miR-122靶向调节SIRT1对糖尿病视网膜病变大鼠炎性反应的影响

樊宗山; 黄海; 胡燕君

doi:10.13898/j.cnki.issn.2097-5252.2025.02.002

miR-122靶向调节SIRT1对糖尿病视网膜病变大鼠炎性反应的影响

Effect of miR-122 targeted regulating SIRT1 on the inflammatory response in rats with diabetic retinopathy

摘要

摘要:
目的探讨微小RNA(miR)-122通过靶向调节沉默信息调节因子1(SIRT1)参与糖尿病视网膜病变(DR)大鼠炎性反应的作用机制。
方法采用腹腔注射链脲佐菌素液建立糖尿病大鼠模型, 造模成功后, 再经玻璃体腔注射血管内皮生长因子(VEGF)构建DR大鼠模型。将造模成功的大鼠随机分为模型组、miR-122 antagomir组、antagomir-NC(阴性对照)组、EX527组(SIRT1抑制剂)、miR-122 antagomir+EX527组, 每组10只；另选取10只正常大鼠作为对照组, miR-122 antagomir组、antagomir-NC组、EX527组、miR-122 antagomir+EX527组分别注射miR-122 antagomir、antagomir-NC、EX527、miR-122 antagomir+EX527干预, 对照组和模型组大鼠注射等体积的0.9%氯化钠溶液, 每组每周干预一次, 连续操作12周。末次干预结束后处死大鼠, HE染色分析大鼠视网膜病理学变化；qRT-PCR法检测视网膜组织miR-122、SIRT1 mRNA表达水平；双荧光素酶实验验证miR-122与SIRT1的靶向关系；酶联免疫吸附(ELISA)法检测大鼠血清中超敏C反应蛋白(hs-CRP)、肿瘤坏死因子α(TNF-α)、白细胞介素-6(IL-6)含量、糖化血红蛋白；全自动生化仪分析相关生化指标尿素氮(BUN)、血肌酐(Scr)、三酰甘油(TG)、总胆固醇(TC)、丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)含量；蛋白免疫印迹(Western blotting)法检测大鼠视网膜组织中SIRT1蛋白表达水平。
结果双荧光素酶实验验证SIRT1为miR-122的直接靶点；与对照组相比, 模型组、antagomir-NC组大鼠视网膜结构均出现水肿, 细胞排布紊乱, 炎症浸润等现象, 大鼠血清中hs-CRP、TNF-α、IL-6水平、血糖水平、糖化血红蛋白、TG、TC、ALT、AST、Scr、BUN水平、视网膜组织中miR-122表达升高(P < 0.01), 视网膜组织中SIRT1 mRNA及蛋白表达下降(P < 0.05~P < 0.01)；与模型组、antagomir-NC组相比, miR-122 antagomir组大鼠视网膜结构相对完整, 水肿、炎症浸润等现象逐渐减轻, 大鼠血清中hs-CRP、TNF-α、IL-6水平、血糖水平、糖化血红蛋白、TG、TC、ALT、AST、Scr、BUN水平、视网膜组织中miR-122表达降低(P < 0.05~P < 0.01), 视网膜组织中SIRT1 mRNA及蛋白表达升高(P < 0.05~P < 0.01), EX527组大鼠血清中hs-CRP、TNF-α、IL-6水平、血糖水平、糖化血红蛋白、TG、TC、ALT、AST、Scr、BUN水平、视网膜组织中miR-122表达增加(P < 0.05~P < 0.01), 视网膜组织中SIRT1 mRNA及蛋白表达降低(P < 0.05~P < 0.01)；与miR-122 antagomir组相比, miR-122 antagomir+EX527组大鼠水肿、炎症浸润加重, 血清中hs-CRP、TNF-α、IL-6水平、血糖水平、糖化血红蛋白、TG、TC、ALT、AST、Scr、BUN水平、视网膜组织中miR-122表达升高(P < 0.05~P < 0.01), 视网膜组织中SIRT1 mRNA及蛋白表达降低(P < 0.05~P < 0.01)；与EX527组相比, miR-122 antagomir+EX527组大鼠水肿、炎症浸润减轻, 血清中hs-CRP、TNF-α、IL-6水平、血糖水平、糖化血红蛋白、TG、TC、ALT、AST、Scr、BUN水平、视网膜组织中miR-122表达降低(P < 0.05~P < 0.01), 视网膜组织中SIRT1 mRNA及蛋白表达显著增加(P < 0.05~P < 0.01)。
结论通过抑制miR-122表达, 可以靶向激活SIRT1, 减轻DR大鼠炎症反应, 阻碍DR的发生发展。

Abstract:
Objective To investigate the mechanism of microRNA (miR)-122 involved in the inflammatory response of diabetic retinopathy (DR) rats through targeted regulation of silent information regulator transcript-1 (SIRT1).
Methods A diabetic rat model was established by intraperitoneal injection of streptozotocin solution.After the model was successfully established, vascular endothelial growth factor (VEGF) was injected into the vitreous cavity to construct a DR rat model.The successfully modeled rats were randomly divided into model group, miR-122 antagomir group, antagomir-NC (negative control) group, EX527group (SIRT1 antagomir) and miR-122 antagomir+EX527group, with 10 rats in each group; in addition, 10 normal rats were selected as the control group.The miR-122 antagomir group, the antagomir-NC group, EX527 group and miR-122 antagomir+EX527 group were injected with miR-122 antagomir, antagomir-NC, EX527, miR-122 antagomir+EX527, respectively, the control group and the model group were injected with an equal volume of 0.9% sodium chloride solution, once a week, continuous operation for 12 weeks.After the last intervention, the rats were sacrificed, and HE staining was used to analyze the pathological changes of retina in rats; qRT-PCR was used to detect the expression levels of miR-122 and SIRT1 mRNA in retina tissue; dual luciferase experiment was used to verify the targeting relationship between miR-122 and SIRT1;enzyme-linked immunosorbent assay (ELISA) was used to detect the contents of hypersensitive C-reactive protein (hs-CRP), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and glycosylated hemoglobin in rat serum.The contents of urea nitrogen (BUN), creatinine (Scr), triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed by automatic biochemical analyzer.Western blotting was used to detect the expression level of SIRT1 protein in rat retina.
Results The dual luciferase experiment verified that SIRT1 was a direct target of miR-122.Compared with the control group, the retinal structure of the rats in the model group and the antagomir-NC group showed edema, disordered cell arrangement, and inflammatory infiltration, the contents of hs-CRP, TNF-α, IL-6, blood glucose content, glycosylated hemoglobin, TG, TC, ALT, AST, Scr, BUN content in serum and the expression of miR-122 in retinal tissue increased (P < 0.01), the expression of SIRT1 mRNA and protein in retinal tissues significantly decreased (P < 0.05 to P < 0.01).Compared with the model group and antagomir-NC group, the retinal structure of rats in the miR-122 antagomir group was relatively complete, and the edema and inflammatory infiltration were gradually reduced, the contents of hs-CRP, TNF-α, IL-6, blood glucose content, glycosylated hemoglobin, TG, TC, ALT, AST, Scr, BUN content in serum and the expression of miR-122 in retinal tissue decreased (P < 0.05 to P < 0.01), the expression of SIRT1 mRNA and protein in retinal tissues significantly increased (P < 0.05 to P < 0.01), the contents of hs-CRP, TNF-α, IL-6, blood glucose content, glycosylated hemoglobin, TG, TC, ALT, AST, Scr, BUN content in serum and the expression of miR-122 in retinal tissue in the EX527 group increased (P < 0.05 to P < 0.01), the expression of SIRT1 mRNA and protein in retinal tissues significantly decreased (P < 0.05 to P < 0.01).Compared with the miR-122 antagomir group, the edema and inflammatory infiltration in the miR-122 antagomir+EX527 group were gradually intensified, the contents of hs-CRP, TNF-α, IL-6, blood glucose content, glycosylated hemoglobin, TG, TC, ALT, AST, Scr, BUN content in serum and the expression of miR-122 in retinal tissue increased (P < 0.05 to P < 0.01), the expression of SIRT1 mRNA and protein in retinal tissues decreased significantly (P < 0.05 to P < 0.01).Compared with the EX527 group, the edema and inflammatory infiltration in the miR-122 antagomir+EX527 group were gradually reduced, the contents of hs-CRP, TNF-α, IL-6, blood glucose content, glycosylated hemoglobin, TG, TC, ALT, AST, Scr, BUN content in serum and the expression of miR-122 in retinal tissue decreased (P < 0.05 to P < 0.01), the expression of SIRT1 mRNA and protein in retinal tissues increased significantly (P < 0.05 to P < 0.01).
Conclusions Inhibiting the expression of miR-122 canbe targeted to activate SIRT1, reduce the inflammatory response in DR rats, and hinder the occurrence and development of DR.

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