基于PI3K/Akt通路探讨脉络宁注射液对早期糖尿病视网膜病变的作用

Mailuoning injection for early diabetic retinopathy by regulating PI3K/Akt pathway

目的基于PI3K/Akt通路探讨脉络宁注射液对早期糖尿病视网膜病变(DR)的作用。方法将30只SD雄性大鼠随机分为正常对照组、糖尿病组、糖尿病脉络宁组,每组10只。除正常对照组外,各实验组大鼠均采用STZ腹腔注射诱导大鼠糖尿病模型。每天脉络宁组大鼠按照0.92 mL/kg剂量进行腹腔给药,为期12周,对照组给予等量生理盐水。给药结束后分离各组大鼠视网膜组织并检测视网膜组织中磷酸化Akt(p-Akt)、Akt总蛋白水平以及PI3K、Caspase3、Bcl-2的蛋白水平及mRNA水平。同时体外培养人视网膜内皮细胞(HRCEC),将细胞分为三组:正常对照组、高糖组、高糖脉络宁组。除正常对照组外,其余组均高糖(25 mmol/L)环境下培养,脉络宁组同时加入脉络宁注射液(0.8μg/mL),处理72 h检测各处理组细胞中的p-Akt的水平。结果体内实验中,与糖尿病组相比脉络宁组大鼠视网膜组织中p-Akt表达明显降低,PI3K及下游Bcl2、Caspase的蛋白表达量显示出与p-Akt相同的变化趋势。Real-time PCR检测发现PI3K及下游Bcl2、Caspase3基因表达情况与蛋白变化情况一致。体外实验中p-Akt的量在高糖组明显增加,脉络宁干预后下降。结论脉络宁注射液通过调控PI3K/Akt信号通路保护糖尿病视网膜细胞,改善糖尿病视网膜病变。

Objective To explore the effect of Mailuoning injection on early diabetic retinopathy(DR)based on PI3K/Akt pathway.Methods Thirty SD male rats were randomly divided into the control group,the model group,and the Mailuoning group,with 10 rats in each group.Rats of DR model were induced by intraperitoneal injection of STZ,except the control group.The Mailuoning group was administered intraperitoneally at a dose of 0.92 mL/kg every day for 12weeks,and the control group was given the same amount of saline.12 weeks later,the retinal tissue of each group rats were isolated and the phosphorylated Akt protein、total Akt,PI3K、Caspase3、Bcl-2 protein level and mRNA expression in the retinal tissue were detected.At the same time,HRCEC cells were cultured in vitro,and the cells were divided into the control group,High-Sugar group and the Mailuoning group.Except the control group,the other groups were cultured under high glucose(25mmol/L)environment.The Mailuoning group was added with Mailuoning injection(0.8μg/mL).Then,all the groups were treated for 72h.At the end of the experiment,pAkt in HRCEC cells were detected.Results In vivo experiments,compared with the model group,the expression of p-Akt in rat retinal tissue was significantly reduced.The total amount of AKT did not change between the three in vitro experiments,but the amount of phosphorylated AKT(p-Akt)increased in the model group,and decreased after the intervention of Mailuoning;the expression of PI3K and downstream Bcl2 and Caspase showed the same changes as phosphorylated AKT.Real-time PCR showed that the gene expression was consistent with the amount of protein change.Conclusion Mailuoning can protect the retinal cell of DR rats through PI3K/Akt signal path.