益景汤调控IL-6/STAT3/ERK途径抑制高糖条件下iBRB模型损伤的研究  

Study on Yijing Decoction Regulating IL-6/STAT3/ERK Pathway to Inhibit Damage to an iBRB Model under High-glucose Conditions

作　　者：何建忠 黄婷 程艳芳 罗丽梅 潘铭东[1] 刘光辉[1] 郑永征[1] HE Jianzhong;HUANG Ting;CHENG Yanfang;LUO Limei;PAN Mingdong;LIU Guanghui;ZHENG Yongzheng(The People's Hospital Affiliated to Fujian University of Traditional Chinese Medicine,Fuzhou 350004,China)

机构地区：[1]福建中医药大学附属人民医院,福州350004 [2]福建中医药大学第一临床医学院,福州350004

出　　处：《中国中医眼科杂志》2024年第12期1101-1107,1113,共8页China Journal of Chinese Ophthalmology

基　　金：国家自然科学基金项目(81774359);福建省自然科学基金项目(2022J01352)。

摘　　要：目的探讨益景汤对高糖条件下体外血-视网膜屏障(iBRB)模型损伤的影响及作用机制。方法大鼠视网膜微血管周细胞-内皮细胞(rRMPs-rRMECs)共培养构建体外iBRB模型,分为对照组(CG)、模型组(MG)和益景汤组(YG),在培养基中分别加入5.5 mmol/L葡萄糖、33.0 mmol/L葡萄糖、4 mg/mL益景汤+33.0 mmol/L葡萄糖培养48 h。细胞计数试剂-8(CCK-8)法观察视网膜微血管细胞活性,原位末端转移酶标记技术(TUNEL)观察视网膜微血管细胞凋亡情况,实时荧光定量PCR(RT-qPCR)法检测白细胞介素-6(IL-6)、信号传导和转录激活因子3(STAT3)、细胞外信号调节激酶(ERK)mRNA的表达,Western Blot法检测IL-6、p-STAT3、p-ERK及紧密连接蛋白的表达,激光共聚焦显微镜观察视网膜三维微血管模型形态。结果(1)细胞活性:MG组细胞活性低于CG组(t=3.016,P=0.009),YG组细胞活性高于MG组(t=11.810,P=0.000),差异均有统计学意义。(2)细胞凋亡:MG组细胞凋亡率高于CG组(t=54.609,P=0.000),YG组细胞凋亡率低于MG组(t=31.455,P=0.000),差异均有统计学意义。(3)相关mRNA:MG组rRMECs-rRMPs的IL-6、STAT3、ERK mRNA表达量均高于CG组(t_(IL-6)=13.097、t_(STAT3)=29.032、t_(ERK)=11.005,均P=0.000),YG组rRMECs-rRMPs的IL-6、STAT3、ERK mRNA表达量均低于MG组(t_(IL-6)=4.594,P=0.004;t_(STAT3)=20.695,P=0.000;t_(ERK)=4.066,P=0.020),差异均有统计学意义。(4)相关蛋白:MG组rRMECs-rRMPs的IL-6、p-STAT3、p-ERK蛋白表达水平高于CG组(t_(IL-6)=6.009,P=0.001;t_(p-ERK)=7.816、tp-STAT3=11.741,均P=0.000),ZO-1、Occludin、Claudin-5蛋白表达水平低于CG组(t_(ZO-1)=6.818、t_(Occludin)=6.710,均P=0.001;t_(Claudin-5)=11.766,P=0.000),差异均有统计学意义;YG组rRMECs-rRMPs的IL-6、p-STAT3、p-ERK蛋白表达水平低于MG组(t_(IL-6)=2.973,P=0.025;tp-STAT3=6.685,P=0.001;t_(p-ERK)=3.859,P=0.008),ZO-1、Occludin、Claudin-5蛋白表达水平高于MG组(t_(ZO-1)=2.640,P=0.039;t_(Occludin)=3.397,P=0.016;t_(Claudin-5)=6.241,P=0.001),差异均有�OBJECTIVE To investigate the effect and mechanism of Yijing Decoction on an in vitro blood-retinal barrier(iBRB)model under high-glucose conditions.METHODS An in vitro iBRB model was established through co-culture of rat retinal microvascular pericytes and endothelial cells(rRMPsrRMECs).The study included three groups,control group(CG),model group(MG),and Yijing Decoction group(YG).The medium for each group contained 5.5 mmol/L glucose,33.0 mmol/L glucose,and 4 mg/mL Yijing Decoction+33.0 mmol/L glucose,respectively,and the cells were cultured for 48 hours.Cell viability was assessed by the Cell Counting Kit-8(CCK-8)assay.Retinal microvascular cell apoptosis was observed using terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL).The mRNA expression levels of interleukin-6(IL-6),signal transducer and activator of transcription 3(STAT3),and extracellular signal-regulated kinase(ERK)were measured by real-time quantitative PCR(RT-qPCR).Protein expression levels of IL-6,phosphorylated STAT3(pSTAT3),phosphorylated ERK(pERK),and tight junction proteins were determined by Western Blot.The morphology of the retinal three-dimensional microvascular model was observed by laser confocal microscopy.RESULTS(1)Cell viability:The cell viability in the MG was significantly lower than that in the CG(t=3.016,P=0.009),whereas it was significantly higher in the YG compared to the MG(t=11.810,P=0.000).(2)Cell apoptosis:The apoptosis rate in the MG was significantly higher than that in the CG(t=54.609,P=0.000),while it was significantly lower in the YG compared to the MG(t=31.455,P=0.000).(3)mRNA expression:The mRNA expression levels of IL-6,STAT3,and ERK in rRMECs-rRMPs were significantly higher in the MG than in the CG(t_(IL-6)=13.097,t_(STAT3)=29.032,t_(ERK)=11.005,all P=0.000).The expression levels of IL-6,STAT3,and ERK mRNA were significantly lower in the YG compared to the MG(t_(IL-6)=4.594,P=0.004;t_(STAT3)=20.695,P=0.000;t_(ERK)=4.066,P=0.020).(4)Protein Expression:The protein expression levels of IL-6,p-STAT3,and

关 键 词：益景汤 糖尿病视网膜病变 血-视网膜屏障 IL-6/STAT3/ERK 

分 类 号：R774.13[医药卫生—眼科] R285.5[医药卫生—临床医学]