDL,ROCK1 siRNA with ox-LDL,wtROCK1 with ox-LDL.Podocytes were incubated with DiI labeled ox-LDL for 4 h and fluorescence microscope was used to analyze lipid distribution.Results Compared with control group,ox-LDL increased cell cholesterol accumulation,activated ROCK along with decreased nephrin,LC3-Ⅱ (P ＜ 0.05),and increased p62,and p-ULK1 expression (P ＜ 0.05).Over-expression of ROCK1 significantly decreased the expression of nephrin and LC3-Ⅱ,but up-regulated the levels of p52,p-ULK1 and cell cholesterol accumulation in ox-LDL stimulated podocytes (P ＜ 0.05).In contrast,Inhibition of ROCK1 protected podocyte by improved lipophagy.Conclusion ROCK1 mediated disfunction of lipophagy contributes to the ox-LDL induced podocyte injury.