电针对帕金森病大鼠中脑黑质Sirt3/NLRP3/GSDMD信号通路的影响

Effects of electroacupuncture on Sirt3/NLRP3/GSDMD signaling pathway in the substantia nigra of midbrain of rats with Parkinson’s disease

目的:观察电针“风府”“太冲”“足三里”对帕金森病(PD)模型大鼠中脑黑质酪氨酸羟化酶(TH)、α-突触核蛋白(α-syn)及沉默信息调节因子同源蛋白3(Sirt3)、NOD样受体3(NLRP3)、消皮素D(GSDMD)的影响,探讨电针治疗PD的机制。方法:SD大鼠随机分为对照组、模型组、电针组,每组10只。采用颈背部皮下注射鱼藤酮复制PD大鼠模型。电针组给予电针“风府”“太冲”“足三里”,30 min/次,1次/d,连续治疗28 d。采用旷场实验检测大鼠自主运动总路程;爬杆实验检测大鼠肢体协调能力;免疫组织化学法检测大鼠中脑黑质组织中TH的阳性表达;实时荧光定量PCR法检测大鼠黑质组织中α-syn、Sirt3、NLRP3、GSDMD mRNA表达水平;Western blot法检测大鼠黑质组织中NLRP3、接头蛋白-凋亡相关斑点样蛋白(ASC)、半胱氨酸蛋白酶-1(Caspase-1)蛋白表达水平。结果:与对照组比较,模型组大鼠旷场实验自主运动总路程缩短(P<0.01);爬杆实验评分升高(P<0.01);中脑黑质TH阳性表达减少(P<0.01);Sirt3 mRNA表达水平降低(P<0.01),α-syn、NLRP3、GSDMD mRNA表达水平升高(P<0.01);NLRP3、ASC、Caspase-1蛋白表达水平升高(P<0.01)。与模型组比较,电针组旷场实验自主运动总路程延长(P<0.01);爬杆实验评分降低(P<0.05);中脑黑质TH阳性表达增多(P<0.01);Sirt3 mRNA表达水平升高(P<0.01),α-syn、NLRP3、GSDMD mRNA表达水平降低(P<0.01);NLRP3、ASC、Caspase-1蛋白表达水平降低(P<0.01,P<0.05)。结论:电针“风府”“太冲”“足三里”可修复PD大鼠神经元损伤,清除中脑黑质异常聚集的α-syn,改善线粒体损伤,其机制可能与调控中脑黑质Sirt3/NLRP3/GSDMD信号通路进而抑制神经炎性反应有关。

Objective To observe the effects on tyrosine hydroxylase(TH),α-synaptic nucleoprotein(α-syn),sirtuin 3(Sirt3),NOD-like receptor 3(NLRP3)and gasdermin-D(GSDMD)in the substantia nigra of midbrain after electroacupuncture(EA)at“Fengfu”(GV16),“Taichong”(LR3)and“Zusanli”(ST36)in rats of Parkinson’s disease(PD),so as to explore the mechanism of EA in treatment of PD.Methods SD rats were randomly divided into control,model and EA groups,with 10 rats in each group.The PD model was established by injecting rotenone into the neck and back,lasting 28 days.In the EA group,EA was applied to GV16,LR3 and ST36,30 min each time,once daily,consecutively for 28 days.The open-field test was adopted to detect the total distance of autonomic movement of rats,and the pole climbing test was used to detect the body coordination ability of rats.In the substania nigra of midbrain,the positive expression of TH was determined using immunohistochemistry,the mRNA expression levels ofα-s yn,Sirt3,NLRP3 and GSDMD were detected by quantitative real-time fluorescence PCR,and the protein expression levels of NLRP3,apoptosis-associated speck-like protein containing a caspase-recruitment domain(ASC)and cysteinyl aspartate specific proteinase(Caspase)-1 were detected by Western blot.Results Compared with the control group,the total distance of autonomous movement was decreased(P<0.01)in the model group,and the score of pole climbing experiment was increased(P<0.01);in the midbrain substantia nigra the positive expression of TH was decreased(P<0.01);the mRNA expression level of Sirt3 was decreased(P<0.01),and those ofα-syn,NLRP3 and GSDMD were increased(P<0.01);while the protein expression levels of NLRP3,ASC and Caspase-1 were increased(P<0.01).When compared with the model group,the total distance of autonomous movement in open field experiment was increased(P<0.01)in the EA group and the score of pole climbing experiment was lower(P<0.05);in the midbrain substantia nigra the positive expression of TH was increased(P<0.01);the mRNA expression level of Sirt3 in the midbrain substantia nigra was increased(P<0.01),and those ofα-syn,NLRP3 and GSDMD were reduced(P<0.01);while the protein expression levels of NLRP3,ASC and Caspase-1 decreased(P<0.01,P<0.05).Conclusion EA at“GV16”“LR3”and“ST36”can repair the neuronal injury,clear the abnormal accumulation ofα-syn in the substania nigra of midbrain,and ameliorate mitochondrial damage in PD rats,which may be obtained by regulating Sirt3/NLRP3/GSDMD signaling pathway,so as to delay the occurrence and development of Parkinson’s disease.