过表达TBK1通过调控RIPK1信号通路对糖尿病大鼠视网膜神经节细胞凋亡的抑制作用

目的　探讨TANK结合激酶1（TBK1）对糖尿病大鼠视网膜神经节细胞(RGC)凋亡的作用机制。方法　按60 mg·kg^-1腹腔一次性注射链脲佐菌素制备糖尿病模型。模型制备完成后，随机分成三组:糖尿病(DM)组、TBK1过表达(TBK1-OE)组 (大鼠玻璃体内单次注射TBK1过表达慢病毒载体5 μL，病毒滴度为 1.0×10⁹ IU·mL^-1)、TBK1空载体(TBK1-NC)组(玻璃体内注射等剂量病毒包装的阴性对照液)，另取正常大鼠作为对照(CON)组，每组15只。12周后，免疫荧光染色检测大鼠视网膜TBK1蛋白表达定位，Hoechst 33342染色检测大鼠视网膜RGC凋亡，ELISA检测大鼠视网膜肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)含量，Western blot检测大鼠视网膜TBK1、受体相互作用蛋白激酶1（RIPK1）、Caspase-3蛋白相对表达量。结果　TBK1在大鼠RGC中特异表达。与CON组相比，DM组、TBK1-NC组大鼠RGC凋亡率，TNF-α、IL-6、IL-1β含量，RIPK1、Caspase-3蛋白相对表达量均明显增加，TBK1蛋白相对表达量均明显降低(均为P<0.01)；TBK1-OE组各指标均增加（均为P<0.01);而与DM组相比，TBK1-OE 组大鼠RGC凋亡率，TNF-α、IL-6、IL-1β含量，RIPK1、Caspase-3蛋白相对表达量均明显降低，TBK1蛋白相对表达量明显增加(均为P<0.01)。而DM组与TBK1-NC组大鼠之间RGC凋亡率，TNF-α、IL-6、IL-1β含量，TBK1、RIPK1、Caspase-3蛋白相对表达量差异均无统计学意义(均为P>0.05)。结论　过表达TBK1对糖尿病大鼠RGC凋亡具有抑制作用，其机制与阻断RIPK1信号通路激活有关。

Inhibitory effect of TANK-binding kinase 1 overexpression on apoptosis of retinal ganglion cells in diabetic rats by regulating receptor-interacting protein kinase 1 signaling pathway

Objective　To investigate the effect of TANK-binding kinase 1 (TBK1) on the apoptosis of retinal ganglion cells (RGC) in diabetic rats. Methods　Diabetic models were prepared by intraperitoneal injection of 60 mg·kg^-1 streptozotocin in rats. After the models were prepared, they were randomly divided into three groups: diabetes mellitus (DM) group, TBK1 overexpression (TBK1-OE) group (rats were intravitreally injected with 5 μL of TBK1 overexpression lentivirus vector. The virus titer was 1.0×10⁹ IU·mL^-1), and TBK1 empty vector (TBK1-NC) group (rats were intravitreally injected with an equal dose of virus packaged negative control solution), with 15 rats in each group. Another 15 normal rats were selected as the control (CON) group. After 12 weeks, the expression and localization of TBK1 protein in the retina were detected by immunofluorescence staining, apoptosis of RGC was detected by Hoechst 33342 staining, the content of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-1β in the retina was detected by ELISA, and the relative expression of TBK1, receptor-interacting protein kinase 1 (RIPK1) and Caspase-3 proteins in the retina were detected by Western blot. Results　TBK1 was specifically expressed in RGC. Compared with the CON group, the RGC apoptosis rate, the content of TNF-α, IL-6 and IL-1β, and the relative expression of RIPK1 and Caspase-3 proteins were significantly increased, and the relative expression of TBK1 protein was significantly reduced in the DM and TBK1-NC groups (all P<0.01), while all these indicators were significantly increased in the TBK1-OE group (all P<0.01). Compared with the DM group, the RGC apoptosis rate, the content of TNF-α, IL-6 and IL-1β, and the relative expression of RIPK1 and Caspase-3 proteins were significantly decreased, while the relative expression of TBK1 protein was significantly increased in the TBK1-OE group (all P<0.01). There was no significant difference in the RGC apoptosis rate, the content of TNF-α, IL-6 and IL-1β, and the relative expression of TBK1, RIPK1 and Caspase-3 proteins between the DM group and the TBK1-NC group (all P>0.05). Conclusion　Overexpression of TBK1 has an inhibitory effect on RGC apoptosis in diabetic rats, which may be related to its blocking the activation of the RIPK1 signaling pathway.