摘要
目的探讨特女贞苷对高糖诱导人视网膜微血管内皮细胞(hRMECs)损伤的抑制作用及其机制。方法将hRMECs分为正常对照组、高渗组、高糖组、高糖+低浓度特女贞苷组、高糖+中浓度特女贞苷组和高糖+高浓度特女贞苷组,分别用含5.5 mmol/L葡萄糖、5.5 mmol/L葡萄糖+24.5 mmol/L甘露醇、30 mmol/L葡萄糖、30 mmol/L葡萄糖+25、50、100μmol/L特女贞苷的培养液培养24 h。另将hRMECs分为高糖+小干扰RNA阴性序列(si-NC)组、高糖+si-叉头框转录因子O4(FOXO4)组、高糖+特女贞苷+pcDNA组、高糖+特女贞苷+pcDNA-FOXO4组,转染相应试剂后分别用含30 mmol/L葡萄糖或100μmol/L特女贞苷+30 mmol/L葡萄糖的培养液培养24 h。采用流式细胞术检测hRMECs细胞凋亡情况;采用硫代巴比妥酸法检测细胞中丙二醛(MDA)质量浓度;采用黄嘌呤氧化酶法检测细胞中超氧化物歧化酶(SOD)活性;采用ELISA法检测细胞培养上清液中IL-1β和TNF-α质量浓度;采用Western blot法检测细胞中FOXO4蛋白表达水平。结果正常对照组、高渗组、高糖组、高糖+低浓度特女贞苷组、高糖+中浓度特女贞苷组和高糖+高浓度特女贞苷组细胞凋亡率分别为(7.32±0.72)%、(7.44±0.70)%、(23.96±1.32)%、(19.84±1.09)%、(14.13±0.85)%和(9.84±0.70)%。各组细胞凋亡率、MDA质量浓度、SOD活性值、IL-1β质量浓度、TNF-α质量浓度和FOXO4蛋白相对表达量总体比较,差异均有统计学意义(F=498.545、1186.693、516.629、654.247、638.238、472.655,均P<0.001),其中与高糖组相比,高糖+低、中、高浓度特女贞苷组细胞凋亡率、MDA浓度、IL-1β和TNF-α质量浓度、FOXO4蛋白相对表达量均明显降低,SOD活性值明显升高,且呈剂量依赖性;与高糖+si-NC组相比,高糖+si-FOXO4组FOXO4蛋白相对表达量、细胞凋亡率、MDA浓度、IL-1β和TNF-α质量浓度降低,SOD活性值升高;与高糖+特女贞苷+pcDNA组相比,高糖+特女贞苷+pcDNA-FOXO4组细胞凋亡率、MDA浓度、IL-1β和TNF-α质量浓度、FOXO4蛋白相对表达量均明显升高,SOD活性值明显降低,差异均有统计学意义(均P<0.05)。结论特女贞苷可保护hRMECs免受高糖损伤,作用机制与其下调FOX4表达抑制hRMECs凋亡、氧化应激和炎症反应有关。
Objective To investigate the effect of specnuezhenide on high glucose-induced human retinal microvascular endothelial cells(hRMECs)injury and its mechanism.Methods The hRMECs were divided into a normal control group cultured in a culture medium containing 5.5 mmol/L glucose,a hypertonic group cultured in a culture medium containing 5.5 mmol/L glucose+24.5 mmol/L mannitol,a high glucose group cultured in a culture medium containing 30 mmol/L glucose,as well as high glucose+low-,medium-,and high-dose specnuezhenide groups cultured in culture media containing 30 mmol/L glucose+25,50,100μmol/L specnuezhenide for 24 hours,respectively.In addition,hRMECs were divided into a high glucose+small interfering RNA-negative control(si-NC)group cultured in a culture medium containing 30 mmol/L glucose,a high glucose+si-forkhead box O4(FOXO4)group cultured in a culture medium containing 30 mmol/L glucose,a high glucose+specnuezhenide+pcDNA group cultured in a culture medium containing 100μmol/L specnuezhenide+30 mmol/L glucose,and a high glucose+specnuezhenide+pcDNA-FOXO4 group cultured in a culture medium containing 100μmol/L specnuezhenide+30 mmol/L glucose for 24 hours after transfection by corresponding reagents.Cell apoptosis was detected by flow cytometry.The malondialdehyde(MDA)concentration and superoxide dismutase(SOD)activity in cells were detected by the thiobarbituric acid method and xanthine oxidase method,respectively.The concentrations of interleukin(IL)-1βand tumor necrosis factor(TNF)-αin the cell culture supernatant were detected by enzyme linked immunosorbent assay.The relative expression level of FOXO4 protein in cells was determined by Western blot.Results The apoptosis rates of normal control group,hypertonic group,high glucose group,high glucose+low-,medium-and high-dose specnuezhenide groups were(7.32±0.72)%,(7.44±0.70)%,(23.96±1.32)%,(19.84±1.09)%,(14.13±0.85)%and(9.84±0.70)%,respectively.There were significant differences in cell apoptosis rate,MDA concentration,SOD activity,the concentration of IL-1β,the concentration of TNF-α,and the relative expression level of FOXO4 protein among the six groups(F=498.545,1186.693,516.629,654.247,638.238,472.655;all at P<0.001).Compared with high glucose group,the apoptosis rate,MDA concentration,IL-1βand TNF-αconcentration,FOXO4 protein expression level were significantly decreased in high glucose+low-,medium-and high-dose specnuezhenide groups,and SOD activity was significantly increased in a dose-dependent manner.Compared with high glucose+si-NC group,the expression level of FOXO4 protein,cell apoptosis rate,MDA concentration,IL-1βand TNF-αmass concentrations were decreased in high glucose+si-FOXO4 group,while the SOD activity was increased.Compared with high glucose+specnuezhenide+pcDNA group,the apoptosis rate,MDA concentration,IL-1βand TNF-αconcentrations,FOXO4 protein expression level of hRMECs in high glucose+specnuezhenide+pcDNA-FOXO4 group were significantly increased,and SOD activity was significantly decreased(all at P<0.05).Conclusions Specnuezhenide can protect hRMECs from high glucose-induced apoptosis,oxidative stress and inflammatory response by down-regulating FOXO4.
作者
刘茜
冯效梅
刘长庚
李海军
杨潇远
任静
张颖
Liu Qian;Feng Xiaomei;Liu Changgeng;Li Haijun;Yang Xiaoyuan;Ren Jing;Zhang Ying(Department of Ophthalmology,Henan Provincial People's Hospital,Henan Eye Hospital,Henan Eye Institute,People's Hospital of Zhengzhou University,Zhengzhou 450003,China)
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2023年第4期312-320,共9页
Chinese Journal Of Experimental Ophthalmology
基金
国家自然科学基金项目(U1404812)
河南省医学科技攻关计划省部共建项目(SBGJ2018083)
河南省立眼科医院基础研究专项项目(21JCQN005)。
