二氢杨梅素抑制坏死性凋亡信号通路改善帕金森病小鼠认知功能障碍的作用机制

Mechanisms of Dihydromyricetin Mediated Necroptosis Signaling Pathway in Improving Cognitive Dysfunction in Mice with Parkinson's Disease

目的研究二氢杨梅素(DHM)对帕金森病(PD)小鼠认知功能障碍的影响及分子机制。方法将30只C57BL/6雄性小鼠随机分为正常组、模型组、DHM组,每组10只。模型组、DHM组采用腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP,25 mg·kg^(-1)),1 h后皮下注射丙磺舒(250 mg·kg^(-1)),每间隔3.5 d注射1次,共注射10次,以复制慢性PD小鼠模型。模型复制后,DHM组按100 mg·kg^(-1)剂量灌胃给药,每日1次,每周5次,持续8周。采用Morris水迷宫评估小鼠认知功能;微板法检测小鼠海马组织中乳酸脱氢酶(LDH)释放量;Western Blot法检测小鼠纹状体及海马组织中酪氨酸羟化酶(TH)、肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、白细胞介素6(IL-6),磷酸化受体相互作用蛋白激酶1(p-RIPK1)、受体相互作用蛋白激酶1(RIPK1)、磷酸化受体相互作用蛋白激酶3(p-RIPK3)、受体相互作用蛋白激酶3(RIPK3)、混合谱系激酶结构域样假激酶(MLKL)、半胱氨酸蛋白酶8(Caspase-8)、B细胞淋巴瘤因子2(Bcl-2)、Bcl-2相关X蛋白(Bax)及自噬微管相关蛋白轻链3(LC3)和泛素结合蛋白(p62)的蛋白表达水平。结果与正常组比较,模型组小鼠纹状体TH蛋白表达水平明显降低(P<0.05);逃逸潜伏期显著延长(P<0.01),穿越平台次数、穿越靶象限时间、靶象限时间百分比均显著减少(P<0.01),平均游泳速度显著降低(P<0.01);海马组织中TNF-α、IL-1β蛋白表达水平明显升高(P<0.05,P<0.01);LDH释放量及p-RIPK1、p-RIPK3、RIPK3、MLKL蛋白表达水平均明显上升(P<0.05,P<0.01);Bax蛋白表达水平明显上升(P<0.05),Bcl-2蛋白表达水平显著下降(P<0.01);LC3Ⅱ/Ⅰ蛋白表达比值降低(P>0.05),p62蛋白表达水平显著升高(P<0.01)。与模型组比较,DHM组小鼠纹状体TH蛋白表达水平明显升高(P<0.05);逃逸潜伏期明显缩短(P<0.05),穿越平台次数、穿越靶象限时间、靶象限时间百分比均明显增加(P<0.05),平均游泳速度显著提高(P<0.01);海马组织中TNF-α、IL-1β、IL-6蛋白表达水平明显降低(P<0.05,P<0.01);LDH释放量及p-RIPK1、p-RIPK3、RIPK1、RIPK3、MLKL蛋白表达水平均明显下降(P<0.05,P<0.01),Caspase-8蛋白表达水平显著上升(P<0.01);Bax蛋白表达水平明显下降(P<0.05),Bcl-2蛋白表达水平显著上升(P<0.01);LC3Ⅱ/Ⅰ蛋白表达比值显著升高(P<0.01),p62蛋白表达水平显著降低(P<0.01)。结论DHM对PD小鼠认知障碍具有改善作用,其机制可能与DHM抑制神经炎症反应,下调坏死性凋亡信号,同时恢复自噬水平、降低凋亡程度有关。

Objective To investigate the effect and the molecular mechanisms of dihydromyricetin(DHM) on cognitive dysfunction in mice with Parkinson’s disease (PD).Methods Thirty C57BL/6 male mice were randomly divided into normal,model and DHM groups,with 10 mice in each group.The model group and DHM group were injected with MPTP (25 mg·kg^(-1)) intraperitoneally and Probenecid (250 mg·kg^(-1)) subcutaneously 1-hour later,at 3.5 days intervals,for a total of 10 injections to replicate the chronic PD mouse model.After model replication,the DHM group was administered by gavage at a dose of 100 mg·kg^(-1)once daily,5 times a week for8 consecutive weeks.The cognitive function of the mice was assessed by Morris water maze;lactate dehydrogenase(LDH) release was measured in the hippocampal tissue of mice by microplate method;Western Blot was used to detect the protein expressions of tumor necrosis factor α (TNF-α),interleukin 1β (IL-1β),interleukin 6 (IL-6),phosphorylated-receptor interacting protein kinase 1 (p-RIPK1),receptor interacting protein kinase 1 (RIPK1),phosphorylation-receptor interacting protein kinase 3 (p-RIPK3),receptor interacting protein kinase 3 (RIPK3),mixed lineage kinase domain-like pseudokinase(MLKL),cysteine protease 8(Caspase-8),B-cell lymphoma factor 2(Bcl-2),Bcl-2-associated X protein (Bax),autophagic microtubule-associated protein light chain 3 (LC3)and ubiquitin-binding protein (p62).Results Compared with the normal group,TH protein expression level in striatum of mice in model group was significantly decreased (P<0.05);the escape incubation period was significantly prolonged (P<0.01),the times of crossing platform,crossing target quadrant time and percentage of target quadrant time were significantly decreased (P<0.01),and the average swimming speed was significantly decreased(P<0.01);the expression levels of TNF-α and IL-1β in hippocampus were significantly increased (P<0.05,P<0.01).LDH release and protein expression levels of p-RIPK1,p-RIPK3,RIPK3,MLKL were significantly increased (P<0.05,P<0.01);Bax protein expression level was significantly increased (P<0.05) and Bcl-2protein expression level was significantly decreased (P<0.01);LC3Ⅱ/Ⅰ protein expression ratio was decreased (P<0.05) and the p62 protein expression level was significantly increased (P<0.01).Compared with the model group,the striatal TH protein expression level was significantly increased in the DHM group mice (P<0.05);the escape latency was significantly shortened (P<0.05),the times of crossing platforms,the time to cross the target quadrant and the percentage of time in the target quadrant were all significantly increased (P<0.05),and the mean swimming speed was significantly increased (P<0.01);protein expression levels of TNF-α,IL-lβ,and IL-6were significantly decreased (P<0.05,P<0.01);LDH release and protein expression levels of p-RIPK1,pRIPK3,RIPK1,RIPK3,MLKL were significantly decreased (P<0.05,P<0.01),Caspase-8 protein expression level was significantly increased (P<0.01);Bax protein expression level was significantly decreased (P<0.05)and Bcl-2 protein expression level was significantly increased (P<0.01);LC3Ⅱ/Ⅰ protein expression ratio was significantly increased (P<0.01) and p62 protein expression level was significantly decreased (P<0.01).Conclusion DHM has an ameliorating effect on cognitive impairment in PD mice,and the mechanism may be related to DHM inhibiting neuroinflammatory responses and down-regulating necroptosis signaling,while restoring autophagy levels and reducing the degree of apoptosis.