红景天苷抑制高糖诱导大鼠视网膜Müller细胞的凋亡

Salidroside inhibits apoptosis of retinal Müller cells induced by high glucose in rats

背景:有研究发现红景天苷对糖尿病视网膜病变有改善作用,但红景天苷对高糖引起视网膜Müller细胞损伤的保护作用尚不明确。目的:探讨红景天苷对高糖诱导的大鼠视网膜Müller细胞系(rMC-1)氧化应激及细胞凋亡的保护作用及其作用机制。方法:高糖培养rMC-1细胞模拟高糖环境,实验分为4组:正常对照组;高糖损伤组(培养基葡萄糖终浓度为35.5 mmol/L);红景天苷组(加入红景天苷处理4 h,再加入高糖共同孵育);PI3K抑制剂组(红景天苷和PI3K抑制剂共同处理4 h,再加入高糖共同孵育)。48 h后使用CCK8法检测各组细胞活力,DCFH-DA荧光探针检测活性氧水平,采用试剂盒测定超氧化物歧化酶、过氧化氢酶的活力。流式细胞术检测各组细胞凋亡情况,采用Western blot检测PI3K、AKT、磷酸化AKT(P-AKT)、Cleaved caspase-3蛋白表达的变化。结果与结论:①与正常对照组相比,高糖损伤组细胞活力、超氧化物歧化酶和过氧化氢酶活力显著降低,活性氧水平、细胞凋亡率明显增高,Cleaved caspase-3蛋白表达水平显著增加,而P-Akt/Akt蛋白表达比值降低(均为P<0.05);②与高糖损伤组比较,红景天苷组细胞存活率、超氧化物歧化酶和过氧化氢酶活力明显升高,活性氧水平、细胞凋亡率显著降低,Cleaved caspase-3蛋白表达水平显著下降,P-Akt/Akt蛋白表达比值增加(P<0.05);③PI3K抑制剂组各指标与高糖损伤组无显著差异(P>0.05);④结果说明,红景天苷能通过激活PI3K/Akt通路抑制高糖诱导的氧化应激和细胞凋亡,减轻rMC-1细胞损伤。

BACKGROUND:It has been found that salidroside can improve diabetic retinopathy.However,it is not clear whether salidroside can protect retinal Müller cells(rMC-1)against damage induced by high glucose.OBJECTIVE:To investigate the effect and mechanism of salidroside on oxidative stress and apoptosis induced by high glucose in rat rMC-1 cells.METHODS:The rMC-1 cells were divided into four groups:normal control group,high glucose group(culture medium with a final glucose concentration of 35.5 mmol/L),salidr oside group(treatment with salidroside for 4 hours followed by co-culture with high glucose)and PI3K inhibitor group(treatment with salidroside and PI3K inhibitor for 4 hours followed by co-culture with high glucose).The viability of the rMC-1 cells was measured by cell counting kit-8 assay.Reactive oxygen species production was detected by DCFH-DA.The superoxide dismutase and catalase activities were tested by specific kits.Cell ap optosis was detected by flow cytometry.Western blot was used to detect the expression levels of Cleaved caspase-3,PI3K,AKT and phosphorylated AKT.RESULTS AND CONCLUSION:Compared with the normal control group,the cell viability and the activities of antioxidant enzymes(superoxide dismutase,catalase)in the high glucose group were significantly decreased,the production of reactive oxygen species,apoptotic rate and the level of Cleaved caspase-3 were significantly increased,and the phosphorylated AKT/AKT ratio was down-regulated(P<0.05).Compared with the high glucose group,salidroside significantly enhanced the cell viability and increased the activities of antioxidant enzymes(superoxide dismutase,catalase),decreased the production of reactive oxygen species,reduced the apoptotic rate of the cells and downregulated the expression level of Cleaved caspase-3(P<0.05).Salidroside also activated the phosphorylation of Akt(P<0.05).Whereas,the addition of LY294002,a pharmacological inhibitor of PI3K,showed similar results in the high glucose group(P>0.05).To conclude,salidroside can protect rMC-1 cells through inhibiting apoptosis and oxidative stress induced by high glucose.The main mechanism responsible for the inhibition of oxidative stress is the activation of the PI3K/Akt pathway.