Wu Mei Pill Mitigates Dopaminergic Neuron Damage-Induced Parkinson's Disease in Mice Through Modulation of the Gut-Brain-Microbiota Axis

doi:10.21203/rs.3.rs-4957349/v1

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Research Article

Wu Mei Pill Mitigates Dopaminergic Neuron Damage-Induced Parkinson's Disease in Mice Through Modulation of the Gut-Brain-Microbiota Axis

https://doi.org/10.21203/rs.3.rs-4957349/v1

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Objective

The aim of this research is to delve into the preventive and therapeutic effects of Wu Mei Pill (WMP) on Parkinson's disease (PD), with a special emphasis on its capability to counteract the PD-induced imbalance in gut microbiota and unveil its underlying mechanisms.

Methods

In this investigation, a PD mouse model induced by 6-OHDA was employed to study the impact of WMP. Following the establishment of the PD model, a range of evaluations including behavioral assessments, immunohistochemistry, Western Blot (WB), and enzyme-linked immunosorbent assay (ELISA) were executed to assess neurological functions and the influence of WMP on PD. Fecal samples and brain tissues were analyzed for microbiome and transcriptome studies.

Results

Administration of WMP improved neurological function, elevated the number of TH + cells, and enhanced the dopamine levels in the brain. The damage to dopaminergic neurons induced by 6-OHDA was associated with an upsurge in pro-inflammatory bacteria (Bacteroides), and bacteria involved in tryptophan metabolism (Azospirillum_sp.47_25 and unclassified_Bacteroidia) and cholesterol metabolism (unclassified[Eubacterium]_coprostanoligenes_group), along with a reduction in anti-inflammatory bacteria (Roseburia). WMP addressed these microbial shifts and key metabolite alterations (L-Tryptophan and Bambuterol) in the brain.

Conclusion

The alterations in the microbiome triggered by damage to dopaminergic neurons have the potential to aggravate PD symptoms. WMP was able to correct significant microbial and metabolic disturbances in the brain, thus reducing the loss of dopaminergic neurons, boosting dopamine levels, and enhancing neurological functions.

Wumei Pill

Parkinson's disease

Inflammation

Kynurenine pathway

gut-brain axis

6-OHDA

Parkinson's disease (PD) ranks as the second most prevalent neurodegenerative disorder after Alzheimer's disease, manifesting primarily through motor dysfunctions such as tremors and muscle rigidity [1]. As of 2016, Parkinson's disease afflicted over six million individuals globally, with projections indicating a doubling of this number in the coming generation [2]. The condition is primarily marked by the demise of dopaminergic neurons within the substantia nigra and the aggregation of α-synuclein within neurons. Motor symptoms predominate in PD patients, including muscle stiffness, slowed movements, resting tremors, and muscle spasms, in addition to non-motor symptoms like sleep disruptions, depression, and cognitive deficits [3,4]. The most effective treatment for PD to date is dopamine replacement therapy, especially with Levodopa [5]. Yet, these dopaminergic medications only offer symptomatic relief and do not arrest or reverse the disease's progression, highlighting the critical need for new therapeutic avenues and drugs. Recent advances in understanding PD's pathogenesis have led to the identification of new therapeutic targets. For example, P2B001, a novel drug formulation combining sub-therapeutic doses of Pramipexole (DA) with Rasagiline (MAOB-I), demonstrated promise in phase II trials by minimizing risks and side effects linked to dopaminergic therapies [6]. Additionally, Tavapadon (PF-06649751), a selective D1/D5 dopamine receptor agonist, has shown potential in reducing blood pressure and impulse control disorder (ICD) side effects[7]. These clinical trials of novel drugs open up vast prospects for the discovery of new PD medications with unique therapeutic benefits.

For years, the intricate relationship between the gastrointestinal (GI) tract and the brain has been the focus of extensive research. The gut-brain axis, a term that describes the complex communication network between the GI tract and the central nervous system (CNS), is gaining recognition for the pivotal role gut microbiota play in this bidirectional communication. This interaction is thought to influence neural development, affect neurotransmission, and alter behavior, thereby contributing to the onset and progression of neurodevelopmental, neuropsychiatric, and neurodegenerative disorders [8]. Clinical observations have revealed that PD patients frequently experience gut microbiota dysbiosis, alterations in microbial metabolites, among other changes [9–12]. Research by Cheperjans et al. indicated a significant reduction in Prevotellaceae in PD patients' guts compared to healthy controls, with a noted association between Enterobacteriaceae abundance and PD motor symptoms [13]. Qian et al.'s exploration into the fecal microbiome of Chinese PD patients uncovered an enrichment of certain bacteria and identified correlations between specific bacterial genera and disease duration and cognitive impairments [14]. Evidence suggests that fecal microbiota transplantation could curb TLR4/TBK1/NF-κB/TNF-α-mediated intestinal and neurological inflammation, diminish microglia and astrocyte activation, replenish SCFAs, and elevate DA and 5-HT levels in PD mice, thus offering symptom improvement and potential disease prevention[15].

Chinese herbal medicine (CHM) has a long history of treating various ailments, showing promise in terms of efficacy, safety, and cost-effectiveness. Wumei Pill, a traditional Chinese medicine with over a thousand years of history [16], has demonstrated anti-inflammatory properties and the ability to enhance intestinal microbiota [17–19]. It has been shown to quell intestinal inflammation through the MAPK and NF-κB pathways and suppress pro-inflammatory factors [20]. Moreover, Wumei Pill can adjust the diversity of intestinal microbiota in obese mice towards a healthier composition [21], improve the gut flora in diarrhea-predominant irritable bowel syndrome rat models [22], and, when combined with Liraglutide, significantly alter the microbial landscape in obese type 2 diabetes patients [23]. While the effects of WMP on PD have yet to be fully explored, its pharmacological profile suggests a potential role in PD prevention and treatment. This research, employing a PD mouse model induced by dopaminergic neuron damage, utilizes behavioral, molecular biology, and multi-omics techniques to examine Wumei Pill's preventive capabilities against PD. Furthermore, it delves into the pill's regulatory impact on the dysbiosis of the gut-brain axis microbiota triggered by PD. This approach aims to uncover new targets and strategies for the clinical prevention and treatment of Parkinson's disease, leveraging the potential of Wumei Pill to contribute to these efforts.

2.1 Animals

A cohort of 50 healthy male C57BL/6 mice, aged 10 weeks and weighing between 25–30 grams, was procured from the Animal Experiment Center at Kunming Medical University. Prior to the commencement of the study, these mice were allowed a week-long acclimatization period under standard conditions. The mice were then allocated into four groups: Sham, Sham + WMP (Wu Mei Pill), PD model, and PD + WMP, with each group comprising 10 mice. They were subjected to the modeling process and received intragastric doses of WMP as illustrated in Fig. 1A. Ethical clearance for this study was granted by the Animal Experiment Ethics Committee of Kunming Medical University, under the approval code KMMU20231408. The methodologies employed throughout this research adhered rigorously to the ethical standards and guidelines delineated in the Guide for the Care and Use of Laboratory Animals, affirming the ethical and compassionate treatment of all animal subjects involved.

2.2 Model Creation and Wu Mei Pill (WMP) Administration

6-OHDA Unilateral Lesion Procedure: Prior to the procedure, mice designated for the model group were subjected to a 24-hour fasting period, although they were allowed unrestricted access to water. Following anesthesia, these mice underwent a craniotomy, during which 6-OHDA (catalog number H197233, sourced from Aladdin, China) at a volume of 2µl and concentration of 5mg/ml, was meticulously injected into the left striatum (coordinates: AP: +0.3; ML: +2.3; DV: -2.9) at a rate of 0.5µl/min using a precision stereotaxic apparatus. Post-operative care involved keeping the mice warm for two hours before their return to their respective cages for recovery monitoring.

WMP Treatment Protocol

Wu Mei Pill (WMP) (procured from KUNZHONGYAO, China) was administered intragastrically at a dosage of 0.1mg/g body weight, delivered in a 200µl volume, on a daily basis starting one week prior to the modeling procedure and continuing after the modeling until the conclusion of the study period, as delineated in Figs. 1A and B.

APO Rotation Assessment

Commencing in the second week following surgery, the mice were administered an intraperitoneal injection of apomorphine (APO) solution (catalog number A276058, from Aladdin, China) at a dosage of 0.5µg/g body weight. The subjects were then placed in a sterile enclosure, and their rotations towards the side opposite to the lesion were recorded for a duration of 30 minutes. A criterion of more than 7 rotations per minute was established as indicative of successful lesion modeling. This assessment was performed on a weekly basis until a week prior to the collection of samples for analysis, as shown in Fig. 1B.

2.3 Preparation of Brain Tissue Samples

Brain tissues from ten mice in each experimental group underwent fixation through perfusion. Additionally, brains from another subset of five mice per group were carefully separated into their left and right hemispheres. These brain samples were then lysed using RIPA lysis buffer (Catalog No. P0013J, from Beyotime, China), which included a 1% solution of PMSF (Catalog No. P0100, Solarbio, China) for preservation. Post-lysis, the tissues were stored at a temperature of -80°C for further analysis.

2.4 Conduct of Behavioral Assessments

Pole Climbing Test

Mice were individually placed atop a pole with a height of 55 centimeters, and the duration required for each mouse to descend was meticulously recorded. To ensure accuracy, this procedure was repeated three times for each mouse, with the mean of these three trials calculated and recorded as the data point for the experiment.

Rota-Rod Performance

Prior to any testing, mice were allowed a 30-minute period to familiarize themselves with the test environment. In preparation for the modeling process, the mice participated in adaptive training sessions over three consecutive days, with three sessions daily. These sessions involved maintaining constant speeds of 4 rotations per minute (r), 8 rotations per minute (r), and a progressive acceleration from 4 to 16 rotations per minute (r), each session lasting five minutes. The duration each mouse managed to stay on the rotating rod was documented for subsequent analysis.

Cylinder Test

A 30-minute acclimatization period to the test environment was provided for the mice before commencing the test. During the test, each mouse's interactions with the wall, specifically the number of touches within a 10-minute timeframe, were counted and recorded. This assessment was performed three times for each mouse, and the average value of these trials was computed and utilized as the individual data point for that mouse.

2.5 Immunohistochemical Analysis

Mouse brain specimens were preserved in formaldehyde, then subjected to dehydration through an automated dehydration process, embedded within paraffin, and sectioned. These sections were deparaffinized and rehydrated before undergoing heat-mediated antigen retrieval in a sodium citrate solution (Catalog No. D0081, Beyotime, China). To quench endogenous peroxidase activity, a Peroxidase Blocking Solution (Catalog No. P0100A, Beyotime, China) was applied. To prevent non-specific antibody binding, the tissue sections were treated with 10% goat serum (Catalog No. C-0005, Bioss, China) at ambient temperature for 1 hour. These were then incubated with a primary antibody against TH (Catalog No. T55731, Abmart, China) in a controlled environment at 4°C overnight. The subsequent day, after washing with PBS, the sections were incubated with a secondary antibody (Catalog No. PV9001, ZSGB-BIO, China) at 37°C for 30 minutes. For visualization, DAB (3,3'-Diaminobenzidine) staining was conducted using a DAB Staining Kit (Catalog No. ZLI-9018, ZSGB-BIO, China), followed by a rinse under tap water. Hematoxylin (Catalog No. G1120, Solarbio, China) was used for counterstaining the sections for 1 minute. Following these steps, the sections underwent a final dehydration, clearing, and mounting process. Observations and analyses of the stained sections were performed using a Leica microscope (Model DMi8, Germany).

2.6 Western Blotting

Protein extracts were combined with a 5x loading buffer and subjected to heating at 100°C for a duration of 10 minutes to ensure proper denaturation. The samples were then separated through SDS-PAGE to resolve the proteins based on their molecular weight. After electrophoresis, the proteins were transferred onto a PVDF membrane to facilitate antibody binding. The membrane was then incubated with primary antibodies directed against TH (Catalog No. 66334-1-Ig, Proteintech, China) and β-actin (Catalog No. AF2815, Beyotime, China) to specifically label the proteins of interest. This was followed by the application of a compatible secondary antibody to enhance the detection signal.

2.7 ELISA Assay

The concentrations of dopamine (DA) neurotransmitters present in brain samples were quantified utilizing ELISA kits (Catalog No. RXJ302756, RUIXIN Biotech, China). This process was conducted in strict accordance with the provided instructions of the kit. Optical density (OD) values necessary for quantification were obtained using a microplate reader.

2.8 Microbiome and Metabolome Analysis

At the seventh week following the modeling procedure, fresh fecal specimens were collected from the mice in the Sham, PD model, and PD + WMP groups, ensuring six samples per group for the purpose of microbiome study. Concurrently, brain tissue samples from these groups were also gathered for metabolomic investigation. These samples were then dispatched to Biomarker Technologies (Beijing, China) for comprehensive microbiome and metabolome analyses.

The microbiome datasets were initially processed using Trimmomatic v0.33 to refine the raw sequence data. Primer sequences were identified and excised using the cutadapt software version 1.9.1, resulting in the generation of clean sequences devoid of any primer sequences. The DADA2 algorithm, as implemented within the QIIME2 2020.6 software suite [24,25], facilitated the denoising of the data, thereby producing a final collection of sequences that were free from chimeric artifacts. The processed data was then subjected to various analyses, including but not limited to, the classification of microbial features (e.g., Operational Taxonomic Units [OTUs], Amplicon Sequence Variants [ASVs]), diversity assessments, differential abundance testing, correlation studies, and predictive functional analyses.

3.1 Improvement of PD Symptoms through Wumei Pill (WMP) Treatment

Relative to the Sham group, mice in the PD group demonstrated a marked increase in the duration required to descend the climbing pole (P < 0.01 compared to Sham, as illustrated in Fig. 1A), a significant reduction in the duration of stay on the rotarod (P < 0.05 compared to Sham, depicted in Fig. 1B), and a significant decrease in the number of wall touches with the contralateral forelimb (P < 0.01 compared to Sham, shown in Fig. 1C). Conversely, mice in the PD + WMP group, after receiving Wumei Pill treatment, exhibited a notable reduction in climbing pole descent time (P < 0.01 compared to the PD group, as seen in Fig. 1A), an appreciable increase in the duration on the rotarod (P < 0.05 compared to the PD group, as indicated in Fig. 1B), and an increase in the frequency of wall touches with the contralateral forelimb (P < 0.01 compared to the PD group, as presented in Fig. 1C).

3.2 Wumei pill prevents PD by protecting substantia nigra and its dopamine synthesis function

Immunohistochemistry employing TH antibody staining highlighted varying expressions across the study groups (Fig. 2A). Both the Sham and Sham + WMP groups showed strong TH-positive staining, indicating healthy dopamine synthesis. In stark contrast, a notable decrease in TH expression was observed in the right substantia nigra of mice from the PD model group. Intriguingly, the PD + WMP treatment group presented a uniform TH expression across both hemispheres of the substantia nigra, pointing towards the pill's protective effects on dopaminergic neurons.

Furthermore, analyses revealed a significant reduction in both TH protein and dopamine levels in the brains of PD-afflicted mice (P < 0.01 compared to Sham, Fig. 1B and C). However, administration of Wumei Pill led to a notable increase in the levels of both TH protein and dopamine (P < 0.01 compared to PD, Fig. 1B and C), underscoring its potential in mitigating PD-related biochemical changes.

3.3 Integral analysis of gut flora after treatment by Wumei pill in PD model

Recent studies have documented the alterations in the microbial community as a consequence of Parkinson's Disease (PD). In our investigation, we assessed the impact of Wumei Pill on gut microbiota. The analysis indicated minimal differences between the PD and PD-Wumei treated groups. However, a comparison with the Sham group revealed a significant reduction in total Operational Taxonomic Units (OTUs) in both PD and PD-Wumei treated groups (Fig. 3A). Nonmetric Multidimensional Scaling (NMDS) analysis demonstrated that the PD and PD-Wumei samples clustered more closely together, suggesting a higher resemblance between them (Fig. 3B). Notably, the analysis of species distribution revealed a distinct difference in microbial abundance between the PD and PD-Wumei groups. Specifically, the PD group showed increased levels of Alloprevotella, highlighting its potential link to Parkinson's disease. In contrast, the PD-Wumei group exhibited a notable increase in Lachnospiraceae_NK4A136_group, suggesting its association with the therapeutic benefits of Wumei Pill in PD (Fig. 3C). The ternary phase diagram analysis revealed that Bacteroidota positioned at the center showed a shift towards the Ctrl and PD groups, whereas Proteobacteria leaned towards the Ctrl group, and Firmicutes towards the PD-Wumei group (Fig. 3D). Additionally, graph-based phylogenetic analysis confirmed Bacteroidota, Firmicutes, and Proteobacteria as the principal phyla (Fig. 3E). The FUNGuild functional prediction highlighted that PD-Wumei treatment specifically increased xylanolysis-associated microbiota, in contrast, PD condition enhanced fermentation-related microbiota (Fig. 3F). Collectively, these findings suggest that PD disrupts the normal composition of gut flora, a disturbance that Wumei Pill treatment can significantly ameliorate.

3.4 Identification of Gut Flora Genus Modified by Wumei Pill in PD Model

Further analysis utilizing ANOVA and Rank sum tests identified key genera that differentiated among groups. Notably, unclassified_Gastranaerophilales, unclassified_[Eubacterium]_coprostanoligenes_group, and Defluviitaleaceae_UCG_011 were significantly more prevalent in the PD group. Conversely, the PD-Wumei group showed significant increases in Enterobacter and Acetatifactor (Fig. 4A). Additional analysis of genus abundance revealed distinctive enrichment of Alloprevotella, Bacteroides, and unclassified_Oscillospiraceae in the PD group, whereas unclassified_Lachnospiraceae, Dubosiella, Lachnospiraceae_NK4A136_group, and Parasutterella were notably abundant in the PD-Wumei group. Intriguingly, unclassified_Muribaculaceae, which decreased in the PD group, showed an inverse increase in both the PD-Wumei and Sham groups, suggesting its pivotal role related to PD and its restoration through Wumei treatment (Fig. 4B). Furthermore, taxa such as Bacteroides and unclassified_Muribaculaceae (grouped under Bacteroidota) along with Alloprevotella and Paraprevotella, and those like unclassified_Oscillospiraceae, unclassified_Lachnospiraceae, Dubosiella, and Lachnospiraceae_NK4A136_group (classified under Firmicutes) were highlighted for their categorical significance (Fig. 4C). The correlation network analysis illustrated interconnections among NO.4 (Lachnospiraceae_NK4A136_group), NO.28 (Parasutterella), NO.46 (unclassified_Lachnospiraceae), NO.48 (unclassified_Muribaculaceae), and NO.47 (Dubosiella), all predominantly found in the PD-Wumei group, indicating a complex interaction network influenced by Wumei treatment (Fig. 4D).

3.5 Integrated Metabolomic and Microbiota Analysis

In an effort to further uncover the mechanisms behind the Wumei Pill's effect on gut microbiota in Parkinson's Disease (PD), we undertook a detailed examination by conducting module-to-module analyses between metabolites and microbiotas (Fig. 5A). This was complemented by Canonical Correspondence Analysis (CCA), coinertia analysis, and a metabolite-genus network approach to identify central components (Fig. 5B-D). The associations between specific metabolites and microbiotas were then detailed through a heatmap representation (Fig. 5E). Our findings identified Peptococcus, unclassified_Vermiphilaceae, Negativibacillus, unclassified_Enterobacteriaceae, Enterobacter, Roseburia, unclassified_Tepidisphaeraceae, and Mycoplasma as potential targets influenced by Wumei treatment. Metabolites such as PE(24:1(15Z)/18:1(12Z)-2OH(9,10)), Adenosine 3',5'-diphosphate, and 8-Amino-7-oxononanoic acid were significantly elevated in the PD-Wumei group. Notably, L-Tryptophan levels were increased in the PD group but were found to be reduced in the PD-Wumei group (Fig. 5E), highlighting the impact of Wumei Pill treatment on specific metabolic pathways and microbiota.

The quest for effective Parkinson's disease (PD) treatments remains challenging, with current therapies unable to decelerate or stop the disease's progression [1]. Additionally, long-term application of dopamine replacement therapy may lead to side effects or diminished efficacy[26,27]. PD is characterized by the depletion of dopaminergic neurons in the substantia nigra and the accumulation of α-synuclein-rich Lewy bodies and Lewy neurites in surviving neurons. In our study, methods such as immunohistochemistry, Western blotting, and ELISA assays (Fig. 2A, B, and C) have confirmed that Wumei Pills (WMP) can safeguard dopaminergic neurons in the substantia nigra from damage in a 6-hydroxydopamine-induced PD mouse model, with behavioral assessments (Fig. 1) supporting WMP's preventive and therapeutic potential in PD.

Gut microbiota dysbiosis in PD is marked by an increase in pro-inflammatory bacteria and a decrease in anti-inflammatory ones, with both gut and neuroinflammation critically contributing to PD's development [28–30]. Meta-analysis of over 2000 samples indicated significant alterations in bacteria/genes/pathways associated with inflammation in PD patients [31]. Our cross-analysis of gut microbiota metagenomics and brain tissue metabolomics identified an increase in the anti-inflammatory bacterium Roseburia in the PD + WMP group (Fig. 5E), consistent with global studies showing reduced Roseburia levels in PD patients [32]. Roseburia, a butyrate-producer, alongside other short-chain fatty acids (SCFAs) like acetate and propionate, has been shown to decrease in PD [33,34]. Prevotellaceae_UCG_001 also showed recovery in the PD + WMP group (Fig. 4B), another SCFA-producing family reported to decrease in PD patients [35,36](Qin et al., 2010; Scheperjans et al., 2015).

We observed an increase in Bacteroides in the PD model, which is known to induce TNF-α secretion via LPS [37]. WMP treatment was found to lower Bacteroides abundance (Fig. 4B). Azospirillum_sp._47_25 also followed a similar trend and was correlated with inflammatory cytokines [38]. Additionally, Bambuterol, an anti-inflammatory metabolite, increased in the PD + WMP group (Fig. 5E), highlighting WMP's role in enhancing anti-inflammatory genera and reducing pro-inflammatory ones.

Correlation analysis between metabolomic and microbiological profiles showed variations in Azospirillum_sp._47_25, unclassified_Bacteroidia, and unclassified_Acidobacteriales, all of which were less abundant in the WMP treatment group but more so in the PD model (Fig. 5E). A significant metabolite, L-Tryptophan, was higher in the PD model but reduced in the PD + WMP group (Fig. 5E), indicating alterations in the kynurenine pathway, a major metabolic route for L-Tryptophan.

The presence of coprostanoligenes was significantly higher in the PD group but lower in the control and WMP treatment groups (Fig. 5E), suggesting its role in cholesterol metabolism and its potential influence on PD risk. Our findings align with other studies noting specific probiotic genera's inverse correlation with PD symptoms, underscoring the importance of gut microbiota in PD pathology and treatment.

WMP offers a promising treatment option for PD by protecting dopaminergic neurons and modifying gut microbiota composition. This study not only suggests a novel therapeutic strategy but also highlights the importance of the gut-brain axis in PD management, opening new avenues for future treatments focused on gut microbiota regulation.

As depicted in the summarized diagram (Fig. 6), the induction of dopaminergic neuron damage by 6-OHDA disrupts the gut microbiome through the gut-brain axis, leading to an enhanced presence of pro-inflammatory bacteria and changes in the bacterial genera involved in serotonin and cholesterol metabolism, such as Bacteroides, Azospirillum_sp.47_25, unclassified_Bacteroidia, and unclassified [Eubacterium]_coprostanoligenes_group. This damage coincides with a diminished abundance of the anti-inflammatory genus Roseburia. Wumei Pill (WMP) treatment, however, effectively counteracts these adverse changes, rebalancing the aforementioned bacterial genera towards their normal levels. Concurrently, in the WMP-treated group, there's a normalization of the kynurenine pathway's substrate L-Tryptophan and an elevation of the anti-inflammatory metabolite Bambuterol within brain tissues. Similarly, the effects of dopaminergic neuron damage and alterations in dopamine levels tend to revert towards those observed in the sham-operated group upon WMP treatment. Moreover, behavioral tests indicate a notable amelioration of motor dysfunctions seen in the model group, showcasing the therapeutic potential of WMP treatment.

Data availability

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgements

We would like to express our sincere gratitude to BETTER BIOTECH Research Institute for providing the research platform for the immunohistochemistry (IHC), mouse animal models, and other experimental platforms has been invaluable to the advancement of our work. Their expertise and resources have greatly contributed to our understanding and findings.

Funding

This research received support from the Natural Science Foundation of Chongqing, China (Grant No. cstc2021jcyj-msxmX0856), and the Science and Technology Planning Project of Jiangjin, Chongqing, China (Grant No. Y2023019).

Author Contributions

Zuowen Zhang led the study's conceptualization, methodology, and manuscript writing, conducting behavioral tests, immunohistochemistry, and Western Blot experiments. Lan Shen contributed to behavioral tests, while Yinyou Bai handled microbiota-related data analysis. Shishuang Li focused on ELISA experiment. Shumei Wang provided guidance and oversaw manuscript refinement as the corresponding author.

Conflict of Interest

The authors declare no conflicts of interest.

Ethics statements

The ethical clearance for this investigation was granted by the Animal Experiment Ethics Committee at Kunming Medical University, under the approval code KMMU20231408. The methodologies employed throughout this study adhered rigorously to the ethical standards and guidelines delineated in the Guide for the Care and Use of Laboratory Animals, affirming the ethical and compassionate treatment of all animal subjects involved.

Patient consent for publication

Not applicable.

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No competing interests reported.

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Wu Mei Pill Mitigates Dopaminergic Neuron Damage-Induced Parkinson's Disease in Mice Through Modulation of the Gut-Brain-Microbiota Axis

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Abstract

Objective

Methods

Results

Conclusion

Figures

Introduction

Material and Methods

2.1 Animals

2.2 Model Creation and Wu Mei Pill (WMP) Administration

2.3 Preparation of Brain Tissue Samples

2.4 Conduct of Behavioral Assessments

2.5 Immunohistochemical Analysis

2.6 Western Blotting

2.7 ELISA Assay

2.8 Microbiome and Metabolome Analysis

Results

3.1 Improvement of PD Symptoms through Wumei Pill (WMP) Treatment

3.2 Wumei pill prevents PD by protecting substantia nigra and its dopamine synthesis function

3.3 Integral analysis of gut flora after treatment by Wumei pill in PD model

3.4 Identification of Gut Flora Genus Modified by Wumei Pill in PD Model

3.5 Integrated Metabolomic and Microbiota Analysis

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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