针灸对帕金森病Thy1-αSyn转基因小鼠脑黑质PINK1/Parkin信号通路的影响

Effects of acupuncture and moxibustion on PINK1/Parkin signaling pathway in substantia nigra of Thy1-αSyn transgenic mice with Parkinson disease

目的:观察针灸对帕金森病(PD)Thy1-α突触核蛋白(αSyn)转基因模型小鼠中脑黑质磷酸酶及张力蛋白同源物诱导的蛋白激酶1(PINK1)/帕金蛋白(Parkin)信号通路的影响。方法:将24只Thy1-αSyn转基因小鼠随机分为模型组、针刺组、针灸组和西药组;同窝野生型小鼠6只作为野生组。针刺组取百会和阳陵泉进行针刺;针灸组在针刺组的基础上加灸关元穴;西药组按10 mg/(kg·bw)剂量予雷帕霉素腹腔注射;野生组与模型组给与固定捆绑,不干预。观测各组小鼠总体转棒实验(ORP)评分;采用免疫组织化学法检测各组小鼠脑黑质的酪氨酸羟化酶(TH)阳性神经元;免疫荧光化学法检测αSyn;免疫印迹法检测αSyn、微管相关蛋白轻链3(LC3)-Ⅱ/LC3-Ⅰ、自噬蛋白sequestosome-1(SQSTM-1/p62)、PINK1、Parkin及泛素特异性蛋白酶30(USP30)蛋白表达;荧光定量聚合酶链反应法检测LC3B、p62、PINK1、Parkin及USP30 mRNA表达水平。结果:与野生组相比,模型组小鼠ORP评分及p62、PINK1与Parkin蛋白表达水平显著降低(P<0.01),PINK1 mRNA表达水平降低(P<0.05),USP30蛋白和mRNA表达水平上升(P<0.05)。与模型组相比,针刺组和针灸组的ORP评分上升(P<0.05),针灸组和西药组LC3-Ⅱ/LC3-Ⅰ蛋白表达上升(P<0.05),针刺组、针灸组及西药组p62、PINK1及Parkin蛋白表达上升(P<0.05),USP30蛋白表达显著降低(P<0.01);针刺组和针灸组Parkin mRNA表达水平增高(P<0.05),针灸组USP30 mRNA表达水平降低(P<0.05)。结论:针灸可调节Thy1-αSyn转基因PD模型小鼠PINK1/Parkin信号通路相关分子表达,促进αSyn自噬降解,发挥多巴胺能神经元的保护作用。

Objective:To observe the effects of acupuncture and moxibustion on phosphatase and PTEN-induced putative kinase 1(PINK1)/Parkin signaling pathway in the midbrain substantia nigra of Thy1-αsynuclein(αSyn)transgenic model mice with Parkinson disease(PD).Methods:Twenty-four Thy1-αSyn transgenic mice were randomly divided into a model group,an acupuncture group,an acupuncture+moxibustion group,and a Western medicine group.Six wild-type mice in the same litter were used as the wild-type group.In the acupuncture group,Baihui(GV20)and Yanglingquan(GB34)were selected for acupuncture.In the acupuncture+moxibustion group,Guanyuan(CV4)was added on the basis of the acupuncture group.The Western medicine group was given rapamycin intraperitoneal injection at a dose of 10 mg/(kg·bw).The wild-type group and the model group were fixed without intervention.The overall rod performance(ORP)score of mice was observed in each group.The immunohistochemical method was used to detect the tyrosine hydroxylase(TH)positive neurons in the substantia nigra of mice in each group.TheαSyn was detected by the immunofluorescence chemical method.The expression levels ofαSyn,microtubule-associated protein 1 light chain 3(LC3)-Ⅱ/LC3-Ⅰ,autophagy protein sequestosome-1/protein 62(SQSTM-1/p62),PINK1,Parkin,and ubiquitin-specific protease 30(USP30)proteins were detected by Western blotting assay.The expression levels of LC3B,p62,PINK1,Parkin,and USP30 mRNAs were detected by fluorescence quantitative polymerase chain reaction.Results:Compared with the wild-type group,the ORP score,the p62,PINK1,and Parkin protein expression levels decreased significantly(P<0.01),the PINK1 mRNA expression level decreased(P<0.05),while the protein and mRNA expression levels of USP30 increased(P<0.05)in the model group.Compared with the model group,the ORP score in the acupuncture group and the acupuncture+moxibustion group increased(P<0.05);the expression level of LC3-Ⅱ/LC3-Ⅰprotein in the acupuncture+moxibustion group and the Western medicine group increased(P<0.05);the protein expression levels of p62,PINK1,and Parkin increased(P<0.05),while the USP30 protein expression level decreased significantly(P<0.01)in the acupuncture group,the acupuncture+moxibustion group,and the Western medicine group;the Parkin mRNA expression level in the acupuncture group and the acupuncture+moxibustion group increased(P<0.05);the USP30 mRNA expression level in the acupuncture+moxibustion group decreased(P<0.05).Conclusion:Acupuncture and moxibustion regulate the related molecule expression levels of PINK1/Parkin signaling pathway in the Thy1-αSyn transgenic PD model mice and promote the autophagy degradation ofαSyn to exert the protective effect of dopaminergic neurons.