ghrelin-GHSR-1a对高糖环境下视网膜血管内皮细胞的保护作用  

Protective effects of ghrelin and growth hormone secretin receptor 1a on retinal microvascular endothelial cells under high glucose conditions

作　　者：李蓉[1] 姚国敏[1] 周凌霄[1] 张敏[2] 闫瑾 LI Rong;YAO Guomin;ZHOU Lingxiao;ZHANG Min;YAN Jin(Department of Ophthalmology,the First Affiliated Hospital of Xi’an Medical University,Xi’an 710077,Shaanxi Province,China;Department of Endocrinology,the First Affiliated Hospital of Xi’an Medical University,Xi’an 710077,Shaanxi Province,China;Department of Optometry Teaching and Research Section,College of Medical Technology,Xi’an Medical University,Xi’an 710021,Shaanxi Province,China)

机构地区：[1]西安医学院第一附属医院眼科,陕西省西安市710077 [2]西安医学院第一附属医院内分泌科,710077 [3]西安医学院医学技术学院眼视光教研室,陕西省西安市710021

出　　处：《眼科新进展》2022年第3期205-209,共5页Recent Advances in Ophthalmology

基　　金：西安市科技局医学研究项目(编号:21YXYJ0127)。

摘　　要：目的观察ghrelin及其受体生长激素分泌素受体1a(GHSR-1a)在高糖环境下对视网膜血管内皮细胞的保护作用。方法将体外培养的人视网膜血管内皮细胞(HRMECs)分为对照组和高浓度葡萄糖组。对照组细胞在含5.5 mmol·L^(-1)葡萄糖的M199培养基中培养48 h,高浓度葡萄糖组细胞在含30.0 mmol·L^(-1)葡萄糖的M199培养基中培养48 h,采用免疫荧光染色法检测两组细胞中GHSR-1a的表达。然后另取HRMECs随机分为5组:正常对照(NC)组、高糖(HG)组、HG+ghrelin组、HG+ghrelin+siGHSR-1a组和HG+ghrelin+NC-siGHSR-1a组,孵育48 h。NC组处理同对照组,HG组处理同高浓度葡萄糖组,HG+ghrelin组细胞在含30.0 mmol·L^(-1)葡萄糖、10.0 nmol·L^(-1) ghrelin的M199培养基中培养,HG+ghrelin+siGHSR-1a组细胞先转染GHSR-1a-特异性siRNA,24 h后在含30.0 mmol·L^(-1)葡萄糖和10.0 nmol·L^(-1) ghrelin的M199培养基中培养,HG+ghrelin+NC-siGHSR-1a组细胞先转染非特异性序列,24 h后在含30.0 mmol·L^(-1)葡萄糖和10.0 nmol·L^(-1) ghrelin的M199培养基中培养。分别采用CCK-8和Annexin-V/FITC-PI凋亡检测试剂盒检测各组HRMECs细胞活力和细胞凋亡情况。Western blot检测凋亡相关蛋白Bax和Bcl-2的表达。结果高浓度葡萄糖组的GHSR-1a表达低于对照组(P<0.05)。NC组、HG组、HG+ghrelin组、HG+ghrelin+siGHSR-1a组和HG+ghrelin+NC-siGHSR-1a组的细胞活力分别为1.00、69.87%±0.68%、92.31%±3.62%、75.98%±4.67%和90.87%±1.95%,总体比较差异有统计学意义(P<0.001);两两比较显示,除HG+ghrelin组与HG+ghrelin+NC-siGHSR-1a组的细胞活力差异无统计学意义(P>0.05)外,其余各组细胞活力比较差异均有统计学意义(均为P<0.05)。NC组、HG组、HG+ghrelin组、HG+ghrelin+siGHSR-1a组和HG+ghrelin+NC-siGHSR-1a组的细胞凋亡率分别为5.76%±0.36%、20.53%±0.38%、10.84%±0.52%、14.93%±0.18%和11.03%±0.62%,总体比较差异有统计学意义(F=468.88,P<0.001);两两比较显示,除HG+ghrelin组与HG+ghrelin+NC-sObjective To investigate the protective effects of ghrelin and its receptor,the growth hormone secretin receptor 1a(GHSR-1a),on retinal microvascular endothelial cells under high glucose conditions.Methods Human retinal microvascular endothelial cells(HRMECs)cultured in vitro were divided into the control and high glucose groups.Cells in the control group were cultured in M199 medium containing 5.5 mmol·L^(-1) glucose for 48 h,while cells in the high glucose group were cultured in M199 medium containing 30.0 mmol·L^(-1) glucose for 48 h.The expression of GHSR-1a in each group was detected by immunofluorescence staining.Additional HRMECs were randomly divided into five groups and incubated for 48 h:normal control(NC)group,high glucose(HG)group,HG+ghrelin group,HG+ghrelin+siGHSR-1a group,and HG+ghrelin+NC-siGHSR-1a group.Cells in the NC group were treated the same as the control group,and cells in the HG group were treated the same as the high glucose group.Cells in the HG+ghrelin group were cultured in M199 medium containing 30.0 mmol·L^(-1) glucose and 10.0 nmol·L^(-1) ghrelin.Cells in the HG+ghrelin+siGHSR-1a group were first transfected with GHSR-1a-specific siRNA,and 24 h later,cultured in M199 medium containing 30.0 mmol·L^(-1) glucose and 10.0 nmol·L^(-1) ghrelin.Cells in the HG+ghrelin+NC-siGHSR-1a group were transfected with non-specific sequence,and 24 h later,cultured in M199 medium containing 30.0 mmol·L^(-1) glucose and 10.0 nmol·L^(-1) ghrelin.The viability and apoptosis of HRMECs were detected by CCK-8 and Annexin-V/FITC-PI kit,respectively.Western blot was used to detect the expression of apoptosis-related proteins Bax and Bcl-2.Results The expression of GHSR-1a in the high glucose group was lower than that in the control group(P<0.05).The cell viability in the NC,HG,HG+ghrelin,HG+ghrelin+siGHSR-1a,and HG+ghrelin+NC-siGHSR-1a groups were 1.00,(69.87±0.68)%,(92.31±3.62)%,(75.98±4.67)%,and(90.87±1.95)%,respectively,and the overall difference was statistically significant(P<0.001).The cell

关 键 词：GHRELIN 生长激素分泌素受体1a 糖尿病视网膜病变 高糖 视网膜血管内皮细胞 

分 类 号：R774.1[医药卫生—眼科]