维生素D_(3)抑制高糖诱导视网膜血管内皮细胞凋亡分子机制研究  

Molecular mechanism of vitamin D_(3) inhibiting apoptosis of retinal vascular endothelial cells induced by high glucose

作　　者：刘晓[1] 兰图 刘芳静 蹇京宴 甘榆讯 LIU Xiao;LAN Tu;LIU Fang-jing;JIAN Jing-yan;GAN Yu-xun(Department of Ophthalmology,Zigong First People′s Hospital,Zigong 643000,China)

机构地区：[1]自贡市第一人民医院眼科,四川自贡643000

出　　处：《创伤与急危重病医学》2021年第5期349-355,共7页Trauma and Critical Care Medicine

基　　金：四川省卫生厅科研课题(130855)。

摘　　要：目的探讨维生素D_(3)(VD_(3))对高糖(HG)诱导的视网膜血管内皮细胞(hRECs)的作用及其分子机制。方法体外培养hRECs,分为正常浓度(NG)组、高渗组、HG组、HG+阴性对照(NC)组、HG+小干扰RNA靶向的硫氧还蛋白相互作用蛋白(siTXNIP)组、VD_(3)+HG组、VD_(3)+HG+NC组、VD_(3)+HG+硫氧还蛋白相互作用蛋白(TXNIP)组、N-乙酰半胱氨酸(NAC)+HG组。NG组采用含有5.0 mmol/L D-葡萄糖的培养基培养;高渗组采用含有50.0 mmol/L甘露醇的培养基培养;其余7组用含有50.0 mmol/L的D-葡萄糖培养基培养。其中,HG+NC组、VD_(3)+HG+NC组转染阴性对照无序序列(pcDNA3.1-scramble);HG+siTXNIP组转染pcDNA3.1-siTXNIP;VD_(3)+HG+TXNIP组转染pcDNA3.1-TXNIP质粒;VD_(3)+HG组、VD_(3)+HG+NC组、VD_(3)+HG+TXNIP组在培养基中加入50.0 nmol/L浓度的VD_(3);NAC+HG组加入细胞内活性氧簇(ROS)清除剂NAC,浓度为10.0 mmol/L。各组hRECs均培养48 h。采用CCK-8法、流式细胞术检测细胞的增殖和凋亡情况。采用酶联免疫吸附法检测细胞中丙二醛(MDA)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、白细胞介素1β(IL-1β)、白细胞介素18(IL-18)含量。采用免疫荧光法检测ROS含量。采用蛋白印迹法检测含NLR家族Pyrin域蛋白3(NLRP3)炎症小体相关蛋白表达情况。免疫共沉淀法检测TXNIP和NLRP3蛋白相互作用。结果HG环境下,随着VD_(3)浓度的升高,hRECs细胞存活率逐渐升高,差异有统计学意义(P<0.05)。HG组hRECs细胞凋亡率为(38.65%±4.79%)、VD_(3)+HG组hRECs细胞凋亡率为(17.50%±3.69%),均显著高于NG组的(2.70%±0.78%)、高渗组的(3.65%±0.84%),但VD_(3)+HG组hRECs细胞凋亡率显著低于HG组,差异有统计学意义(P<0.05)。与NG组、高渗组比较,HG组细胞ROS荧光表达量升高;与HG组比较,VD_(3)+HG组、NAC+HG组细胞ROS荧光表达量降低,差异有统计学意义(P<0.05)。HG组细胞培养上清中SOD、GSH-Px含量显著低于NG组、高渗组,MDA、IL-1β、IL-18含�Objective To investigate the effect of vitamin D_(3)(VD_(3))on retinal vascular endothelial cells(hRECs)apoptosis induced by high glucose(HG)and its molecular mechanism.Methods hRECs were cultured in vitro,and divided into normal glucose(NG)group,hypertonic group,HG group,HG+negative control(NC)group,HG+small interfering RNA targeting TNXNIP(siTXNIP)group,VD_(3)+HG group,VD_(3)+HG+NC group,VD_(3)+HG+thioredoxin interacting protein(TXNIP)group,N-acetylcysteine(NAC)+HG group.hRECs in NG group were cultured with 5.0 mmol/L D-glucose,hRECs in hypertonic group were cultured with 50.0 mmol/L mannitol,and hRECs in the other seven groups were cultured with 50.0 mmol/L D-glucose.hRECs in HG+NC group,VD_(3)+HG+NC group were infected with pcDNA3.1-scrable,hRECs in HG+siTXNIP group were infected with pcDNA3.1-siTXNIP,hRECs in VD_(3)+HG+TXNIP group were infected with pcDNA3.1-TXNIP.hRECs in VD_(3)+HG group,VD_(3)+HG+NC group and VD_(3)+HG+TXNIP group were cultured with 50.0 nmol/L VD_(3).hRECs in NAC+HG group were treated with reactive oxygen species(ROS)scavenger NAC and its concentration was 10.0 mmol/L.hRECs in each group were cultured for 48 hours.CCK-8 and flow cytometry were used to detect cell proliferation and apoptosis.The levels of malondialdehyde(MDA),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),interleukin 1β(IL-1β)and interleukin 18(IL-18)were detected by enzyme-linked immunosorbent assay.Immunofluorescence assay method was used to detect the level of ROS.Western blotting was used to detect the expression of NLR family pyrin domain containing protein 3(NLRP3)associated with inflammatory corpuscles.The interaction between TXNIP and NLRP3 protein was detected by immunoprecipitation method.Results The survival rate of hRECs cells increased with the increase of VD_(3) concentration in HG group(P<0.05).The apoptosis rate of hRECs in HG group[(38.65%±4.79%)],VD_(3)+HG group[(17.50%±3.69%)]were significantly higher than those in NG group[(2.70%±0.78%)]and hypertonic group[(3.65%±0.84%)],but the apoptos

关 键 词：维生素D_(3) 视网膜血管内皮细胞 氧化应激 炎症反应 

分 类 号：R774.1[医药卫生—眼科] R587.2[医药卫生—临床医学]