调节性核糖核酸酶1对高糖诱导的视网膜神经节细胞损伤的保护作用  

Protective effect of regnase-1 on retinal ganglion cell injury induced by high glucose

作　　者：窦方方 曹素平 邓志峰 DOU Fangfang;CAO Suping;DENG Zhifeng(Department of Ophthalmology,Heze Hospital of Traditional Chinese Medicine,Heze,Shandong 274000,China)

机构地区：[1]菏泽市中医医院眼科,山东菏泽274000

出　　处：《安徽医药》2023年第4期754-759,共6页Anhui Medical and Pharmaceutical Journal

摘　　要：目的探讨调节性核糖核酸酶1(Reg1)对高糖诱导的视网膜神经节细胞损伤的保护作用及机制。方法2018年8月至2020年2月构建高糖诱导的RGC-5视网膜神经节细胞损伤模型,实验分为四组:Con组(正常糖+未转染的RGC-5细胞)、HG组(高糖+未转染的RGC-5细胞)、pcDNA3-Reg1组(高糖+转染Reg1过表达质粒的RGC-5细胞)、pcDNA3-NC组(高糖+转染对照质粒的RGC-5细胞)。实时荧光定量聚合酶链式反应检测Reg1、白细胞介素(IL)-1β、IL-6、IL-12、肿瘤坏死因子-α(TNF-α)的表达;蛋白质免疫印迹检测Reg1、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、活化的胱天蛋白酶3(cleaved caspase-3)、磷酸化p65(p-p65)、磷酸化核因子κB抑制蛋白α(p-IκBα)的表达;免疫荧光染色检测Reg1、p-p65的表达;TUNEL染色检测凋亡水平;酶联免疫吸附试验检测细胞上清液中各炎性因子的含量。结果Con组中Reg1的相对荧光强度、mRNA相对表达量、蛋白质相对表达量分别为1.00±0.21、1.00±0.15、1.00±0.18,HG组Reg1的相对荧光强度、mRNA相对表达量、蛋白质相对表达量分别为2.49±0.36、3.86±0.42、3.17±0.39,pcDNA3-Reg1组Reg1的相对荧光强度、mRNA相对表达量、蛋白质相对表达量分别为6.77±0.72、8.18±0.67、6.30±0.51,pcDNA3-NC组Reg1的相对荧光强度、mRNA相对表达量、蛋白质相对表达量分别为2.75±0.40、3.59±0.38、2.85±0.34,HG组、pcDNA3-NC组Reg1相对荧光强度、mRNA和蛋白质相对表达量高于Con组,且低于pcDNA3-Reg1组(P<0.05)。Con组TUNEL+细胞比例、Bcl-2、Bax、cleaved caspase-3分别为(1.73±0.22)%、1.00±0.28、1.00±0.20、1.00±0.17,HG组TUNEL+细胞比例、Bcl-2、Bax、cleaved caspase-3分别为(14.79±0.83)%、0.29±0.11、5.25±0.66、4.93±0.52,pcDNA3-Reg1组TUNEL+细胞比例、Bcl-2、Bax、cleaved caspase-3分别为(3.77±0.34)%、0.58±0.43、2.51±0.45、2.58±0.33,pcDNA3-NC组TUNEL+细胞比例、Bcl-2、Bax、cleaved caspase-3分别为(14.55±0.90)%、0.21±0.14Objective To explore the protective effect and mechanism of regulatory ribonuclease 1(Reg1)on retinal ganglion cell injury induced by high glucose.Methods From August 2018 to February 2020,a high glucose-induced RGC-5 retinal ganglion cell injury model was constructed.There were 4 groups for the experiment:Con group(normal glucose+untransfected RGC-5 cells),HG group(high glucose+untransfected RGC-5 cells),pcDNA3-Reg1 group(high glucose+RGC-5 cells transfected with Reg1 overexpression plasmid),pcDNA3-NC group(high glucose+RGC-5 cells transfected with control plasmid).Quantitative real-time polymerase chain reaction was used to detect the expressions of Reg1,interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-12(IL-12),and tumor necrosis factor-α(TNF-α);Western blotting was used to detect the expressions of Reg1,B cell lymphoma-2(Bcl-2),Bcl-2 related X protein(Bax),cleaved cysteine-containing aspartate proteolytic enzyme 3(cleaved caspase-3),phosphorylated p65(p-p65),and phosphorylated nuclear factorκB inhibitoryα(p-IκBα);Immunofluorescence staining was used to detect the expressions of Reg1 and p-p65;TUNEL staining was used to detect the level of apoptosis;Enzyme-linked immunosorbent assay was used to detect the content of various inflammatory factors in the cell supernatant.Results The relative fluorescence intensity,relative mRNA expression and relative protein expression of Reg1 in Con group were 1.00±0.21,1.00±0.15 and 1.00±0.18,respectively.The relative fluorescence intensity,relative mRNA expression and relative protein expression of Reg1 in HG group were 2.49±0.36,3.86±0.42 and 3.17±0.39,respectively.The relative fluorescence intensity,relative mRNA expression and relative protein expression of Reg1 in pcDNA3-Reg1 group were 6.77±0.72,8.18±0.67,and 6.30±0.51,respectively.The relative fluorescence intensity,relative mRNA expression and relative protein expression of Reg1 in pcDNA3-NC group were 2.75±0.40,3.59±0.38 and 2.85±0.34,respectively.The relative fluorescence intensity,relative m

关 键 词：高血糖症 调节性核糖核酸酶1 糖尿病视网膜病变 炎症 视网膜神经节细胞 核因子κB 细胞凋亡 

分 类 号：R774.1[医药卫生—眼科] R587.2[医药卫生—临床医学]