欧前胡素调节HMGB1-RAGE-NF-κB信号通路对高糖诱导的视网膜内皮细胞损伤的影响

Effect of Imperatorin on High Glucose Induced Retinal Endothelial Cell Injury by Regulating the HMGB1-RAGE-NF-κB Signaling Pathway

该研究探讨欧前胡素(IMP)调节高迁移率族蛋白B1(HMGB1)-晚期糖基化终产物受体(RAGE)-核转录因子κB(NF-κB)信号通路对高糖诱导的视网膜血管内皮细胞损伤的影响。使用10~320μmol/L的IMP处理高糖诱导的视网膜血管内皮细胞hRECs,筛选最佳药物浓度;将视网膜血管内皮细胞hRECs细胞分为正常糖组(NG组,5.5 mmol/L葡萄糖)、高糖组(HG组,25 mmol/L葡萄糖)、低浓度欧前胡素组(IMP-L组,25 mmol/L葡萄糖+20μmol/L IMP)、中浓度欧前胡素组(IMP-M组,25 mmol/L葡萄糖+40μmol/L IMP)、高浓度欧前胡素组(IMP-H组,25 mmol/L葡萄糖+80μmol/L IMP)、高浓度欧前胡素+重组HMGB1蛋白组(IMP-H+rHMGB1组,25 mmol/L葡萄糖+80μmol/L IMP+200μg/L rHMGB1)。CCK-8试剂盒检测hRECs细胞活性;流式细胞仪检测hRECs细胞凋亡的情况;管腔形成实验检测细胞血管生成;酶联免疫吸附法(ELISA)检测细胞IL-6、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、MDA水平和SOD水平;Western blot检测HMGB1-RAGE-NF-κB信号通路相关蛋白表达情况。结果显示,IMP可以促进高糖诱导的hRECs增殖,且呈浓度依赖性,选择浓度为20、40、80μmol/L的IMP进行后续实验;与NG组比较,HG组细胞存活率、SOD活性降低,细胞凋亡率,管腔形成数,IL-6、TNF-α、IL-1β、MDA水平及HMGB1、RAGE、NF-κB p65蛋白表达水平升高(P<0.05);与HG组比较,IMP-L组、IMP-M组、IMP-H组细胞存活率、SOD活性升高,细胞凋亡率,管腔形成数,IL-6、TNF-α、IL-1β、MDA水平及HMGB1、RAGE、NF-κB p65蛋白表达水平降低,且呈浓度依赖性(P<0.05);与IMP-H组比较,IMP-H+rHMGB1组细胞存活率、SOD活性显著降低,细胞凋亡率,管腔形成数,IL-6、TNF-α、IL-1β、MDA水平及HMGB1、RAGE、NF-κB p65蛋白表达水平显著升高(P<0.05)。结果表明,IMP可能通过抑制HMGB1-RAGENF-κB信号通路,降低炎症反应和细胞凋亡水平,进而减轻高糖诱导的视网膜血管内皮细胞损伤。

This study investigated the effect of IMP(imperatorin) on high glucose induced retinal endothelial cell injury by regulating the HMGB1(high-mobility group protein box 1)-RAGE(receptor for advanced glycation end products)-NF-κB(nuclear factor kappa-B) signaling pathway.High glucose induced hRECs in retinal endothelial cells were treated with IMP at a concentration of 10-320 μmol/L to screen for the optimal drug concentration.hRECs(human retinal endothelial cells) were separated into normal glucose group(NG group,5.5 mmol/L glucose),high glucose group(HG group,25 mmol/L glucose),low concentration imperatorin group(IMP-L group,25 mmol/L glucose+20 μmol/L IMP),medium concentration imperatorin group(IMP-M group,25 mmol/L glucose+40 μmol/L IMP),high concentration imperatorin group(IMP-H group,25 mmol/L glucose+80 μmol/L IMP),and high concentration imperatorin+recombinant HMGB1 protein group(IMP-H+rHMGB1 group,25 mmol/L glucose+80 μmol/L IMP+200 μg/L rHMGB1).CCK-8assay kit was applied to detect the activity of hRECs cells.Flow cytometry was applied to detect the apoptosis of hRECs cells.The cell angiogenesis was detected by the lumen formation test.ELISA(enzyme linked immunosorbent assay) was applied to detect levels of IL-6,TNF-α(tumor necrosis factor-α),IL-1β(interleukin-1β),MDA,and SOD in cells.Western blot was applied to detect the expression of proteins related to the HMGB1-RAGE-NF-κB signaling pathway.The results showed that IMP was able to promote the proliferation of high glucose induced hRECs in a concentration dependent manner,subsequently,IMP concentrations of 20,40,and 80 μmol/L were selected for subsequent experiments.Compared with the NG group,the cell survival rate and SOD activity in the HG group were decreased;the apoptosis rate,the number of lumen formation,the levels of IL-6,TNF-α,IL-1β,MDA,and the expression of HMGB1,RAGE,NF-κB p65 proteins were increased(P<0.05).Compared with the HG group,the cell survival rate and SOD activity were increased in the IMP-L group,IMP-M group,and IMP-H group;the apoptosis rate,the number of lumen formation,the levels of IL-6,TNF-α,IL-1β,MDA,and the expression of HMGB1,RAGE,NF-κB p65 proteins were decreased and in a concentration dependent manner(P<0.05).Compared with the IMP-H group,the cell survival rate and SOD activity in the IMP-H+rHMGB1group were significantly reduced;the apoptosis rate,the number of lumen formation,the levels of IL-6,TNF-α,IL-1β,MDA,and the expression of HMGB1,RAGE,NF-κB p65 proteins were significantly increased(P<0.05).The results suggest that IMP may reduce inflammatory response and cell apoptosis by inhibiting the HMGB1-RAGE-NF-κB signaling pathway,thereby alleviating high glucose induced injury to retinal endothelial cells.