Mechanisms of Electroacupuncture in Alleviating Visceral Hypersensitivity in Post-Infectious Irritable Bowel Syndrome Mice: The Role of GDNF Signaling Pathway and Gut Microbiota

Irritable bowel syndrome (IBS) is a chronic functional gastrointestinal disorder characterized by recurring abdominal pain, bloating, and abnormal bowel movement [1]. Acute infectious gastroenteritis can be caused by various pathogens, such as bacteria, viruses, and parasites [2, 3]. Notably, even after the pathogen is cleared, patients continue experiencing abdominal pain and intestinal dysfunction, a condition is defined as post-infectious irritable bowel syndrome (PI-IBS) [4‒6]. Visceral hypersensitivity as a distinguishing clinical feature of PI-IBS is the main manifestation of a decreased threshold for visceral pain and discomfort elicited by intraluminal mechanical or chemical stimuli, it’s a core pathological mechanism of PI-IBS [7‒10]. Gut microbiota dysbiosis, immune system dysregulation, and gastrointestinal motility disorders are all important factors that contribute to PI-IBS [11‒13].

The gut microbiota plays an essential role in shaping the intestinal environment. Due to the host intestinal microbiota being important to both immune response and digestive function, it has been associated with a number of health advantages [14]. The emergence of omics technologies such as transcriptomics, proteomics, metabolomics, and genomics has made significant progress in microbiology [15, 16]. These technologies have promoted the continuous improvement and development of the field of microbiological research and are crucial in understanding and elucidating the intricate dynamics of host-microbial interactions [17]. In recent years, microbiomes have demonstrated the significance role of intestinal microbiota in the development of PI-IBS in an increasing number of clinical and animal studies [18, 19]. The change in both the abundance and composition of Bifidobacterium, Lactobacillus, and Enterococcus faecalis is a significant risk factor leading to abdominal pain and gut dysfunction in PI-IBS patients [18].

Glial cell line-derived neurotrophic factor (GDNF) is secreted by enteric neuroglial cells and is highly expressed in the gut [20]. GDNF plays an essential role in maintenance of barrier integrity and regulation of the intestinal inflammatory response [21]. Researchers found that GDNF can cause intestinal stem cell and enterochromaffin (EC) cells differentiation, which leads to visceral hypersensitivity. It is also involved in the pathophysiology of PI-IBS and a key factor in changing the sensory aspects of visceral pain [22, 23]. Electroacupuncture (EA) stimulation at specific acupoints to regulate the gut microbiota can alleviate abdominal pain and abnormal defecation symptoms in patients with PI-IBS and is now an important treatment for visceral hypersensitivity in PI-IBS [24]. Existing studies have shown that EA has the capacity to activate the GDNF signaling and modulate the gut microbiota composition [25, 26]. Nevertheless, the potential therapeutic mechanisms of EA in PI-IBS have not been clarified, especially the specific mechanisms related to the modulation of GDNF signaling and the balance of the gut microbiota. Therefore, the objective of the study was to observe the impact of EA treatment on the GDNF signaling and gut microbiota in mice with 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced visceral hypersensitivity. The research aimed to clarify the potential therapeutic effects of EA to alleviate visceral hypersensitivity by regulating GDNF signaling and gut microbiota, therefore offering new perspectives on the use of EA for treatment of PI-IBS.

The C57BL/6J male mice (20–25 g) used in this study were obtained from the Model Animal Center of Nanjing University. The mice were housed in controlled environments with a 12-h light/dark cycle, an ambient temperature of 20–22°C, a relative humidity of 40–60%, and free access to food and drink ad libitum. The animals used in the experiment followed the Guidelines for the Care and Use of Laboratory Animals issued by the National Research Council. The welfare of the animals was prioritized, ensuring minimal suffering and considering their best interests.

To evaluate the efficacy of acupuncture, mice were randomly divided into two groups: a control group (n = 7) and a model preparation group (n = 35). The model preparation group was given TNBS enema, and after 28 days of TNBS administration, the mice were randomly divided into the model group, EA group, traditional acupuncture (TA) group, GDNF group, and EA + GDNF group (each group n = 7).

The visceral hypersensitivity model of PI-IBS was induced by colon enema of TNBS [27]. Fasting for 12 h before modeling was required, but drinking was not forbidden in order to reduce feces residue in the mice’s intestine. The mice were anesthetized with isoflurane (oxygen at 0.5 L/min accompanied by 3% isoflurane for induction and 1.5% isoflurane for maintenance). After inserted into a polyethylene catheter into the colon, the mice were administered 0.1 mL of TNBS (at a dose of 80 mg/kg) dissolved in a 50% ethanol solution to induce colitis. The visceral sensitivity of PI-IBS mice was assessed by abdominal withdrawal reflex (AWR) 4 weeks post-infection.

The present study used GDNF agonists (Peprotech, USA) in mouse models to study diseases related to GDNF. To enhance GDNF signaling expression, the GDNF agonist was mixed in a 0.9% saline solution and administered intraperitoneally at a dose of 10 μg/kg daily to the visceral hypersensitivity model mice. The mice in the control group were given an equivalent dose of saline solution in order to compare. Visceral sensitivity was assessed through AWR after being administered for 7 consecutive days of the drugs.

The animals received daily EA or TA stimulation for 7 consecutive days. Previous studies showed that acupuncture alleviated visceral hypersensitivity at Zusanli (ST36) and Tianshu (ST25) [28, 29]. Therefore, the acupoints ST25 and ST36 were used in this experiment. The mice were anesthetized by inhaling isoflurane. After 75% alcohol was used to disinfect the skin, disposable stainless steel acupuncture needles (0.19 × 10 mm; Suzhou Medical Supplies Factory Co., Ltd., Jiangsu, China) were rapidly and vertically punctured mice at a depth of 3 mm at ST25 and ST36. The needle handles were connected to a nerve acupoint stimulator (HANS-200A, Nanjing Jinshun Medical Science and Technology Co., Ltd.) for EA stimulation at a frequency of 2/15 HZ and an intensity of 0.5 mA for a duration of 15 min. The only difference between the EA group and the TA group is that the latter was unconnected electric current, after the 7-day intervention, assessed colon sensitivity of mice.

AWR scores were used to assess visceral sensitivity induced by colorectal distention. An AWR test was performed on mice after a 24-h fasting period during which they were not deprived of water. Mice were lightly anesthetized with isoflurane. After the uninflated balloon was inserted into the mouse colon, mice were placed in a transparent observation box, unable to turn around but able to forward and backward only. Following a period of 30 min, after they were completely alert, a solution of saline was administered into a balloon. Saline (0, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, and 0.22 mL) was injected into the balloon for 20 s at 5-min intervals to grade colorectal distention. This procedure was repeated three times, and the mean score was calculated to assess intestinal sensitivity. The following was the AWR scores: 0 = no behavioral changes; 1 = occasional head writhing with no obvious abdominal discomfort; 2 = contraction of abdominal muscles without abdominal lifting; 3 = abdominal lifting; 4 = abdominal and pelvic lift [30].

CO₂ inhalation euthanasia was used to sacrifice mice, and all possible efforts were made to minimize their suffering. Longitudinally incised abdominal cavities were opened in mice. The colonic fecal sample was collected and then transferred to a pre-cooled Eppendorf tube. Samples were quickly frozen with dry ice and stored at −80°C in order for the subsequent 16S ribosomal RNA (16S rRNA) analysis.

16S rRNA gene sequencing was used for the identification and analysis of gut microbiota. 16S rRNA has the advantages of low mutation rate, low cost, and low risk of contamination, which is an efficient method to analyze the composition of intestinal microbiota [31, 32]. The study used the E.Z.N.A. Soil DNA Kit (Omega Bio-Tek, Inc., USA) for DNA extractions from fecal samples, following the manufacturer’s recommendations. The DNA V3-V4 region was amplified by PCR using primers 338F (5′-ACT​CCT​ACG​GGG​AGG​CAG​CAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). After library construction, PCR amplification was used to enrich the library fragments and quantify their size and concentration. Sequencing was subsequently performed using Illumina Miseq technology (Illumina, USA). This sequencing aimed to analyze the diversity and abundance of the microbiota as well as the composition of the microbiome community.

The gene sequences were classified as OUT and analyzed by bioinformatics according to the similarity level of 97%. The sample sequences from the OUT dataset were used for taxonomic identification with the BLAST algorithm against the Silva138 database to determine the species composition. The indicator of Alpha diversity assesses microbial community diversity by analyzing the variety of microbial species and the relative abundance in a given community. It can reflect the evenness and richness of the species. In addition, beta diversity is an indicator that quantifies the differences in species composition between the microbial communities. It shows differences in species composition in number, similarity, and spatiality of species. Finally, the data between groups in this study were statistically analyzed to calculate the number of species with significance.

All statistical analyses were analyzed by SPSS version 26.0 (SPSS, Chicago, USA). The images were generated using Prism 7.0 software (Prism, CA, USA). The research data were expressed as mean ± standard error of the mean (mean ± SEM). The comparison between the two groups was performed using independent-samples t test or Mann-Whitney test. This study used QIIME software to analyze data and the beta diversity of microbiota was analyzed by calculating the distance matrix. The similarity or dissimilarity in the composition of the sample communities was explored by plotting the Principal Co-ordinates Analysis based on the distance matrix using R software. Subgroup comparisons were performed using LEfSe analysis, and the species with significant differences in abundance were calculated by multivariate statistical analysis. Values of p < 0.05 were considered statistically significant.

To evaluate the efficacy of acupuncture in the treatment of PI-IBS, the AWR scores were used to assess different groups of mice post-intervention. Compared with the control group of mice, the AWR scores of the model group were significantly increased, demonstrated the success of the TNBS-induced visceral hypersensitivity of the PI-IBS model. The AWR score was significantly decreased after acupuncture treatment in the sensitivity analysis of the intestinal. Compared with the TA group, the AWR score was significantly decreased in the EA group. These results showed that EA had a significant advantage of alleviating visceral hypersensitivity in PI-IBS (Fig. 1a). Compared with the control group, the AWR score significantly increased with the group of GDNF agonists, which suggested the GDNF played an important role in the development of visceral hypersensitivity in the colon. After EA treatment, there was a significant decrease in the AWR score. Further showing of the treatment effect of EA (Fig. 1b). The above data showed that GDNF plays an important role in the development of visceral hypersensitivity, that EA can via GDNF relieve symptoms of visceral discomfort in PI-IBS mice, and that GDNF is an important therapeutic target for alleviating visceral hypersensitivity in PI-IBS.

The diversity of gut microbiota has a strong association with visceral hypersensitivity in PI-IBS. The Chao1 and Shannon indexes are important indicators of alpha diversity measurement. The Shannon value was calculated with the mothur software, and the OTU clustering results was used in this study. The study used dilution curves to evaluate the microbial diversity of all samples at different sequencing depths. The result showed that the curves were flat and reached a saturation point, showing that the high sequencing depth and the sufficient amount of sample data were capable of detecting the majority of the species under study (Fig. 2a). Furthermore, the model and GDNF agonist groups showed a significant decrease in both the Chao1 and Shannon indices. It demonstrated a decrease in microbial diversity. However, after the EA intervention, there was a reversal in diversity, indicating that EA had a positive effect on microbiota richness (Fig. 2b, c). Beta diversity is analyzed through Principal Co-ordinates Analysis to determine primary components affected in the community composition of samples. The results showed that the community in the model and GDNF groups had uniqueness and complexity. Following EA treatment, the community composition was more similar to the control group, and the species were decreased (Fig. 2d). The above results showed that EA has the potential to regulate the diversity of microbial communities in the intestinal tract.

To further investigate what microbes contribute to the apparent differences in the diversity of gut microbiota and whether changes in gut microbiota were essential to EA in attenuating visceral hypersensitivity, we thoroughly analyzed the alteration in microorganisms at the levels of phylum, class, order, family, genus, and species.

The linear discriminant analysis effect size (LEfSe) analysis was used to analyze the differential abundances in the bacterial microbiota between groups, with a linear discriminant analysis threshold of 3. As shown in Figure 3a, compared with the control group, Actinobacteriota at the phylum level and Coriobacteriia at the class level were significantly more abundant. At the order level, Coriobacteriales and Bifidobacteriales were significantly higher, while Atopobiaceae, Bifidobacteriaceae, Coriobacteriaceae, and Parvibacter were enriched at the family level. Similarly, at the genus level, the population of Ileibacterium, Actinobacteria, Bifidobacterium, and Dubosiellas were significantly increased. Ileibacterium valens and Bifidobacterium pseudolongum had relatively higher levels at the species level.

Compared to those in the model mice in Figure 3b, EA could decrease the population of Actinobacteriota at the phylum level, Coriobacteriia at the class level, Coriobacteriales and Bifidobacteriales at the order level, Atopobiaceae, Bifidobacteriaceae, Coriobacteriaceae, and Parvibacter at the family level, Actinobacteria and Bifidobacterium at the genus level, and B. pseudolongum at the species level. In addition, after EA treatment, Deinococcota and Verrucomicrobiota at the phylum level, Verrucomicrobiae at the class level, Deinococcales, Frankiales, and Verrucomicrobiales at the order level, Weeksellaceae, Mycobacteriaceae, Deinococcaceae, and Akkermansiaceae at the family level, Mycobacterium and Deinococcus, Akkermansia at the genus level, and Adllercreutzia caecicoia, Bifidobacterium breve, Deinococcus grandis, Mycobacterium sp at the species level were also significantly reduced. However, Verrucomicrobiota and Firmicutes at the phylum level, Akkermansiaceae at the family level, and Akkermansia, Prevotella, Allobaculum, Dubosiella, Robinsoniella at the genus level showed significant changes in the TA group than that of the EA group (Fig. 3c).

Subsequently, to further investigate the relationship between the microbiome and GDNF, and whether GDNF was essential to the effect of EA by regulating the microbiome, we treated with recombinant GDNF. Compared with the GDNF group, the EA + GDNF group showed a higher abundance of Firmicutes at the phylum level and Bacilli at the class level. The abundances of Erysipelotrichales and Lactobacillales were significantly higher at the order level, and also, Erysipelotrichaceae, Thermotaleaceae, Lactobacillaceae, and Rikenellaceae at the family level had significantly more abundant. Similarly, at the genus level, Dubosiella, Cronobacter, Turicibacter, Thermotalea, Neglecta, Holdemanella, Lactobacillus, Eubacterium_fissicatena, Robinsoniella, and Enterorhabdus was significantly enriched in the EA + GDNF mice. Cronobacter dublinensis, Pseudomonas veronii, Eubacterium fissicatena, and Robinsoniella peoriensis at the species levels were also enriched (Fig. 3d). These results suggest that EA can influence the gut microbiota by regulating the GDNF signaling to alleviate the visceral hypersensitivity of PI-IBS.

PI-IBS seriously affects the mental health and occupational performance of patients [33]. Pharmaceutical treatments are currently the main therapy for PI-IBS [34]. Nevertheless, these medical treatments have various levels of side effects [35]. Acupuncture as a non-pharmacological medical adjunct therapy, demonstrated significant efficacy in alleviating visceral hypersensitivity of PI-IBS [36].

AWR is an important method used to assess visceral sensitivity. A higher AWR score showed higher intestinal sensitivity, represented a more serious condition. Compared to the control group, the model group mice had higher intestinal sensitivity, which corresponds to the clinical manifestation of patients suffering from visceral hypersensitivity [37]. EA decreased AWR scores in mice and played an important role in alleviating visceral hypersensitivity.

The pathophysiology behind visceral hypersensitivity is still unclear. As is well known, the secretion (5-HT) released from EC cells is an important factor in the development of intestinal hypersensitivity [11, 38]. Previous studies showed that GDNF was widely present in the colonic epithelium of IBS patients and played an important role in EC cells development [39]. The results of this study showed that GDNF agonists had the ability to enhance visceral hypersensitivity in mice. GDNF is an important factor in inducing visceral hypersensitivity. Continuous EA stimulation effectively decreases visceral hypersensitivity induced by GDNF agonists in mice, consequently alleviating symptoms of PI-IBS.

The gut microbiota is crucial for the regulation of the immune system [36]. The imbalance of the gut microbiota could result in visceral hypersensitivity, which is an important factor in PI-IBS. Research has identified that the intestinal microbiota can be effectively regulated to alleviate visceral hypersensitivity [40]. Existing studies have shown that the gut microbiota can prompt producing the GDNF [41, 42]. Chao1 and Shannon indices were important indicators of microbial diversity. Therefore, we inferred that the gut microbiota of visceral hypersensitive animals was influenced by GDNF signaling. Actually, our results showed that activating GDNF signaling decreased not only the Chao1 and Shannon indices of the microbiota but also the microbiota’s abundance, resulting in disruptions in the structure of the flora. These results showed that GDNF signaling could regulate visceral hypersensitivity by modulating the composition and abundance of gut microbial abundance.

This study has several limitations. First of all, the functionally relevant assays about the gut microbiota were only performed by 16S rRNA, with no further analysis of the microbiota activity using macrogenome sequencing. Second, the gut microbiota can be influenced by a range of factors, and this research did not control for relevant factors such as feed intake; finally, the study was concentrated on the effects of EA and GDNF on the gut microbiome, ignoring other potential mechanisms and influencing factors. In the future, our study will extend the substrate scope and focus on exploring the association between the gut microbiome and health from different aspects.

In summary, this research investigation has demonstrated that electroacupuncture has the potential to enhance the abundance of beneficial microbiota of visceral hypersensitivity mice suffering PI-IBS, the improvement is likely achieved through the regulation of GDNF signaling. These results suggested that EA may be a prospective treatment for the future clinical management of PI-IBS.

The animals utilized in this experiment were performed following the rules for the care and application of laboratory animals established by the National Research Council. These guidelines were approved by the Ethics Committee of Nanjing University of Chinese Medicine (No. 012071003694).

The authors have no conflicts of interest to declare.

This project was funded by Jiangsu Province Traditional Chinese Medicine Technology Development Plan Project (QN202101), the National Natural Science Fund (82274630), Chongming Bird Program Special Research Fund (HYCMP-2021005), Luo Linxiu Teacher Development Fund of Nanjing University of Chinese Medicine (LLX202304), and the Graduate Student Research and Innovation Project of Jiangsu Province (KYCX22_1933). The funding bodies provided financial support, and the awardees performed the research.

S.-Y.J. conceived the study and drafted the manuscript; L.-X.P. and L.C. supervised the study; S.-Y.J. and Y.-F.S. performed the experiments; L.C. and Y.-F.S. analyzed data; S.-Y.J., L.-X.P., and J.-H.S. contributed to the discussion of the findings; L.-X.P., L.C., J.-H.S., and S.-Y.J. revised the manuscript; and all authors approved the final manuscript.

Raw data have been deposited in the Sequence Read Archive (SRA) of the NCBI database. Further inquiries can be directed to the corresponding author.