摘要
目的探究补阳还五汤(BYHW)对高糖培养的人视网膜微血管内皮细胞(HRCECs)自噬影响和血管形成的干预作用。方法将HRCECs随机分为对照组(CG)、模型组(MG)、BYHW组、自噬抑制剂3-甲基腺嘌呤(3-MA)组。除CG组外,其余各组细胞于25 g/L葡萄糖环境中培养建立高糖损伤模型,BYHW组在MG组的基础上加入5 mg/mL的BYHW水提液,3-MA组加入40μM的3-MA,各组细胞均孵育24 h。采用血管形成实验检测HRCECs血管生成情况,划痕实验检测细胞迁移能力,实时荧光定量PCR(RT-qPCR)和Western Blot法分别检测细胞中微管相关蛋白1A/1B-轻链3(LC3)、苄氯素1(Becline-1)的mRNA和蛋白表达水平。结果(1)血管形成:与CG组比较,MG组血管形成数升高(t=7.747,P=0.000);与MG组比较,BYHW、3-MA组血管形成数降低(t_(BYHW)=-6.303,P=0.000;t_(3-MA)=-4.596,P=0.002),差异均有统计学意义。而BYHW、3-MA组血管形成数比较,差异无统计学意义(P>0.05)。(2)迁移能力:与CG组比较,MG组HRCECs迁移率升高(t=14.035,P=0.000);与MG组比较,BYHW、3-MA组HRCECs迁移率降低(t_(BYHW)=-9.247,t_(3-MA)=-5.358,均P=0.000);与BYHW组比较,3-MA组HRCECs迁移率升高(t=-3.890,P=0.003),差异均有统计学意义。(3)自噬:与CG组比较,MG组LC3 mRNA、蛋白表达水平升高(t_(mRNA)=8.249,t_(蛋白)=14.890,均P=0.000),Beclin-1蛋白表达水平升高(t=7.114,P=0.000);与MG组比较,BYHW、3-MA组的LC3 mRNA和蛋白表达水平均降低(BYHW:t_(mRNA)=-9.298,t_(蛋白)=-11.727,均P=0.000。3-MA:t_(mRNA)=-3.718,P=0.006;t_(蛋白)=-7.511,P=0.000),BYHW组的Beclin-1蛋白表达水平下降(t=-4.115,P=0.003);与BYHW组比较,3-MA组HRCECs的Beclin-1蛋白表达水平升高(t=-2.448,P=0.038),差异均有统计学意义。结论BYHW延缓非增殖期糖尿病视网膜病的发展可能与其调控HRCECs自噬减少血管形成和迁移有关。
OBJECTIVE To explore the effect of Buyang Huanwu Decotion(BYHW)on autophagy and angiogenesis of human retinal capillary endothelial cells(HRCECs)cultured in high glucose.METHODS The HRCECs was randomly divided into control group(CG),model group(MG),BYHW group and 3-MA group.Except for the control group,the cells in other groups were cultured in 25 g/L glucose to establish high glucose damage model;Beyond that,5 mg/mL BYHW water extract was added in BYHW group and 40μM 3-MA was added in 3-MA group,the cells of each group were incubated for 24 h.Angiogenesis was detected by tube-forming experiments,migration ability by scratch experiments;RT-qPCR and Western Blot were used to detect LC3 and Becline-1 mRNA and protein expression respectively.RESULTS(1)Angiogenesis:Compared with the CG,the number of blood vessels formed in the MG increased(t=7.747,P=0.000);Compared with the MG,the number of blood vessels formed in the BYHW and 3-MA groups decreased(t_(BYHW)=-6.303,P=0.000;t_(3-MA)=-4.596,P=0.002),the differences were statistically significant.There was no significant difference in the number of blood vessels formation between BYHW and 3-MA groups(P>0.05).(2)Migration ability:Compared with the CG,the migration rate of HRCECs in the MG increased(t=14.035,P=0.000);Compared with the MG,the migration rate in the BYHW and 3-MA groups decreased(t_(BYHW)=-9.247,t_(3-MA)=-5.358,both P=0.000);Compared with the BYHW group,the migration rate in the 3-MA group increased(t=-3.890,P=0.003),and the differences were statistically significant.(3)Autophagy:Compared with the CG,the expression levels of LC3 mRNA and protein in the MG increased(t_(mRNA)=8.249,t_(protein)=14.890,both P=0.000),and the expression level of Beclin-1 protein increased(t=7.114,P=0.000);Compared with the MG,the expression levels of LC3 mRNA and protein in BYHW and 3-MA groups decreased(BYHW:t_(mRNA)=-9.298,t_(protein)=-11.727,both P=0.000;3-MA:t_(mRNA)=-3.718,P=0.006;t_(protein)=-7.511,P=0.000),the expression level of Beclin-1 protein in BYHW group decreased(t=-
作者
陈凯铭
陈子扬
石颖
胡艳红
胡俊
陈胜
CHEN Kaiming;CHEN Zi-yang;SHI Ying;HU Yanhong;HU Jun;CHEN Sheng(Fujian University of Traditional Chinese Medicine,Fuzhou 350122,China)
出处
《中国中医眼科杂志》
2023年第2期105-110,共6页
China Journal of Chinese Ophthalmology
基金
福建省自然科学基金项目(2022J01834)
福建省中医药科研项目计划(2021ZYJC08)
福建省卫生健康委员会科技计划项目(2020CXB034)。
关键词
补阳还五汤
高糖
人视网膜微血管内皮细胞
自噬
血管形成
糖尿病视网膜病变
Buyang Huanwu Decoction
high glucose
human retinal capillary endothelial cells
autophagy
angiogenesis
diabetic retinopathy
