柴胡加龙骨牡蛎汤对帕金森病伴发抑郁模型大鼠的神经保护作用及对AMPK/mTOR信号通路的影响

Neurorotective Effect of Chaihu Jia Longgu Mulitang on Parkinson’s Disease with Depression Model Rats and Its Mechanism Based on AMPK/m TOR Signaling Pathway

目的:观察柴胡加龙骨牡蛎汤(CLMT)对帕金森病伴发抑郁(PDD)模型大鼠多巴胺能神经元的保护作用,并基于腺苷酸活化蛋白激酶/哺乳动物雷帕霉素靶蛋白(AMPK/m TOR)信号通路探讨其作用机制。方法:80只雄性SD大鼠,随机选取10只作为正常组,其余采用长期低剂量颈背部皮下注射鱼藤酮联合慢性不可预见性温和应激(CUM)建立PDD大鼠模型,将造模成功的PDD大鼠随机分为模型组、西药组(多巴丝肼0.032 g·kg^(-1)+盐酸氟西汀0.002 g·kg^(-1))、CLMT低、中、高剂量组(5、10、20 g·kg^(-1)),每组10只,正常组和模型组予以等体积生理盐水灌胃,连续4周。旷场实验、爬杆实验评估各组大鼠行为学变化;高效液相色谱法(HPCL)测定脑脊液中多巴胺(DA)、5-羟色胺(5-HT)含量;苏木素-伊红(HE)染色法观察各组大鼠黑质DA能神经元病理改变;免疫组化法(IHC)检测黑质酪氨酸羟化酶(TH)阳性表达;免疫荧光法(IF)检测黑质α-突触核蛋白(α-synuclein)表达;蛋白免疫印迹法(Western blot)检测纹状体微管相关蛋白1轻链3(LC3)、AMPK、磷酸化AMPK(p-AMPK)、m TOR、磷酸化m TOR(p-m TOR)蛋白表达。结果:与正常组比较,模型组大鼠旷场实验水平运动总距离及中央区域活动时间均明显减少(P<0.05,P<0.01),爬杆时间明显缩短(P<0.01),评分升高(P<0.01),脑脊液中DA、5-HT含量减少(P<0.05,P<0.01);与模型组比较,CLMT高剂量组和西药组水平运动总距离明显增加及中央区域活动时间均增加(P<0.05,P<0.01),爬杆时间延长(P<0.05),评分下降(P<0.05,P<0.01),DA及5-HT含量增加(P<0.05,P<0.01)。与正常组比较,模型组大鼠黑质DA能神经元数量明显减少,胞体缩小且排列疏松,α-synuclein荧光表达显著增强(P<0.01),TH阳性表达显著降低(P<0.01);与模型组比较,CLMT高剂量组和西药组黑质DA能神经元数量增加,胞体增大,α-synuclein荧光表达明显减弱(P<0.05,P<0.01),TH表达显著增加(P<0.01)。与正常组比较,模型组纹状体LC3Ⅱ/Ⅰ、p-AMPK/AMPK表达明显降低(P<0.05,P<0.01)、p-m TOR/m TOR表达显著增加(P<0.01);与模型组比较,CLMT高剂量组和西药组LC3Ⅱ/Ⅰ、p-AMPK/AMPK表达均明显增加(P<0.05,P<0.01)、p-m TOR/m TOR表达显著降低(P<0.01)。结论:CLMT可抑制鱼藤酮神经毒性,发挥神经保护作用,提高DA水平,从而改善帕金森病大鼠抑郁状态,其机制可能与调控AMPK/m TOR信号通路,激活自噬,促进异常聚集的α-synuclein降解有关。

Objective:To observe the protective effect of Chaihu Jia Longgu Mulitang(CLMT)on dopaminergic neurons in Parkinson’s disease with depression(PDD)model rats,and to explore the mechanism based on adenosine monophosphate-activated protein kinase/mammalian target of rapamycin(AMPK/m TOR)signaling pathway.Method:Among the 80 male SD rats,10 were randomly selected as normal group and the rest were treated with long-term low-dose subcutaneous injection of rotenone in the neck and back combined with chronic unpredictable mild stress(CUMS)to establish PDD rat model.The successfully modeled PDD rats were randomly divided into model group,western medicine group(madopar 0.032 g·kg^(-1)+fluoxetine hydrochloride0.002 g·kg^(-1)),CLMT low-dose,medium-dose and high-dose groups(5,10 and 20 g·kg^(-1)),10 rats in each group.Normal group and model group were administrated with the same amount of normal saline by gavage for 4consecutive weeks.Behavioral changes of rats in each group were evaluated by open field test and pole climbing test.The content of dopamine(DA)and 5-hydroxytryptamine(5-HT)in cerebrospinal fluid was determined by high performance liquid chromatography(HPCL).The pathological changes of dopaminergic neurons in substantia nigra of rats were observed by hematoxylin-eosin(HE)staining.The positive expression of tyrosine hydroxylase(TH)and expression ofα-synuclein in substantia nigra were detected by immunohistochemistry(IHC)and immunofluorescence(IF),repsectively.The protein expression of microtubule-associated protein 1light chain 3(LC3),adenosine monophosphate-activated protein kinase(AMPK),phosphorylated AMPK(p-AMPK),mammalian target of rapamycin(m TOR)and phosphorylated m TOR(p-m TOR)was detected by Western blot.Result:Compared with the conditions in normal group,the total horizontal distance and the activity time in the central region in open field test and the content of DA and 5-HT in cerebrospinal fluid were decreased(P<0.05,P<0.01),and the time of pole climbing was shortened(P<0.01),with increased score(P<0.01)in model group.Compared with model group,CLMT high-dose group and western medicine group increased the total horizontal distance and activity time in the central region and the content of DA and 5-HT(P<0.05,P<0.01),and extended the time of climbing pole(P<0.05),with decreased score(P<0.05,P<0.01).Compared with those in normal group,the number of dopaminergic neurons in the substantia nigra was reduced,with narrowed and loosely arranged cell body.The fluorescence expression ofα-synuclein was enhanced(P<0.01),and the positive expression of TH was decreased(P<0.01)in model group.Compared with model group,CLMT high-dose group and western medicine group showed elevated number of dopaminergic neurons in the substantia nigra,with enlarged cell body,and decreased fluorescence expression ofα-synuclein,and enhanced the positive expression of TH(P<0.05,P<0.01).Compared with normal group,model group had lowered expression of LC3Ⅱ/Ⅰ,p-AMPK/AMPK in striatum(P<0.05,P<0.01)and increased expression of p-m TOR/m TOR(P<0.01).Compared with those in model group,LC3Ⅱ/Ⅰand p-AMPK/AMPK expression were increased(P<0.05,P<0.01)and p-m TOR/m TOR expression was decreased(P<0.01)in CLMT high-dose group and western medicine group.Conclusion:CLMT exerts a neuroprotective effect by inhibiting rotenone neurotoxicity.It enhances the level of DA,and thus improves the depression condition in rats with Parkinson’s disease.The underlying mechanism may be related to the regulation of AMPK/m TOR signaling pathway,activation of autophagy,and promotion of degradingα-synuclein.