Improvement effect of acupuncture on locomotor function in Parkinson disease via regulating gut microbiota and inhibiting inflammatory factorrelease

针刺通过调节肠道微生物和抑制炎症因子释放发挥对帕金森病运动功能的改善效应

Objective To observe the effect of acupuncture on the intestinal flora in Parkinson disease(PD)model mice and explore the mechanism of acupuncture in improving the locomotor function in PD.Methods Thirty-two C57BL/6 mice were randomly divided into a control group,a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)group,a MPTP+acupuncture group(MPTP+A),and a MPTP+madopar group(MPTP+M),with 8 mice in each group.Except for the control group,the other groups were intraperitoneally injected[25 mg/(kg·bw)]with MPTP to establish PD mouse models.After successful modeling,the MPTP group received no intervention,the MPTP+A received acupuncture at Tianshu(ST25),Guanyuan(CV4),and Zusanli(ST36),and the MPTP+M was given madopar[125 mg/(kg·bw)]by intragastric gavage.After consecutive 10-day interventions,the intestinal function and behaviors of the mice were detected.The 16S rRNA gene sequence was used to analyze the composition of fecal intestinal flora in each group.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and enzyme-linked immunosorbent assay were used to detect the expression levels of inflammatory cytokines in the brain and serum.The expression levels of tyrosine hydroxylase(TH)andα-synuclein in the substantia nigra(SN)were detected by immunohistochemical staining.Toll-like receptor(TLR)2 and lipopolysaccharide receptor CD14(CD14)in the SN were determined by RT-qPCR.Myeloid differentiation factor(MyD)88,nuclear factor kappa-B(NF-κB)and Akt1 in the SN were detected by Western blotting.Results After the intervention,compared with the control group,the intestinal motility,fecal water content,and the expression of TH in the SN were significantly decreased in the MPTP group(P<0.05),along with an increasedα-synuclein expression(P<0.05).Additionally,the results of the fecal microflora test showed that the alpha diversity of the MPTP decreased,and the levels of inflammatory cytokines[tumor necrosis factor(TNF)-α,inducible nitric oxide synthase(iNOS),interleukin(IL)-1β,and IL-6]in the serum and SN,and the expression of NF-κB in the SN were significantly increased(P<0.05).Compared with the MPTP group,acupuncture intervention significantly enhanced the autonomous horizontal movement and coordination ability of PD mice(P<0.05);acupuncture and madopar interventions significantly reduced the levels ofα-synuclein,inflammatory cytokines(TNF-α,iNOS,IL-1β,and IL-6)in the serum and SN,and the NF-κB expression in the SN,along with significantly increased alpha diversity richness index(P<0.05).In addition,the relative abundance of Bacteroides increased significantly in the MPTP+A(P<0.05),while the relative abundance of Firmicutes and Cyanobacteria decreased significantly(P<0.05).Conclusion Acupuncture intervention can improve locomotor function,reduceα-synuclein aggregation and inflammatory factors expression,and increase the Akt signaling pathway in PD mice.In addition,acupuncture intervention can benignly regulate the intestinal flora of PD mice.Therefore,it suggests that acupuncture intervention can protect PD model mice probably by regulating intestinal flora and activating Akt signaling pathway.

目的观察针刺对帕金森病(PD)模型小鼠肠道菌群的影响,探讨针刺改善PD神经功能障碍的机制.方法将32只C57BL/6小鼠随机分为对照组、1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)组、MPTP+针刺(MPTP+A)组和MPTP+美多巴(MPTP+M)组,每组8只.除对照组外,其余各组按每公斤体质量25 mg腹腔注射MPTP建立PD小鼠模型.造模成功后,MPTP组不予干预,MPTP+A组予以针刺天枢、关元和足三里,MPTP+M组按每公斤体质量125 mg进行美多巴灌胃.连续干预10 d后对小鼠进行肠道功能及行为学检测.采用16S rRNA基因序列分析各组小鼠粪便肠道菌群的组成.采用实时荧光定量逆转录聚合酶链反应(RT-qPCR)法和酶联免疫吸附测定法检测脑及血清中炎性细胞因子表达.免疫组化染色检测脑黑质(SN)中酪氨酸羟化酶(TH)及α-突触核蛋白表达.RT-qPCR法测定脑SN中Toll样受体(TLR)2和脂多糖受体CD14(CD14).免迹印迹法检测脑SN中髓样分化因子(MyD)88、核因子κB(NF-κB)和Akt1.结果干预结束后,与对照组比较,MPTP组小鼠肠道动力、粪便含水量及SN中TH表达均降低(P<0.05),α-突触核蛋白表达显著升高(P<0.05).粪便菌群测试结果显示MPTP组小鼠的alpha多样性呈下降趋势,血清及SN中炎性细胞因子[肿瘤坏死因子(TNF)-α、诱导型一氧化氮合酶(iNOS)、白介素(IL)-1β和IL-6]水平及SN中NF-κB的表达显著提高(P<0.05).与MPTP组相比,针刺干预显著增强了小鼠的自主水平运动和协调能力(P<0.05);针刺和美多巴干预显著降低了α-突触核蛋白,血清及SN中炎性细胞因子(TNF-α、iNOS、IL-1β和IL-6)及SN中NF-κB的表达(P<0.05);并且显著增加了alpha多样性丰富度指数(P<0.05).此外,MPTP+A组拟杆菌门的相对丰度显著增加(P<0.05),同时厚壁菌门和蓝藻菌门的相对丰度显著降低(P<0.05).结论针刺干预可改善PD模型小鼠运动功能障碍,降低α-突触核蛋白聚集及炎性因子表达并激活Akt信号通路.此外,针刺干预可良性调节PD小鼠的肠道菌群.因此,针刺干预可能是通过调节肠道菌群以及激活Akt信号通路保护PD模型小鼠.