微小RNA-204对糖尿病视网膜病变大鼠模型的保护作用及其机制

Protective effect of microRNA-204 on diabetic retinopathy in rats and its mechanism

目的探讨微小RNA(miR)-204对糖尿病视网膜病变大鼠模型的保护作用及其机制。方法30只SD大鼠随机数字表格法分为对照组、模型组和miR-204组,每组10只,其中miR-204组大鼠经尾静脉注射过表达miR-204的腺相关病毒;对照组和模型组大鼠注射等体积对照腺相关病毒。注射30d后,模型组和miR-204组大鼠通过高脂饲料喂养+腹腔注射链脲佐菌素建立糖尿病视网膜病变模型。治疗4周后,分析3组大鼠视网膜血管通透性和视网膜厚度;采用酶联免疫吸附试验(ELISA)检测3组大鼠外周血清肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)水平;采用原位缺口末端标记法(TUNEL)染色分析3组大鼠视网膜细胞凋亡;蛋白质印迹法(Westernblot)分析3组大鼠视网膜组织中半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3和高迁移率族蛋白B1(HMGB1)的表达水平。组间比较采用单因素方差分析。结果miR-204组大鼠视网膜组织miR-204表达水平(2.12±0.17)明显高于对照组和模型组miR-204表达水平(1.05±0.11、0.93±0.05),差异有统计学意义(t=16.480、21.130,P<0.05)。miR-204组大鼠视网膜厚度[(147.99±9.09)μm]明显高于模型组[(126.63±9.15)μm],差异有统计学意义(t=5.236,P<0.05)。miR-204组大鼠视网膜厚度[(15.41±2.39)μg/g]明显低于对照组[(27.61±1.87)μg/g],差异有统计学意义(t=12.770,P<0.05)。miR-204组大鼠血清炎性因子TNF-α和IL-6表达水平[(40.51±4.04)、(39.47±4.70)ng/ml]明显低于模型组[(77.54±7.55)、(54.63±4.27)ng/ml],差异有统计学意义(t=13.670、7.907,P<0.05)。miR-204组大鼠视网膜细胞凋亡率[(8.78±0.81)%]明显低于模型组[(19.94±2.34)%],差异有统计学意义(t=14.222,P<0.05)。miR-204组大鼠视网膜组织Caspase-3和HMGB1蛋白表达水平(1.64±0.12、1.83±0.19)明显低于模型组(1.64±0.12、1.83±0.19),差异有统计学意义(t=6.719、8.409,P<0.05)。结论miR-204可显著抑制视网膜血管通透性,抑制炎症和细胞凋亡,可能与抑制HMGB1和细胞凋亡通路有关。

Objective To investigate the protective effect of microRNA(miR)-204 on diabetic ret-inopathy in rats and its mechanism.Methods Totally,30 SD rats were randomly divided into control group,model group and miR-204 group by random number methods(10 rats per group).The miR-204 group was injected with adeno-associated virus overexpressing miR-204 via the tail vein.Tin the control group and the model group were injected with the same volume of control adeno-associated virus.After 30 days of injection,the model group and miR-204 group were given high-fat diet+intraperitoneal injection of Streptozotocin to establish diabetic retinopathy models.After 4 weeks of treatment,the retinal vascular permeability and retinal thickness of three groups were analyzed.The levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were analyzed by enzyme linked immunosorbent assay(ELISA)in the peripheral blood of three groups.The apoptosis of retinal cells in three groups rats was analyzed by termi-nal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)staining.The expression levels of cysteinyl aspartate-specific protease(Caspase)-3 and high mobility group protein BI(HMGBI)in the reti-nal tissues of three groups were detected by Western blotting.The comparison of inter group econometric data was conducted using one-way analysis of variance.Results The expression level of miR-204 in the retina tissue of rats in the miR-204 group(2.12±0.17)was significantly higher than that in the control group and model group(1.05±0.11,0.93±0.05,t=16.480,21.130,P<0.05).Retinal thickness of miR-204 group[(147.99±9.09)μm]was significantly higher than that of model group[(126.63±9.15)μm,t=5.236,P<0.05].Retinal thickness of miR-204 group[(15.41±2.39)μg/g] was significantly lower than that of the control group[(27.61±1.87)μg/g,t=12.770,P<0.05].The expression levels of serum inflammatory factors TNF-α and IL-6 in the miR-204 group[(40.51±4.04),(39.47±4.70)ng/ml]were significantly lower than those in the model group[(77.54±7.55),(54.63±4.27)ng/ml,t=13.670,7.907,P<0.05].The apoptosis rate of retinal cells in the miR-204 group[(8.78±0.81)%]was significantly lower than that in the model group[(19.94±2.34)%,t=14.222,P<0.05].The expression levels of Caspase-3 and HMGB1 proteins in the retina of rats in the miR-204 group(1.64±0.12,1.83±0.19)were significantly lower than those in the model group(1.64±0.12,1.83±0.19,t=6.719,8.409,P<0.05).Conclusion MiR-204 can significantly inhibit retinal vascular permeability,inflammation and apoptosis,which may be related to the inhibition of HMGBI and apoptosis pathway.