摘要目的 探讨维生素D受体(vitamin D receptor,VDR)在蟾蜍灵保护阿霉素诱导的足细胞损伤中的作用.方法 (1)体外实验:乳酸脱氢酶(LDH)释放试验观察不同浓度蟾蜍灵(10-9、10-8、10-7、10-6mol/L)对正常足细胞的毒性作用;阿霉素诱导体外小鼠足细胞损伤,使用10-7 mol/L蟾蜍灵干预48 h,Annexin V-FITC测定足细胞凋亡的变化;RT-PCR法测定VDR基因表达的改变;Western印迹测定VDR和nephrin蛋白水平变化,结合VDRsiRNA技术观察VDR在蟾蜍灵保护足细胞损伤中的作用.(2)体内试验:24只SD大鼠随机分为3组:对照组、阿霉素组、阿霉素+蟾蜍灵组,每组8只,使用TUNEL法检测肾组织足细胞细胞凋亡,间接免疫荧光法测定各组大鼠nephrin表达及分布,透射电镜观察大鼠足突和超微结构变化.结果 浓度不大于10-7 mol/L蟾蜍灵对正常足细胞无明显毒性.体内实验和体外实验显示阿霉素可诱导足细胞损伤,细胞凋亡数目增加,nephrin表达减少(P＜0.05),足突广泛融合,下调VDRmRNA和蛋白表达(P＜0.05);蟾蜍灵干预可上调VDR mRNA和蛋白表达(P＜0.05),减少足细胞凋亡,足突融合减轻,nephrin表达得到一定程度恢复(P＜0.05).VDR特异性siRNA下调VDR后可抑制蟾蜍灵的足细胞保护作用.结论 蟾蜍灵可能通过上调VDR的表达来发挥抗阿霉素诱导的足细胞损伤作用.

abstractsObjective To investigate the role of vitamin D receptor (VDR) in the protection of bufalin on podocyte injury induced by adriamycin (ADR).Methods (1) In vitro:the toxic effect of different concentrations of bufalin (10-9,10-8,10-7,104 mol/L) on podocyte was evaluated by lactate dehydrogenase (LDH) test;Annexin V-FITC and RT-PCR were utilized for podocyte apoptosis and VDR mRNA level respectively.Western blotting was used to analyze the protein expression of VDR and nephrin.SiRNA intervene was also applied to evaluate the role of VDR in bufalin's protective effect on podocyte injury induced by ADR.(2) In vitro:24 SD rats were randomly divided into three groups:control group,ADR group and ADR+bufalin group.TUNEL assay was applied to detect the apoptosis of podocytes in the kidney.Immunofluorescence and transmission electron microscope (TEM) were applied to analyze the expression of VDR and the ultrastructure of the glomerulus.Results Bufalin concentration lower than 10-7 mol/L had no toxicity on normal podocyte.Bufalin reduced the urinary protein excretion (P ＜ 0.05),alleviated the removal of podocyte foot processes and attenuated the changes in nephrin expression in the glomerulus of the adriamycin (ADR) rats (P ＜ 0.05).Bufalin notably inhibited the down-regulation of VDR in protein levels on the glomerulus of the ADR rats.Additionally,bufalin inhibited the down-regulation of VDR in both mRNA levels and protein levels (P ＜ 0.05),nephrin protein expression (P＜ 0.05),and apoptosis induced by ADR in cultured podocytes.Additionally,VDR specific siRNA intervene abolished the protective effect of bufalin in ADR-induced podocyte injury.Conclusion Bufalin can alleviate ADR-induced podocyte injury via enhancing VDR expression.