Mechanism of anti-HuD autoantibody inducing enteric neuronal apoptosis of irritable bowel syndrome and its potential for targeted intervention

Abstract

BACKGROUND

Irritable bowel syndrome (IBS) is a common disorder of gut-brain interaction and is characterized by chronic abdominal pain and altered bowel habits. Current evidence indicates that immune activation and autoantibody production contribute to IBS pathogenesis. However, the mechanisms by which autoantibodies affect the enteric nervous system and contribute to IBS-related symptoms are poorly understood.

AIM

To investigate the role of anti-HuD autoantibodies in enteric neuronal apoptosis in an IBS animal model.

METHODS

A passive-transfer rat model of IBS was generated by intraperitoneal administration of HuD autoantibodies. Gastrointestinal motility, visceral sensitivity, fecal output, and water content were assessed. Enteric neuronal apoptosis in intestinal tissues and primary enteric neurons was analyzed by immunofluorescence. Mechanisms were examined using quantitative reverse transcription polymerase chain reaction, western blotting, and confocal microscopy. Interventions included recombinant HuD, immunoglobulin, 5-hydroxytryptamine receptor modulators, and protein kinase C (PKC) agonists.

RESULTS

Administration of HuD autoantibodies induced IBS-like phenotypes in rats. We observed increased fecal output, elevated fecal water content, accelerated intestinal transit, and enhanced visceral hypersensitivity. HuD autoantibody exposure significantly increased enteric neuronal apoptosis in vivo and in vitro and suppressed the expression of special AT-rich sequence-binding protein 1 (SATB1). Mechanistically, HuD autoantibodies disrupted HuD-mediated RNA regulation, leading to SATB1 downregulation and inhibition of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway. PKC activation restored HuD and SATB1 expression, reactivated PI3K-AKT signaling, and significantly reduced neuronal apoptosis. Treatment with immunoglobulin and 5-hydroxytryptamine receptor agonists showed limited or inconsistent protective effects.

CONCLUSION

HuD autoantibodies induced enteric neuronal apoptosis through disruption of the HuD-SATB1-PI3K-AKT signaling axis and contributed to IBS-like gastrointestinal dysfunction. Activation of PKC may be a potential therapeutic strategy for IBS.

Key Words: Irritable bowel syndrome; Anti-HuD autoantibody; Enteric neuron; Apoptosis; Intervention

Core Tip: Current evidence indicates that immune dysregulation contributes to the pathogenesis of irritable bowel syndrome (IBS). However, the direct effects of autoantibodies on the enteric nervous system are unclear. This study established an antibody-mediated IBS model and demonstrated that anti-HuD autoantibodies directly induced enteric neuronal apoptosis by disrupting HuD-mediated RNA regulation. HuD autoantibodies reduced the stability of downstream mRNA and suppressed phosphatidylinositol 3-kinase-protein kinase B signaling, leading to neuronal loss and IBS-like gastrointestinal dysfunction. Pharmacological activation of protein kinase C restored HuD expression and alleviated neuronal apoptosis, highlighting a potential targeted therapeutic strategy for IBS associated with autoimmune-mediated enteric neuropathy.

INTRODUCTION

Irritable bowel syndrome (IBS) is a chronic and recurrent disorder of gut-brain interaction. It is characterized by abdominal pain related to defecation and altered bowel habits according to the latest Rome IV criteria[1,2]. IBS affects 5%-10% of the global population[3] and substantially impairs quality of life. It is frequently accompanied by psychological comorbidities[4,5]. Although IBS has traditionally been regarded as a functional gastrointestinal disorder, accumulating evidence indicates that immune dysregulation, low-grade inflammation, and enteric nervous system (ENS) abnormalities are important contributors to the pathophysiology of IBS[6]. Currently, the mechanisms linking immune activation to enteric neuronal dysfunction in IBS are incompletely understood.

Autoimmune processes may participate in IBS pathogenesis. Low-grade mucosal inflammation and increased humoral immune responses and immunoglobulin production have been reported in patients, particularly those with diarrhea-predominant IBS (IBS-D)[7-9]. Notably, anti-neuronal antibodies targeting enteric neurons have been detected more frequently in patients with IBS compared with healthy controls. Histopathological analyses have demonstrated lymphocytic infiltration and neuronal degeneration within the enteric plexus[10,11].

Wood et al[12] quantified a significantly higher prevalence of anti-enteric neuronal antibodies (AENA) in IBS cohorts through systematic immunolabeling of whole-mount preparations of the ENS. A recent study of 293 patients with IBS revealed elevated AENA seropositivity rates relative to healthy controls[13]. Among the neuronal autoantigens, HuD or embryonic lethal abnormal vision 4 (ELAVL4), a neuron-specific RNA-binding protein essential for neuronal survival and maintenance[14], has emerged as a potential immune target. Through the utilization of the HuProt™ human proteome microarray and IBS-focused microarray and result validation by ELISA, we found an elevated median optical value of ELAVL4 IgG in the sera of patients with IBS compared with controls[15].

Patients with anti-HuD-positive sera have been shown to induce myenteric neuronal apoptosis in vitro[16], and anti-HuD autoantibodies directly activate enteric neurons and visceral sensory fibers, potentially triggering cytotoxic effects[17]. Several downstream RNA targets of the HuD protein were found to be closely associated with neuronal growth, such as growth-associated protein-43 (GAP-43), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF). The phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway is also closely associated with neuronal growth and apoptosis. Currently, no investigations have determined if HuD autoantibodies directly disrupt HuD-mediated RNA regulation in enteric neurons, thereby inducing neuronal apoptosis and contributing to IBS-related gastrointestinal dysfunction.

In the present study we established a passive-transfer rat model of IBS using HuD autoantibodies to investigate the pathogenic role of autoantibody-mediated enteric neuropathy. We aimed to determine whether HuD autoantibodies directly induced apoptosis of enteric neurons and to elucidate the underlying molecular mechanisms with a particular focus on HuD-mediated RNA regulation and downstream signaling pathways. We also explored potential targeted intervention strategies to counteract antibody-induced neuronal apoptosis. By integrating in vivo and in vitro approaches, this study provided mechanistic insights into autoimmune-mediated enteric neuronal injury and identified proof-of-concept therapeutic targets for IBS.

MATERIALS AND METHODS

Cell line and cell culture

The SH-SY5Y human neuroblastoma cell line was purchased from the Cell Resource Center, Institute of Biochemistry and Cell Biology at the Chinese Academy of Science (Shanghai, China). The cell line was cultured in RPMI1640 medium (Keygen Biotech, Nanjing, China) supplemented with 10% (v/v) fetal bovine serum (ABW, Shanghai, China) at 37 °C and 5% CO₂.

Passive transfer modeling of IBS

Five-week-old male Sprague-Dawley rats weighing 180-200 g were purchased from Beijing Vital River Laboratory Animal Technology Company (Beijing, China) and maintained in pathogen-free conditions. Animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals with the approval of the Animal Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, No. TJH-202204019. No accidental deaths occurred during the study. All rats were injected intraperitoneally with 1% sodium pentobarbital (55 mg/kg) before sacrifice.

All rats were housed in a controlled environment (20-24 °C, relative humidity of 35%, 12-hour light/dark cycle), housed individually in single cages, provided with sufficient shelter and free access to water and standard chow. After 2 weeks of acclimation and quarantine inspection, all rats were weight-matched (ranging between 315-345 g when the experiments were performed). These rats were randomly assigned to three groups, namely the HuD autoantibody group (12 rats) as the experimental group, the normal saline group (6 rats) as the negative control group, and the blank control group (6 rats) to ensure that the studied variables did not result in biased data within each group. For the IBS model (HuD autoantibody group), 10 μg of HuD autoantibodies (human HuD neuronal protein, A21271; Mouse/IgG2b kappa; Thermo Fisher Scientific, Waltham, MA, United States) (the same antibody was used for the in vitro treatment) diluted with normal saline were intraperitoneally injected into the rats for 3 consecutive days. For the normal saline group, the same volume of normal saline diluted with an isotype control antibody (Mouse IgG2b kappa Isotype Control, 11-4732-42; Thermo Fisher Scientific, Waltham, MA, United States) was injected intraperitoneally into the rats for 3 consecutive days to minimize Fc-mediated nonspecific immune activation. The blank control group remained untreated.

For 7 days after the antibody injection, the weights of the rats were measured at 9 am, and the feces produced during 9-10 am and 9-10 pm were collected into sealable plastic bags. Afterward, the total weight of the feces and the dry weight of the feces (desiccated in a microwave oven) were measured within 30 minutes. The water content of the feces was calculated by the following formula: Water content (%) = [total weight of feces (g) - dry weight of feces (g)]/total weight of feces (g) × 100%. On day 11 of the experiment, the abdominal withdrawal reflex experiment was performed[18]. On day 12 after 24 hours of fasting, the intestinal transit experiment was carried out by gavage administration of 10% active carbon suspension. After 45 minutes the rats were sacrificed after intraperitoneal injection of 55 mg/kg sodium pentobarbital, and the entire gut was removed. Then, the length of the small intestine and large intestine and the distance of active carbon suspension intestinal propulsion were measured. The intestinal transit rate was calculated as follows: Intestinal transit rate (%) = distance of active carbon intestinal propulsion (cm)/[small intestine length (cm) + colon length (cm)] × 100%. All rats were monitored according to the Institutional Animal Care and Use Committee guidelines and sacrificed if any excessive deterioration in health was observed.

Full-thickness whole-mount preparation of rat intestine

The protocol used was previously described in detail by Mazzuoli and Schemann[19,20]. Male rats were sacrificed after intraperitoneal injection of 55 mg/kg sodium pentobarbital. The ileum and colon were quickly removed and further dissected in Krebs’ solution aerated with 5% CO₂ carbogen and containing: 117 mmol/L NaCl; 4.7 mmol/L KCl; 1.2 mmol/L MgCl₂; 1.2 mmol/L NaH₂PO₄; 25 mmol/L NaHCO₃; 2.5 mmol/L CaCl₂; and 11 mmol/L glucose. This preparation (10 mm × 10 mm) was pinned onto a silicone-embedded dish and perfused with 37 °C carbogen Krebs’ solution at least three times until clean. The dish was covered in paraformaldehyde overnight for fixation. The mucosa, the submucosa, and the circular muscle layer of the gut were gently removed under an anatomic microscope to obtain a longitudinal muscle-myenteric plexus preparation.

Isolation and culture of primary enteric neurons

The isolation of primary myenteric neurons was performed[21]. These cells were cultured in DMEM-F12 medium (Keygen Biotech, Nanjing, China) supplemented with 10% (v/v) fetal bovine serum (ABW, Shanghai, China), 10 ng/mL NGF (13257-019; Thermo Fisher Scientific, Waltham, MA, United States), and 1% penicillin-streptomycin-gentamicin (Solarbio, Beijing, China) at 37 °C and 5% CO₂.

Plasmid and small interfering RNA transient transfection

The complementary DNA (cDNA) encoding special AT-rich sequence-binding protein 1 (SATB1) was subcloned into the pcDNA3.1 vector (Umine Biotechnology, Guangzhou, China), and the resultant vector was named pcDNA3.1-SATB1. A blank pcDNA3.1 vector served as a control. Transfection with plasmids (1 ng/mL) was performed with Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, United States). The SATB1 small interfering RNA (siRNA) (sense-5’-GCUACAGAGAAUCUUCAUATT-3’; antisense-5’-UAUGAAGAUUCUCUGUAGCTT-3’) (20 nM) was transfected with Lipofectamine 3000. The negative control siRNA (sense-5’-UUCUCCGAACGUGUCACGUTT-3’; antisense-5’-ACGUGACACGUUCGGAGAATT-3’) was used in the negative control group. Transfection was carried out according to the manufacturer’s instructions. Western blot was used to assess the knockdown efficiency at 48-72 hours after transfection.

Lentivirus production, transduction, and plasmid construction

The pGMLV-CMV-H_ELAVL4-3 × Flag-PGK-Puro vector and relevant control vector were constructed by Genomeditech (Shanghai, China). These vectors and a lentiviral vector packaging system (ObiO Technology, Milpitas, CA, United States) were then co-transfected into 293 T cells using Lipofectamine 3000. These lentiviruses were designed to express HuD. SH-SY5Y cells were infected with these lentiviruses in the presence of polybrene (Sigma-Aldrich, St Louis, MO, United States) and selected by puromycin. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot were used to detect changes of HuD levels in cells.

RNA isolation and qRT-PCR

Total RNA was extracted from the colon and ileum specimens using Trizol reagent (Takara, Shiga, Japan) and reverse transcribed into cDNA using 5 × ABScript III RT Mix (Abclonal, Wuhan, China). Then, qRT-PCR was performed using SYBR Premix (Q711-02; Vazyme, Nanjing, China). A standard curve for each gene was generated from serially diluted standards, and values for unknown samples were extrapolated. All standards and samples were assayed in triplicate. The expression of the downstream mRNA targets of the HuD protein (GAP-43, NGF, BDNF, neuroserpin, neurotrophic factor-3, and SATB1) was determined. Primer sequences are detailed in Supplementary Table 1.

Western blot analysis

Samples harvested from the rat colons or cell lines (30 μg of total protein) were resolved on 10% polyacrylamide sodium-dodecyl-sulfate gels and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk, incubated with appropriate primary antibodies and horseradish peroxidase-conjugated suitable secondary antibodies, and detected with enhanced chemiluminescence reagents (Pierce Chemical, Rockford, IL, United States). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control; all blots were normalized according to GAPDH. Monoclonal mouse anti-GAPDH was used as an internal control. Antibodies involved are listed in Supplementary Table 2.

Immunofluorescence and terminal-deoxynucleotidyl transferase mediated nick end labeling

The terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) assay utilized an In Situ Cell Death Detection Kit (11684795910; Roche, Indianapolis, IN, United States). For the immunofluorescent analysis, the cell and tissue sections were incubated with primary antibodies, followed by incubation with secondary antibodies according to the experimental design. PGP9.5 antibodies (1:50 dilution) were used to indicate neurons in primary cells. The signals were detected by fluorescein isothiocyanate and Cy3 channels. Cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI).

All immunostaining, including TUNEL, was conducted with three or six biological replicates. To ensure objectivity and statistical power, a rigorous sampling strategy was employed. For each biological replicate, three non-consecutive tissue sections were selected for imaging. Within each section three random, non-overlapping fields were captured using a 40 × objective. A cell was defined as positive only if the TUNEL signal or cleaved caspase 3 (both green fluorescence) and the PGP9.5 (red fluorescence) colocalized precisely with the nuclear counterstain (DAPI, blue fluorescence) and exhibited a distinct morphology characteristic of the neuron. Cells with only cytoplasmic staining or diffuse, non-nuclear signal were excluded.

To objectively define a positive signal and minimize background noise, a consistent intensity thresholding method was applied across all images. Before thresholding, a rolling-ball background subtraction algorithm (radius = 50 pixels) was applied to each channel to correct for uneven illumination. The intensity threshold for the TUNEL and immunofluorescence channels was determined using Otsu’s method applied to the entire image stack. This method maximized the interclass variance between the signal and background noise. The final threshold value was recorded and applied uniformly to all experimental images.

Images were obtained with an LSM880 confocal microscope (Zeiss, Oberkochen, Germany). All image analyses were performed using ImageJ/Fiji software (National Institutes of Health, Bethesda, MD, United States) by two independent, masked observers who were blinded to the experimental group assignments. Antibodies involved are listed in Supplementary Table 2.

In vitro and in vivo targeted intervention experiments

To investigate potential intervention strategies for counteracting the effects of autoantibodies, primary neuron cells were preincubated with the HuD autoantibody (1:1000), followed by treatment with 1 μg/mL recombinant HuD protein, 20 mg/mL immunoglobulin, 1 μM protein kinase C (PKC) agonist Bryostatin1 (HY-105231; MCE, United States), 1 μM 5-hydroxytryptamine (5-HT₄) receptor agonist Velusetrag hydrochloride (HY-10457A, MedChem Express, Monmouth Junction, NJ, United States), and 1 μM 5-HT₄ receptor antagonist (HY-100170; MedChem Express, Monmouth Junction, NJ, United States).

For the in vivo experiments, a PKC agonist and immunoglobulin were administered to evaluate their effects on the apoptosis rate of ileal neurons. Each group consisted of three Sprague-Dawley rats. All groups received an intraperitoneal injection of the HuD autoantibody for 3 consecutive days as described previously. The two experimental groups were injected with the PKC agonist or immunoglobulin the day after the last injection of the HuD autoantibody. The control group received an injection of normal saline of the same volume. Based on the weights of the rats, the PKC agonist (30 μg/kg) was injected into rats intraperitoneally, whereas the intravenous immunoglobulin (1 g/kg) was administered for another 3 consecutive days. Rats were sacrificed on day 12 after the first injection of the antibody.

Statistical analysis

All experiments were carried out at least twice independently. Data are presented as mean ± SD for each experiment; error bars represent the standard deviation. Statistical analysis was performed using R (v4.0.2) and GraphPad Prism 9.0 software. We chose appropriate statistical tests according to the data type. Normally distributed data were analyzed by the Student’s t-test and one-way or two-way analysis of variance with Tukey’s multiple comparison test. A P < 0.05 indicated statistical significance.

RESULTS

IBS model established by the passive transfer of commercial HuD autoantibody

To explore the role of HuD autoantibodies in IBS, we developed an antibody-induced IBS model via passive transfer. After a 7-day observation period, no significant differences in body weight gain were observed among the HuD autoantibody group, the normal saline group, and the blank control group (Figure 1A). Compared with the normal saline and blank control groups, the HuD autoantibody group exhibited a significantly higher total number of fecal pellets over 24 hours on days 3, 4, and 6 during the observation period (Figure 1B). Additionally, the 2-hour fecal water content in the HuD autoantibody group was significantly greater than that in the negative control and blank control groups on days 1, 3, and 4 (Figure 1C).

Figure 1 Indicators of the irritable bowel syndrome model established by passive transfer of a commercial anti-HuD autoantibody. A: Changes in body weight of the three groups of rats during the observation period; B: Changes in the number of fecal pellets of the three groups of rats during the observation period; C: Changes in the water content of feces of the three groups of rats during the observation period; D: Abdominal withdrawal reflex scores of three groups of rats under different pressures; E: Recorded pressure of three groups of rats during abdominal withdrawal reflex in which 3 represents the visceral pain threshold; F: Comparison of the intestinal transit rate of the three groups of rats; G: Representative image of the propelling distance of activated carbon powder in the intestine shown by the arrow. Data are expressed as mean ± SD. B-D were analyzed by two-way analysis of variance with Tukey’s multiple comparison test; E and F were analyzed by one-way analysis of variance with Tukey’s multiple comparison test. For A-F, n = 6 for the normal saline group and blank control group and n = 12 for the HuD autoantibody group. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001.

On day 11 the abdominal withdrawal reflex test was performed, and the scores were recorded to assess gut sensitivity. The rats in the HuD autoantibody group exhibited significantly higher scores compared with those in the negative control group and the blank control group under external pressures of 60 mmHg and 80 mmHg (Figure 1D). Using an abdominal withdrawal reflex score of 3 as the visceral pain threshold, the minimum pressure required by the HuD autoantibody group was significantly lower than that of the negative control group and the blank control group (Figure 1E).

In the intestinal transit experiment performed on day 12, the intestinal transit function of rats in the HuD autoantibody group was markedly enhanced compared with that of the negative control group and the blank control group (Figure 1F). A representative image showing the propelling distance of activated carbon powder in the intestine is indicated by the arrow (Figure 1G). Based on a comprehensive evaluation of these indicators, the establishment of the IBS rat model using a commercial HuD autoantibody was successful.

HuD autoantibody induced primary neuronal apoptosis

Primary enteric neurons were isolated and cultured to investigate the specific effect of HuD autoantibodies on intestinal neurons in vitro. Following incubation with the HuD autoantibody at a dilution of 1:1000 for 48 hours, the expression levels of Bax and cleaved caspase-3 proteins were significantly upregulated in the HuD autoantibody-treated group. Conversely, the expression of Bcl-2 protein was slightly downregulated in exposed primary enteric neurons compared with the negative control group (Figure 2). During the initial 48-hour period without antibody exposure, both groups exhibited consistent morphology characterized by large, intact nerve fibers with strong refraction and well-formed neural network structures as observed under the microscope. After culturing the cells with the antibody for an additional 48 hours, apoptosis occurred more frequently in the antibody-treated group (Figure 2B).

Figure 2 Anti-HuD autoantibody induced apoptosis of primary enteric neurons. A: Expression of apoptotic proteins detected by western blot; B: Morphological changes after 48 hours of antibody incubation. More apoptotic cells with vacant cytoplasm, partial atrophy, and reduced refraction were observed in the treated group. The yellow arrows indicate apoptotic neurons. Scale bar: 60 μm; C: Immunofluorescence detected the expression of cleaved caspase 3, and anti-PGP 9.5 indicated neurons; D: Immunofluorescence and terminal-deoxynucleotidyl transferase mediated nick end labeling staining detected nuclei cleavage. Anti-PGP 9.5 indicated neurons. The yellow arrows indicate apoptotic neurons. Scale bar: 20 μm; E: Statistical analysis of the relative protein expression of Bcl-2, Bax, and cleaved caspase 3; F: Statistical analysis of the percentage of apoptosis using cleaved caspase 3 immunostaining; G: Statistical analysis of the percentage of apoptosis using terminal-deoxynucleotidyl transferase mediated nick end labeling immunostaining. Data are expressed as mean ± SD; Student’s t-test was performed, n = 6 for each group. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. DAPI: 4’,6-diamidino-2-phenylindole; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; TUNEL: Terminal-deoxynucleotidyl transferase mediated nick end labeling.

Immunofluorescence and TUNEL were conducted to quantify cell apoptosis. The percentage of apoptotic primary neurons was analyzed using triple staining with anti-PGP 9.5/anti-cleaved caspase-3/DAPI or anti-PGP 9.5/TUNEL/DAPI. Notably, the HuD autoantibody-treated group demonstrated significantly increased cleaved caspase-3 protein expression and enhanced TUNEL fluorescence signals compared with the control group (Figure 2C, D, F, and G). Incubation with HuD autoantibodies induced apoptosis in primary neuronal cells, altered their morphology, and increased the expression of apoptotic proteins and nuclear DNA fragmentation.

HuD protein counteracted the antibody to alleviate apoptosis of SH-SY5Y cells

To further elucidate the role of the HuD protein in neuronal apoptosis, the human-derived neuroblastoma cell line SH-SY5Y was utilized, and lentiviral-mediated overexpression of ELAVL4 was performed to enhance HuD protein expression. Compared with the control group, incubation with the HuD autoantibody led to increased levels of cleaved caspase 3 and Bax, decreased Bcl-2 expression, and a reduced Bcl-2/Bax ratio. These findings were consistent with those observed in primary neuronal cells. The effects of the HuD autoantibody were antagonized by lentiviral transfection, leading to elevated HuD protein expression. Compared with the negative and blank controls, the levels of cleaved caspase 3 and Bax expression were markedly reduced, whereas Bcl-2 expression was relatively increased, resulting in an elevated Bcl-2/Bax ratio in the group with HuD upregulation (Figure 3A).

Figure 3 HuD protein counteracted the anti-HuD autoantibody to alleviate apoptosis in SH-SY5Y cells. A: Western blot analysis showing the effect of HuD plasmid transfection in counteracting HuD autoantibody-induced expression of apoptotic proteins in SH-SY5Y cells ; B: Terminal-deoxynucleotidyl transferase mediated nick end labeling showing the effect of HuD plasmid transfection antagonizing the HuD autoantibody on nuclei cleavage in SH-SY5Y. Neurons are indicated by PGP 9.5; C: Immunofluorescence showing the effect of HuD plasmid transfection in counteracting HuD autoantibody-induced cleaved caspase 3 expression in SH-SY5Y cells. Neurons are indicated by PGP 9.5; D: Statistical analysis of the mean fluorescence intensity per cell using terminal-deoxynucleotidyl transferase mediated nick end labeling immunostaining; E: Statistical analysis of the mean fluorescence intensity per cell using cleaved caspase 3 immunostaining. Data are expressed as mean ± SD. One-way and two-way analysis of variance with Tukey’s multiple comparison test were performed, and n = 3 for each group. Scale bar: 50 μm. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001. DAPI: 4’,6-diamidino-2-phenylindole; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; TUNEL: Terminal-deoxynucleotidyl transferase mediated nick end labeling.

In the TUNEL assay the fluorescent signal in the cell group treated with the HuD autoantibody was stronger than that in the blank control group, corroborating the results obtained from primary neuronal cells. Conversely, when the HuD autoantibody was antagonized by lentiviral transfection to increase HuD protein expression, the TUNEL fluorescent signal was significantly attenuated compared with the negative and blank control groups (Figure 3). In the immunofluorescence experiments, incubation with the HuD autoantibody led to an increased expression of cleaved caspase 3 protein compared with both the blank control group and the negative control group, corroborating the findings observed in primary neuronal cells.

Conversely, when the HuD autoantibody was antagonized by upregulating HuD protein expression, the expression of cleaved caspase 3 was significantly reduced relative to the negative and blank control groups (Figure 3). These results indicated that the HuD autoantibody group exhibited a higher degree of apoptosis compared with the negative and blank control groups. In contrast, the group with HuD upregulation demonstrated lower expression of proapoptotic proteins, higher expression of antiapoptotic proteins, diminished nuclear DNA fragmentation, and reduced levels of apoptosis in neuronal cells.

HuD autoantibody induced neuronal apoptosis in the IBS rat model

Immunofluorescent staining and TUNEL were conducted to examine the effect of the HuD autoantibody on neuronal apoptosis in the ileum or colon of rats. Given the technical challenges associated with TUNEL staining on whole-mount sections, paraffin-embedded sections were utilized for TUNEL analysis. Triple staining with anti-PGP 9.5/TUNEL/DAPI on paraffin sections of rat ileo-colons revealed a relatively modest increase in the intensity of TUNEL fluorescent signals and a significantly elevated apoptotic rate in the HuD autoantibody group compared with the negative control and blank control groups (Figure 4A and B). Nevertheless, no significant difference was observed in neuronal apoptosis between the ileum and colon within the HuD autoantibody group (Figure 4A and B).

Figure 4 Anti-HuD autoantibody induced neuronal apoptosis in an irritable bowel syndrome rat model. A: Terminal-deoxynucleotidyl transferase mediated nick end labeling of neurons in the ileal plexus of the three groups of rats with neurons indicated by PGP 9.5; B: Cleaved caspase 3 staining of neurons in the ileal plexus of the three groups of rats with enteric neurons indicated by PGP 9.5; C: Percentage of apoptotic neurons by terminal-deoxynucleotidyl transferase mediated nick end labeling; D: Percentage of apoptotic neurons by cleaved caspase 3 staining. The yellow arrows indicate apoptotic enteric neurons. Scale bar: 20 μm. Data are expressed as mean ± SD. C and D were analyzed by two-way analysis of variance with Tukey’s multiple comparison test, and n = 3 for each group. ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001. DAPI: 4’,6-diamidino-2-phenylindole; TUNEL: Terminal-deoxynucleotidyl transferase mediated nick end labeling.

Subsequently, the myenteric plexus and submucosal plexus were isolated from the rat ileum, and triple staining with anti-PGP 9.5/anti-cleaved caspase 3/DAPI was performed on whole-mount sections to assess the level of apoptosis. The HuD autoantibody group exhibited a modest increase in the expression of cleaved caspase 3 and a significant increase in the apoptotic rate compared with the negative and blank control groups (Figure 4C and D). However, no significant difference was detected in the percentage of neuronal apoptosis between the myenteric plexus and submucosal plexus in the rat ileum (Figure 4C and D).

PKC agonist reduced neuronal apoptosis in vitro and in vivo

The addition of recombinant HuD protein to neutralize HuD autoantibodies led to a slight reduction in cleaved caspase 3 and Bax expression, a modest increase in Bcl-2 expression, and a significant increase in the Bcl-2/Bax ratio compared with the negative control group (Figure 5A). Treatment with the PKC agonist, 5-HT₄ receptor agonist and antagonist, and immunoglobulin resulted in reduced cleaved caspase-3 and Bax expression as well as increased Bcl-2 expression. Notably, only the groups treated with the PKC agonist and immunoglobulin showed significant differences from the blank control group, demonstrating a markedly elevated Bcl-2/Bax ratio (Figure 5B).

Figure 5 Effect of intervention in primary enteric neurons and the irritable bowel syndrome rate model. A: Effect of neutralizing antibodies by recombinant HuD protein on enteric neurons; B: Effect of protein kinase C agonist, Bryostatin1, 5-hydroxytryptamine agonist and antagonist, and immunoglobulin on enteric neurons; C: PGP 9.5/terminal-deoxynucleotidyl transferase mediated nick end labeling/4’,6-diamidino-2-phenylindole staining of neurons in ileal paraffin sections from the protein kinase C agonist group, the intravenous immunoglobulin group, and the anti-HuD autoantibody group; n = 3 for each group. Scale bar: 20 μm. Data are expressed as mean ± SD. One-way and two-way analysis of variance with Tukey’s multiple comparison test were performed. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IVIG: Immunoglobulin; PKC: Protein kinase C; TUNEL: Terminal-deoxynucleotidyl transferase mediated nick end labeling; DAPI: 4’,6-diamidino-2-phenylindole.

Based on the in vitro cell experiments, the PKC agonist and immunoglobulin were administered in the rat model to evaluate their effects on the apoptosis rate of ileal neurons. The findings indicated that the injection of the PKC agonist significantly decreased the apoptosis rate of ileal neurons in rats. However, the immunoglobulin-treated group exhibited only a marginal improvement that was not statistically significant (Figure 5C).

HuD autoantibody induced neuronal apoptosis associated with the SATB1-PI3K-AKT pathway

To explore the potential role of the HuD protein, we determined the RNA expression levels of downstream proteins regulated by HuD using qRT-PCR. RNA was reverse transcribed into cDNA from samples collected from the ileum and colon of rats. The results indicated that the expression levels of SATB1, GAP-43, NGF, and BDNF were significantly reduced, whereas no significant differences were observed in the expression levels of neuroserpin and neurotrophic factor-3 (Figure 6). Additionally, western blot analysis and confocal laser immunofluorescence were performed on rat specimens. The expression levels of SATB1 and HuD were significantly higher in the PKC agonist group compared with those in the HuD autoantibody, blank control, and negative control groups (Figure 7A). Confocal laser immunofluorescence further demonstrated that the HuD protein and SATB1 protein were colocalized in the nuclei of intestinal neurons with high expression levels in the PKC agonist (Figure 7B). These results highlight a close association between SATB1 and HuD protein in enteric neurons.

Figure 6 mRNA expression of the downstream targets of HuD in rat ileum and colon. A: Special AT-rich sequence-binding protein 1; B: Nerve growth factor; C: Brain-derived neurotrophic factor; D: Neurotrophic factor-3; E: Growth-associated protein-43; F: Neuroserpin. Data are expressed as mean ± SD. One-way analysis of variance with Tukey’s multiple comparison test was performed and n = 3. ^aP < 0.05, ^bP < 0.01, and ^dP < 0.0001. SATB1: Special AT-rich sequence-binding protein 1; NGF: Nerve growth factor; BDNF: Brain-derived neurotrophic factor; NT-3: Neurotrophic factor-3; GAP-43: Growth-associated protein-43.

Figure 7 Anti-HuD autoantibody induced neuronal apoptosis through the special AT-rich sequence-binding protein 1-phosphatidylinositol 3-kinase-protein kinase B pathway. A: Western blot showing the elevated expression of HuD and special AT-rich sequence-binding protein 1 (SATB1) in the Bryostatin1 group (n = 3); B: Confocal immunofluorescence of HuD and SATB1 in rat ileal neurons (n = 3); C: Western blot showing HuD and SATB1 upregulation attenuated apoptosis through the phosphatidylinositol 3-kinase-protein kinase B pathway; D: Confocal immunofluorescence of HuD and SATB1 in SH-SY5Y neurons; E: Western blot showing that SATB1 was necessary for HuD autoantibody-induced apoptosis within the phosphatidylinositol 3-kinase-protein kinase B pathway. Data are expressed as mean ± SD. Scale bar: 30 μm. Two-way analysis of variance with Tukey’s multiple comparison test was performed. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001. DAPI: 4’,6-diamidino-2-phenylindole; SATB1: Special AT-rich sequence-binding protein 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; p-AKT: Phosphorylated protein kinase B.

The SH-SY5Y cell line was utilized to investigate the roles of HuD and SATB1 in the PI3K-AKT signaling pathway and in the regulation of apoptosis. Stable cell lines with high expression of HuD and SATB1 were successfully constructed. The findings demonstrated that HuD could enhance the expression of SATB1 protein. Furthermore, overexpression of HuD alone or co-overexpression of both HuD and SATB1 resulted in increased phosphorylation of AKT (p-AKT/AKT), reduced expression of proapoptotic proteins, cleaved caspase 9, Bax, cleaved caspase 3, and elevated expression of the antiapoptotic protein Bcl-2. Conversely, treatment with PI3K inhibitors abrogated these effects, leading to decreased p-AKT/AKT levels, upregulation of proapoptotic proteins, cleaved caspase 9, Bax, cleaved caspase 3, and downregulation of Bcl-2 expression (Figure 7C). Additionally, colocalization of HuD and SATB1 proteins was observed in SH-SY5Y cells (Figure 7D). These results suggest that elevated expression of HuD and SATB1 inhibits neuronal apoptosis. Moreover, PI3K inhibitors may impair the survival-promoting functions of HuD and SATB1, thereby inducing neuronal apoptosis.

siRNA was used to knock down SATB1 in SY-SY5Y cells, and SH-SY5Y cells with SATB1 overexpression were treated with the HuD autoantibody. The SATB1 knockdown group exhibited a proapoptotic profile characterized by the significant elevation of cleaved caspase 9, Bax, and cleaved caspase 3 alongside downregulation of antiapoptotic indicators Bcl-2 and p-AKT/AKT, recapitulating the proapoptotic phenotype observed following HuD autoantibody exposure. Conversely, the SATB1 overexpression group showed an opposite trend for these apoptotic markers (Figure 7E). This functional validation strongly suggested that SATB1 was critically involved in HuD autoantibody-induced apoptosis and that restoring SATB1 expression could effectively counteract the proapoptotic signals downstream of HuD inhibition. Collectively, these findings indicated that the PI3K-AKT axis is strongly associated with enteric neuron survival with HuD and SATB1 serving as key upstream regulators.

DISCUSSION

This study provided novel insights into the role of the HuD autoantibody in the pathogenesis of IBS. We successfully established a rat model mimicking IBS via the passive transfer of a commercially purified HuD autoantibody. The fecal particle count and fecal water content were increased, reproducing clinical features of IBS-D. The immunofluorescent analyses revealed an elevated level of neuronal apoptosis in both the IBS rat model and primary enteric neurons isolated from healthy rats. Furthermore, we demonstrated that the SATB1-PI3K-AKT signaling axis may play a role in IBS. The in vivo intervention experiment indicated that a PKC agonist and immunoglobulin therapy may be therapeutic candidates.

The passive transfer model is an established approach for modeling autoimmune diseases[22]. Recently, this model has been applied to animal studies on autoimmune neuropathies, including autoimmune encephalitis[23], Tourette syndrome, and small fiber neuropathy[24]. Specific antibodies derived from patients or animal models or purified IgG extracted from serum can directly engage the target antigens to replicate the pathophysiological mechanisms associated with the disease. Successful modeling indicates that the serum antibodies play a role in the disease’s pathogenesis[23]. Administration of a HuD autoantibody induced intestinal motility acceleration and visceral hypersensitivity in the passive transfer rat model of IBS. This in vivo model can be used to elucidate immune-mediated mechanisms underlying IBS and to identify possible therapeutic targets.

A critical consideration in modeling autoimmune diseases is the source of the autoantibodies. Patient-derived materials, such as sera or purified IgG, inherently have substantial inter-individual variability in antibody titers, antigen specificity, epitope recognition, and immunoglobulin subclasses. This heterogeneity and the limited availability of sufficient volumes of well-characterized, high-titer patient samples[25] pose significant challenges for achieving experimental reproducibility, dose standardization, and clear mechanistic interpretation in animal models. Therefore, we strategically employed a commercially available monoclonal antibody. This approach guaranteed experimental consistency, defined antigen specificity, and reproducibility in the in vivo and in vitro assays.

Multiple autoantibodies have been identified in patients with IBS, including gluten-related antibodies[26], a nondescript ribonucleoprotein, and small nuclear ribonucleoprotien polypeptide A[12]. Therefore, the establishment of the IBS passive transfer model was crucial. However, it is noteworthy that no significant weight loss was observed during the modeling period, suggesting that the IBS model rats did not exhibit reduced food intake, abnormal behavior, or other manifestations. Consequently, this model may not adequately capture psychological comorbidities (e.g., anxiety and depression) frequently observed in patients with IBS.

Meanwhile, our experimental results corroborated previous findings, demonstrating that the HuD autoantibody mimicked certain IBS symptoms. Specifically, the study revealed that the HuD autoantibody could excite primary afferent nerves to induce peripheral sensitization, thereby leading to visceral hypersensitivity in rats[17]. Still, the underlying mechanism of the HuD autoantibody in IBS remained poorly understood. While previous work demonstrated the acute effects of autoantibodies on synaptic dynamics and the excitatory-inhibitory balance[27,28], our study extended these findings by focusing on the downstream consequences.

Our team previously demonstrated that AENA derived from the serum of patients with IBS induced apoptosis of myenteric neurons in guinea pigs[13,15,29]. The current study further revealed that neuronal apoptosis triggered by the HuD autoantibody specifically affected neurons within the enteric plexus of the ENS in the rat model with no significant differences observed across various gut layers or regions. The importance of the ENS in the pathogenesis of IBS-D was highlighted by a previous study[30], showing that heterotypic chronic and acute stress induced morphological alterations in enteric neurons, correlating with motility and secretion abnormalities.

Building upon this foundation, this study solidified the role of ENS dysfunction by identifying a specific, nonstress-related mechanism (i.e., autoantibody-mediated neuronal apoptosis) that directly drives the observed clinical phenotype. Together, these studies established ENS pathology as a common and crucial pathway through which diverse etiologies, including both stress and autoimmunity, converge to cause IBS-D. The ENS is often referred to as the little brain of the gut. It plays a critical role in regulating normal gastrointestinal physiology, including movement and secretion. The ENS is an autoimmune target in other diseases with gastrointestinal symptoms such as delayed colonic transit[31,32]. Our previous findings also showed that the prevalence of headaches and sleep disorders was significantly higher in patients positive for both anti-cerebral neuronal antibodies (known as ACNA) and AENA[29]. The autoimmune process targeting the central nervous system (CNS) may directly impact the ENS, reflecting the intricate interplay within the gut-brain axis. Given that IBS is classified as a disorder of gut-brain interaction, it is crucial to thoroughly investigate the role of the ENS using relevant IBS rat models.

HuD, an RNA-binding protein, plays a pivotal role in the pathogenesis of nervous system diseases[33]. As a member of the ELAVL/Hu protein family, HuD is specifically expressed in nerve cells and is crucial for neuronal differentiation, maturation, synaptic growth, and survival[34]. We previously identified HuD autoantibody as the most significantly altered IgG in patients with IBS and hypothesized that it could serve as a serum biomarker for IBS diagnosis[15]. HuD participates in myelin restoration in mouse models of multiple sclerosis through posttranscriptional regulation of gene expression[35]. Our study revealed a significant reduction in the mRNA levels of SATB1, GAP-43, NGF, and BDNF within ileocolonic specimens from the IBS rat model. Downregulation of these proteins critically involved in neuronal growth and survival strongly suggests a detrimental influence exerted by the HuD autoantibody on enteric neuronal integrity[36,37]. However, whether these proteins directly induce neuronal apoptosis and the underlying pathways involved remain unknown.

HuD autoantibodies can antagonize HuD protein during incubation, thereby reducing the stability of downstream HuD mRNA and rendering it more susceptible to degradation or reduced expression. Our study demonstrated that incubation with HuD autoantibodies induced apoptosis in SH-SY5Y neuroblastoma cells. Apoptosis observed in primary enteric neurons following exposure to the HuD autoantibody closely resembled human neuroblastoma cells. Currently, there is no established enteric neuron cell line. Therefore, we used the SH-SY5Y cell line to investigate the mechanism of apoptosis induced by the HuD autoantibody. Our finding aligned with a previous neuroblastoma study that revealed that HuD promoted autophagy by inhibiting mTORC1 activity and upregulating ARL6IP1, thereby enhancing cell survival through negative regulation of apoptosis[38].

A recently published study further indicated that HuD levels increased under oxidative stress conditions, contributing to neuromuscular junction dysfunction and acting downstream of FUS mutations in sporadic and familial amyotrophic lateral sclerosis[39]. Previous studies highlighted the role of HuD in regulating autophagy, but its precise involvement in other programmed cell death pathways, particularly apoptosis, remained largely unexplored. Our current study addressed this gap by demonstrating a direct molecular link between HuD autoantibodies and enteric neuronal apoptosis, thereby extending the known regulatory functions of HuD beyond autophagy.

Upregulation of HuD effectively counteracted the effects of the HuD autoantibody and mitigated antibody-induced neuronal apoptosis. This finding underscored the critical role of the HuD protein in neuronal development, and its absence may serve as a key factor contributing to neuronal apoptosis, aligning with previous literature[34]. While the critical role of the HuD protein has been extensively characterized in the CNS, its function within the ENS has remained largely elusive. Consequently, future strategies could involve exogenous supplementation of HuD protein, enhancing HuD protein expression, or neutralizing antibodies through immunoglobulin injection.

Our results demonstrated that treatment with recombinant HuD protein, a PKC agonist, and immunoglobulin significantly reduced primary enteric neuron apoptosis, thereby providing neuroprotection. The PKC agonist and immunoglobulin were utilized in the animal experiments to investigate potential therapeutic interventions for reducing ileal neuron apoptosis, revealing that the PKC agonist reduced enteric neuron apoptosis and immunoglobulin exhibited protective effects without statistical significance. Changes in the number of enteric neurons in the ENS correlated with alterations in defecation and intestinal transit function. Our findings provided novel insights into the pathogenic role of HuD in the gut and expanded the known functional scope of HuD from the CNS to the ENS.

According to the literature, intravenous immunoglobulin is commonly used as a first-line therapy in the treatment of autoimmune neurological diseases such as Guillain-Barré syndrome, myasthenia gravis, and demyelinating diseases[40]. Animal modeling experiments have demonstrated that intravenous immunoglobulin can mitigate neurodegeneration in animal models of neuromyelitis[41]. However, the limited in vivo neuroprotective effect of immunoglobulin in our study may be attributed to the small sample size, which represents a primary limitation of this work. Other contributing factors may include a potential mismatch between the immunoglobulin preparations and specific antibody types, suboptimal dosage, or frequency of administration. To further validate our findings and address the concerns regarding statistical power, we plan to repeat these experiments with a larger cohort.

Currently, PKC agonists and inhibitors have been extensively applied in clinical studies for various conditions, including cancers, infectious diseases, cardiovascular diseases, and neurological disorders, and have shown neuroprotective potential[42]. Notably, clinical trials are actively exploring the application of PKC agonists in the treatment of Alzheimer’s disease with significant improvements in patients’ cognitive function[43,44]. The potential of PKC agonists in treating neuropathic diseases appears promising. Furthermore, studies in animal models of Alzheimer’s disease revealed that PKC agonists reversed neuronal synaptic loss and promoted synaptic maturation[45,46]. Mechanistically, PKC agonists upregulate the expression of HuD and BDNF to facilitate synapse formation and to exert antiapoptotic and pro-growth effects[46,47]. Our findings suggest that PKC agonists mitigate enteric neuron apoptosis, thereby offering a proof-of-concept pharmacological candidate for alleviating IBS symptoms.

Rat brain neurons can internalize HuD autoantibodies to directly induce neuronal death[48]. Although direct evidence that HuD autoantibodies enter enteric neurons requires further confirmation, mounting evidence shows that autoantibodies reach patients’ intracellular targets and elicit pathogenic functional consequences[49]. We further revealed a correlation between HuD and SATB1 in the ENS, and the PKC agonist enhanced the expression of both HuD and SATB1 in intestinal neurons. Previous studies indicate that HuD can upregulate SATB1 expression in CNS neurons, and SATB1 as a transcription factor reciprocally promotes HuD expression through positive feedback regulation[50]. Both HuD and SATB1 may contribute to the proliferation and development of SH-SY5Y cells while inhibiting apoptosis. Previous studies highlighted the critical roles of HuD and SATB1 in the growth and development of various neurons in the CNS, suggesting that deletion or reduced expression of these proteins may underlie the pathogenesis of multiple neurological disorders[51]. However, the functions and mechanisms of these two proteins in the peripheral nervous system are poorly understood.

A PI3K inhibitor can abrogate the growth-promoting and apoptosis-inhibiting effects mediated by HuD and SATB1, thereby inducing neuronal apoptosis, similar to the effect of HuD autoantibodies. Consequently, this study indicated that HuD autoantibodies might interfere with the HuD-PI3K-AKT signaling pathway, leading to downstream alterations that promote apoptosis in enteric neurons. The PI3K-AKT signaling pathway is a canonical pathway regulating cell survival, suppressing apoptosis, and playing a pivotal role in the development and maintenance of nerve cells[52]. Dysregulation of signaling molecules within this pathway is a critical factor contributing to the initiation and progression of neuroblastoma cells[53]. Current research on this pathway predominantly focuses on the CNS, but emerging studies have highlighted its crucial role in the peripheral nervous system[54]. We provided evidence supporting a previously unrecognized molecular cascade in the ENS. HuD may play a critical role in IBS as an upstream regulator that links to a key transcription factor: SATB1. The association of the HuD autoantibody and a major survival pathway provides a novel insight for understanding enteric neuronal integrity.

Limitations

We used a small number of animals in this study because of the exploratory and proof-of-concept focus[55]. Further research with larger cohorts is warranted to validate our findings, especially for the intervention experiment of the PKC agonist and immunoglobulin. The model of IBS induced by the HuD autoantibody was an acute model for proof-of-concept and did not fully mimic all IBS symptoms. The quantitative method used in immunostaining, particularly for primary enteric neurons, relied on counting triple-stained cells by a second observer, which may introduce variability.

We recognize that utilizing the SH-SY5Y cell line to determine mechanism represents a necessary trade-off between experimental feasibility and physiological fidelity. Given the current constraints, the absence of a stable, immortalized enteric neuronal cell line, and the limited accessibility of primary enteric neurons, the SH-SY5Y cells provided a robust and scalable system for studying the molecular effects of HuD autoantibodies. However, as a non-enteric, transformed cell line, SH-SY5Y cells may not fully recapitulate the specific molecular profile or complex signaling pathways of mature enteric neurons. Therefore, future studies employing primary ENS cultures or induced pluripotent stem cell-derived enteric neurons are warranted to validate and extend these findings.

The evidence of direct binding between HuD and SATB1 mRNA is lacking in this study. Binding assays such as RNA immunoprecipitation and cross-linking and immunoprecipitation in primary enteric neurons are needed. The therapeutic efficacy of PKC agonists and the role of SATB1-PI3K-AKT signaling also require further exploration using a larger cohort and meticulously designed experiments. An intestine-specific HuD knockdown model to validate the role of HuD in the ENS and SATB1-PI3K-AKT signaling and their correlation with IBS is warranted.

CONCLUSION

We established a rat model of IBS in which injection of a HuD autoantibody significantly elevated the apoptosis rate of enteric neurons. We propose that these antibodies induce neuronal apoptosis by disrupting the binding of HuD to its downstream target mRNAs, leading to a reduction in the quantity and stability of key transcripts, such as SATB1 mRNA. Furthermore, SATB1 suppression-induced apoptosis may involve the PI3K-AKT signaling pathway. Collectively, these findings provide evidence for the mechanism of HuD autoantibody-induced IBS and suggest that pharmacological intervention with PKC agonists could serve as a promising therapeutic candidate.

ACKNOWLEDGEMENTS

We sincerely thank the Experimental Medicine Research Center for its instrumental support.