HYOU1对高糖环境施万细胞自噬和凋亡的影响及其机制

Effects of HYOU1 on autophagy and apoptosis of Schwann cells in high glucose environment and its mechanism

目的观察内质网分子伴侣复合物缺氧上调基因1(HYOU1)对高糖环境施万细胞自噬和凋亡的影响,并基于ROS/PI3K/AKT通路探讨相关机制。方法培养RSC96细胞,构建HYOU1重组表达质粒,采用随机数字表法将细胞分为高糖组、高糖+对照质粒组和高糖+HYOU1质粒组,三组均以高糖培养,其中高糖+对照质粒组和高糖+HYOU1质粒组分别转染对照质粒和pEGFP-HYOU1质粒。采用Western blotting法检测各组细胞中的自噬相关蛋白(LC3、ATG5、ATG12、Beclin1、p62)、JAK/STAT通路相关蛋白(JAK1、JAK2、STAT1、STAT3、STAT5、STAT6)、ROS/PI3K/AKT通路相关蛋白(PI3K、AKT、mTOR、pPI3K、pAKT、pmTOR),采用流式细胞术测算细胞凋亡率,采用RT-PCR法检测凋亡相关基因Bax、Bcl-2,采用DCFH-DA染色检测各组ROS水平。结果高糖+HYOU1质粒组LC3、ATG5、ATG12、Beclin1蛋白表达低于高糖组和高糖+对照质粒组,p62蛋白表达高于高糖组和高糖+对照质粒组(P均<0.05)。高糖+HYOU1质粒组凋亡率、Bax mRNA表达低于高糖组和高糖+对照质粒组,Bcl-2 mRNA表达及JAK1、JAK2、STAT1、STAT3、STAT5、STAT6蛋白表达高于高糖组和高糖+对照质粒组(P均<0.05)。高糖+HYOU1质粒组PI3K、AKT、mTOR、pPI3K、pAKT、pmTOR蛋白表达高于高糖组和高糖+对照质粒组,ROS水平低于高糖组和高糖+对照质粒组(P均<0.05)。结论HYOU1过表达可下调高糖环境施万细胞的自噬和凋亡,作用机制可能与调控ROS/PI3K/AKT通路有关。

Objective To explore the effects of hypoxia up-regulated 1(HYOU1)on autophagy and apoptosis of Schwann cells in the high glucose environment and to explore the related mechanism based on ROS/PI3K/AKT pathway.Methods RSC96 cells were cultured,HYOU1 recombinant expression plasmid was constructed,and the cells were randomly divided into the high glucose group,high glucose+control plasmid group,and high glucose+HYOU1 plasmid group.Cells in the three groups were all cultured with high glucose,and then cells in the high glucose+control plasmid group and high glucose+HYOU1 plasmid group were transfected with control plasmid and pEGFP-HYOU1 plasmid,respectively.Western blotting was used to detect autophagy-related proteins(LC3,ATG5,ATG12,Beclin1,p62),JAKSTAT pathway-related proteins(JAK1,JAK2,STAT1,STAT3,STAT5,STAT6),and ROS/PI3K/AKT pathway-related proteins(PI3K,AKT,mTOR,pPI3K,pAKT,pmTOR).Flow cytometry was used to detect the apoptosis rate.The apoptosis-related genes Bax and Bcl-2 were detected by RT-PCR,and the ROS level in each group was detected by DCFH-DA staining.Results Compared with the high glucose group and high glucose+control group,LC3,ATG5,ATG12 and Beclin1 protein levels decreased and p62 protein level increased in the high glucose+HYOU1 group(all P<0.05).Compared with the high glucose group and high glucose+control group,the apoptosis rate and Bax mRNA expression decreased,while Bcl-2 mRNA expression and the protein expression levels of JAK1,JAK2,STAT1,STAT3,STAT5 and STAT6 increased in the high glucose+HYOU1 group(all P<0.05).Compared with the high glucose group and high glucose+control group,PI3K,AKT,mTOR,pPI3K,pAKT and pmTOR protein levels increased,and ROS level decreased in the high glucose+HYOU1 group(all P<0.05).Conclusion HYOU1 overexpression can down-regulate autophagy and apoptosis of Schwann cells in the high glucose environment,and the mechanism may be related to the regulation of ROS/PI3K/AKT pathway.