Effect And Mechanism Of Electroacupuncture In The Treatment Of Dyskinesia In Parkinson’s Disease Based On The "microbe-gut-brain" Axis

Objective: Parkinson’s disease(PD)is a common neurodegenerative disease in old-aged people,which is mainly manifested by movement disorders such as tremor,muscle tonus,and bradykinesia,seriously affecting the quality of patients’ survival and increases their psychological and social burdens.In the clinical study,primary PD patients were used as the research object to observe the regulatory effects of electroacupuncture on dyskinesia,Chinese medicine symptom score and serum GLP-1 in PD patients.In the experimental study,MPTP-induced PD model mice were used as research subjects to explore the mechanism of electroacupuncture to inhibit neuroinflammation and enhance the activity of dopaminergic neurons by regulating the microbe-gut-brain axis and activating the PI3K-AKT-GSK3β signalling pathway.Methods:1.The clinical study was conducted using a randomised controlled method,in which the 80 patients included were randomly divided into 40 cases each in the treatment group and the control group.The UPDRS total score and UPDRS III score were used as the primary outcome indicators,and Berg balance scale,get-up-and-walk test,gait analysis,serum GLP-1 concentration,and Chinese medicine evidence points scale were used as the secondary outcome indicators to observe the improvement of the indicators of the PD patients after the electroacupuncture intervention,and to apply subjective scale,objective serum indexes,and gait analysis techniques to comprehensively assess the clinical efficacy of electroacupuncture in improving PD.2.Forty C57BL/6 mice were randomly divided into control group,model group,electroacupuncture group and drug group,ten mice in each group.The mice were subjected to electroacupuncture under anaesthesia with isoflurane(flow rate of0.8L/min,concentration of 1.5%),and acupuncture was performed on Baihui,Zu Sanli(double)and Taichong(double).The Zu Sanli-Taichong on the same side was used as the electroacupuncture connection pair of points for 20 min/time,one time/day,and the intervention was carried out for 14 consecutive days.The mental state,fur colour,reactivity and activity of mice in each group were observed.The improvement of the mice’s locomotor ability after electroacupuncture intervention was evaluated by the open field test,pole test,rotarod test and traction test.Immunohistochemical method was used to observe the expression of nigral tyrosine hydroxylase(TH)in mice in each group.To clarify the improvement of motor impairment and pathological changes in PD by electroacupuncture.The 16 S r DNA technique was applied to analyse the intestinal flora of the cecum contents of mice in the blank,model and electroacupuncture groups to analyse the modulating effect of electroacupuncture on the intestinal microorganisms in PD mice,and to find the key flora after electroacupuncture intervention.Targeted metabolomics analysis of mouse cecum contents by GC-MS technology was performed to analyse the regulatory effects of electroacupuncture on SCFAs.Real-time fluorescence quantitative PCR,immunofluorescence and ELISA were used to detect the expression of GPR40/GPR43 gene,GLP-1 expression and serum GLP-1 concentration of SCFAs receptor in mouse colon.Western blot and real-time fluorescence quantitative PCR were used to detect the expression of GLP-1R,downstream pathway proteins(PI3K,p-PI3 K,AKT,p-AKT,GSK3β,p-GSK3β),and inflammatory factors(TNF-α,IL-6,IL-1β)in mouse substantia nigra.Results:The total clinical effectiveness rate of electroacupuncture for Parkinson’s disease was 86.11%,which was better than that of the control group.Comparison within the group revealed that the total UPDRS score,UPDRS III score,and the length of up-and-over walking test of the patients in the treatment group decreased compared with the pre-treatment period(P < 0.05),and the score of Berg Balance Scale increased(P < 0.05),while in the control group,only the UPDRS III score decreased statistically compared with the pre-treatment period(P < 0.05),and the gait speed of the treatment group in the gait analysis test increased compared with the pre-treatment period(P < 0.05),stride time and double-support phase decreased compared to pre-treatment(P < 0.05),and serum GLP-1 concentration decreased in the treatment group compared to pre-treatment(P < 0.05).Comparison between the groups revealed that UPDRS total score,UPDRS III score,and the length of the rise-and-walk test decreased in the treatment group compared with the control group(P < 0.05),Berg Balance Scale score increased in the treatment group compared with the control group(P < 0.05),and in the gait analysis,the step speed of the treatment group was higher than that of the control group(P < 0.05),and the concentration of serum GLP-1 in the treatment group was higher than that of the control group(P <0.05).It indicated that the electroacupuncture group had some advantages in improving movement disorder,gait abnormality and serum GLP-1 concentration in PD patients.Behavioural results showed that the electroacupuncture group and the drug group could significantly improve the dyskinesia of the PD model mice in the open field test,the rotarod test,the pole test and the traction experiment.The results of brain histopathological experiments showed that the electroacupuncture group could significantly enhance the expression of nigrostriatal TH cells and increase the activity of dopaminergic neurons in PD model mice.Analysis of intestinal flora results showed that MPTP-induced PD model mice showed reduced intestinal flora diversity and disrupted species structure,and electroacupuncture increased Chao1,Shannon and Simpson indices of the flora in PD model mice.Electroacupuncture increased the number of OUTs in the cecum contents,regulated the diversity and abundance of the intestinal flora,and increased the expression level of beneficial bacteria to reduce the expression level of harmful bacteria,and the results of the species difference analysis showed that the levels of Proteobacteria,Ruminococcaceae,Desulfovibrio,and Odoribacter were elevated in the PD mice(P<0.05),while the levels of Actinobacteria,S24-7,Coprococcus,and Ruminococcus were decreased(P<0.05),and this trend was reversed after electroacupuncture intervention(P<0.05).Metabolomics results of the targeted SCFAs showed that MPTP induced a significant decrease in the levels of acetic acid,propionic acid,and butyric acid in PD mice(P <0.05),whereas the levels of acetic acid,propionic acid,and butyric acid were significantly increased after electroacupuncture intervention(P < 0.05).Colonic pathology results showed that electroacupuncture significantly increased the expression of SCFAs receptor GPR40/43 genes and GLP-1 in the colon of PD model mice,and serum ELISA results showed that electroacupuncture increased the concentration of GLP-1 in the serum of PD model mice,indicating that electroacupuncture could promote the release of GLP-1 by regulating intestinal microorganisms.The nigrostriatal pathology results showed that electroacupuncture increased the nigrostriatal GLP-1R expression and the ratios of p-PI3K/PI3 K and p-AKT/AKT,decreased the ratio of p-GSK3 β/GSK3β,and further inhibited the expression of the inflammatory factors TNF-α,IL-6,and IL-1β in the nigra in the mice with PD model.Conclusion:1 On the basis of Medopa treatment,combined with electroacupuncture therapy can significantly improve the dyskinesia of PD patients,increase the balance ability of the patients,adjust the abnormal gait,improve the stability during walking,reduce the risk of falling,reduce the Chinese medicine evidence points,promote the release of GLP-1,and increase the level of GLP-1 in the serum.2 Electroacupuncture plays a role in inhibiting neuroinflammation,enhancing the activity of dopaminergic neurons,and improving movement disorders in PD by regulating the microbe-gut-brain axis and activating the PI3K-AKT-GSK3 βsignalling pathway.