丝裂原激活蛋白激酶信号通路抑制剂对高糖培养视网膜微血管内皮细胞的影响

Influence of inhibitors of mitogen-activated protein kinase signaling pathway on high glucose cultured retinal microvascular endothelial cells

目的研究丝裂原激活蛋白激酶(MAPK)信号通路抑制剂对高糖培养大鼠视网膜微血管内皮细胞(RMECs)的作用。方法体外培养RMECs,将细胞分为5组:空白组、模型组、SB203580(p38抑制剂)组、U0126(胞外调节蛋白激酶抑制剂)组和SP600125(应激活化蛋白激酶抑制剂)组,各抑制剂组加入相应抑制剂10μmol·L^(-1)预孵育1 h后,除空白组外,其他4组高糖处理24 h。以免疫荧光实验检测细胞中过氧化物酶体增殖物激活受体γ(PPAR-γ)的表达分布,用实时荧光定量-聚合酶链式反应及免疫印迹法检测PPAR-γmRNA与蛋白的表达水平,流式细胞仪检测细胞线粒体膜电位(MMP)。结果空白组、模型组、SB203580组、U0126组和SP600125组的PPAR-γmRNA表达量比值分别为1. 00±0. 01,0. 79±0. 06,1. 01±0. 04,1. 19±0. 11和0. 73±0. 12;这5组MMP分别为0. 40±0. 02,0. 22±0. 01,0. 41±0. 01,0. 45±0. 01和0. 21±0. 01。模型组与空白组比较,上述指标均显著降低,差异均有统计学意义(均P <0. 05);SB203580组和U0126组与模型组比较,上述指标均升高,差异均有统计学意义(均P <0. 05); SP600125组与模型组比较,差异均无统计学意义(均P> 0. 05);抑制剂组中,U0126组的上述指标升高最显著,差异均有统计学意义(均P <0. 05)。PPAR-γ的蛋白水平变化与mRNA水平变化一致。结论在高糖环境中,抑制剂U0126可上调RMECs的PPAR-γ表达并减轻其线粒体损伤。

Objective To investigate the roles of inhibitors of mitogen - activated protein kinase (MAPK) signaling pathway on high glucose cultured rat retinal microvascular endothelial cells(RMECs) .Methods RMECs were randomly divided into 5 groups: blank group,model group, SB203580(p38 inhibitor) group,U0126 (extracellular regulated kinase inhibitor) group and SP600125 (c-Jun N-terminal kinase inhibitor) group.The cells of each inhibitor groups were pretreated with inhibitor 10 μmol·L^-1 for 1 h.In addition to the blank group,the remaining four groups were collected after 24 h of high glucose treatment.The expression of peroxisome proliferator-activated receptor-γ(PPAR-γ) in cells was detected by cell immunofluorescence.The mRNA and protein expression of PPAR-γ in each cell was detected by real-time quantitative PCR and Western blotting.The mitochondria membrane potential (MMP) was assessed by flow cytometry.Results The main indexes in blank,model,SB203580,U0126 and SP600125 groups were compared: the mRNA expression of PPAR-γ were 1.00±0.01,0.79±0.06,1.01±0.04, 1.19±0.11 and 0.73±0.12; the MMP levels were 0.40±0.02,0.22±0.01,0.41±0.01,0.45±0.01 and 0.21±0.01.Compared with blank group,the above indicators of model group decreased significantly(all P<0.05) ; compared with model group, the above indicators of SB203580 and U0126 groups increased significantly (all P<0.05) ; while no significant change was observed in SP600125 group(P > 0.05) ; the above indicators of U0126 group was the highest in inhibitor groups(P<0.05) .Changes of PPAR-γ in protein results were consistent with the changes in mRNA levels.Conclusion In high glucose-induced RMECs,U0126 inhibitor can increase the expression of PPAR-γ and reduce the destruction of mitochondrion.