Parabacteroides distasonis ameliorates methylphenidate hydrochloride-induced colonic inflammatory responses and associated gastrointestinal distress

Abstract

Background

Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder in childhood, with methylphenidate hydrochloride (MPH) as its first-line pharmacotherapy. Though effective, MPH is associated with underrecognized gastrointestinal adverse events warranting further investigation. Recently, the gut microbiota has attracted attention for treating gastrointestinal distress, but research on its role in alleviating MPH-induced gastrointestinal reactions remains scarce.

Methods

Herein, Wistar-Kyoto rats (WKY) served as controls alongside spontaneously hypertensive rats (SHR) to explore MPH-induced gastrointestinal adverse drug reactions. The gastrointestinal phenotypes, colonic histopathological characteristics, and inflammatory factor levels were evaluated, and the gut microbiota profiles were analyzed via 16S rRNA gene sequencing. The protective effect of Parabacteroides distasonis (P. distasonis) was further verified by supplementing the treated rats with this strain.

Results

We demonstrated MPH induced diarrhea and colonic inflammatory responses in rats. 16S rRNA gene amplicon sequencing revealed that MPH-treated rats had significantly decreased gut microbiota health index, increased dysbiosis index, and reduced P. distasonis abundance, suggesting MPH-induced gastrointestinal reactions may correlate closely with reduced P. distasonis. Further experiments confirmed that P. distasonis supplementation effectively alleviated diarrhea and colonic inflammatory responses in MPH-treated rats.

Conclusions

Collectively, this is the first study verifying that P. distasonis supplementation mitigates MPH-induced gastrointestinal adverse reactions, providing a novel intervention strategy to improve tolerance of ADHD pharmacotherapy.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12876-026-05079-8.

Introduction

Attention-deficit/hyperactivity disorder (ADHD), also known as hyperactivity disorder or attention deficit disorder, is a neurodevelopmental disorder characterized primarily by behavioral and learning impairments [1]. Its core clinical manifestations include age-inappropriate attention deficits, hyperactivity, and impulsive behavior, often accompanied by impaired social functioning [2]. The disorder typically occurs during childhood, with symptoms persisting into adolescence or even adulthood in some cases [3]. ADHD is the most common mental disorder among children and adolescents, with a prevalence rate higher than any other childhood mental disorder [4]. Epidemiological studies indicate that the global prevalence of ADHD among children and adolescents ranges from 5.3% to 7.2%, with approximately 9.8% in the United States and 6.2% to 7.0% in China [1, 5, 6]. In recent years, the prevalence of ADHD has been on the rise, making it one of the most significant public health issues globally [6]. ADHD can impair patients’ academic performance, social skills, and cognitive functions to varying degrees, and impose significant economic burdens on families, society, and healthcare systems [7]. In clinical treatment, pharmacological intervention is the primary treatment modality, with central nervous system stimulants being the first-line treatment option [8].

Methylphenidate hydrochloride (MPH) is a typical central nervous system stimulant widely used in the treatment of ADHD in adolescents and adults [9–14]. The drug is absorbed through the gastrointestinal tract into the bloodstream, crosses the blood-brain barrier, and inhibits dopamine transporters, thereby increasing dopamine concentrations in the synaptic cleft. This improves core ADHD symptoms and cognitive dysfunction [14, 15]. Clinically, MPH can improve attention and classroom performance while reducing hyperactivity, impulsivity, and disruptive behavior [16]. Although it has a relatively favorable risk-benefit profile, certain safety concerns remain [17]. Studies indicate that approximately 30% of patients exhibit poor response to MPH or cannot tolerate it due to side effects [18]. Serious adverse reactions include psychotic symptoms and mood disorders, occurring at a rate of approximately 3%; additionally, approximately one-quarter of children and adolescents experience non-serious adverse events after medication, such as sleep disturbances, decreased appetite, abdominal pain, and diarrhea [19, 20]. The drug label also notes that in multiple open-label clinical trials, MPH can cause gastrointestinal adverse reactions such as diarrhea, abdominal discomfort, and abdominal pain [20].

The gut is an important open ecosystem in the human body, home to over 100 trillion microorganisms, including bacteria, fungi, and viruses, collectively forming the gut microbiota [21, 22]. Under normal conditions, the gut microbiota maintains host health by preserving the integrity of the gut barrier, regulating energy metabolism, and participating in immune defense [23]. However, various factors can disrupt the stability of the gut microbiota, with drug use being a common contributing factor, which can lead to dysbiosis by directly damaging beneficial microbial communities [24]. Given that the gastrointestinal tract is the primary absorption site for MPH, previous studies have suggested that it may induce structural disruption of the gut microbiota [25]. Considering the common gastrointestinal adverse reactions associated with MPH, it is speculated that its effects on the gut microbiota may be closely linked to these adverse reactions. Herein, we used Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) as experimental models to evaluate MPH-induced gastrointestinal adverse reactions and their potential mechanisms. The experimental results revealed that SHR rats treated with MPH exhibited diarrhea and colonic inflammatory responses, and gut microbiota analysis showed a significant decrease in the relative abundance of P. distasonis. Additionally, supplementation with Parabacteroides bacteroides (P. distasonis) effectively alleviated MPH-induced colonic inflammatory responses and associated gastrointestinal distress. The findings of this study provide new experimental evidence and potential clinical application strategies for intervening in adverse reactions associated with ADHD medications.

Materials and methods

Experimental animals

The experimental animals were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., including 3-week-old (approximately 50 g in weight) healthy male Spontaneously Hypertensive Rat (SHR) and their control strain, Wistar-Kyoto (WKY). The animals were housed in an environment maintained at a temperature of 23 ± 1 °C and relative humidity of 50 ± 10%, with a 12/12-hour light-dark cycle following the natural circadian rhythm. During the experiment, the animals were allowed to eat and drink freely, with five rats per cage. All experimental animals underwent a one-week acclimatization period prior to the formal experiment. At the end of all behavioral experiments and immediately prior to tissue collection, all rats were anesthetized via right intraperitoneal injection of tribromoethanol (Avertin) at a dose of 10 µL/g body weight. An adequate depth of anesthesia was confirmed by the complete absence of the corneal reflex and paw withdrawal reflex. Rats were then humanely euthanized by cervical dislocation to ensure rapid, painless death. The animal experiments were conducted in strict accordance with the “Regulations on the Management of Experimental Animals” of China and the ARRIVE 2.0 guidelines, and were approved by the Animal Ethics Committee of Chongqing Medical University (Approval No. IACUC-CQMU-2021-072).

Cultivation and enrichment of P. distasonis

P. distasonis (strain number B81085, DSM 20701 = ATCC 8503) was cultured anaerobically at 37 °C on CMC medium (KDM150) or blood agar plates (TSA supplemented with 5% defibrinated sheep blood). The media were supplemented with 0.1% vitamin K₁ (Aladdin #V104148) and hemin (Aladdin #H421920) in an anaerobic atmosphere consisting of 80% N₂, 10% CO₂ and 10% H₂. After incubation, bacterial cells were harvested, washed three times with sterile PBS and centrifuged. The bacterial suspension was adjusted to an optical density at 600 nm (OD₆₀₀) of 2.0, equivalent to 1 × 10⁸ colony-forming units (CFU)/mL.

Drug administration and grouping

We adopted a two-stage experimental design, and the detailed protocols were described as follows.

Experiment 1: MPH intervention

After one week of acclimatization, 10 WKY rats were designated as the control group, and 20 SHR rats were randomly divided into a model group and a treatment group, with 10 rats in each group. Rats in the control group and model group were administered 0.9% sterile saline solution via gavage daily, and the treatment group received oral gavage of MPH solution (4.85 mg/kg) [26] for 3 days. MPH (Xi’an Janssen Pharmaceutical Co., Ltd.) was dissolved in sterile saline and converted to the equivalent dose for rats based on the weight of children and clinical dosage.

Experiment 2: P. distasonis supplementation

After one week of acclimatization, 20 SHR rats were randomly divided into a control group and an experimental group, with 10 rats in each group. Both groups of rats were first gavaged with MPH solution (4.85 mg/kg) for 3 consecutive days. Subsequently, the control group received daily oral administration of sterile glycerol, while the experimental group was gavaged with P. distasonis bacterial solution diluted with sterile glycerol (2 × 10⁸ CFU/mL) for 14 days.

Behavioral experiments

Behavioral tests were performed using an animal behavioral video analysis system and the movement trajectories and behavioral data were recorded. Prior to the test, rats were placed in the testing room for at least 1 h to acclimate to the environment. All tests were conducted under dim light and quiet conditions to ensure data accuracy. To eliminate cross-interference, feces and urine were removed from the apparatus, and the surface was thoroughly cleaned with 75% ethanol between each test.

Open field test

The open field test was conducted to evaluate spontaneous locomotor activity, exploratory behaviors and anxiety-like behaviors as previously described [28]. Each rat was gently placed in the central zone of the open field apparatus and allowed to explore freely for 5 min. The movement trajectory was automatically recorded by a video tracking system. The main behavioral parameters included total distance, central square during and average speed.

Elevated plus maze

The elevated plus maze was performed to evaluate impulsive, hyperactive, and anxiety-related behaviors. This test was carried out as reported previously [27–29]. The EPM apparatus was positioned 50 cm above the floor and 1 m away from the wall, consisting of closed arms and open arms. Each rat was placed in the center of the maze facing an open arm and allowed to explore freely for 5 min. The movement trajectory was automatically recorded by a video tracking system. Behavioral characteristics measured by the instrument include total distance, number of entries into the open arms, and time spent in the open arm region.

Tissue collection and organ coefficient calculation

The animals were anesthetized by intraperitoneal (i.p.) injection of tribromoethanol (Avertin, 10 µL/g body weight) and then euthanized by cervical dislocation. The spleen, cecum, and colon were rapidly dissected on ice. The length of the colon was measured and photographed for documentation. The weights of the spleen and cecum were recorded, and the number of fecal pellets retained in the colon was counted. The organ coefficients were calculated to normalize organ weight to body weight using the following formulas:

Hematoxylin-eosin staining (H&E)

Tissue samples were fixed in 4% paraformaldehyde, routinely dehydrated, and embedded in paraffin wax. Paraffin Sect.  (4 μm) were incubated at 65 °C for 1 h, dewaxed in xylene, and rehydrated through a graded ethanol series. Subsequently, the sections were stained with hematoxylin for 5 min, differentiated with 1% hydrochloric acid ethanol, and blued with tap water. Then, the sections were counterstained with eosin for 2 min. After staining, the sections were dehydrated in ascending ethanol concentrations, cleared in xylene, and mounted with neutral balsam. The histopathological morphology was observed and imaged under a light microscope after 24 h.

RNA extraction and quantitative polymerase chain reaction

Total RNA was extracted from colon tissue samples using TRIzol reagent following the manufacturer’s protocol. Reverse transcription was conducted to synthesize complementary DNA (cDNA) with 5× PrimeScript™ RT Master Mix (TaKaRa, Japan). Quantitative real-time PCR (qRT-PCR) was performed on a Bio-Rad CFX Manager 3.1 system using Blast Taq™ 2× qPCR Master Mix with gene-specific primers. All primer sequences were derived from previously published studies and synthesized by Sangon Biotech (Shanghai, China) [30–33]. The detailed sequences are provided in Supplementary Table 1. The qRT-PCR reaction system (10 µL total volume) was prepared as follows: 5 µL TB Green, 0.25 µL forward primer (10 µM), 0.25 µL reverse primer (10 µM), 1 µL cDNA template, and 3.5 µL DEPC-treated water.

Bacterial DNA extraction

Genomic DNA was extracted from rat feces using the TIANamp Fecal DNA Kit (TIANGEN#DP328) in strict accordance with the manufacturer’s instructions. The concentration of the extracted DNA was measured using a Nanodrop spectrophotometer. After adjusting to a uniform concentration, the DNA samples were stored at − 20 °C. DNA amplification was performed via qPCR on the Bio-Rad CFX Manager 3.1 using Blast Taq™ 2× qPCR Master Mix (Abm#G891) and gene-specific primers. The 16 S rRNA gene was used as the internal reference gene, and the relative abundance fold difference of the target bacterial gene was calculated using the 2^−ΔΔCt method.

16S rRNA gene amplicon sequencing and gut microbiota data analysis

Fresh rat fecal samples were collected after the designed treatments, and genomic DNA was extracted using the PF Mag-Bind Fecal DNA Kit (Omega). The concentration, purity, and integrity of the extracted DNA were simultaneously determined using a NanoDrop 2000 spectrophotometer. PCR amplification was conducted with barcode-labeled primers 338 F/806R, and the resulting amplicons were purified using a PCR Clean-Up Kit. The purified products were quantified with a Qubit 4.0 fluorometer (Thermo Fisher Scientific, USA). Subsequently, library construction was performed using the NEXTFLEX Rapid DNA-Seq Kit, and high-throughput sequencing was conducted on the Illumina NextSeq 2000 platform (Shanghai Majorbio Biotechnology Co., Ltd.). After quality filtering and denoising, the number of valid sequences per sample ranged from 41,028 to 67,490, with an average sequencing depth of 51,289 sequences per sample. To eliminate bias from uneven sequencing depth, all samples were rarefied to 30,005 sequences per sample prior to diversity analysis. Rarefaction curves based on the Shannon index reached a plateau for all samples, confirming that the sequencing depth was sufficient to cover the full microbial community diversity. The raw sequencing data were deposited in the NCBI SRA database under the accession number SRP609942.

All downstream bioinformatic and statistical analyses were performed using the Majorbio Cloud Platform (https://cloud.majorbio.com). Alpha diversity indices, including Chao1 and Shannon, were calculated with mothur (version 1.30.2) based on the rarefied ASV abundance table, and between-group differences in alpha diversity were assessed using the Wilcoxon rank-sum test. Principal coordinate analysis (PCoA) was performed based on the Bray–Curtis distance matrix using the vegan package in R (version 3.3.1) to visualize structural differences in microbial communities among groups. The Gut Microbiome Health Index (GMHI) was computed according to the method described by Gupta et al. [34]. To evaluate the severity of microbial dysbiosis in each sample, the Microbial Dysbiosis Index (MDI) was calculated as MDI = log₁₀[(total abundance of genera increased in the disease group) / (total abundance of genera decreased in the disease group)], as previously reported [35]. Venn analysis was performed to identify shared and unique species/genes/metabolites among groups, and Venn diagrams were generated using the VennDiagram package in R (version 3.3.1). Linear discriminant analysis effect size (LEfSe) was performed with default parameters (LDA score > 2, P < 0.05) to identify bacterial taxa with significantly differential abundance from phylum to genus level.

Statistical analysis

All statistical data were expressed as the mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software Inc., La Jolla, CA, USA). Normality and homogeneity of variance were tested for all datasets. Comparisons between two groups were conducted using Student’s t-test, and comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. A p-value < 0.05 was considered statistically significant. The significance levels were marked as *p < 0.05, **p < 0.01, and ***p < 0.001.

Results

MPH improves ADHD-like behaviors in SHR rats

To verify the intervention effect of MPH on ADHD‑like behaviors, we established an MPH‑intervened rat model (Fig. 1a) and systematically evaluated the neurobehavioral phenotypes of rats through behavioral tests. In the open field test, compared with the WKY control group, SHR model rats exhibited significantly increased spontaneous activity distance, time spent in the central region, and average speed. MPH-treated SHR rats showed significant decreases in the above indicators compared with the model group (Fig. 1b-f). In the elevated plus maze test, SHR model rats exhibited significantly higher total activity distance, number of entries into the open arms, and time spent in the open arm region compared to the control group, demonstrating typical ADHD-like hyperactive behaviors. Following MPH treatment, these abnormal behavioral indicators were significantly improved. These results indicate that MPH can effectively improve hyperactive and impulsive-like behaviors in SHR rats, suggesting that it has a significant alleviating effect on ADHD-like behaviors (Fig. 1g-k).

Fig. 1.

MPH improves ADHD-like behaviors in SHR Rats. a WKY rats were gavaged with 0.9% sterile saline for 3 days, while SHR rats were gavaged with either 0.9% sterile saline or MPH for 3 days. Subsequenty,behavioral experiments were conducted, and tissues were harvested. b-e Total distance,centralsquare duration, and average speed in the open field test among rats in the control group, model group, and treatment group. f The representative movement trajectories after designed treatment in the open field test. g-j Total distance, entries in open arm, and time spent in open arm in the elevated plus maze test among rats in the control group,model group, and treatment group following MPH administration. k The representative track maps after designed treatment in the elevated plus maze test. Data are presented as mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, no significant diference

Effects of MPH on intestinal function

To assess the effects of MPH on gastrointestinal adverse reactions in SHR rats, rats were euthanized after behavioral experiments, and indicators such as spleen weight and cecum length were measured. Fecal morphology was also observed to evaluate related phenotypic characteristics. During MPH gavage, we observed that rats in the treatment group exhibited obvious diarrhea symptoms, with yellowish-brown soft feces that were sticky and soft in texture; obvious water stains could be observed at the edge of the feces when placed on sterile filter paper. By contrast, rats gavaged with sterile saline showed no obvious abnormalities, with dark brown, regular oval or nearly round granular feces and no water stain diffusion on sterile filter paper (Fig. 2a). Histomorphological comparison results showed that compared with the control group, the spleen index, colon length, and number of fecal particles in the colon of rats in the model group were significantly decreased, and there was no statistically significant difference in the cecum index between the two groups. After MPH treatment, the colon length and the number of fecal particles in the colon of SHR rats were increased. There were no statistically significant differences in the spleen index between the MPH treatment group and the control group or the model group, and no statistically significant difference was observed in the cecum index among the three groups (Fig. 2b–f).

Fig. 2.

Effects of MPH on intestinal function. a Photographs of feces from rats in the control group, model group, and treatment group during the experimental period. b Spleen index in Control, Model, and Treatment groups. Spleen index (mg/g) = spleen weight (mg) / rat body weight (g). c Cecum index in Control, Model, and Treatment groups. Cecum index (mg/g) = cecum weight (mg) / rat body weight (g). d Colon length in Control, Model, and Treatment groups. e Number of fecal pellets in the colon of rats in the control group, model group, and treatment group. f Representative images of rat cecum, colon, and spleen. g The histological changes of colonic tissue were observed by H&E staining. h-k The mRNA expression levels of II6, Tnf, Il10 and Ocln in the colon tissue. Data are presented as mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, no significant difference.

The H&E staining results of the colon showed that the colonic mucosal structure of the model group rats was intact, with tightly arranged crypts, normal crypt depth, and no ulcers or necrotic pathological changes; the colonic mucosa of the MPH-treated group rats showed inflammatory cell infiltration and an increase in the number of goblet cells (Fig. 2g). Further qPCR analysis of inflammatory factor expression levels revealed that the mRNA expression levels of pro-inflammatory factors Il6 and Tnf were significantly elevated in the colon tissue of MPH-treated rats, while the expression level of the anti-inflammatory factor Il10 was upregulated (Fig. 2h-j). Additionally, the mRNA expression levels of the tight junction gene Ocln was significantly reduced (Fig. 2k). These results indicate that MPH treatment can induce intestinal inflammatory responses in rats, accompanied by impaired intestinal barrier function.

Effects of MPH on the gut microbiota

Diarrhea and intestinal inflammation are common and important clinical manifestations of gastrointestinal diseases, which are often associated with gut microbiota dysbiosis [36, 37]. Therefore, we performed 16S rRNA gene amplicon sequencing analysis on rat fecal samples. α-diversity and β-diversity analyses revealed significant differences in gut microbiota structure between WKY rats and SHR rats, while there was no statistically significant difference between the MPH treatment group and the model group (Fig. 3a-g). Further analysis of the sequencing data from the model group and treatment group showed that the Venn diagram displayed distinct ASV composition between the two groups, with 29 unique ASVs in the model group and 57 unique ASVs in the treatment group (Fig. 3h). Analysis of microbial community characterization indices indicated that the treatment group had lower microbial health indices and higher dysbiosis indices compared with the model group (Fig. 3i-j).

Fig. 3.

16S rRNA gene amplicon sequencing of rat fecal samples. a-f Analysis of a-diversty in the control group, model group, and treatment group. g Analysis of β-diversity in the control group, model group, and treatment group. h Venn diagram showing shared and unique ASVs between the model group and treatmen group. i-j Gut microbiome heath index and micobial dysbiosis index in the model group and treatment group. Data are presented as mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, no significant difference

MPH treatment alters the relative abundance of P. distasonis in the rat gut

In genus-level species comparison, the relative abundance of the Parabacteroides genus was significantly reduced in the treatment group (Fig. 4a). Baseline comparison further revealed that Parabacteroides abundance was already significantly lower in SHR rats compared with WKY rats (Fig. 4b). These results suggest that MPH treatment can induce gut microbiota dysbiosis in SHR rats, accompanied by a decrease in the relative abundance of Parabacteroides genus bacteria. Given that the Parabacteroides genus includes multiple species, we further extracted bacterial DNA from the feces of rats in the model group and treatment group and performed qPCR quantitative analysis targeting known common Parabacteroides species to identify the specific key species affected species. The results showed that compared with the model group rats, the relative abundance of P. distasonis in the feces of the treatment group rats was significantly reduced; there were nosignificant differences in the abundances of Parabacteroides goldsteinii, Parabacteroides johnsonii, and Parabacteroides gordonii; while the relative abundance of Parabacteroides merdae was increased after MPH treatment (Fig. 4c-g).

Fig. 4.

MPH treatment induces a decrease in Parabacteroides relative abundance in rats. a Wlcoxon rank-sum test comparing diferences in genus-level microbiota between model group and treatment group. b 16S rRNA gene sequencing analyzing the relative abundance of the genus Parabacteroides in fecal samples from WKY and SHR rats. c Relative abundance of Parabacteroides distasonis in feces of rats in the model group and treatment group. d Relative abundance of Parabacteroides goldsteinii in feces of rats in the model group and treatment group. e Relative abundance of Parabacteroides johnsonii in feces of rats in the model group and treatment group. f Relative abundance of Parabacteroides gordonii in feces of rats in the model group and treatment group. g Relative abundance of Parabacteroides merdae in rat feces from the model group and treatment group. Data are presented as mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, no significant difference

P. distasonis supplementation exerts no significant effect on ADHD-like behaviors

Preliminary experimental results indicated that MPH treatment may reduce the relative abundance of P. distasonis in the intestines of SHR rats. Based on this, we further investigated whether supplementation with P. distasonis could ameliorate MPH-induced intestinal dysfunction.

To ensure the scientific validity and reliability of subsequent animal intervention experiments, P. distasonis was first cultured under anaerobic conditions in vitro. The growth status of the strain was monitored in real time, and the growth curve was plotted (Fig. 5a). Subsequently, the viable count was performed using the plate coating method. The number of colonies was counted after gradient dilution, coating inoculation and constant-temperature culture, so as to optimize and determine the optimal concentration and dosage of P. distasonis suspension for oral gavage in rats (Fig. 5b). After the establishment of the P. distasonis supplementation model (Fig. 5c), fecal samples were collected from rats for bacterial DNA extraction. qPCR was performed to detect the relative abundance of P. distasonis in the intestine of rats between the two groups, which verified the successful intestinal colonization of the supplemented strain (Fig. 5d).

Fig. 5.

Supplementation with P. distasonis has no effect on the efficacy of MPH treatment for ADHD. a Growth curve of P. distasonis cultured under anaerobic conditions, monitored by OD₆₀₀ values over 38 h. b Colony counts of P. distasonis on agar plates after serial dilution (10⁻⁶ to 10⁻⁹), used to determine the optimal concentration for in vivo gavage administration. c SHR rats were gavaged with either glycerol or MPH for 3days. Subsequently, behavioral experiments were conducted, and tissues were collected. d Quantitative PCR was used to validate the colonization of P. distasonis in the control and experimental groups. e-g Total distance traveled, time spent in the central area, and average speed in the open field test among rats in the control and experimental groups following MPH or glycerol administration. h The representative track maps after designed treatments in the open field test. i-k Total distance traveled, number of entries into the open arms, and time spent in the open arm region in the elevated plus maze among rats in the control and experimental groups following MPH or glycerol administration. l The representative track maps after designed treatments in the elevated plus maze. Data are presented as mean ± S.E.M. * p < 0.05, ** p < 0.01, ***p < 0.001. ns, no significant difference

We then conducted behavioral tests on both groups of rats to assess whether P. distasonis supplementation affected the therapeutic efficacy of MPH. Results from the open field test and elevated plus maze showed no significant differences in ADHD-related behavioral indicators between the control group and the P. distasonis experimental group (Fig. 5e-l). These results suggest that P. distasonis may not directly participate in the regulation of ADHD-like behaviors.

P. distasonis supplementation ameliorates MPH-induced colonic inflammatory responses and gastrointestinal distress

After supplementation with P. distasonis, rat organ indices were observed, and the results showed that compared with the control group, the cecum index and colon length of the experimental group rats were significantly increased, while there was no significant difference between the two groups in terms of spleen index and the number of fecal in colon (Fig. 6a-e). Additionally, we observed that the diarrhea symptoms of rats were significantly ameliorated after supplementation with P. distasonis, with fecal morphology and consistency restored to a nearly normal physiological state (Fig. 6f).

Fig. 6.

Supplementation with P. distasonis alleviates gastrointestinal adverse reactions caused by MPH. a Spleen index in control and experimental groups. b Cecum index in control and experimental groups. c Colon length in control and experimental groups. d Number of fecal particles in the colon of rats in the control and experimental groups. e Images of rat cecum, colon, and spleen. f Photographs of feces from rats in the control and experimental groups during the experimental period. g The histological changes of colonic tissue were observed by H&E staining. h-k The mRNA expression levels of II6, Tnf, Il10, and Ocln in the colon tissue. Data are presented as mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, no significant difference

H&E staining results of rat colons showed that, compared with the control group, rats supplemented with P. distasonis had more intact intestinal mucosal structures, ordered and dense crypts, restored normal crypt depth, and no ulcers or necrosis were observed (Fig. 6g). Inflammatory factor qPCR results indicated that the mRNA expression levels of pro-inflammatory factors Il6 and Tnf were significantly downregulated in the experimental group, while the expression level of anti-inflammatory factor Il10 was upregulated. Concurrently, the mRNA expression level of Ocln was also increased (Fig. 6h-k).

These results suggest that supplementation with P. distasonis can effectively alleviate MPH-induced gastrointestinal inflammation and structural damage, and alleviate intestinal dysfunction.

Discussion

This study aimed to explore gastrointestinal adverse reactions induced by MPH, a first-line drug for ADHD, with a focus on the potential regulatory role of the gut microbiota. MPH treatment alleviated ADHD-like behaviors in rats but simultaneously triggered obvious colonic inflammatory responses and gastrointestinal dysfunction. 16S rRNA sequencing analysis further revealed that MPH treatment also caused gut microbiota dysbiosis in rats, characterized by reduced microbiota health indices, increased dysbiosis indices, and decreased relative abundance of P. distasonis. Supplementation with P. distasonis significantly alleviated diarrhea symptoms and intestinal inflammatory responses in SHR rats. These results suggest that the reduced relative abundance of this bacterial species may be a key factor in MPH-induced gastrointestinal adverse reactions.

ADHD is one of the most common neurodevelopmental disorders in childhood. Epidemiological studies have revealed that ADHD is associated with various comorbidities, including obesity, sleep disorders, and asthma, as well as otitis media, allergic rhinitis, movement disorders, urinary symptoms, migraines, and celiac disease [38–41]. However, robust evidence is lacking to indicate that the incidence of diarrhea and colonic inflammatory responsesis significantly higher in untreated ADHD patients than in the general population [42]. While some studies have reported that ADHD patients may experience non-specific gastrointestinal functional issues such as dyspepsia and chronic constipation, no significant increase in the risk of inflammatory bowel disease (IBD) or idiopathic diarrhea has been observed in untreated ADHD patients [43]. This study demonstrated that SHR rats treated with MPH exhibited significant diarrhea and colonic inflammation phenotypes, suggesting that these specific gastrointestinal symptoms are more likely to be direct side effects of MPH exposure rather than inherent clinical manifestations of ADHD.

MPH, as a commonly used medication, effectively improves ADHD-like symptoms but may also cause adverse drug reactions such as sleep disorders, decreased appetite, weight loss, and gastrointestinal abnormalities [44, 45]. Clinical observations indicate that non-severe adverse events associated with MPH are common, yet research on the gastrointestinal abnormalities it induces and their intervention strategies remains limited. Gastrointestinal side effects caused by MPH include diarrhea, upper abdominal pain, and dyspepsia, yet the specific regulatory mechanisms underlying these effects remain unclear [46]. The present study demonstrated that SHR rats treated with MPH exhibited gastrointestinal adverse drug reactions such as diarrhea and colonic inflammatory responses, which is consistent with the MPH package insert and previous studies. Notably, a literature review indicated that differences in the type, dose, or duration of administration did not significantly affect the risk of gastrointestinal adverse events, suggesting that the occurrence of side effects may not solely depend on drug exposure levels [20]. Current mechanistic studies propose that MPH-related gastrointestinal dysfunction is associated with multiple factors, including excessive gastric acid secretion leading to heartburn and indigestion, stimulant-mediated disruption of normal gastrointestinal motility resulting in delayed gastric emptying, bloating, and constipation, as well as direct irritation of the gastrointestinal mucosa causing inflammation and abdominal discomfort [47]. However, these mechanisms cannot fully explain the complexity of MPH-induced gastrointestinal side effects, implying the involvement of additional regulatory factors.

In recent years, the role of the gut microbiota in drug-induced adverse reactions has garnered significant attention, as drugs may disrupt gut microbiota homeostasis and trigger gastrointestinal symptoms. In this study, we observed that Parabacteroides, as a core symbiotic genus, exhibited high abundance in the intestines of normotensive WKY rats, whereas its abundance was significantly reduced in SHR rats. This difference may be associated with the gut dysbiosis accompanying spontaneous hypertension, and previous studies have confirmed that SHR rats commonly exhibit intestinal microbial imbalance [48, 49]. Furthermore, MPH treatment further decreased the abundance of P. distasonis in the gut of SHR rats, and this change was closely correlated with the occurrence of gastrointestinal discomfort symptoms; supplementation with this strain significantly alleviated these symptoms. These results suggest that MPH may induce intestinal adverse reactions by exacerbating the pre-existing dysbiosis in SHR rats, particularly through the suppression of P. distasonis. P. distasonis is a normal intestinal microorganism isolated and preserved in 1933. It is a Gram-negative, non-spore-forming, rod-shaped, strictly anaerobic bacterium widely present in the gastrointestinal tract of humans and animals [50, 51]. Due to its potential anti-inflammatory, anti-obesity, and intestinal barrier protective effects, it has become a promising candidate for next-generation probiotic research [52]. Multiple studies have confirmed that P. distasonis has positive regulatory effects on gastrointestinal and liver diseases, including inflammatory bowel disease, colorectal cancer, liver fibrosis, and fatty liver disease. Its mechanisms mainly depend on metabolites such as short-chain fatty acids, amino acid derivatives, and bile acids, which participate in host immune regulation, intestinal barrier maintenance, and metabolic homeostasis [53]. The metabolites of P. distasonis primarily include short-chain fatty acids, amino acid derivatives, bile acids (BAs), and other bioactive compounds, which participate in regulating various physiological processes [54, 55]. P. distasonis possesses multifaceted physiological functions. In terms of immune regulation, it promotes immune cell differentiation, stimulates the secretion of anti-inflammatory factors, inhibits pro-inflammatory factors, and enhances anti-tumor immunity. Its metabolic products regulate macrophage polarization and Th17 cell differentiation, exerting anti-inflammatory effects. In terms of maintaining intestinal barrier integrity, it can upregulate tight junction protein expression, increase goblet cell numbers, and enhance mucus secretion. Its metabolic products activate pathways such as the aryl hydrocarbon receptor and G protein-coupled receptors, thereby enhancing intestinal barrier integrity and reducing plasma endotoxin levels [52, 56, 57]. Notably, we also observed that MPH treatment significantly increased the relative abundance of Parabacteroides merdae to investigate whether Parabacteroides merdae contributed to the MPH-induced disruption of intestinal homeostasis, we simultaneously examined the abundance of Parabcteroides merdae following P. distasonis supplementation (data not shown). The results demonstrated that P. distasonis supplementation did not significantly alter the abundance of Parabacteroides merdae, suggesting that Parabacteroides merdae is unlikely to be a direct contributor to the MPH-induced disruption of intestinal homeostasis. Furthermore, existing literature indicates that Parabacteroides merdae is primarily involved in bile acid transformation, enhances host resistance to pathogen infection, and cooperates with other bacterial strains to exert beneficial effects, such as anti-epileptic and anti-atherosclerotic activities. To date, no study has suggested a direct association between Parabacteroides merdae and colonic inflammation or intestinal adverse reactions [48, 58, 59]. Nonetheless, the functions of Parabacteroides merdae within the intestinal microbial network are highly complex, and its multiple roles in host health, as well as its potential interactions with P. distasonis, remain important directions for future investigation.

Inflammatory factors and tight junction proteins such as occludin play a crucial role in maintaining intestinal health. Elevated levels of pro-inflammatory factors such as Il6 and Tnf directlyreflect the presence of intestinal inflammation. As a key anti-inflammatory factor, elevated Il10 may reflect early compensatory or homeostatic anti-inflammatory responses. The expression level of occludin is closely associated with intestinal barrier function, and reduced occludin expression increases intestinal permeability, leading to intestinal dysfunction [60, 61]. Notably, MPH-induced diarrhea in rats was accompanied by increased colon length and colonic fecal pellet count relative to the SHR model group. This finding is closely linked to the inherent pathological features of SHR rats, including colonic wall stiffness, thickened smooth muscle, and abnormal submucosal collagen deposition, which reduce intestinal mechanical compliance [62]. The elevated colon length and fecal pellet count induced by MPH may not reflect physiological repair, but rather result from MPH-triggered colonic inflammation, which should be interpreted together with pathological and molecular indicators. Although chronic inflammation typically causes colon shortening, acute inflammatory mucosal congestion and edema can lead to transient intestinal elongation, while enhanced intestinal peristalsis accelerates fecal excretion and increases fecal pellets [63, 64]. The compensatory upregulation of Il10 in MPH-treated rats further supports that the colon is in the early inflammatory stage, and Il10 elevation serves as a protective response against MPH-induced local inflammation.In this study, MPH intervention caused elevated inflammatory factors and downregulated occludin expression in SHR rats, while supplementation with P. distasonis simultaneously inhibited inflammatory factor release and upregulated Ocln expression, forming a synergistic “anti-inflammatory-barrier repair” effect. This aligns with previous research findings, where P. distasonis demonstrated anti-inflammatory effects both in vitro and in vivo in studies related to colitis. The study confirmed that supplementation with P. distasonis reduces levels of inflammatory factors such as Il1β, Il4, Il8, and Tnf, while also enhancing the expression of intestinal tight junction proteins Zonula occludens-1 and Occludin. In colitis-related studies, pre-treatment with P. distasonis whole-cell lysate alleviated DSS-induced colitis; cell culture models showed that P. distasonis can restore and strengthen the epithelial barrier; mice fed a diet containing P. distasonis exhibited significantly increased expression of tight junction proteins Zonula occludens-1 and Occludin [65–69]. This further demonstrates the anti-inflammatory properties of P. distasonis.

While MPH has proven efficacy, its gastrointestinal side effects and lack of superior alternative treatments lead to reduced treatment adherence in some patients. Existing studies have primarily focused on developing alternative medications or behavioral therapy interventions, but strategies to alleviate MPH-induced drug side effects remain unexplored. This study is the first to address this critical issue and successfully proposes and validates an intervention strategy to alleviate MPH-induced gastrointestinal adverse reactions by targeting the regulation of the gut microbiota, providing a new research direction for the clinical management of MPH. This study further investigated the effects of the P. distasonis strain on core behavioral characteristics of ADHD. Notably, supplementation with P. distasonis did not significantly affect ADHD-related behavioral phenotypes. This finding confirms the safety of P. distasonis in the context of ADHD-related animal models, and indicates that this strain can alleviate MPH-induced gastrointestinal side effects without altering the inherent ADHD-related behavioral phenotypes, supporting its promising clinical translation potential.

This study also has certain limitations. First, the depth of the mechanism study is insufficient. Although it has been confirmed that P. distasonis can regulate inflammatory factors and the tight junction protein occludin, the specific molecular pathways remain unclear. Second, the study only focused on the effects of a single P. distasonis strain and did not explore the synergistic effects of other microbial community members. Furthermore, the intervention period in this study was relatively short, and the long-term gastrointestinaladverse reactions of MPH exposure were not evaluated. Additionally, this study lacks synchronized dynamic monitoring data on tissue concentrations of MPH, changes in gut microbiota composition, and intestinal inflammatory responses across different anatomical regions of the gastrointestinal tract and at multiple key time points.All these aspects warrant further systematic investigation.

Conclusion

This study demonstrated that MPH improves ADHD-like behaviors in SHR rats while inducing diarrhea and colonic inflammation, a mechanism closely related to the decreased relative abundance of P. distasonis. Supplementing with P. distasonis can alleviate MPH-induced gastrointestinal adverse reactions by inhibiting colonic inflammatory factors and upregulating the expression of tight junction protein Occludin. This study provides new insights into the microbiota mechanisms underlying the alleviation of MPH drug adverse reactions and lays the experimental foundation for developing probiotic intervention strategies based on P. distasonis. Future research should focus on elucidating the specific molecular pathways through which MPH regulates P. distasonis.

Authors’ contributions

Jinwen Yao: Methodology, Investigation, Writing-Original Draft, Formal analysis; Xia Gao: Methodology, Investigation; Lin Zhu: Supervision, Formal analysis, Validation; Linlin Wang: Methodology, Data Curation; Qian Chen: Methodology; Weijuan Liang: Data Curation, Validation; Huaili Feng: Methodology; Ting You: Data Curation; Changli Wei: Supervision, Formal analysis; Yuqi Liu: Formal analysis; Hongyang Zhang: Project administration, Writing-Review & Editing; Chengzhi Chen: Funding acquisition, Project administration, Conceptualization; Li Chen: Project administration, Funding acquisition.

Funding

This research was supported by National Key R&D Program of China (2022YFC2705205), the Chongqing’s Technological Innovation and Application Development Sichuan Chongqing Joint Implementation Key R&D projects (No. CSTB2022TIAD-CUX0003), and the Chongqing Postgraduate Research Innovation Project (CYS240324).

Data availability

The raw sequencing data were deposited in the NCBI SRA database under the accession number SRP609942.

Declarations

Ethics approval and consent to participate

This work was approved by the ethics committee of Chongqing Medical Univeristy (Approval No. IACUC-CQMU-2021-072).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.