基于TLR4/NF-κB信号通路探讨银杏二萜内酯对糖尿病大鼠视网膜病变的影响

Effect of ginkgo diterpenoid lactone on retinopathy in diabetic rats based on TLR4/NF-κB signal pathway

关键词:  银杏二萜内酯  糖尿病  视网膜  TLR4/NF-κB信号通路  炎症  凋亡

Key words:  ginkgo diterpene lactone  diabetic mellitus  retina  TLR4/NF-κB signal pathway  inflammation  apoptosis

摘要:

[目的] 研究银杏二萜内酯（GDL）对糖尿病大鼠视网膜病变的影响，并基于Toll样受体4/核因子-κB（TLR4/NF-κB）信号通路探索其可能的作用机制。[方法] 采用高糖高脂饮食加腹腔注射30 mg/kg链脲佐菌素（STZ）的方法构建糖尿病视网膜病变大鼠模型，设模型组、GDL（2.3 mg/kg）组、TAK242（TLR4抑制剂，3 mg/kg）组、GDL+TAK242（2.3 mg/kg+3 mg/kg）组；另设正常对照组。各组分别每日1次腹腔注射给药治疗14 d后，测定空腹血糖（FBG）水平、视网膜厚度和视网膜血管通透性，通过苏木精-伊红（HE）染色、TUNEL染色观察视网膜病理改变和细胞凋亡，酶联免疫吸附测定（ELISA）法检测视网膜炎症因子[白细胞介素-1β（IL-1β）、IL-6、IL-8、肿瘤坏死因子-α（TNF-α）]含量，Western blot法检测视网膜TLR4、NF-κB、p-NF-κB、NF-κB抑制因子（IκBα）、p-IκBα、高迁移率族蛋白B1（HMGB1）、B淋巴细胞瘤-2基因（Bcl-2）、Bcl-2相关X蛋白（Bax）表达。[结果] 与模型组相比，GDL组、TAK242组和GDL+TAK242组FBG水平和血管通透性降低，视网膜厚度增加（P < 0.05）；视网膜神经节细胞层和内核层细胞数目减少、排列紊乱、分层不清、水肿、炎性浸润等病理改变明显减轻，细胞凋亡状况明显改善；视网膜IL-1β、IL-6、IL-8、TNF-α含量明显降低（P < 0.05），TLR4、p-NF-κB、p-IκBα、HMGB1、Bax表达量及p-NF-κB/NF-κB、p-IκBα/IκBα、Bax/Bcl-2比值降低（P < 0.05），Bcl-2表达量升高（P < 0.05）。GDL+TAK242组对糖尿病大鼠上述指标的调控作用明显优于GDL组和TAK242组（P < 0.05）。[结论] GDL可抑制糖尿病大鼠视网膜炎症反应和细胞凋亡，降低视网膜血管通透性，减轻视网膜病理改变，其机制可能与抑制TLR4/NF-κB信号通路活化有关。

Abstract:

[Objective] To investigate the effect of ginkgo diterpene lactone (GDL) on retinopathy in diabetic mellitus rats, and explore its mechanism based on toll like receptor 4/nuclear factor-κB (TLR4/NF-κB) signal pathway.[Methods] The diabetic mellitus retinopathy rat model was established by intraperitoneal injection of 30 mg/kg streptozotocin after high sugar and high fat diet, and set model group, GDL (2.3 mg/kg) group, TAK242 (TLR4 inhibitor, 3 mg/kg) group and GDL+TAK242 (2.3 mg/kg+3 mg/kg) group; and another normal group was setup. After 14 days of intraperitoneal injection of the drugs once a day in each group, the fasting blood glucose (FBG), retinal thickness, retinal vascular permeability were measured; the pathological changes and cell apoptosis of retina were observed by HE staining and TUNEL staining; the content of inflammatory factor [interleukin-1β (IL-1β), IL-6, IL-8, tumor necrosis factor α (TNF-α)] in retinal were detected by ELISA method; the expression of TLR4, NF-κB, p-NF-κB, NF-κB Inhibitor (IκBα), p-IκBα, high mobility group protein B1(HMGB1), B-lymphomatoma-2 gene(Bcl-2), Bcl-2 related X protein(Bax) were detected by Western blot.[Results] Compared with the model group, the FBG level, and vascular permeability were decreased in GDL group, TAK242 group and GDL+TAK242 group, the retinal thickness was increased(P < 0.05);the pathological changes such as cells number decreased, arrangement disordered, unclear stratification, edema and inflammatory infiltration of retinal retinal ganglion cell layer and inner nuclear layer were significantly reduced, and retinal cells apoptosis was significantly improved; the content of IL-1β, IL-6, IL-8, TNF-α in retinal were decreased(P < 0.05);the expression of TLR4, p-NF-κB, p-IκBα, HMGB1, Bax and the ratio of p-NF-κB/NF-κB, p-IκBα/IκBα, Bax/Bcl-2 were decreased(P < 0.05);the expression of Bcl-2 was increased(P < 0.05). The regulating effect of GDL+TAK242 group on the indexes above of diabetic mellitus rats was significantly better than that of GDL group and TAK242 group(P < 0.05).[Conclusion] GDL can inhibit the inflammatory reaction and cell apoptosis, reduce the retinal vascular permeability and alleviate the pathological changes of the retina in diabetic mellitus rats, which mechanism may be related to the inhibition of TLR4/NF-κB signal pathway activation.