SC79对高糖诱导RPE细胞凋亡的拮抗作用及其对AKT-XIAP信号通路的调控机制

Antagonistic effect of SC79 on high glucose-induced apoptosis of RPE cells and its regulatory mechanism on AKT-XIAP signaling pathway

目的探讨特异性AKT激活剂SC79对体外高糖诱导人视网膜色素上皮(ARPE)-19细胞凋亡的拮抗作用及其潜在机制。方法将体外培养的ARPE-19细胞分别置于含5、10、20μg/ml SC79的高糖(30 mmol/L葡萄糖)培养液中培养6、12、24 h,根据细胞增生率探索最佳的作用浓度和作用时间。将细胞分为4个组,其中正常对照组、甘露醇组和高糖组、分别于含5.6 mmol/L葡萄糖正常培养液、含5.6 mmol/L葡萄糖和24.4 mmol/L甘露醇培养液、高糖培养液中培养48 h,高糖+SC79组细胞于含10μg/ml SC79正常培养液中培养12 h,然后于高糖培养液中继续培养36 h。采用MTS法检测细胞的增生率,采用流式细胞仪检测各组细胞凋亡率,采用Western blot法检测磷酸化的蛋白激酶B(p-Akt)、X连锁凋亡抑制蛋白(XIAP)、caspase-9、caspase-3及活化片段active-caspase-3的相对表达量。将细胞分为Neg-shRNA组、AKT shRNA组和空白对照组分别加入相应转染复合物和无血清培养基,采用实时荧光定量PCR检测各转染组Akt mRNA表达。将转染细胞分为Neg-shRNA+SC79组和AKT shRNA+SC79组,参照高糖+SC79组处理方式进行培养,采用流式细胞仪检测各组细胞凋亡率。结果不同浓度SC79处理不同时间细胞中以10μg/ml SC79预处理12 h细胞的增生率最高。高糖组细胞增生率明显低于正常对照组、甘露糖组和高糖+SC79组,差异具有统计学意义(均P<0.01)。高糖组细胞凋亡率为(52.27±3.21)%,明显高于正常对照组的(3.90±0.71)%和高糖+SC79组的(20.70±3.62)%,差异均有统计学意义(均P<0.01)。高糖组p-Akt、XIAP、caspase-9及caspase-3蛋白相对表达量明显低于正常对照组和高糖+SC79组,active-caspase-3蛋白相对表达量明显高于正常对照组和高糖+SC79组,差异均具有统计学意义(均P<0.05)。正常对照组、Neg-shRNA组和AKT shRNA组AKT mRNA相对表达量分别为0.60±0.07、0.59±0.03和0.11±0.10,总体比较差异有统计学意义(F=30.44,P<0.01)。AKT shRNA+SC79组细胞凋亡率明显高于高糖+SC79组和Neg-shRNA+SC79组,差异均有统计学意义(均P<0.001)。结论SC79可以部分拮抗高糖诱导的ARPE-19细胞凋亡,其机制可能与活化AKT/XIAP通路,抑制凋亡执行蛋白caspase家族有关。

Objective To investigate the antagonistic effect and potential mechanism of specific AKT activator SC79 on the apoptosis of human retinal pigment epithelial(ARPE)-19 cells induced by high glucose in vitro.Methods The ARPE-19 cells were cultured in high glucose medium(containing 30 mmol/L glucose)plus 5,10 or 20μg/ml SC79,respectively.After 6-,12-and 24-hour culture,the optimal experimental concentration and timing were determined according to cell proliferation rate.Then ARPE-19 cells were divided into four groups,normal control group cultured in normal medium containing 5.6 mmol/L glucose for 48 hours,mannitol group cultured in medium containing 5.6 mmol/L glucose and 24.4 mmol/L mannitol for 48 hours,high glucose group cultured in high glucose medium for 48 hours,and high glucose+SC79 group cultured in normal medium containing 10μg/ml SC79 for 12 hours plus in high glucose medium for 36 hours.The proliferation rate of APRE-19 cells was detected by MTS assay.The apoptosis rate was measured by flow cytometry.The relative expression levels of phosphorylated protein kinase B(p-Akt),X-linked inhibitor of apoptosis protein(XIAP),caspase-9,caspase-3 and its active fragments(active-caspase-3)were assayed by Western blot.The ARPE-19 cells were divided into Neg-shRNA group,AKT shRNA group and blank control group and were treated with the corresponding transfection complex and serum-free medium.The AKT mRNA expression was detected by real-time PCR.The transfected ARPE-19 cells were divided into Neg-shRNA+SC79 group and AKT shRNA+SC79 group and were cultured according to the culturing method of high-glucose+SC79 group.The apoptosis rate of the two groups was tested by flow cytometry.Results Among different concentrations of SC79 and treatment times,the proliferation rate of cells treated with 10μg/ml SC79 for 12 hours was the highest.The proliferation rate of ARPE-19 cells in high-glucose group was significantly lower than that in normal control group,mannitol group and high-glucose+SC79 group,and the differences were statistically significant(all at P<0.01).The apoptosis rate of cells in the high-glucose group was(52.27±3.21)%,which was significantly higher than(3.90±0.71)%in normal control group and(20.70±3.62)%in high-glucose+SC79 group(both at P<0.01).The relative expression levels of p-Akt,XIAP,caspase-9 and caspase-3 were significantly lower and the relative expression level of active-caspase-3 was significantly higher in high glucose group than those in normal control group and high-glucose+SC79 group(all at P<0.05).The relative expression level of AKT mRNA in normal control group,Neg-shRNA group and AKT shRNA group was 0.60±0.07,0.59±0.03 and 0.11±0.10,respectively,showing a statistically significant difference among the groups(F=30.44,P<0.01).The apoptosis rate of cells in the AKT shRNA+SC79 group was significantly higher than that in high-glucose+SC79 group and Neg-shRNA+SC79 group(both at P<0.001).Conclusions SC79 can partially antagonize the apoptosis of ARPE-19 cells induced by high glucose,which is related to the activation of AKT/XIAP pathway and the inhibition of the caspase family.