高迁移率蛋白1的小干扰RNA对高糖环境下视网膜血管内皮细胞凋亡的影响

目的　探讨高迁移率蛋白1（high-mobility group box-1，HMGB1）的小干扰RNA（small interfering RNA，siRNA）对视网膜血管内皮细胞的影响。方法　采用siRNA对HMGB1的表达进行抑制，采用CCK8检测试剂盒、Hochest33342染色、流式细胞仪检测，观察高糖环境下HMGB1 siRNA对视网膜血管内皮细胞的影响，同时通过Western blot检测凋亡相关蛋白的表达。结果　HMGB1转染后，siRNA组的蛋白表达与正常对照组相比减少了73%（P<0.05），siRNA组的蛋白表达与非特异性siRNA（scr-siRNA）组相比减少了75%（P<0.05），而正常对照组和scr-siRNA组之间差异无统计学意义（P>0.05）。流式细胞仪检测结果显示，正常对照组、高糖组、scr-siRNA组和siRNA组细胞的总凋亡率分别为（0.40±0.03）%、（49.80±3.50）%、（47.60±1.98）%和（23.60±2.40）%。与正常对照组相比，高糖组、scr-siRNA组、siRNA组的细胞凋亡率均明显升高（均为P<0.05）；与scr-siRNA组相比，siRNA组的细胞凋亡率明显降低，差异有统计学意义（P<0.05）；而scr-siRNA组和高糖组的细胞凋亡率差异无统计学意义（P>0.05）。与正常对照组相比，高糖组和scr-siRNA组的cleaved-caspase 3蛋白（半胱天冬酶3）表达分别增加了（233±10）%、（266±22）%（均为P<0.05）；与scr-siRNA组相比，siRNA组的cleaved-caspase 3蛋白表达减少了（43±3）%（P<0.05）；而高糖组和scr-siRNA组的蛋白表达差异无统计学意义（P>0.05）。与正常对照组相比，高糖组和scr-siRNA组的B淋巴细胞瘤-2（B-cell lymphoma-2，Bcl-2）蛋白表达分别减少了（32±2）%、（29±3）%（均为P<0.05）；与scr-siRNA组相比，siRNA组的Bcl-2蛋白表达增加了（42±2）%（P<0.05）；而高糖组和scr-siRNA组的蛋白表达差异无统计学意义（P>0.05）。结论　HMGB1 siRNA可以通过抑制cleaved-caspase 3的活化和增加Bcl-2蛋白的表达减轻高糖环境下视网膜血管内皮细胞的凋亡。

Effects of HMGB1 siRNA on the apoptosis of retinal vascular endothelial cells in high glucose environment

Objective　To investigate the effects of HMGB1 small interference RNA （siRNA） on retinal vascular endothelial cells.Methods　siRNA was used to inhibit the expression of HMGB1，followed by the application of CCK8 assay，Hochest33342 staining and flow cytometry to observe the effects of HMGB1 siRNA on retinal vascular endothelial cells in high glucose environment.Meanwhile，the expression of proteins related to apoptosis was detected by Western blot.Results　The transfection of HMGB1 siRNA down-regulated the protein expression level of HMGB1 by 73% in siRNA group compared with normal control （NC） group （P<0.05），and the protein expression level of HMGB1 in siRNA group was decreased by 75% compared with scr-siRNA group （P<0.05）.There was no significant difference between NC group and scr-siRNA group （P>0.05）.The total apoptotic rate of NC group，high-glucose group，scr-siRNA group and siRNA group was （0.40±0.03）%，（49.80±3.50）%，（47.60±1.98）% and （23.60±2.40）% by flow cytometry.Compared with NC group，the apoptotic rates of high-glucose group，scr-siRNA group and siRNA group were increased （all P<0.05）.Compared with scr-siRNA group，the apoptotic rate of HRECs in siRNA group was reduced，with significant statistical difference （P<0.05）.There was no significant difference in the rate of cell apoptosis between scr-siRNA group and high-glucose group （P>0.05）.Compared with the NC group，the protein expression level of cleaved-caspase3 protein in high-glucose group and scr-siRNA group were increased by （233±10）% and （266±22）%，respectively （both P<0.05）；compared with scr-siRNA group，the protein expression level of cleaved-caspase 3 in siRNA group was reduced by （43±3）% （P<0.05）；and there was no significant difference in the protein expression of cleaved-caspase 3 in high-glucose group and scr-siRNA group （P>0.05）.Compared with the NC group，the protein expression of B-cell lymphoma-2 （Bcl-2） in high-glucose group and scr-siRNA group was decreased by （32±2）% and （29±3）%，accordingly （both P<0.05）；compared with scr-siRNA group，the protein expression level of Bcl-2 in siRNA group was increased by （42±2）%（P<0.05）；and there was no significant difference in the protein expression of Bcl-2 in high-glucose group and scr-siRNA group （P>0.05）.Conclusion　HMGB1 siRNA can reduce the apoptosis of retinal vascular endothelial cells in high glucose environment by inhibiting the activation of cleaved-caspase3 and increasing the expression of Bcl-2.