| Objective: The aim of this study was to investigate the changes of systemic behaviour,intestinal flora and striatal neurotransmitters in MPTP-induced subacute PD animal models,and the effects of electro-acupuncture at different acupoints on motor behavior,intestinal flora diversity and striatal neurotransmitter levels in PD mice,to establish a systematic,efficient and accurate behavioral index system for evaluating the early efficacy of PD and PD,to explore the relationship between intestinal flora diversity and the incidence of PD,and to clarify the intervention characteristics of electro-acupuncture points?Baihui,GV-20;Dazhui,GV-14?and electro-acupuncture points on extremities?Hegu,LI-4;Taichong,LR-3?on PD mice,so as to treat PD with clinical electro-acupuncture.It provides new ideas and experimental evidence.Methods:1.The animal models of Parkinson's disease in mice were made by intraperitoneal injection of MPTP?30mg/kg?for 7 consecutive days.The indexes of climbing rod,rotating rod and suspension behavior were detected immediately after the model was established.The traditional animal behavior test method was used to evaluate the establishment of Parkinson's animal model.After the successful establishment of the model,the corresponding intervention measures were given to each group.The control group and the model group were fed with normal diet.The model + positive drug?Madopa?group was given Madopa by gavage.The dosage was converted into clinical human dosage.The weight of 6.25mg/kg mice was once a day for two consecutive weeks.Model + electro-acupuncture at GV-20 and GV-14 for 2 weeks;model + electro-acupuncture at limb acupoints?LI-4 and LR-3?for 2 weeks.The gait,climbing rod,rotating rod and hanging behavior of mice in each group were evaluated by dynamic detection system at 1 and2 weeks of continuous electro-acupuncture.After 2 weeks of intervention,PD animals were anesthetized with 1% sodium pentobarbital by intraperitoneal injection of 35 mg/kg body weight.After anesthesia,the brain was isolated and bilateral striatum was taken.The levels of dopamine andacetylcholine in striatum were measured by ELISA,and the effects of electro-acupuncture at different acupoints on PD mice were observed.It is influenced by transmitter.After 2 weeks of intervention,PD animals were anesthetized and bilateral striatum was taken.At the same time,colonic tissues of experimental mice were intercepted to analyze the taxonomy,diversity,community composition and species difference of intestinal flora,and to clarify the characteristics of intestinal flora diversity of PD animals and the effects of electro-acupuncture intervention.Result:1.Establishment of Parkinson's animal model and analysis of its behavioral characteristics1.1 General behavioral observations of PD miceAfter 5-30 minutes of MPTP injection,the mice developed restlessness symptoms such as erect hair,tremor,tail warping,upper back arch and hind limb opening.After 30 minutes,salivation occurred.After 1 hour,the mice began to move less,their eyes were dull,their reactions were slow and their movement became slower.After 2h,the symptoms gradually disappeared and returned to normal after 6hours.As the frequency of administration increased,activity decreased,exercise slowed down and reaction became slow.1.2 Results of PD mice climbing rod testAfter 7 consecutive days of intraperitoneal injection of MPTP,rod climbing behavior was tested.The results showed that compared with the control group,the upper half time,the lower half time and the total time?the sum of the upper half time and the lower half time?of the test rod in the model group were prolonged to varying degrees?P < 0.01,P < 0.001,P < 0.001?.1.3 Suspension test results of PD miceIn the suspension test,C57BL/6 mice were scored according to the scoring criteria.The higher the score,the firmer the mice grasped the wires.It indicated that the better the coordination of limb movements in mice,and vice versa,it indicated that the situation of tremor and muscle stiffness in mice was serious.It was found that there was a significant difference between the control group and the PD model group?P <0.05?.In addition,the fall latency of the model group was significantly lower than that of the control group?P <0.01?.1.4 Results of PD mice rotating rod testIn order to study the effect of MPTP intraperitoneal injection on the motor ability of mice,the rotating rod method was used to detect the balance and coordination ability of PD mice.Rotary rod test is the most commonly used method in evaluating the motor coordination ability of rodents.Rotary rod experiment was conducted in mice for one week before the experiment.The results showed that the residence time?latency?of mice on the rotating rod was significantly shortened 7 days after the last injection of MPTP?P < 0.05?.The behavioral results of all PD mice were consistent with the symptoms of Parkinson disease.2.Effect of different electroacupuncture acupoints on behavioral improvement of Parkinson's disease animal models2.1 The results of behavioral experiment in mice of each group for 1 week after stimulation by electroacupuncture stimulation2.1.1 Experimental results of gait behavior in each group of mice after electroacupuncture for 1 weeksThe results showed that the average body rotation angle,single support phase,double support phase,average footprint strength,contralateral coordination,speed,walking cycle,support phase,instantaneous speed and propulsion index of Parkinson's disease animal model had significant abnormal changes.After one week of treatment,electro-needle points,limb points and positive drug Madopa showed significant changes in the average body rotation angle,single support phase,double support phase,average footprint intensity,contralateral coordination,speed,walking cycle,support phase,instantaneous speed and propulsion index.The gait function has been improved obviously,especially the number and degree of improvement of gait index after electro-acupuncture stimulation of limb acupoints are better than those of electro-needle acupoint group.2.1.2 Experimental results of climbing rod behavior in each group after 1weeks of electroacupuncture stimulationCompared with the control group,the lower half time and the total time of climbing pole in the model group were significantly prolonged?P < 0.01,P < 0.01?.Compared with the model group,there was no significant improvement in the positive drug group,the electro-needle acupoint group and the electro-acupuncture limb acupoint group.2.1.3 Results of suspension test in each group after 1 weeks of electroacupuncture stimulationCompared with the control group,the drop latency and hanging score of the model group decreased significantly?P < 0.05,P < 0.01?after one week of electro-acupuncture stimulation.Compared with the model group,there were no significant changes in the drop latency and hanging score of the mice in the electro-needle acupoint group,the electro-acupuncture limb acupoint group and the positive drug group.2.1.4 The results of rotating rod test in each group after 1 weeks of electroacupuncture stimulationCompared with the control group,the latency of the model group was significantly shortened?P < 0.05?.Compared with the model group,there was no significant change in the latency of the mice in the electro-acupuncture point group,the electro-acupuncture limb point group and the positive drug group.2.2 Results of behavioral experiment in mice of 2 weeks after stimulation by electroacupuncture stimulation2.2.1 Experimental results of gait behavior in each group of mice after electroacupuncture for 2 weeksThe results showed that there are significant differences in the average body turning angle,left-posterior single support time,right-posterior single support time,left-anterior-posterior double support time,left-posterior-anterior double support time,left-anterior-posterior-right anterior,left-anterior footprint average area,left-posterior footprint average area,right-anterior footprint average area and right-posterior footprint average area right-rear coordination with the left front and the opposite side,the right-front coordination with the left rear,the speed,the average walking cycle,the support phaseleft front,the support phaseleft rear,the support phaseright front,the support phaseright rear,the instantaneous velocityleft front,the instantaneous velocityleft rear,the instantaneous velocityright front,the instantaneous velocityright front and the instantaneous velocityright rear of Parkinson's disease animal model.The results showed that the gait function of Parkinson's disease animal model had obvious dysfunction 2 weeks after modeling.The gait function of electro-needle acupoints,limb acupoints and positive drug Madopa had obvious improvement 2 weeks after treatment.Especially,the number and degree ofimprovement of gait index after electro-needle acupoint treatment were significantly better than that of electro-acupuncture limb acupoint group.2.2.2Experimental results of climbing rod behavior in each group after 2weeks of electroacupuncture stimulation2 weeks after electroacupuncture stimulation,Compared with the control group,the turning time,the first half time,the second half time and the total climbing time of the model group were significantly prolonged?P < 0.001,P < 0.01,P < 0.001,P <0.01?.Compared with the model group,the turning time,the first half time and the total climbing time of the needle group were significantly shortened.?P < 0.05,P <0.05,P < 0.05?,the first half time and the second half time of the electro-acupuncture limb acupoint group were significantly shortened?P < 0.05,P < 0.05?,and the turn-around time and the second half time of the positive drug group were significantly shortened?P < 0.05,P < 0.05?.2.2.3 Results of suspension test in each group after 2 weeks of electroacupuncture stimulationCompared with the control group,the drop latency of the model group was significantly shortened?P < 0.01?after 2 weeks of electro-acupuncture stimulation.Compared with the model group,the drop latency of the mice in the electro-acupuncture point group,the electro-acupuncture limb acupoint group and the positive drug group were significantly prolonged?P < 0.05,P < 0.01,P < 0.05?.Compared with the control group,the hanging test scores in the model group were significantly reduced?P < 0.05?,and the hanging test scores in the electro-needle acupoint group and the positive drug group were significantly increased?P < 0.05,P< 0.05?.2.2.4 The results of rotating rod test in each group after 2 weeks of electroacupuncture stimulationCompared with the control group,the latency of the model group was significantly shortened?P < 0.01?,and the latency of the model group was significantly prolonged?P < 0.05?.3.Effects of Electroacupuncture on neurotransmitter levels in brain tissue of animals with Parkinson's disease3.1 Effect of Electroacupuncture on dopamine level in striatum of Parkinson miceThe striatal dopamine level in model group was significantly lower than that in control group?P < 0.001?.Compared with model group,the level of dopamine in electro-needle acupoint group,electro-acupuncture limb acupoint group and positive drug group increased significantly?P < 0.001,P < 0.05,P < 0.001?.3.2 Effect of Electroacupuncture on acetylcholine level in brain striatum of Parkinson miceThe striatum acetylcholine level in model group was significantly lower than that in control group?P < 0.01?.Compared with model group,the level of acetylcholine in electro-needle acupoint group,electro-acupuncture limb acupoint group and positive drug group increased significantly?P < 0.05,P < 0.05,P < 0.05?.4 Effect of Electroacupuncture on intestinal flora in Parkinson's disease animal4.1 Taxonomic analysis of intestinal floraRank-Abundance curve of intestinal flora showed that the control group had a small curve width and a small range on the transverse axis;the model group had a large Rank-Abundance curve width and a large range on the transverse axis;and the EA group had a Rank-Abundance curve between the control group and the model group.4.2 Pan/Core species analysis resultsPan curve of intestinal flora showed that the OTU number of intestinal flora increased with the increase of the number of samples in three groups.When the number of samples in each group increased to 4,the increase of OTU number tended to be flat,indicating that the number of samples in each group was sufficient.In addition,the total number of OTU in the model group was the largest,while that in the control group was less than that in the electroacupuncture group.Core curve of intestinal flora showed that the total number of OTUs in intestinal flora of three groups decreased with the increase of the number of samples.When the number of samples in each group increased to 5,the decreasing trend of the number of OTUs tended to be gentle,indicating that the number of samples in each group was sufficient.In addition,when the sample size of each group reached 6,the number of OTUs in the model was the largest,followed by the control group,and the lowest in the electro-acupuncture limb acupoint group.4.3 Alpha diversity analysis of intestinal floraThe results of community richness index analysis showed that the ace,Chao and sobs indexes of intestinal flora in the model group increased compared with the control group,but the difference was not statistically significant?P > 0.05?.There was no significant difference between the four limbs acupoint group and the model group?P > 0.05?.Compared with the model group,the electro-acupuncture limb acupoint group decreased to a certain extent,but there was no significant difference?P > 0.05?.The results of community diversity index analysis showed that the Shannon of intestinal flora in model group and EA group increased to a certain extent,while the Simpson index decreased significantly,but there was no significant difference between the two groups?P > 0.05?.4.4 Beta diversity analysis of intestinal floraThe first two coordinate data?PC1 and PC2?with matrix eigenvalues are selected for principal component analysis?PCA?.The results showed that at the OTU classification level,the model group and the control group were located in different regions.The demarcation was clear and far from each other,while the electro-acupuncture group and the model group were overlapped and the demarcation was not clear,and the distance between the two groups was close.However,the distance between Electroacupuncture of the acupoints and the control group was close to that between the model group and the control group.Principal co-ordinates analysis?PCo A?based on the eigenvalues of distance matrix in the first few places,The results showed that at OTU classification level,the distribution area of each sample in the control group was different from that in the model group and the electro-acupuncture limb acupoint group,and the demarcation was clearer and the distance was longer.The distribution range of the samples in the electro-acupuncture limb acupoint group was closer to the normal group than that in the model group.Non-metric multidimensional scaling?NMDS?was performed at OTU classification level.The results showed that the distribution of samples in the control group was concentrated,while the spatial distance between the other two groups and the control group was farthest,with the model group being the farthest;the distribution areas of acupoints in the model group and electro-acupuncture limbs overlapped.4.5 Composition analysis of intestinal microbial communityAt phylum classification level,the community structure of intestinal flora was analyzed.The results showed that among the detected flora types,Firmicutes,actinobacteria,Deferribacteres and Verrucomicrobi were found in the model group as compared with the control group.A)the proportion has increased significantly.The proportion of Bacteroidetes decreased;compared with the model group,the proportion of Firmicutes,actinobacteria,Deferribacteres and Verrucomicrobia decreased in the electro-acupuncture limb acupoint group,while that of Bacteroidetes decreased in the electro-acupuncture limb acupoint group.The proportion has increased.4.6 Species diversity analysis of intestinal floraAt the taxonomic level of selected genera,the intestinal flora with the highest community abundance ranked in the top 15 were tested for significant differences among groups.The results showed that: norankf<sub>BacteroidalesS24-7group ?Lactobacillus ? Alistipes ? Turicibacter ? Faecalibaculum ?LachnospiraceaeNK4A136group ? LachnospiraceaeNK4A136group ?Bifidobacterium ? CoriobacteriaceaeUCG-002 ? Clostridiumsensustricto1 ?unclassifiedf<sub>Erysipelotrichaceae ? PrevotellaceaeUCG-001 ?DefluviitaleaceaeUCG-011 ? unclassifiedo<sub>Bacteroidales ?unclassifiedf<sub>Peptostreptococcaceae ? Burkholderia-Paraburkholderia There were significant differences between the 15 groups?P < 0.05 or P < 0.01?.Compared with the control group,CoriobacteriaceaeUCG-002 and Clostridiumsensustricto1 in Parkinson's model group increased significantly?P < 0.05 or P < 0.01?,while the level of CoriobacteriaceaeUCG-002 and Clostridiumsensustricto1 in electro-acupuncture limb acupoint group decreased significantly?P < 0.05 or P <0.01?.Compared with the control group,Turicibacter,DefluviitaleaceaeUCG-011 and unclassifiedoBacteroidales in the Parkinson model group had significant changes?P < 0.05 or P < 0.01?.Electro-acupuncture at limb acupoints had some improvement effect,but there was no statistical difference.Conclusion:1.Electro-needle points?GV-20 and GV-14?and electro-needle points?LI-4 and LR-3?on extremities can effectively improve the motor dysfunction of PD mice and the neurotransmitters closely related to PD.Moreover,electro-acupuncture points and electro-acupuncture points on extremities have their own advantages in the treatmentof PD,which provides new ideas and experimental basis for clinical electro-acupuncture treatment of PD.2.The automatic gait analysis system overcomes the shortcoming that the traditional classic behavior evaluation method cannot accurately and sensitively capture the subtle changes in the animal behaviors of Parkinson's disease in the early stage,and minimizes the damage and stimulation to experimental animals.It may replace or partially replace the current classic behavior evaluation method of Parkinson's animal experiments and become a more objective,comprehensive,efficient and accurate new method,providing new methods for exploring and evaluating the acupuncture treatment of Parkinson's disease.3.MPTP-induced intestinal flora changes in PD mice.Electro-acupuncture can promote the recovery of abnormal changes in intestinal flora diversity in PD mice.It is preliminarily confirmed that intestinal flora diversity is closely related to the pathogenesis of PD.Electro-acupuncture may alleviate the motor dysfunction of PD mice by regulating intestinal flora diversity. |
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