益景汤经PI3K/AKT/eNOS信号通路拮抗高糖诱导的视网膜血管内皮细胞损伤

Yijing Decoction inhibits retinal microvascular endothelial cell injury induced by high glucose through PI3K/AKT/eNOS signal pathway

目的探讨益景汤对高糖诱导大鼠视网膜微血管内皮细胞损伤的拮抗作用及机制。方法将大鼠视网膜微血管内皮细胞随机分为5组:对照组(CG)、高糖组(HG)、低浓度益景汤组(LYG)、中浓度益景汤组(MYG)、高浓度益景汤组(HYG)。细胞生长至80%~90%时,往高糖组及益景汤组培养基中加入33 mmol/L葡萄糖培养3 d造高糖内皮细胞损伤模型,第4天按分组加入相应正常糖培养基及各浓度益景汤培养24 h。使用CCK-8试剂盒检测内皮细胞活性,Transwell法观察内皮细胞迁移,荧光聚合酶链式反应(PCR)检测细胞间紧密连接蛋白ZO-1及内皮细胞磷脂酰肌醇三羟基激酶(PI3K)、苏氨酸蛋白激酶(AKT)、内皮型一氧化氮合酶(eNOS)的信使RNA(mRNA)表达。结果高糖条件下,大鼠视网膜微血管内皮细胞活性降低,与对照组比较,差异有统计学意义(均P<0.05);与高糖组比较,低、中、高浓度益景汤组细胞活性均高于高糖组,差异均有统计学意义(均P<0.05)。高糖条件下,大鼠视网膜微血管内皮细胞迁移增强,与对照组比较,差异有统计学意义(P<0.05);与高糖组比较,低、中、高浓度益景汤组细胞迁移均低于高糖组,差异均有统计学意义(均P<0.05)。高糖条件下,大鼠视网膜微血管内皮细胞间连接蛋白ZO-1及内皮细胞PI3K、AKT、eNOS的mRNA表达量均降低,与对照组比较,差异均有统计学意义(均P<0.05);与高糖组比较,低、中、高浓度益景汤组细胞间连接蛋白ZO-1及内皮细胞PI3K、AKT、eNOS的mRNA表达量均高于高糖组,差异均有统计学意义(均P<0.05)。结论益景汤能经PI3K/AKT/eNOS信号通路抑制高糖诱导视网膜微血管内皮细胞迁移,提高细胞活性,减轻细胞损伤。

Objective To investigate the protective efct and mechanism of Yijing Decoction on retinal vascular endothe-lial cell injury induced by high glucose in rats.Methods Rat retinal microvascular endothelial cells were randomly divided into five groups:control group(CG),high glucose group(HG),low concentration Yijing Decoction group(LYG),medium concen-tration Yjing Decoction group(MYG)and high concentration Yijing Decoction group(HYG).When the cells grew to 80%-90%,the high glucose endothelial cell injury model was established by adding 33 mmol/L glucose to the culture medium of high glucose group and Yijing Decoction groups for three days,and the corresponding normal glucose medium and Yiing Decoction of various concentrations were added for 24 hours on the 4th day.Endothelial cell viability was detected by CCK-8 kit,endothelial cell migration was observed by Transwell method,and the mRNA expression of intercellular connexin ZO-1 and endothelial cell PI3K,AKT and eNOS was detected by fluorescent PCR.Results Under the condition of high glucose,the activity of retinal.microvascular endothelial cells in the control group was significantly lower than that in the control group(all P<0.05).The cell activity in the low,middle and high concentration Yijing Decoction groups were significantly higher than that in the high glucose group(all P<0.05).The migration of retinal microvascular endothelial cells in the high glucose group was significantly higher than that in the control group(P<0.05),and the cell migration in the low,middle and high concentration Yijing Decoction groups were significantly lower than that in the high glucose group(all P<0.05).Under the condition of high glucose,the mR-NA expression of ZO-1,PI3K,AKT and eNOS in retinal microvascular endothelial cells were significantly lower than those in the control group.Compared with the high glucose group,the mRNA expression of intercellular connexin ZO-1 and endothelial cell PI3K,AKT and eNOS in the low,middle and high concentration Yiing Decoction groups were significantly higher than those in the high glucose group(all P<0.05).Conclusion Yiing Decoction can antagonize the expression of PI3K,AKT,eNOS and ZO-1mRNA in retinal microvascular endothelial cells of rats injured by high glucose,reduce endothelial cell migra-tion,increase endothelial cell activity and reduce microvascular endothelial cell injury.