褪黑素减轻高糖诱导的人脐静脉内皮细胞损伤的机制  

Mechanism of melatonin alleviating human umbilical vein endothelial cell injury induced by high glucose

作　　者：刘婉卿 但章勇 周青[1] 汪渊[1] 王怡 朱华庆[1] Liu Wanqing;Dan Zhangyong;Zhou Qing;Wang Yuan;Wang Yi;Zhu Huaqing(Dept of Biochemistry and Laboratory of Molecular Biology of Anhui Medical University,Key Laboratory of Gene Utilization for Severe Diseases of Anhui Province,Hefei 230032;Dept of Bioengineering, College of Life Sciences, Anhui Medical University,Hefei 230032)

机构地区：[1]安徽医科大学分子生物学实验室(省部共建教育部重要遗传病基因资源利用重点实验室),合肥230032 [2]安徽医科大学生命科学学院生物工程系,合肥230032

出　　处：《安徽医科大学学报》2022年第5期764-770,共7页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:82170484);安徽省重点研究与开发计划项目(编号:202004b11020025)。

摘　　要：目的探究褪黑素(MLT)对高糖诱导的人脐静脉内皮细胞(HUVEC)损伤的影响及其可能的分子机制。方法培养HUVEC并分为3组:对照组、模型组和治疗组。对照组用葡萄糖浓度为5.5 mmol/L的DMEM培养基处理,模型组用高糖(葡萄糖浓度为30 mmol/L)的DMEM培养基处理,治疗组用含有100μmol/L MLT的高糖DMEM培养基处理。MTT法检测高糖及MLT对HUVEC增殖的影响,试剂盒法测定细胞培养上清液中乳酸脱氢酶(LDH)的释放量,流式细胞术检测活性氧(ROS)的生成,Hoechst 33342/PI双染检测细胞凋亡和坏死情况,Western blot检测高糖及MLT对HUVEC内质网应激和细胞焦亡相关蛋白表达的影响。结果与对照组相比,模型组的细胞增殖水平下降;细胞培养上清液中LDH释放量增加;HUVEC中ROS生成增加;双染试验显示,蓝色荧光和红色荧光增加,GRP78、ATF4、CHOP、IRE1α、PERK、ATF6α、NLRP3、cleaved caspase-1、IL-1β蛋白表达量增加。与模型组相比,治疗组的细胞增殖水平升高;细胞培养上清液中LDH释放量减少;HUVEC中ROS生成减少;双染试验中蓝色荧光和红色荧光减少,GRP78、ATF4、CHOP、IRE1α、PERK、ATF6α、NLRP3、cleaved caspase-1、IL-1β蛋白表达量减少。结论MLT可以减轻高糖诱导的HUVEC损伤,其机制可能与调控内质网应激和细胞焦亡有关。Objective To explore the effect of melatonin(MLT)on human umbilical vein endothelial cell(HUVEC)injury induced by high glucose and to explore its probable molecular mechanism.Methods HUVEC were cultured and divided into three groups:control group,model group and treatment group.The control group(Ctrl group)was treated with DMEM medium at a normal low glucose level(5.5 mmol/L glucose),and the model group(HG group)was treated with DMEM medium at a high glucose level(30 mmol/L glucose).Treatment group(HG+MLT group)was treated with DMEM medium at a high glucose level containing 100μmol/L melatonin.The method of MTT was used to detect the effects of high glucose and melatonin on HUVEC proliferation.The release of lactate dehydrogenase(LDH)in cell culture supernatant was determined.The production of reactive oxygen species(ROS)was detected by flow cytometry.Hoechst 33342/PI double-staining was used to detect cell apoptosis and necrosis.Western blot was used to detect the effects of high glucose and melatonin on endoplasmic reticulum stress and cell pyroptosis related protein expression in HUVEC.Results Compared with the control group,cell proliferation level of the model group decreased,LDH releasing in cell culture supernatant increased,ROS production increased,the blue and red fluorescence increased in the double-staining,the expressions of GRP78,ATF4,CHOP,IRE1α,PERK,ATF6α,NLRP3,cleaved caspase-1,IL-1βincreased.Compared with model group,cell proliferation level of the treatment group increased,LDH releasing in cell culture supernatant decreased,ROS production decreased,the blue and red fluorescence decreased in the double-staining,the expressions of GRP78,ATF4,CHOP,IRE1α,PERK,ATF6α,NLRP3,cleaved caspase-1,IL-1βdecreased.Conclusion MLT can alleviate HUVEC damage induced by high glucose,and the probable mechanism may be related to the regulation of endoplasmic reticulum stress and cell pyroptosis.

关 键 词：高糖 人脐静脉内皮细胞 褪黑素 内质网应激 细胞焦亡 

分 类 号：R34[医药卫生—基础医学]