Abstract
Objective:Parkinson’s disease (PD) is a chronic neurodegenerative disease involving non-motor symptoms,of which gastrointestinal disorders are the most common. In light of recent results,intestinal dysfunction may be involved in the pathogenesis of PD. Electroacupuncture (EA) has shown potential effects,although the underlying mechanism remains mostly unknown. We speculated that EA could relieve the behavioral defects of PD,and that this effect would be associated with modulation of the gut microbiota.
Methods:Mice were randomly divided into three groups:control,PD +MA (manual acupuncture),and PD +EA. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) was used to establish the mouse model of PD. Rotarod performance tests,open field tests,and pole tests were carried out to assess motor deficiencies. Immunohistochemistry was conducted to examine the survival of dopaminergic neurons. 16S ribosomal RNA (rRNA) gene sequencing was applied to investigate the alterations of the gut microbiome. Quantitative real-time polymerase chain reaction (PCR) was performed to characterize the messenger RNA (mRNA) levels of pro-inflammatory and anti-inflammatory cytokines.
Results:We found that EA was able to alleviate the behavioral defects in the rotarod performance test and pole test,and partially rescue the significant loss of dopaminergic neurons in the substantia nigra (SN) chemically induced by MPTP in mice. Moreover,the PD +MA mice showed a tendency toward decreased intestinal microbial alpha diversity,while EA significantly reversed it. The abundance of Erysipelotrichaceae was significantly increased in PD +MA mice,and the alteration was also reversed by EA. In addition,the pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α were substantially increased in the SN of PD +MA mice,an effect that was reversed by EA.
Conclusion:These results suggest that EA may alleviate behavioral defects via modulation of gut microbiota and suppression of inflammation in the SN of mice with PD,which provides new insights into the pathogenesis of PD and its treatment.
Keywords
electroacupuncture,Erysipelotrichaceae,gut microbiota,inflammation,MPTP,Parkinson’s disease
Introduction
Parkinson’s disease (PD) is a chronic neurodegenerative disease that often occurs in middle-aged and elderly people. Studies have shown that the incidence of this disease in the population over the age of 60 is approximately 1% and,as age increases,so does the incidence.1 There are many factors in the pathogenesis of PD,with environmental factors being predominant,while genetic factors are implicated in less than 10%.2 PD is characterized by tremor,muscle rigidity,and motor retardation,which are its main clinical manifestations. The major pathological features of PD include degeneration,deletion and reduction of dopamine (DA) neurons,deposition of neuronal alpha-synuclein (αsynuclein),and formation of Lewy bodies in the substantia nigra (SN). The commonly used reagents for establishing PD animal models are 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP),6-hydroxydopamine (6-OHDA),and rotenone. The MPTP mouse model remains the most commonly used animal model of PD and there are three types of MPTP treatment schemes:acute,subacute,and chronic. Considering advantages of the duration and the degree of similarity to PD,the subacute model has often been employed to assess the validity of effective treatments,3 and so we chose the subacute model for our study. In a 1997 study by Tatton and Kish,mice were injected with a total cumulative dose of 150 mg/kg of MPTP delivered over 5 days,and it was found that this dose resulted in the death of 30%–40% of the dopaminergic neurons,with apoptotic cell death being initiated within 72 h of the first injection of the neurotoxin and peaking 24 h after the final MPTP injection.4 In addition to motor symptoms,patients with PD often have many non-motor symptoms,of which gastrointestinal complaints,including constipation and delayed gastric emptying,are the most common.5 Moreover,Lai et al.6 found that gastrointestinal dysfunction and intestinal pathology manifest prior to motor dysfunction in a mouse model of PD. Therefore,we speculated that dysfunction of the intestines may be involved in the pathogenesis of PD. Since gut microbiota play an important role in normal intestinal function,we aimed to examine for changes in the gut microbiota of PD model mice using 16S ribosomal RNA (rRNA) gene sequencing.
Acupuncture has a long history of use for the treatment of various diseases of both the peripheral and central nervous systems and has shown positive effects with very few side effects. As a practical and effective traditional medical treatment method,acupuncture has received an increasing amount of attention from psychiatrists and patients. More and more patients are willing to try acupuncture to treat diseases of the nervous system. Electroacupuncture (EA) is a major innovation based on manual acupuncture (MA). Compared with traditional acupuncture,EA is easier to objectively evaluate and accurately operate,so it is more suitable for experimental research and clinical applications. The parameters of EA includeselection of needling sites (traditional acupuncture point locations),stimulation frequency,current intensity,and treatment course. The choice of EA parameters is closely related to the achieved effect. Currently,the main treatment for PD is medication,with the most commonly used drug being levodopa.7 However,after taking the drug for 3–5 years,patients can experience side effects,such as wearing off,“on-off” phenomena,and movement disorders. Acupuncture intervention in PD has potential advantages as a non-drug treatment and has been shown to be feasible.8 Moreover,a previous study showed that EA at ST36 (Zusanli) can lead to production of DA,the very neurotransmitter that is reduced in PD patients,in the adrenal medulla.9 In fact,EA has been considered a potential nondrug therapy for PD.10 Nevertheless,the mechanism underlying the effect of EAin PD remains to be discovered.
One previous study showed that EA treatment can mitigate obesity by modulation of gut microbiota,revealing that there was a significant alteration of gut microbiota in obese versus control mice,and that this alteration could be reversed by EA.11 Another study reported that EA was an effective way of treating ulcerative colitis,and that its effect was associated with modulation of gut microbiota.12 These studies suggest that gut microbiota may be a target for EA treatment and,as they are also involved in the pathogenesis of PD,we speculate that EA can alleviatedyskinesiain PD via modulation of gut microbiota. Actually,in our study,the abundance of Erysipelotrichaceae was significantly increased in PD model mice receiving MA,and this alteration was reversed byEA. The brain–gut axis,a communication system between the central nervous system and the gastrointestinal system,is known to play a crucial role in mutual regulation of the two systems.13,14 Specifically,gut microbiota can affect the development and function of the brain.15,16 As Erysipelotrichaceae may be correlated with inflammation,17 we speculated that high relative abundance of Erysipelotrichaceae causes inflammation,which exacerbates the loss of dopaminergic neurons in the SN and the behavioral defects of PD model mice. Therefore,we hypothesized that EA would reduce the high abundance of Erysipelotrichaceae and thereby alleviate the inflammation,lessen the severity of dopaminergic neuron loss in the SN,and reduce the behavioral defects after these conditions had been chemically induced by MPTP in mice.
In order to verify this hypothesis,MPTP was used to establish the subacute PD model and EA was performed. Rotarod performance,open field,and pole tests were carried out to assess MPTP-induced motor deficiencies. Immunohistochemistry was conducted to examine the effect of MPTP and EA on survival of dopaminergic neurons in the SN. 16S rRNA gene sequencing was applied to evaluate alterations of the gut microbiome in PD mice. Finally,quantitative real-time polymerase chain reaction (PCR) was performed to characterize the messenger RNA (mRNA) levels of pro-inflammatory and anti-inflammatory cytokines in order to examine the effect of EA on the inflammation in the SN of PD model mice.
Methods
Experimental procedure
As shown in Figure 1(a),mice were allowed a 1-week habituation period before any experimental manipulation. Then,mice were randomly divided into three groups:control,PD +MA,and PD +EA groups (n =8 each). The PD +MA and PD +EA mice were injected with MPTP intraperitoneally once a day for five consecutive days,while the control mice were injected with normal saline. Concurrently,the PD +EA mice received EA treatment,and the PD +MA mice received MA treatment once a day for 5 days. On days 12 and 13,the rotarod test was performed and,on day 13,the open field test and pole test were conducted. On day 14,the mice were euthanized with an overdose of 4% pentobarbital sodium (200 mg/kg) and the brains,together with the contents of the cecal sections,of the control,PD +MA,and PD +EA mice were collected.
Animals
For all experiments,male C57BL/6J mice (7–8 weeks old,from Shanghai Lingchang Biotechnology Co.,Ltd.,Shanghai,China) were used. They were fed ad libitum,allowed a 1-week habituation period before experimental manipulation,and housed at 23 ±2°C under a 12h light/ dark cycle (lights on at 07:00). This study was approved by the animal ethics committee of Shanghai University of Medicine and Health Sciences (ref. 2018-GZR-20) and carried out in accordance with the National Institutes of Health “Guide for click here the Care and Use of Laboratory Animals.”
MPTP administration
The PD +MA mice and PD +EA mice were injected with MPTP (30 mg/kg; Shanghai ABCONE Biotechnology Co.,Ltd.,Shanghai,China) intraperitoneally once a day for 5 days,while the control mice were injected with normal saline as vehicle.
EA treatment
ST36,located on the tibialis anterior muscle,is one of the most important treatment targets in clinical acupuncture,whereas acupuncture at GV20 (Baihui),located on the scalp,is widely used in the treatment of brain diseases.18 Acupuncture at ST36 and GV20 has been shown to improve the symptoms of PD in previous clinical and animal studies,18–20 and thus,these traditional acupuncture points were also chosen in our study. We made an apparatus to immobilize mice. It consisted of a wooden pole and some ropes. The PD +MA mice and PD +EA mice were fixed using the ropes and stayed awake in the process of intervention. In contrast,nothing was applied to the control mice. EA was performed by inserting needle electrodes to a depth of about 3 mm at ST36 and GV20. ST36 is located on the tibialis anterior muscle,2 mm lateral to the anterior tubercle of the tibia and 4 mm distal to the lower point of the knee joint,in the proximity of the common peroneal and tibial branches of the sciatic nerve. GV20 is located at the intersection of the median line at the top of the head and a line connecting the two ear tips. The alternating stimulation frequencies were 2/100 Hz. Current intensities were 0.5,1.0,and 1.5 mA. They were increased step by step with each one lasting for 10 min. The total treatment time was 30 min. The PD +EA mice underwent EA treatment once a day for 5 days. The PD +MA mice underwent MA intervention using the same procedure,but without the electrical stimulation.
Rotarod performance test
On day 12,the rotarod performance test was conducted after 5 days of MPTP injections. Training was performed three times at 09:00 on the first day. Furthermore,training and testing were both performed once at 09:00 on day 13. The starting speed was 4 rpm and the maximum speed was 40 rpm,which was reached with an acceleration of 0.12 rpm after 5 min,which was the limit of both the training and testing time. Two interdependent values were measured during the test. The performance time was defined as the latency to fall from the rod and it was recorded together with the speed of the rod at the time of falling. The test was performed with rotarod instrumentation and analyzed by software (Rota Rod Rotamex 5; Columbus Instruments,Columbus,OH,USA).
Open field test
On day 13,the open field test was performed at 14:00 as described previously by our group.21 It followed the rotarod test,which finished at 10:00. The open box apparatus for mice was 40 cm ×40 cm ×40 cm. The size of the center zone was 20 cm ×20 cm. Mice were individually placed in the center of the arena and allowed to explore for 5 min. Total distance traveled and time spent in the center zone were recorded. Locomotor activity was tracked by a video recorder and analyzed by software (EthoVisionXT,Noldus,Netherlands).
Pole test
On day 13,the pole test was performed at 16:00. The apparatus consisted of a wooden pole with a wooden ball at the top. The wooden pole was 55 cm high and 1 cm in diameter and wrapped with gauze to prevent slipping. Plastic foam was placed underneath in case of slippage. The mice were pre-trained with the pole three times to make sure that all of them would turn their head down once they were put on the ball with their head upward. The total time taken for a mouse to get from the top to the bottom was recorded.
Immunohistochemistry
Immunohistochemistry was performed as previously described.22 After the behavioral tests were finished,brains were collected,fixed in 4% paraformaldehyde (PFA) overnight,and dehydrated,first with 20% sucrose then 30% sucrose. Brain slices were then cut using a cryostat (Leica Microsystems,Wetzlar,Germany),incubated in 5% donkey serum with 0.3% Triton-X 100 for 2 hat room temperature (RT) and then incubated overnight at 4°C with anti-tyrosine hydroxylase (anti-TH) antibody (1:200,ab112; Abcam,Cambridge,UK). The sections were subsequently washed in 0.01 M phosphate buffered saline (PBS) and incubated with Cy3-labeled goat anti-rabbit IgG (H +L) (1:500,A0516; Beyotime,Shanghai,China) for 2 hat RT. The sections were analyzed using a fluorescence microscope (Leica) and the numbers of positive cells were quantified.
16S rRNA gene sequencing
The major steps of 16S rRNA gene sequencing included collecting samples,extracting the gut microbial genome,amplifying the 16S rRNA fragments by PCR,determining all the amplified product sequences by high-throughput sequencing,and,finally,analyzing the bacteria tissue biomechanics represented by each sequence. After the behavioral tests were finished,the contents of the cecal sections of the control,PD +MA,and PD +EA mice were aseptically collected in a sterile cryotube,immediately stored in liquid nitrogen,and sent directly to Shanghai Majorbio Biopharm Technology Co.,Ltd. for 16S rRNA gene sequencing detection.
Quantitative real-time PCR
SN tissue was homogenized using Trizol. Total RNA was then reverse-transcribed using iScript™ complementary DNA (cDNA) synthesis kits (Bio-Rad,Hercules,CA,USA) and the resulting cDNA was analyzed by quantitative polymerase chain reaction (qPCR). SYBR green detection was used according to the manufacturer’s protocol (Bio-Rad). The 2−ΔΔCT method was then used to convert ΔCT values to mRNA fold changes,relative to the control group. The mRNA levels of interleukin (IL)-6,tumor necrosis factor (TNF)-α,IL-4,and IL-10 were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels to exclude the effects of variable RNA amounts. Primer sequences were as follows:IL-6,forward:GATACC ACT CCC AAC AGAC,reverse:CTTTTCTCATTTCCACGA T; TNF-α,forward:ACT CTG ACC CCT TTA CTC TG,reverse:GAGCCATAATCCCCTTTCTA; IL-4,forward:CCA TGA ATG AGT CCA AGT CC,reverse:TGA TGC TCT TTAGGC TTT CC; IL-10,forward:GGGAAGAGA AAC CAG GGAGA,reverse:GGG GAT GAC AGTAGG GGAAC; GAPDH,forward:AAA TGG TGAAGG TCG GTG TG,reverse:AGG TCA ATGAAG GGG TCG TT.
Statistical analysis
Data are presented as mean ±standard error of the mean (SEM). Statistical analyses were performed and graphs were plotted using GraphPad Prism 6.01 (GraphPad Software Inc.,San Diego,CA,USA). Statistical analysis of intestinal microbial species was performed using Kruskal– Wallis H tests. Correlation analysis was performed using Pearson’s product-moment test. Other results were analyzed using one-way analysis of variance (ANOVA). Then,statistical analysis of changes in the intestinal microbial alpha diversity was performed using Bonferroni’s multiple comparisons test,and other results were performed using Tukey’s multiple comparisons test. The statistical significance level was set top <0.05. Results MPTP induced behavioral defects in the rotarod test and pole test in PD +MA mice and EA protected against these behavioral defects As shown in Figure 1(b) and (c),after 5 days of MPTP administration,the performance time and the rod speed at fall of the PD +MA mice in the rotarod test was significantly decreased as compared to the control mice,while such behavioral defects did not occur in the EA-treated group. However,5 days of MPTP administration did not induce behavioral defects in PD mice in the open field test. Figure 1(d) shows heat maps of the motion trajectory for the control,PD +MA,and PD +EA mice in the open field test. There was no difference in the total distance traveled among the three groups (Figure 1(e)). In the pole test,the total time of the PD +MA mice was significantly increased as compared to the control mice,while EA treatment shortened this time (Figure 1(f)). These results suggest that EA can improve motor deficiencies induced by MPTP in mice. MPTP induced a significant loss of dopaminergic neurons in the SN of PD +MA mice and EA was able to reduce that loss to a certain extent To examine the effect of EA on survival of dopaminergic neurons in the SN,we characterized TH (a dopaminergic neuron marker) expression by immunohistochemistry staining. The staining in the SN revealed a significant loss of dopaminergic neurons in PD +MA mice,an effect that was partially rescued by EA (Figure 2(a) and (b)). This result demonstrates that the neuroprotective effects of EA involve suppression of dopaminergic neuron loss in SN,such as the one induced by MPTP in mice. The PD +MA mice showed a tendency toward decreased intestinal microbial alpha diversity The diversity of microorganisms in the environment can be analyzed by alpha diversity,which uses a series of statistical analysis indices to reflect the abundance of the species and diversity of local microbial communities. The commonly used indices reflecting community richness are the abundance-based coverage estimator (ACE) and Simpson diversity indices. The ACE index is used to estimate the number of operational taxonomic units (OTUs) in a community. It is commonly used to estimate the total number of species in ecology,and the higher the ACE index,the greater the richness of the community. The Simpson index,on the other hand,estimates the probability that two individuals randomly selected from a sample will belong to the same species. Thus,the higher the Simpson index,the lower the richness of the community. As shown in Figure 3,although there was no difference in the ACE index across the three groups (Figure 3(a) and (b)),the PD +MA mice showed a tendency toward an increased Simpson index (p <0.1) and EA significantly reversed it (Figure 3(c) and(d)). These results indicate that 5 days of MPTP administration affects the intestinal microbial alpha diversity and that EA may reverse this effect. Analysis of differences in intestinal microbial species for the control,PD +MA,and PD +EA mice The percentage of community abundance on genus level of the control,PD +MA,and PD +EA mice is shown in Figure 4(a). As evidenced in Figure 4(b),the abundance of Erysipelotrichaceae was Endodontic disinfection significantly increased in PD +MA mice,and the alteration was reversed by EA. We speculate that Erysipelotrichaceae may play an important role both in the formation of behavioral defects induced by MPTP as well as in the neuroprotective effect of EA. Consistently,correlation analysis (Figure 4(c)) revealed a significantly non-zero slope of the linear relationship between the performance time in the rotarod test and the corresponding proportion of sequences of Erysipelotrichaceae in the control,PD +MA,and PD +EA groups.
MPTP considerably up-regulated proinflammatory cytokines in the SN of PD +MA mice and EA was able to suppress the inflammation
To examine the effect of EA on inflammation in the SN of PD mice,we characterized the mRNA levels of proinflammatory (IL-6 and TNF-α) and anti-inflammatory (IL-4 and IL-10) cytokines by quantitative real-time PCR. The results revealed that the pro-inflammatory cytokines IL-6 (Figure 5(a)) and TNF-α (Figure 5(b)) were notably increased in PD +MA mice,an effect that was reversed by EA. However,MPTP and EA treatment did not alter the mRNA levels of the anti-inflammatory cytokines IL-4 (Figure 5(c)) and IL-10 (Figure 5(d)). This result demonstrates that the neuroprotective effects of EA involve suppression of the inflammation in SN,such as the one induced by MPTP in mice.
Discussion
To establish a PD model,mice underwent daily administration of MPTP (30 mg/kg,i.p.) for 5days,as described previously in multiple studies.23–25 According to these prior publications,PD mice exhibit motor dysfunction,reduced striatal DA and serotonin content,as well as degenerate dopaminergic neurons and TH levels in SN. In our study,the performance time and the rod speed at time of fallin the rotarod test were significantly decreased for PD +MA mice as compared to the control mice. Besides,the total time in the pole test of the PD +MA mice was significantly increased. However,there was no difference between these groups in the total distance traveled in the open field test. In line with our result,a paper studying subacutely MPTPtreated mice also revealed that the model mice failed todisplay motor impairments in the open field test; thus,it is reasonable to speculate that the subacute MPTP mouse model may not produce visible motor defects in that particulartest. Furthermore,in our study,we observed a significant loss of dopaminergic neurons in PD +MA mice,which could be partially rescued byEA. Taken together,we have found that EA was able to alleviate behavioral defects induced by MPTP,and also partially rescue the significant loss of dopaminergic neurons in the SN of PD mice.
The human gut microbiota has a wide variety and complex structure. The normal gut microbiota can keep metabolism,immunity,and endocrine function of the human body at a relatively stable level. It is known that many peripheral and central nervous system diseases are closely related to dysfunction of gut microbiota. In addition,studies have shown that the brain–gut axis,a communication system between the central nervous system and the gastrointestinal system,plays a crucial role in mutual regulation of these systems.13,14 Gut microbiota can affect the development and function of the brain15,16 by regulating neurotransmitters and neurotrophins,including DA,norepinephrine,5-hydroxytryptamine (5-HT),gamma-aminobutyric acid (GABA),26,27 and brain-derived neurotrophic factor (BDNF).28 Unsurprisingly,the gut microbiota has received much research attention in recent years. In fact,there is evidence that the gut microbiota of PD patients is substantially different from that of the normal population.29 A previous study revealed that gut microbiota may be involved in the production of α-synuclein and in the pathogenesis of PD.30
Currently,16S rRNA sequencing is one of the most popular high-throughput sequencing-dependent methods. It can accurately quantify the species in the gut microbiota,and it is gradually becoming mainstream in studying the abundance of intestinal microbial species. In our study,16S rRNA gene sequencing was applied to investigate the alterations of gut microbiota in PD mice. We found that the PD +MA mice showed a tendency toward decreased intestinal microbial alpha diversity and EA significantly reversed this trend. Besides,Erysipelotrichaceae,Lactobacillus,Bacteroides,Lachnospiraceae,and Ruminococcaceae were significantly altered in our study. A recentreview summarized nine original publications that reported gut microbiota alterations in PD subjects.31 According to this review,changes in the abundance of Erysipelotrichaceae did not occur. However,another recent review revealed that the abundance of Erysipelotrichaceae in PD patients was significantly increased in two studies.32 In our study,its abundance was also increased in PD +MA mice. Consistent with our results,another study using a chronic low-dose MPTP model also indicated that the relative abundance of Erysipelotrichaceae was significantly increased in PD mice,compared with levels found in control mice.6 In addition,one of the aforementioned reviews reported an increased abundance of Lactobacillus in PD subjects across five studies.31 However,a study of Chinese patients with PD found that the abundance of Lactobacillus in PD patients was decreased.33 These results contrasted with our findings in PD +MA mice. Similarly,studies reporting alterations of the abundance of Bacteroides in PD subjects have also been inconsistent.34 In our study,its abundance was decreased in PD +MA mice. In addition,van Kessel and El demonstrated that decreased abundance of Lachnospiraceae in PD patients was reported in six studies,34 which is in total agreement with our study. Another study revealed that the abundance of Ruminococcaceae was significantly heightened in PD patients,35 which contrasted with our results. Thus,the studies of gut microbiota in PD appear complex and inconsistent. We infer that there are two reasons for this. On the one hand,the microbial ecosystem is heavily influenced by individual dietary habits,and environmental factors might also contribute to additional regional differences. On the other hand,the incongruent observations might be in part due to the stage of progression of PD. Hence,further studies are required.
In this study,the abundance of Erysipelotrichaceae was significantly increased in PD +MA mice,and the alteration was reversed byEA. Moreover,correlation analysis revealed a significantly non-zero slope of the linear relationship between the performance time in the rotarod test and the corresponding proportion of sequences of Erysipelotrichaceae of the control,PD +MA,and PD +EA mice. Thus,Erysipelotrichaceae might play a vital role in the pathogenesis of PD. Erysipelotrichaceae is a type of facultative anaerobe,and its imbalance maybe associated with many diseases. Areview providing insight into the role of Erysipelotrichaceae in humans concluded that it may be correlated withinflammation.17 The relative abundance of Erysipelotrichaceae positively correlated with IL-6,36 as well as TNF-α and CD14 levels.37 Besides,a prior study suggested that blockade of cannabinoid receptor 1 (CB1) ameliorates obesity and metabolic disorder by reducing macrophage inflammatory mediators in the serum,such as IL-17,monocyte chemoattractant protein-1 (MCP-1),eotaxin,and macrophage inflammatory protein-1α (MIP-1α),and this effect is associated with decreased Erysipelotrichaceae in the gut.38 Another study of HIV-infected individuals also found that,after Lactobacillus rhamnosus GG (LGG) supplementation,a decrease in intestinal inflammation was detected,along with a reduction of Erysipelotrichaceae in the gut microbiome.39 Chronic pro-inflammatory immune activity is increasingly being recognized as a fundamental element of neurodegenerative disorders with inflammation in the intestine appearing particularlyrelevant in development of PD.40 As Erysipelotrichaceae may be correlated with inflammation,17 we speculate that a high relative abundance of Erysipelotrichaceae causes inflammation,which exacerbates the loss of dopaminergic neurons in the SN and intensifies the behavioral defects of PD +MA mice. Furthermore,as EA was shown to reduce the high abundance of Erysipelotrichaceae,we speculate that EA can alleviate the inflammation,reduce the loss of dopaminergic neurons in the SN,and attenuate the behavioral defects that result from MPTP treatment of mice. As we expected,in our study,EA was able to suppress inflammation in the SN induced by MPTP. This result demonstrates that the neuroprotective effects of EA involve reversal of the heightened abundance of Erysipelotrichaceae and suppression of inflammation in the SN,which may provide new insights into the pathogenesis of PD and contribute to efforts in finding an effective treatment for it. It is reasonable to expect that research into the chemical substances released by the altered intestinal flora will become an increasingly important direction in the study of PD.