背景:越来越多的证据显示了帕金森病(PD)的过程。可能,炎症在它们之间发挥着至关重要的作用。因此,这项研究的目的是分析白细胞介素-6受体(IL-6R)表达对IL-6/信号转导和转录激活因子3(STAT3)/缺氧诱导因子-1α(HIF-1α)炎症信号通路在PD合并2型糖尿病(T2DM)小鼠模型中的影响。
方法:我们选择10周龄的健康野生型C57BL/6J雄性小鼠制备PD合并T2DM的小鼠模型。将过表达IL-6R或AAVIL-6R-shRNA基因的腺相关病毒(AAV)注射到小鼠的黑质(SN)中。极点测试的行为指标用于检查小鼠的运动功能。使用免疫荧光分析,研究了IL-6R对多巴胺能神经元和小胶质细胞酪氨酸羟化酶(TH)和抗离子化钙结合衔接分子1(IBA-1)水平的影响。此外,采用酶联免疫吸附试验(ELISA)检测HIF-1α和炎性细胞因子肿瘤坏死因子-α(TNF-α)的表达,IL-1β,血清中的IL-6和IL-4。在这项研究中,TH的蛋白质表达水平,α-突触核蛋白(α-Syn),IBA-1,IL-6,IL-6R,通过蛋白质印迹测试SN中的磷酸化和总信号转导子和转录激活子3(p-STAT3(Tyr705)和STAT3)和HIF-1α。为了确定TNF-α的mRNA表达,IL-1β,IL-6、IL-4和HIF-1α,我们使用定量实时聚合酶链反应(RT-qPCR)。
结果:根据极点试验结果,IL-6R-shRNA治疗可显著缩短T2DM共病小鼠模型的总PD时间,逆转1-甲基-4-苯基-1,2,3,6-四氢吡啶盐酸盐(MPTP)刺激的TH阳性神经元的减少,并降低小胶质细胞的活化(均p<0.05)。Further,IL-6R-shRNA治疗阻碍了IL-6,p-STAT3(Tyr705)的表达,和SN中的HIF-1α,降低了TNF-α的水平,IL-1β,血清中IL-6、IL-4和HIF-1α,和TNF-α的mRNA表达,IL-1β,SN中的IL-6和HIF-1α(均p<0.05)。相比之下,IL-6R过表达降低TH水平,上调IBA-1,IL-6,p-STAT3(Tyr705)的水平,和HIF-1α,IL-1β水平升高,TNF-α,血清中IL-6、IL-4和HIF-1α(均p<0.05)和SN在PD小鼠模型中与T2DM共病。
结论:通过上调IL-6/STAT3/HIF-1α轴,AAVIL-6R过表达可促进以T2DM为合并症的PD进展。相反,AAVIL-6R-shRNA抑制IL-6/STAT3/HIF-1α通路,减轻神经炎症,从而削弱了PD与T2DM合并症的发展。
BACKGROUND: More and more evidence has shown the process of Parkinson\'s disease (PD). Probably, inflammation exerts a crucial role between them. Therefore, the aim of this study was to analyze the impact of interleukin-6 receptor (IL-6R) expression on the IL-6/signal transducer and activator of transcription 3 (STAT3)/hypoxia-inducible factor-1α (HIF-1α) inflammatory signaling pathway within a mouse model of PD with type 2 diabetes mellitus (T2DM) as co-morbidity.
METHODS: We chose healthy wild-type C57BL/6J male mice at the age of 10 weeks to prepare a mouse model of PD with T2DM co-morbidity. Adeno-associated virus (AAV) overexpressing IL-6R or AAV IL-6R-shRNA genes were injected into the substantia nigra (SN) of the mice. The behavioral indices of the pole test were used for examining the motor function of the mice. Using immunofluorescence analysis, the impacts of IL-6R on the level of tyrosine hydroxylase (TH) and anti-ionized calcium-binding adaptor molecule 1 (IBA-1) on dopaminergic neurons and microglia were examined. Additionally, enzyme-linked immunosorbent assay (ELISA) was adopted for determining the expressions of HIF-1α and inflammatory cytokines like tumor necrosis factor-α (TNF-α), IL-1β, IL-6, and IL-4 in the serum. In this study, the protein expression levels of TH, α-Synuclein (α-Syn), IBA-1, IL-6, IL-6R, phosphorylated and total signal transducer and activator of transcription 3 (p-STAT3 (Tyr705) and STAT3) and HIF-1α in the SN were tested via western blotting. To ascertain the mRNA expressions of TNF-α, IL-1β, IL-6, IL-4, and HIF-1α, we used quantitative Real-Time Polymerase Chain Reaction (RT-qPCR).
RESULTS: IL-6R-shRNA treatment could markedly shorten the total time of PD in the T2DM co-morbidity mouse model based on the pole test results, reverse the decrease in TH-positive neurons stimulated by 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), and lower the activation of microglia (all p < 0.05). Further, IL-6R-shRNA treatment hindered the expression of IL-6, p-STAT3 (Tyr705), and HIF-1α in the SN, lowered the levels of TNF-α, IL-1β, IL-6, IL-4, and HIF-1α in the serum, and mRNA expressions of TNF-α, IL-1β, IL-6, and HIF-1α in the SN (all p < 0.05). In contrast, IL-6R overexpression reduced TH levels, upregulated the level of IBA-1, IL-6, p-STAT3 (Tyr705), and HIF-1α, increased the level of IL-1β, TNF-α, IL-6, IL-4, and HIF-1α (all p < 0.05) in the serum and SN in the PD mouse model with T2DM as a co-morbidity.
CONCLUSIONS: PD progression with T2DM as a co-morbidity can be boosted by AAV IL-6R-overexpression through upregulation of the IL-6/STAT3/HIF-1α axis. Conversely, AAV IL-6R-shRNA treatment suppressed the IL-6/STAT3/HIF-1α pathway and alleviated neuroinflammation, thus weakening the development of PD with T2DM as a co-morbidity.