| 窦方方,曹素平,邓志峰.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[J].安徽医药,2023,27(4):754-759. |
| 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 |
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| DOI：10.3969/j.issn.1009-6469.2023.04.027 |
| 中文关键词: 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 |
| 英文关键词: Hyperglycemia Regnase-1 Diabetic retinopathy Inflammation Retinal ganglion cell Nuclear factorκB Apoptosis |
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| 中文摘要: |
| 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 pcDNA3Reg1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，pcDNA3Reg1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±0.14、5.14±0.59、4.77±0.46，Con组、 pcDNA3-Reg1组 TUNEL+细胞比例、 Bax、cleaved caspase-3蛋白质相对表达量低于 HG组、 pcDNA3-NC组，而 Bcl-2蛋白质相对表达量高于 HG组、 pcDNA3-NC组( P<0.05)。 Con组、 pcDNA3-Reg1组细胞 IL-1β、IL-6、IL-12、TNF-α mRNA相对表达量，及上清液蛋白质含量低于 HG组、 pcDNA3-NC组( P<0.05)。 Con组、 pcDNA3-Reg1组 p-p65相对荧光强度，及 p-p65、p-IκBα蛋白质相对表达量低于 HG组、 pcDNA3-NC组( P<0.05)。结论高糖状态下 RGC-5视网膜神经节细胞高表达 Reg1，上调 Reg1表达可以降低细胞凋亡及炎症，可能与抑制 NFκB信号通路活化有关。 |
| 英文摘要: |
| Objective 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 factor κB inhibitory α (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 exmRNA 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. Therelative 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 mRNA and protein expression levels of Reg1 in HG and pcDNA3-NC groups were higher than those in Con group, but lower than those in pcDNA3-Reg1 group (P<0.05). The proportion of TUNEL + cells, and expression levels of Bcl-2, Bax, cleaved caspase-3 in Con group were (1.73±0.22) %, 1.00±0.28, 1.00±0.20, and 1.00±0.17, respectively. The proportion of TUNEL + cells, and expressionlevels of Bcl-2, Bax and cleaved caspase-3 in HG group were (14.79±0.83) %, 0.29±0.11, 5.25±0.66 and 4.93±0.52, respectively. Theproportion of TUNEL + cells, and expression levels of Bcl-2, Bax, cleaved caspase-3 in pcDNA3-Reg1 group were (3.77±0.34) %, 0.58±0.43, 2.51±0.45, and 2.58±0.33, respectively. The proportion of TUNEL + cells, and expression levels of Bcl-2, Bax, cleaved caspase-3 in pcDNA3-NC group were (14.55±0.90) %, 0.21±0.14, 5.14±0.59, and 4.77±0.46, respectively. The proportion of TUNEL + cells andthe relative expression levels of Bax and cleaved caspase-3 protein in Con and pcDNA3-Reg1 groups were lower than those in HG and pcDNA3-NC groups, while the relative expression level of Bcl-2 protein was higher than those in HG and pcDNA3-NC groups (P<0.05). The relative mRNA expressions of IL-1β, IL-6, IL-12, TNF-α and the protein content of supernatant in Con and pcDNA3-Reg1 groups were lower than those in HG and pcDNA3-NC groups (P<0.05). The relative fluorescence intensity of p-p65 and the relative protein expressions of p-p65 and p-IκBα in Con and pcDNA3-Reg1 groups were lower than those in HG group and pcDNA3-NC group (P<0.05). Conclusion In the state of high glucose, Reg1 is highly expressed in RGC-5 retinal ganglion cells, and up-regulation of Reg1 expression can reduce cell apoptosis and inflammation, which may be related to the inhibition of NFκB signaling pathway activation. |
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