摘要目的 研究过表达叉头状转录因子O1(FoxO1)对高糖条件下小鼠足细胞及线粒体功能的影响及机制.方法 分别采用过表达FoxO1的慢病毒(LV-CA-FoxO1)和空病毒(LV-NC)感染条件永生性小鼠肾足细胞,在不同糖浓度(正常糖5.5 mmol/L、高糖25.0 mmol/L)下培养72 h.实验分为正常糖组、高糖组、FoxO1组(高糖+LV-CA-FoxO1)和NC组(高糖+LV-NC)共4组,分别检测各组细胞FoxO1表达情况、线粒体及细胞功能指标.多组间比较采用方差分析,组内比较采用SNK法.结果(1)与高糖组比较,FoxO1组FoxO1转录活性增加[(0.94±0.06)比(2.72±0.27),q=3.77,P<0.05];活性氧簇和丙二醛量减少(q=9.22、2.44,均P<0.05),线粒体膜电位上升,线粒体结构损伤减轻;(2)FoxO1组较高糖组足细胞蛋白nephrin、podocin和podocalyxin表达增加(q=2.18、5.87、5.65,均P<0.05),单层屏障功能改善,凋亡率下降(q=6.96、8.99,均P<0.05);(3)FoxO1组较高糖组中PTEN诱导激酶1(PINK1)、parkin mRNA水平上升,线粒体动力相关蛋白1 mRNA下降(q=1.46、7.50、1.89,均P<0.05).与高糖组比较,FoxO1组PINK1蛋白表达水平上升(q=2.36,P<0.05).NC组与高糖组各项指标比较,差异均无统计学意义(P>0.05).结论 过表达FoxO1可减轻高糖诱导的小鼠足细胞线粒体及细胞功能损伤,可能通过激活PINK1/parkin途径实现.

abstractsObjective To explore the role and molecular mechanism of forkhead transcription factor O1(FoxO1) on cellular and mitochondrial injury in conditionally immortalized mouse podocyte cell line (CIMPs) under high glucose (HG) conditions. Methods The CIMPs cells were infected by constitutively active FoxO1 lentiviral vector (LV-CA-FoxO1) or empty lentiviral vector (LV-NC), and cultured under different glucose levels (5.5 mmol/L for normal glucose or 25.0 mmol/L for high glucose). The CIMPs were divided into 4 groups:group NG (normal glucose), group HG (high glucose), group FoxO1 (HG+LV-CA-FoxO1) and group NC (HG+LV-NC ). After 72 hours of treatment, the expression of FoxO1, mitochondrial and cellular function, and expression of possible downstream genes of each group were examined. Analysis of variance was used for comparison in multiple groups. SNK method was used for comparison between two groups. Results (1) The transcriptional activity of FoxO1 in group FoxO1 was increased compared with group HG [(0.94 ± 0.06) vs (1.72 ± 0.27), q=3.77, P<0.05]. (2) Mitochondrial functions:compared with group HG, the level of reactive oxygen species and malonaldehyde were decreased and the mitochondrial membrane potential was increased (q=9.22, 2.44, both P<0.05), whereas the mitochondrial ultrastructure injuries were ameliorated in group FoxO1. (3) Cellular functions:compared with group HG, the expression of phenotypic proteins of podocyte including nephrin, podocin and podocalyxin were increased (q=2.18, 5.87, 5.65, all P<0.05), whereas the monolayer barrier function was improved and apoptosis rate was decreased (q=6.96, 8.99, both P<0.05) in group FoxO1. (4) Mitochondria related protein:compared with group HG, the phosphatase and tensin homolog deleted on chromosome ten (PTEN) induced putative kinase 1 (PINK1) mRNA and parkin mRNA level were increased whereas the dynamin-related protein 1 mRNA level was decreased in group FoxO1 (q=1.46, 7.50, 1.89, all P<0.05). Compared with group HG, the expression of PINK1 protein level was increased (q=2.36,P<0.05). All indices of NC group had no statistical difference with those of HG group (all P>0.05). Conclusion Overexpression of FoxO1 alleviates the mitochondrial and cellular injuries induced by high glucose in CIMPs, probably via activation of PINK1/parkin pathway.