坏死性凋亡在帕金森病小鼠抑郁样行为中的作用及运动的抗抑郁效应

关键词：坏死性凋亡  帕金森病  跑台运动  抑郁样行为

Keywords:necroptosis  Parkinson's disease  treadmill exercise  depressive-like behavior

摘要：

      摘要 目的：探讨坏死性凋亡信号在慢性帕金森病（PD）的抑郁样行为中的作用及运动的抗抑郁效应。 方法：40只6周龄健康雄性C57BL/6小鼠随机分为对照组（Control）、模型组（PD）、跑台运动组（PD+Ex）、受体相互作用蛋白激酶1（RIPK1）抑制剂组（PD+Nec-1），每组10只。采用MPTP/p制备慢性PD小鼠模型，PD+Ex组在注射MPTP/p同时进行5周跑台运动训练，PD+Nec-1组进行5周Nec-1腹腔注射。评价模型成功后，采用行为学实验评估Nec-1及运动干预对小鼠抑郁样行为的影响。收集各组小鼠粪便评价便秘情况，采用16SrRNA基因测序评价肠道菌群组成变化，HE、PAS染色观察小鼠结肠形态学改变，免疫荧光染色检测小鼠结肠黏膜屏障完整性。qPCR检测结肠炎症因子mRNA表达水平，Western Blotting检测海马和结肠相关蛋白表达。 结果：①PD小鼠模型成功复刻，出现抑郁样行为，Nec-1、跑台运动干预后均得到显著改善；②与Control组比较，PD组小鼠结肠上皮屏障受损、结肠呈炎性改变，粪便含水量显著下降，肠道菌群多样性减少；③与Control组比较，PD组小鼠海马组织坏死性凋亡相关蛋白显著增高，神经胶质细胞激活；④与PD组比较，PD+Ex组结肠上皮屏障受损改善，粪便含水量增多，肠道菌群多样性增加，结肠坏死性凋亡相关蛋白表达显著降低，促炎因子蛋白表达降低，抗炎因子mRNA表达升高，海马坏死性凋亡、神经胶质细胞激活相关蛋白表达显著降低，抗炎因子蛋白表达显著升高；⑤与PD组比较，PD+Nec-1组结肠上皮屏障损伤、炎症及粪便含水量显著改善，坏死性凋亡相关蛋白表达显著降低，海马坏死性凋亡、神经胶质细胞激活相关蛋白表达降低，促炎因子蛋白表达降低，抗炎因子蛋白表达显著升高；⑥运动干预和Nec-1的抗抑郁效果相当，但运动干预在改善结肠屏障完整性方面效果较好。 结论：跑台运动通过调节肠脑互动的坏死性凋亡途径改善PD小鼠抑郁样行为。

Abstract:

      Abstract Objective： To investigate the role of necroptosis signaling in depression-like behavior in chronic Parkinson's disease （PD） mouse model and to examine the antidepressant effect of treadmill exercise. Method： Forty six-week-old healthy male C57BL/6 mice were randomly assigned to four groups (n=10 per group): control group， PD model group， PD with treadmill exercise group (PD+Ex) and PD with receptor-interacting protein kinase 1（RIPK1） inhibitor group (PD+Nec-1) 10 mice in each group. Chronic PD mouse model was established by using MPTP/probenecid (MPTP/p). The PD+Ex group were given MPTP/P and treadmill training for 5 weeks， and PD+Nec-1 group received intraperitoneal injection of Necrostatin-1 for 5 weeks. After model validation， depression-like behaviors were assessed using behavioral tests. Stools samples were collected to evaluate constipation. Gut microbiota composition was analyzed by 16SrRNA gene sequencing . Hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining was used to observe the morphological changes of mice colon， and immunofluorescence staining was used to detect the integrity of the colonic mucosal barrier. mRNA expression of inflammatory cytokines in the colon was measured by quantitative PCR， and the protein expression related to necroptosis and neuroinflammation in the hippocampus and colon was analyzed by Western Blotting. Result： The PD mice model was successfully replicated and exhibited significant depressive-like behaviors， which were significantly improved after treatment with Nec-1 and treadmill exercise interventions. Compared with control group， PD mice showed impaired colonic epithelial barrier integrity， inflammatory colonic changes， reduced fecal water content and decreased gut microbiota diversity. Additionally, PD mice exhibited significantly increased expression of hippocampal necroptosis related proteins and activation of glial cells. Compared with the PD group， the PD+Ex group demonstrated improved colonic epithelial barrier integrity， increased fecal water content， gut microbiota diversity, reduced expression of necroptosis related proteins and pro-inflammatory cytokines in the colon， increased anti-inflammatory cytokines mRNA expression， significantly reduced necroptosis and microglial activation-related protein expression in the hippocampus， and significantly increased anti-inflammatory cytokines levels. Compared with the PD group, the PD+Nec-1 group showed the similar improvements in the colonic epithelial barrier integrity， inflammation， fecal water content and significant reductions in hippocampal necroptosis related protein and glial cell activation related protein expression, decreased pro-inflammatory factor protein expression and significantly increased anti-inflammatory factor protein expression. Exercise had similar antidepressant effects as Nec-1， but exercise intervention was more effective in improving the integrity of the colonic barrier. Conclusion： Treadmill exercise improves depression-like behavior in PD mice by modulating necroptosis signaling pathway within gut-brain interactions.