迷迭香酸通过调控自噬抑制高糖诱导的HRMEC血管生成  

Rosmarinic acid inhibits high glucose induced angiogenesis of HRMEC by regulating autophagy

作　　者：李蓉[1] 陈琳[1] 李亚[1] 马雅玲[2] 周凌霄[1] LI Rong;CHEN Lin;LI Ya(The First Affiliated Hospital of Xi'an Medical University,Xi'an 710077,China)

机构地区：[1]西安医学院第一附属医院,西安710077 [2]宁夏医科大学总医院,银川750004

出　　处：《中国中医眼科杂志》2020年第10期691-698,共8页China Journal of Chinese Ophthalmology

基　　金：国家自然科学基金(81500726);国家自然科学基金配套基金(XYFYPT-2020-01);陕西省卫生健康科研基金项目(2018D074);西安市科技局医学研究项目(201805097YX5SF31[4])。

摘　　要：目的探讨迷迭香酸(RA)对高糖培养条件下人视网膜微血管内皮细胞(HRMEC)血管生成的影响及与自噬的关系。方法将HRMEC随机分为正常对照组(NC组)、高糖组(HG组)、低浓度RA组(LC组)、中浓度RA组(MC组)、高浓度RA组(HC组)。NC组细胞,在低糖培养基中培养;HG组细胞,在培养基中加入30 mmol/L D-葡萄糖培养;LC组细胞,在培养基中加入25μmol/L RA+30 mmol/L D-葡萄糖培养;MC组细胞,在培养基中加入50μmol/L RA+30 mmol/L D-葡萄糖培养;HC组细胞,在培养基中加入100μmol/L RA+30 mmol/L D-葡萄糖培养。培养48 h。分别采用MTT、Transwell及Matrigel法检测细胞增殖、迁移和管腔形成;电镜下各组细胞内的自噬泡形成情况;采用Western blot法检测各组细胞的自噬相关蛋白微管相关蛋白1轻链3(LC3)及Beclin-1的表达。结果(1)HRMEC增殖:与NC组比较,HG组细胞增殖率增加(t=-98.465,P=0.000);与HG组比较,LC组、MC组和HC组细胞增殖率依次降低(t LC=8.198,P=0.001;t MC=12.427,P=0.000;t HC=44.898,P=0.000),差异有统计学意义。(2)HRMEC迁移:与NC组比较,HG组细胞迁移数均增加(t=-22.958,P=0.000);与HG组比较,LC组、MC组、HC组细胞迁移数依次降低(t LC=17.504,t MC=22.463,t HC=20.330,均P=0.000),差异有统计学意义。(3)HRMEC管腔形成:与NC组比较,HG组细胞管腔形成数增加(t HG=-13.924,P=0.000);与HG组比较,LC组、MC组和HC组细胞管腔形成数依次降低(t LC=3.528,P=0.024;t MC=7.437,P=0.002;t HC=11.213,P=0.000),差异有统计学意义。(4)HRMEC自噬泡:与NC组相比,HG组细胞质内自噬泡增多,LC组、MC组和HC组自噬泡较HG组减少,且RA浓度越高,自噬泡越少。(5)自噬蛋白LC3表达:与NC组比较,HG组LC3-II/LC3-I比值增加(t=-86.500,P=0.000);与HG组比较,LC组、MC组和HC组的LC3-II/LC3-I比值依次降低(t LC=40.000,t MC=35.002,t HC=37.165,均P=0.000),差异均有统计学意义。(6)自噬蛋白Beclin-1表达:与NC组比较,HG组表达增加(t=-85.865,P=0.000);与HG组比较,LC�OBJECTIVE To study the effect of rosmarinic acid(RA)on the angiogenesis of human retinal microvascular endothelial cells(HRMEC)and its relationship with autophagy.METHODS In vitro cultured HRMEC cells were randomly divided into the control group(NC),the high-glucose group(HG),low concentration of RA group(LC),medium concentration of RA group(MC)and high concentration of RA group(HC).In the NC group,cells were cultured in the low glucose medium.In the HG group,cells were cultured with 30 mmol/L D-glucose in the medium.In the LC group,cells were cultured with 25μmol/L of RA and 30 mmol/L D-glucose.In the MC group,cells were cultured with 50μmol/L of RA and 30 mmol/L D-glucose.In the HC group,cells were cultured with 100μmol/L of RA and 30 mmol/L D-glucose.After 48h of culture,MTT,Transwell and Matrigel assays were used to detect cell proliferation,migration and tube formation respectively.The formation of autophagic vacuoles in HRMECs was observed by electron microscopy.Western blot was used to detect the expression of autophagy related proteins microtubule-related protein 1 light chain 3(LC3)and Beclin-1 in each group.RESULTS(1)Proliferation of HRMEC:compared with the NC group,the cell proliferation rate was increased in the HG group(t=-98.465,P=0.000).Compared with the HG group,the cell proliferation rates of LC,MC and HC group was decreased successively with statistically significant differences(t LC=8.198,P=0.001;t MC=12.427,P=0.000;t HC=44.898,P=0.000).(2)Migration of HRMEC:compared with the NC group,the number of migrated cells was increased in the HG group(t=-22.958,P=0.000).Compared with the HG group,the number of migrated cells of LC,MC and HC group was decreased successively(t LC=17.504,t MC=22.463,t HC=20.330,all P=0.000),with statistically significant difference.(3)Tube formation of HRMEC:compared with the NC group,the number of tube formation was increased in the HG group(t HG=-13.924,P=0.000).Compared with the HG group,the number of tube formation of LC,MC and HC group was decreased successively wi

关 键 词：迷迭香酸 自噬 高糖 视网膜内皮细胞 血管生成 

分 类 号：R285.5[医药卫生—中药学]