蛋白质精氨酸甲基转移酶4及其抑制剂在角膜新生血管中的作用及机制  

The effects of protein arginine methyltransferases four and its inhibitors on corneal neovascularization

作　　者：毛介文 高月兰 万珊珊[1] 杨燕宁[1] Mao Jiewen;Gao Yuelan;Wan Shanshan;Yang Yanning(Department of Ophthalmology,Renmin Hospital of Wuhan University,Wuhan 430061,China)

机构地区：[1]武汉大学人民医院眼科中心,武汉430061

出　　处：《中华实验外科杂志》2024年第10期2309-2315,共7页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(82101081、82371023)。

摘　　要：目的:探讨蛋白质精氨酸甲基转移酶4(PRMT4)及其抑制剂在碱烧伤诱导的角膜新生血管(CNV)形成中的作用及机制。方法:选用武汉大学动物实验中心提供的健康清洁级8周龄雄性C57BL/6小鼠90只,采用随机数字表法将40只小鼠分为碱烧伤后1、4、7 d和正常对照组,每组10只,造模后第1、4、7天处死小鼠并取其双眼角膜;另将50只小鼠采用随机数字表法分为碱烧伤组、二甲基亚砜(DMSO)组、TP-064(5 mg/kg)组、TP-064(10 mg/kg)组和正常对照组,每组10只,造模后第7天处死小鼠并取其双眼角膜。采用氢氧化钠(NaOH)碱烧伤法构建CNV模型,分别结膜下注射PRMT4抑制剂TP-064(5 mg/kg和10 mg/kg)和DMSO至碱烧伤第7天,全部小鼠于造模后第1、4、7天行裂隙灯显微镜检查并测量CNV面积;采用组织病理染色法观察角膜结构变化和炎性细胞的表达;采用角膜冰冻切片免疫荧光染色法、实时荧光定量聚合酶链反应(PCR)法及蛋白质印迹法(Western blot)检测角膜组织中PRMT4、血管内皮生长因子A(VEGFA)、NOD样受体蛋白3(NLRP3)、天冬氨酸特异性半胱氨酸蛋白酶-1(Caspase-1)、焦孔素D(GSDMD)及其N端(GSDMD-N)的表达及定位;各组间测量指标总体差异比较采用单因素方差分析,多重比较采用独立样本t检验。结果:碱烧伤组PRMT4、VEGFA、NLRP3、Caspase-1、GSDMD的mRNA表达水平高于正常对照组(4.53±0.09比0.63±0.04,F=582.335,P<0.05;5.27±0.12比0.79±0.12,F=120.371,P<0.05;4.23±0.21比0.42±0.05,F=52.759,P<0.05;4.72±0.19比0.78±0.07,F=32.652,P<0.05;3.78±0.16比0.53±0.20,F=74.133,P<0.05);PRMT4抑制剂TP-064(10 mg/kg和5 mg/kg)组CNV面积低于碱烧伤组、DMSO组及正常对照组(1.83±0.27比2.33±0.49比4.84±0.62比4.76±0.15,F=143.064,P<0.05);免疫荧光染色法表明PRMT4抑制剂TP-064(10 mg/kg和5 mg/kg)组PRMT4、VEGFA及焦亡相关分子的表达低于碱烧伤+DMSO组和正常对照组。结论:PRMT4抑制剂可阻碍碱烧伤诱导的角膜炎性反应及Objective:Exploring the effect and mechanism of protein arginine methyltransferase 4(PRMT4)and its inhibitor in corneal neovascularization(CNV)induced by alkali burns.Methods:Ninety clean male C57BL/6 miceaged 8 weeks provided by the animal experiment center of wuhan university were selected for the study.Forty mice were divided into 1,4,7 days groups after alkali burns and normal control group according to randomized number table,with 10 mice in each group.After modeling,the mice were sacrificed on the 1,4,and 7 day and the corneas of both eyes were harvested;Additionally,fifty mice were randomly divided into groups,including alkali burn group,dimethyl sulfoxide(DMSO)group,TP-064(5 mg/kg)group,TP-064(10 mg/kg)group,and a normal control group,each consisting of 10 mice.The mice were sacrificed on the 7th day after modeling and the corneas of both eyes were harvested.The alkali burn-induced CNV was established using NaOH alkali burn method.PRMT4 inhibitor TP-064(5 mg/kg and 10 mg/kg)and DMSO were injected subconjunctivally until the 7th day post-alkali burns.CNV was examined 1,4,7 days after alkali burns by the slit lamp microscope and the CNV related area in the cornea was calculated.Immunofluorescence staining on corneal frozen sections was utilized to detect the expression and localization of PRMT4,vascular endothelial growth factor A(VEGFA),NOD-like receptor protein 3(NLRP3),and gasdermin D(GSDMD).Tissue pathology staining was utilized to observe corneal structural changes and inflammatory cell expression.Real-time quantitative polymerase chain reaction(PCR)was utilized to measure the relative expression levels of PRMT4,VEGFA,NLRP3,cysteinyl aspartate specific proteinase(Caspase)-1,and GSDMD mRNA in mouse corneal tissues.Western blotting was used to analyze the the expression of PRMT4,VEGFA,NLRP3,Caspase-1,GSDMD,and gasdermin-D N-terminal(GSDMD-N)proteins in the cornea.The overall differences of the measured indicators among the groups were compared using one-way analysis of variance,and multiple comparisons w

关 键 词：甲基转移酶 角膜新生血管 碱烧伤 细胞焦亡 

分 类 号：R779.13[医药卫生—眼科]