| Objective: Parkinson’s disease（PD）is the second most common senile neurodegenerative disease in the world,and its pathogenesis remains unclear.Studies at home and abroad have shown that acupuncture,as a traditional therapy,can effectively prevent and delay the process of PD,the mechanism of which may involve acupuncture stimulating the body’s self-regulatory system and protecting dopaminergic（DA）neurons from apoptosis.The GLP-1R related signaling pathway has been widely studied in recent years to promote cell survival,but there have been few reports on how electroacupuncture plays a neuroprotective role through the GLP-1R/PI3K/AKT/NF-κB signaling pathway.In this experiment,rotenone gavage was used to create Parkinson’s disease mouse model,and the behavioral changes of mice at "fengfu "（GV16）,"Taihong "（LR3）and "Zusanli"（ST36）points before and after treatment were observed,and the changes of TH and α-syn mean optical density values in substansubstania nigra of mice in each group were observed.The expression levels of Gl P-1R,PI3 K,AKT and NF-κB protein in the substantia nigra,the changes of microglia cell marker Iba-1 protein content,and the changes of serum inflammatory factors IL-1β and TNF-α were investigated to explore the possible mechanism of electroacupuncture in the prevention and treatment of PD,and to provide experimental basis for more extensive clinical acupuncture treatment.Methods: A total of 66 SPF male C57BL/6 mice aged 7 weeks with the body weight of 18-22 g were selected and fed adapatively for one week.After that,behavioral tests were conducted for three times,and mice with spontaneous behavioral abnormalities were screened out in the open field experiment.According to random number table method,normal mice were divided into four groups: normal group（n=12）,model group（n=18）,electroacupuncture group（n=18）and inhibitor group（n=18）.Rotenone was dissolved in 4%carboxymethyl cellulose and 1.25% chloroform solution for 10mg/kg/d for 4weeks.The normal group was given 0.9% sodium chloride solution for 4weeks.The electroacupuncture group and inhibitor group had the same modeling method as the model group.After modeling by the above methods,behavioral scores were performed for each group of mice,with the scoring criteria referring to the behavioral scoring system established by Chen Xin et al.Mice with scores in the range of2-8 were classified as successful models,and mice that died and were not included in the score range were excluded.Three successful mouse models with scores in the range of 2-8 were selected for brain biopsy by left ventricular paraformaldehyde perfusion and immunohistochemical examination to evaluate the pathological changes of the brain.After modeling,the remaining mice were divided into model group,electroacupuncture group and inhibitor group according to random number table method,with 12 mice in each group.In the electroacupuncture group,"Fengfu","Taichong" and "Zusanli" were treated with continuous wave,voltage 1.6V,intensity 1 m A,frequency 2 Hz.Local tremor at the acupoint was appropriate,30 min/time,and continuous treatment for 14 days."Fengfu" point is used for acupuncture fixation,while the double points "Taichong" and "Zusanli" are used alternately every other day.The inhibitor group was given ligagliptin（10mg/kg/d）for two weeks.Except for the electroacupuncture group,the other three groups were fixed for the same time every day without electroacupuncture treatment.After the intervention,morphological and behavioral changes of the mice were observed.According to the behavioral scoring criteria of Chen Xin et al.,the autonomous motor function and anxiety and depression of the mice were detected in open field experiments from multiple perspectives and at multiple levels.The average absorbance value of TH and α-syn was detected by immunohistochemistry.After blood was collected from the eyes of the remaining half of the mice,the heads were cut off quickly on the ice surface for brain homogenization.The expression levels of GLP-1R,PI3 K,AKT,NF-κB and the content of microglia marker IBa-1 in the substantia nigra of each group were determined by Western-blot method.Serum inflammatory factors IL-1β and TNF-αwere detected by ELISA.Results:1)Behavioral scores of mice in each group.Compared with normal group,behavioral scores of model group,electroacupuncture group and inhibitor group were significantly higher（all P﹤0.01）;Compared with model group,behavioral score of electroacupuncture group and inhibitor group decreased significantly（P ﹤ 0.01,P ﹤0.05）;Compared with electroacupuncture group,behavioral score of inhibitor group was higher（P﹤0.01）.2)Results of open field experiment of mice in each group.Compared with normal group,model group,the curative group autonomous movement in the total distance,average movement speed,movement duration decreased significantly（P ﹤ 0.01）,voluntary movement breaks group increased significantly（P ﹤ 0.01）,inhibitors should be autonomous movement in the total distance,average movement rate,sustained exercise time reduced（P﹤0.05）;Compared with the model group,the total distance,average speed and duration of spontaneous movement in the reaction box of electroacupuncture group and inhibitor group were significantly increased（all P ﹤ 0.01）,and the rest time of spontaneous movement was significantly decreased（all P﹤0.01）.Compared with the electroacupuncture group,the total distance and average speed of autonomous exercise were decreased,the duration of continuous exercise was decreased（all P﹤0.05）,and the rest time of autonomous exercise was increased（P ﹤ 0.05）in the inhibitor group.3)Experimental results of TH expression level in substantia nigra of each group of mice.Compared with normal group,the average absorbance value of TH in model group,electroacupuncture group and inhibitor group was significantly decreased（all P﹤0.01）;Compared with model group,the average absorbance value of TH in the substantia nigra of mice in electroacupuncture group and inhibitor group was increased（all P ﹤ 0.05）;Compared with electroacupuncture group,the average absorbance value of TH in the substantia nigra of inhibitor group had no significant difference（P>0.05）.4)Experimental results of α-syn expression level in substantia nigra of each group.Compared with normal group,the average absorbance value of α-syn in substantia nigra of model group,electroacupuncture group and inhibitor group was significantly increased（all P﹤0.01）.Compared with model group,the average absorbance value of α-syn in the substantia nigra of mice in electroacupuncture group and inhibitor group was decreased（all P ﹤ 0.05）.Compared with electroacupuncture group,the average absorbance value ofα-syn in substantia nigra of inhibitor mice had no significant difference（P>0.05）.5)Experimental results of relative expression levels of GLP-1R,PI3 K,AKT and NF-κB p65 protein in the substantia nigra of each group.Compared with normal group,GLP-1R/GAPDH,p-PI3K/PI3 K,p-AKT/AKT（all P<0.01）were significantly decreased in model group,while p-NF-κB p65/NF-κB p65 was significantly increased in model group（P ﹤0.01）.Compared with normal group,GLP-1R/GAPDH and p-PI3 K /PI3 K in the substantia nigra of electroacupuncture group were significantly decreased（all P﹤0.01）,p-AKT/AKT had no significant difference（P>0.05）,and P-NF-κB p65/NF-κB p65 were increased（P﹤0.05）.Compared with normal group,GLP-1R/GAPDH,p-PI3K/PI3 K and p-AKT/AKT in inhibitor group were significantly decreased（all P<0.01）,and p-NF-κB p65/NF-κB p65 were significantly increased（P<0.01）.Compared with model group,GLP-1R/GAPDH,p-PI3K/PI3 K and p-AKT/AKT in substantia nigra of electroacupuncture group were significantly increased（all P ﹤ 0.01）,while p-NF-κBp65/NF-κB p65 in electroacupuncture group was significantly decreased（P﹤0.01）.Compared with model group,p-PI3K/PI3 K in inhibitor group was significantly increased（P﹤0.01）,p-NF-κB p65/NF-κB p65 was significantly decreased（P<0.01）,GLP-1R/GAPDH and p-AKT/AKT were increased（P<0.05）.Compared with electroacupuncture group,GLP-1R/GAPDH in inhibitor group was decreased（P<0.05）,p-PI3K/PI3 K was significantly decreased（P﹤0.01）,and the protein content of p-AKT/AKT and p-NF-κB p65/NF-κB p65 had no significant difference compared with electroacupuncture group（P>0.05）.6)Experimental results of GLP-1R,PI3 K,AKT and NF-κB gene expression levels in the substantia nigra of each group.Compared with normal group,the gene expression levels of GLP-1R,PI3 K and AKT in model group were significantly decreased（all P﹤0.01）,NF-κB was significantly increased（P﹤0.01）,the gene expression levels of GLP-1R and PI3 K in electroacupuncture group and inhibitor group were significantly decreased（all P ﹤ 0.01）.The expression of AKT gene was decreased（all P﹤0.05）,and the expression of NF-κB gene was increased（all P ﹤ 0.01）.Compared with model group,the gene expression levels of GLP-1R,PI3 K and AKT in the substantia nigra of electroacupuncture group and inhibitor group were significantly increased（all P﹤0.01）,and the gene expression level of NF-κB was significantly decreased（all P ﹤ 0.01）.Compared with electroacupuncture group,there were no significant differences in GLP-1R,PI3 K,AKT and NF-κB gene expression levels in inhibitor group（all P>0.05）.7)Experimental results of relative expression level of Iba-1 protein in the substantia nigra of each group.Compared with normal group,the protein content of Iba-1 in model group and inhibitor group was significantly increased（P ﹤ 0.01）,and the protein content of Iba-1 in electroacupuncture group was increased（P﹤0.05）;Compared with model group,the protein content of Iba-1 in the substantia nigra of mice in Electroacupuncture group and inhibitor group was significantly decreased（all P ﹤ 0.01）;Compared with Electroacupuncture group,the protein content of Iba-1 in the substantia nigra of inhibitor group was increased（P﹤0.05）.8)Experimental results of serum inflammatory factors IL-1β and TNF-α in each group.Compared with normal group,serum inflammatory factors IL-1β and TNF-α were significantly increased in model group,Electroacupuncture group and inhibitor group（all P ﹤ 0.01）.Compared with model group,serum inflammatory factors IL-1β and TNF-α in electroacupuncture group and inhibitor group were significantly decreased（P ﹤ 0.01）;Compared with electro-acupuncture group,serum inflammatory factors IL-1β and TNF-α were increased in inhibitor group（all P﹤0.05）.Conclusion:1)Electroacupuncture at three points of "Fengfu","Taihong" and "Zusanli" can effectively improve the appearance and autonomous motor function of rotenone-induced Parkinson’s disease model mice.2)Electroacupuncture can effectively reduce the formation and aggregation of α-syn,the pathological product characteristic of PD,thus reducing neurotoxicity.On the other hand,it promotes TH synthesis and protects DA neuronal activity.3)The mechanism of electroacupuncture intervention in the treatment of Parkinson’s disease model mice may be through activating the GLP-1R/PI3K/AKT/NF-κB signaling pathway,inhibiting the overactivation of microglia,reducing the release of inflammatory factors IL-1β and TNF-α,alleviating neuroinflammatory response,promoting cell survival and delaying the pathological process of PD.So as to achieve the purpose of prevention and treatment of PD. |
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