TRIM46对HDAC1泛素化和高糖诱导HRCECs通透性的影响及作用机制  

Effect and Mechanism of TRIM46 on HDAC1 Ubiquitination and High Glucose Induced Permeability of HRCECs

作　　者：许畅 周峰 张炜 李晶晶 Xu Chang;Zhou Feng;Zhang Wei;Li Jingjing(Department of Ophthalmology,Xiangyang Central Hospital,Affiliated Hospital of Hubei University of Arts and Science,Xiangyang 441021,China)

机构地区：[1]湖北文理学院附属医院襄阳市中心医院眼科,襄阳441021

出　　处：《中华眼视光学与视觉科学杂志》2025年第2期96-103,共8页Chinese Journal Of Optometry Ophthalmology And Visual Science

基　　金：襄阳市医疗卫生领域科技计划项目(重点项目)(2020YL27)。

摘　　要：目的:研究人类三重基序蛋白46(TRIM46)对组蛋白去乙酰化酶1(HDAC1)泛素化和高糖(HG)诱导的人视网膜毛细血管内皮细胞(HRCECs)通透性的影响及作用机制。方法:实验研究。2020年3月体外培养HRCECs后,依据葡萄糖培养浓度分为正常组(5.5 mmol/L正常葡萄糖培养)、HG组(25.0 mmol/L高浓度葡萄糖培养)、阴性组(转染空质粒vector或shRNA)、oeTRIM46组(转染TRIM46过表达质粒)、shTRIM46组(转染TRIM46的RNAi序列以下调TRIM46表达)、oeTRIM46+vector组(共转染TRIM46过表达质粒和空质粒vector)、oeTRIM46+HDAC1组(共转染TRIM46过表达质粒和HDAC1过表达质粒)。蛋白免疫印迹分析(Western blot)和实时荧光定量PCR(qRT-PCR)分别检查血管内皮钙黏蛋白(VE-cadherin)、TRIM46、HDAC1等蛋白或TRIM46 mRNA表达;免疫共沉淀法测定HDAC1泛素化表达;跨内皮电阻(TER)检测单层细胞电阻(Ω×cm2);Transwell通透性实验观察HRCECs单层通透性。采用单因素方差分析或t检验比较各组间TRIM46表达、HRCECs单层通透性、HDAC1泛素化表达等数据的差异。结果:与正常组相比,HG处理后可诱导HRCECs中TRIM46表达上调(t=7.06,P<0.001)和TER升高(t=8.13,P=0.001),明显增加HRCECs通透性(t=12.03、6.57、12.38,P<0.05);oeTRIM46组上调TRIM46表达后,在HG条件下,单层HRCECs的TER和异硫氰酸荧光素葡聚糖FD-20通透性增加,反之亦然(均P<0.05)。TRIM46过表达可促进HDAC1泛素化,敲低则显示相反的效果,在oeTRIM46组和HG组中,HDAC1泛素化较正常组均明显增加(均P<0.05);此外,与阴性组相比,oeTRIM46组p-VE-cadherinTyr731表达水平、单层细胞TER以及FD-20荧光强度均明显增加,TRIM46表达下调后上述变化相反(均P<0.001),而HDAC1表达上调后可改善TRIM46过表达对HRCECs通透性的影响(P<0.001)。结论:HG可诱导HRCECs中TRIM46表达水平升高,而高水平TRIM46可促进HDAC1泛素化和降解,并增加HRCECs通透性。Objective:To explore the impact of human triple motif protein 46(TRIM46)on histone deacetylase 1(HDAC1)ubiquitination and the permeability of human retinal capillary endothelial cells(HRCECs)induced by high glucose(HG)conditions,and to elucidate its underlying mechanism.Methods:This was an experimental study.In March 2020,HRCECs were cultured in vitro and subsequently divided into groups based on glucose culture concentration and experimental manipulations.The groups included:the normal group(cultured in 5.5 mmol×L-1 normal-concentration glucose),the HG group(cultured in 25.0 mmol×L-1 high-concentration glucose),the negative group(transfected with empty plasmid vector or shRNA),the oeTRIM46 group(transfected with a TRIM46 overexpressed plasmid),the shTRIM46 group(transfected with a TRIM46-specific RNAi sequence to downregulate TRIM46 expression),the oeTRIM46+vector group(co-transfected with TRIM46 overexpression plasmid and an empty plasmid vector),and the oeTRIM46+HDAC1 group(co-transfected with TRIM46 overexpressed plasmid and HDAC1 overexpressed plasmid).Western blot and quantitative real-time fluorescent PCR(qRT-PCR)were employed to examine the VE-cadherin,TRIM46,and HDAC1 proteins,as well as TRIM46 mRNA expressions.The ubiquitination status of HDAC1 was determined through immunocoprecipitation.Transendothelial electrical resistance(TER)measurements were conducted to assess the resistance(Ω×cm 2)of the monolayer of HRCECs.The permeability of HRCECs monolayer was observed by Transwell permeability assays.Statistical comparisons among groups for TRIM46 expression,HRCECs monolayer permeability,and HDAC1 ubiquitination expression were performed using one-way ANOVA or t-tests.Results:Compared with the normal group,HG treatment significantly upregulated TRIM46 expression(t=7.06,P<0.001)and increased TER values in HRCECs(t=8.13,P=0.001),which consequently led to a marked enhancement in permeability of HRCECs(t=12.03,6.57,12.38,P<0.05).In the oeTRIM46 group,where TRIM46 expression was upregulated,the TER of monol

关 键 词：视网膜细胞 人类三重基序蛋白46 组蛋白去乙酰化酶1泛素化 通透性 

分 类 号：R73[医药卫生—肿瘤]