基于PKA信号通路探讨胆南星对MPTP诱导帕金森病模型小鼠的保护作用

Investigation on the protective effect of Arisaema Cum Bile on MPTP-induced Parkinson’s disease modelmice based on PKA signaling pathway

目的 探讨胆南星对帕金森病(PD)模型小鼠的改善作用及可能机制。方法 将60只雄性C57BL/6J小鼠随机分为正常组、模型组、胆南星低剂量组[0.39 g/(kg·d)]、胆南星高剂量组[1.56 g/(kg·d)]和阳性对照药左旋多巴片组[80 mg/(kg·d)],每组12只。除正常组小鼠注射等体积生理盐水外,其余各组连续5 d腹腔注射1-甲基-4-苯基-1,2,3,6-四氢吡啶[MPTP,35 mg/(kg·d)]建立亚急性PD模型;造模完成后连续给药治疗7 d,于造模前1 d、造模第5天和末次给药后进行爬杆实验和线悬挂测试。采用免疫荧光法检测小鼠脑黑质中酪氨酸羟化酶(TH)阳性神经元数量;采用酶联免疫吸附法检测小鼠血清中白细胞介素1β(IL-1β)和肿瘤坏死因子α(TNF-α)水平以及脑黑质中IL-1β、TNF-α、环氧合酶2(COX-2)和诱导型一氧化氮合酶(iNOS)水平;采用Western blot法检测小鼠脑黑质中cAMP依赖的蛋白激酶催化亚单位α(PKA C-α)、谷胱甘肽过氧化物酶4(GPX4)以及铁蛋白重链多肽1(FTH1)蛋白的表达。结果 末次给药后,与正常组比较,模型组小鼠爬杆时间显著延长(P<0.01),线悬挂评分显著降低(P<0.01),脑黑质中TH阳性神经元数量显著减少(P<0.01),血清中IL-1β、TNF-α水平和脑黑质中IL-1β、TNF-α、COX-2、iNOS水平显著升高(P<0.01),脑黑质中GPX4、PKA C-α和FTH1蛋白表达水平显著降低(P<0.05或P<0.01)。与模型组比较,胆南星高剂量组小鼠上述指标水平均显著回调(P<0.05或P<0.01)。结论 胆南星能够改善MPTP诱导的PD模型小鼠运动能力障碍,减少脑黑质TH神经元死亡,对模型小鼠具有神经保护作用;这可能与其激活PKA信号通路来抑制神经炎症和神经元细胞铁死亡有关。

OBJECTIVE To investigate the improvement effects of Arisaema Cum Bile on Parkinson’s disease(PD)model mice and its potential mechanism.METHODS Sixty male C57BL/6J mice were randomly divided into normal group,model group,Arisaema Cum Bile low-dose group[0.39 g/(kg·d)],Arisaema Cum Bile high-dose group[1.56 g/(kg·d)]and positive control drug Levodopa tablet group[80 mg/(kg·d)],with 12 mice in each group.Except that normal group was given constant volume of normal saline,other groups were given 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine[MPTP,35 mg/(kg·d)]intraperitoneally for 5 consecutive days to induce subacute PD model;after modeling,they were given relevant medicine continuously for 7 d;rod climbing test and line suspension test were performed 1 d before modeling,on the 5th day of modeling and after the last medication.The number of tyrosine hydroxylase(TH)-positive neurons in the substantia nigra of mice were measured by immunofluorescence;the levels of interleukin 1β(IL-1β)and tumor necrosis factorα(TNF-α)in serum and the levels of IL-1β,TNF-α,cyclooxygenase-2(COX-2)and inducible nitric oxide synthase(iNOS)in the substantia nigra of mice were measured by enzyme-linked immunosorbent assay.The expression levels of cAMP-dependent protein kinase catalytic subunitα(PKA C-α),glutathione peroxidase 4(GPX4)and ferritin heavy chain polypeptide 1(FTH1)proteins in the substantia nigra of mice was measured by Western blot.RESULTS After last medicine,compared with the normal group,mice in the model group had significantly longer pole-climbing time(P<0.01),significantly lower line suspension scores(P<0.01),significantly fewer TH-positive neurons in the substantia nigra(P<0.01),significantly higher serum concentrations of IL-1βand TNF-αand nigrostriatal concentrations of IL-1β,TNF-α,COX-2 and iNOS(P<0.01),while lower protein expression levels of GPX4,PKA C-αand FTH1 in the substantia nigra(P<0.05 or P<0.01).Compared with the model group,the above indexes of mice were significantly returned in Arisaema Cum Bile high-dose group(P<0.05 or P<0.01).CONCLUSIONS Arisaema Cum Bile can improve motor impairment and reduce apoptosis of nigrostriatal TH neurons in MPTP-induced PD mice,and has neuroprotective effects on model mice;this may be related to its inhibition of neuroinflammation and the inhibition of ferroptosis by up-regulating PKA signaling pathway.