积雪草苷对足细胞α-actinin-4、synaptopodin蛋白及ERK/JNK通路的影响

Influence of Asiaticoside on Podocyteα-actinin-4,synaptopodin Protein and ERK/JNK Pathway

目的:探讨积雪草苷对阿霉素诱导的足细胞中α-actinin-4、synaptopodin蛋白及ERK/JNK通路的影响。方法:1)MTT法检测积雪草苷对足细胞及阿霉素诱导的足细胞活力的影响。2)选用1μmol/L阿霉素制造足细胞损伤模型,蛋白印迹法(Western blotting)检测足细胞α-actinin-4、synaptopodin蛋白表达。3)免疫荧光检测足细胞骨架及Western blotting检测ERK/JNK信号通路的磷酸化水平。结果:1)低浓度积雪草苷(10~80μg/mL)对足细胞活力无明显影响,而高浓度积雪草苷(>80μg/mL)可降低足细胞活力;2)阿霉素体外可损伤足细胞,降低足细胞活力;积雪草苷浓度依赖性地减轻阿霉素对足细胞的损伤,增加足细胞活力;阿霉素体外可增加足细胞骨架蛋白α-actinin-4的表达,减少足细胞骨架蛋白synaptopodin的表达,而积雪草苷可下调阿霉素诱导的足细胞α-actinin-4蛋白表达,上调阿霉素抑制的足细胞synaptopodin的表达;3)阿霉素(1μmol/L)能升高ERK、JNK的磷酸化水平,与空白对照组比较差异有统计学意义(P<0.05);积雪草苷预处理细胞后,能够有效遏制阿霉素诱导的ERK活化,与模型对照组比较差异有统计学意义(P<0.05),但却不能抑制阿霉素诱导的JNK信号通路中JNK的活化。结论:积雪草苷预处理细胞后可能通过遏制阿霉素诱导的ERK信号通路的活化,减少阿霉素对足细胞骨架的损伤作用,从而改变了α-actinin-4、synaptopodin蛋白表达。

Objective:To explore the influence of asiaticoside on podocyteα-actinin-4,synaptopodin protein and ERK/JNK pathways induced by adriamycin.Methods:1)MTT method was used to detect the effects of asiaticoside on podocyte and podocyte viability induced by adriamycin.2)Podocyte injury model was established by choosing adriamycin at the dose of 1μmol/L,Western blotting to detect the expressions ofα-actinin-4 and synaptopodin protein in podocyte.3)Immunofluorescence to measure podocyte skeleton,Western blotting to measure the phosphorylation level of ERK/JNK pathways.Results:1)Low concentrations of asiaticoside(10 to 80μg/mL)showed no influence on podocyte viability,while high concentrations of asiaticoside(>80μg/mL)could lower podocyte viability;2)Adriamycin in vitro could cause the damage to podocyte and lower podocyte viability;asiaticoside could relieve adriamycin-induced damage to podocyte and increase podocyte viability in dose dependent manner;adriamycin in vitro could increase the expressions of podocyte cytoskeleton proteinα-actinin 4 and lower the expressions of podocyte cytoskeleton protein synaptopodin,while asiaticoside could down regulate the expressions of podocyteα-actinin-4 protein induced by adriamycin,up regulate the expressions of podocyte synaptopodin inhibited by adriamycin;3)Adriamycin(1μmol/L)could lift phosphorylation of ERK and JNK,the difference had statistical meaning when it was compared with blank control group(P<0.05);after preconditioning cells,asiaticoside could effectively inhibit ERK activation induced by adriamycin,the difference showed statistical meaning compared with model control group(P<0.05),while it can not inhibit JNK activation induced by adriamycin.Conclusion:Asiaticoside pretreatment could change the expressions ofα-actinin-4 and synaptopodin protein possibly through inhibiting the activation of ERK signaling pathway induced by adriamycin and reducing adriamycin-induced damage to podocyte cytoskeleton.