杨梅苷对高糖环境下视网膜微血管内皮细胞的保护作用及其调控机制  

Protective effect of myricitrin on retinal microvascular endothelial cells induced by high glucose and its regulation mechanism

作　　者：刘茜 刘长庚 李海军 董仰曾 张颖 Liu Qian;Liu Changgeng;Li Haijun;Dong Yangzeng;Zhang Ying(Department of Ophthalmology,Henan Provincial People's Hospital,People's Hospital of Zhengzhou University,Henan Eye Hospital,Henan Eye Institute,Zhengzhou 450003,China)

机构地区：[1]河南省人民医院眼科,郑州大学人民医院眼科,河南省立眼科医院,河南省眼科研究所,郑州450003

出　　处：《中华实验眼科杂志》2022年第7期623-631,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(21JCQN005);23456河南省人民医院人才工程项目。

摘　　要：目的探讨杨梅苷对高糖诱导视网膜微血管内皮细胞(HRMECs)损伤的抑制作用及其调控机制。方法将HRMECs分为正常对照组、高糖组及12.5μg/ml、25.0μg/ml、50.0μg/ml杨梅苷组,其中杨梅苷组细胞在含杨梅苷的高糖培养基中培养24 h。分别采用pcDNA和pcDNA-circZNF292转染HRMECs后使用含25 mmol/L D-葡萄糖的高糖培养基培养24 h,作为pcDNA组和pcDNA-circZNF292组。分别采用siR-NC和siR-circZNF292转染至HRMECs后加入含50.0μg/ml杨梅苷和25 mmol/L D-葡萄糖的培养基中培养24 h,作为杨梅苷+siR-NC组和杨梅苷+siR-circZNF292组。采用流式细胞术检测细胞凋亡率;利用酶联免疫吸附测定试剂盒检测细胞中丙二醛(MDA)浓度和超氧化物歧化酶(SOD)活性;采用实时荧光定量PCR法检测circZNF292和miR-23b-3p的基因表达量;采用双荧光素酶报告实验检测circZNF292与miR-23b-3p的靶向关系;采用Western blot法检测B细胞淋巴瘤-2(bcl-2)和bcl-2相关蛋白(bax)蛋白表达量。结果正常对照组、高糖组及12.5μg/ml、25.0μg/ml、50.0μg/ml杨梅苷组细胞bax和bcl-2蛋白相对表达量、细胞凋亡率、MDA浓度、SOD活性值以及circZNF292和miR-23b-3p相对表达量总体比较,差异均有统计学意义(F=105.707、111.835、74.515、109.651、135.020、219.919、116.304,均P<0.001)。随着杨梅苷剂量的逐渐增加,细胞中bax蛋白相对表达量、细胞凋亡率、MDA浓度和miR-23b-3p相对表达量逐渐下降,bcl-2蛋白相对表达量、SOD活性值和circZNF292相对表达量逐渐升高,不同剂量杨梅苷组间比较差异均有统计学意义(均P<0.05)。共转染野生型载体WT-circZNF292细胞中,miR-23b-3p组细胞中相对荧光素酶活性值为0.35±0.03,低于miR-NC组的0.96±0.09,差异有统计学意义(t=11.137,P<0.001)。与pcDNA组比较,pcDNA-circZNF292组细胞中circZNF292相对表达量、bcl-2蛋白相对表达量和MDA浓度明显升高,miR-23b-3p相对表达量、bax蛋白相对表达量、细胞凋亡率和SODObjective To explore the effect of myricitrin on the injury of human retinal microvascular endothelial cells(HRMECs)induced by high glucose and its regulation mechanism.Methods HRMECs were divided into normal control group,high glucose group and 12.5μg/ml,25.0μg/ml and 50.0μg/ml myricitrin groups.HRMECs transfected with pcDNA and pcDNA-circZNF292,respectively and then cultured in high-glucose medium containing 25 mmol/L D-glucose for 24 h ours were assigned as pcDNA group and pcDNA-circZNF292 group.HRMECs transfected with siR-NC and siR-circZNF292,respectively and then cultured in medium containing 50.0μg/ml myricitrin and 25 mmol/L D-glucose for 24 h ours were assigned as myricitrin+siR-NC group and myricitrin+siR-circZNF292 group.The cell apoptosis rate was detected by flow cytometry.The concentration of malondialdehyde(MDA)and the activity of superoxide dismutase(SOD)in cells were detected by enzyme-linked immunosorbent assay(ELISA)kits.The expression levels of circZNF292 and miR-23b-3p were detected by real-time fluorescence quantitative PCR.The targeting relationship between circZNF292 and miR-23b-3p was detected by dual-luciferase reporter assay.The relative expression levels of B-cell lymphoma-2(bcl-2)and bcl-2-related X protein(bax)were assayed by Western blot.Results Significant differences were found in the relative expressions of bax and bcl-2 proteins,cell apoptosis rate,MDA concentration,SOD activity,circZNF292 and miR-23b-3p among normal control group,high glucose group and 12.5μg/ml,25.0μg/ml,50.0μg/ml myricitrin groups(F=105.707,111.835,74.515,109.651,135.020,219.919,116.304;all at P<0.001).With the increase of myricitrin concentration,the relative expression levels of bax protein,cell apoptosis rate,MDA concentration and miR-23b-3p in cells gradually decreased,while the relative expression levels of bcl-2 protein,SOD activity and circZNF292 increased,with statistically significant differences among groups with different concentrations of myricitrin(all at P<0.05).In the co-transfected wild

关 键 词：杨梅苷 细胞凋亡 氧化应激 人视网膜微血管内皮细胞 circZNF292 miR-23b-3p 

分 类 号：R285[医药卫生—中药学]