Electroacupuncture at ST36 protects against dextran sulfate sodium-induced ulcerative colitis in mice by enhancing sympathetic nerve innervation in colonic tissue

Abstract

Ulcerative colitis (UC), characterized by diffuse colonic inflammation and enteric nervous system (ENS) alterations, remains a therapeutic challenge. While the role of the parasympathetic nervous system in intestinal inflammation has been extensively documented, understanding of sympathetic nervous system (SNS) function in UC pathogenesis remains limited. Electroacupuncture (EA) at ST36 (Zusanli) is a widely used alternative therapy for UC, yet its regulatory effects on the ENS—particularly the SNS—and the underlying mechanisms remain unclear. Here, we established a 2.5% dextran sulfate sodium (DSS)-induced murine UC model and validated disease phenotypes via histological assessment. Using a non-anesthetic EA intervention protocol mimicking the clinical conditions, we found that EA at ST36 attenuated local inflammatory progression in DSS-induced UC mice and mitigated ENS degeneration. Critically, our results demonstrated that EA at ST36 specifically reversed SNS reduction (tyrosine hydroxylase-positive, TH⁺ neurons) in colonic tissue caused by intestinal inflammation. In conclusion, this study reveals that EA at ST36 restores colonic homeostasis through a sympathetic-dependent mechanism, providing novel insights and therapeutic strategies for the clinical management of UC.

Keywords

electroacupuncture / ST36 (Zusanli) / anti-inflammation / ulcerative colitis / sympathetic nerve systems

1 Introduction

Ulcerative colitis (UC) is a systemic, chronic digestive disease that is characterized by diffuse, chronic inflammation in mucosa located on rectal and colonic intestine [1,2]. Currently, various agents have been prescribed to treat UC but the curative effects are far from ideal due to their disadvantages and economic burden [3,4]. In recent years, accumulating evidence has demonstrated that electroacupuncture (EA) can alleviate both acute and chronic experimental colitis [5,6]. Nevertheless, the specific therapeutic effects of EA on colitis and its underlying mechanisms remain incompletely elucidated, highlighting the necessity for further investigations to address these knowledge gaps.

The precise pathogenesis of UC remains complex, but excessive inflammation is widely recognized as a key driver of disease progression [1,7]. Consequently, controlling elevated inflammatory responses is critical for mitigating gut tissue damage, particularly in UC [8]. The enteric nervous system (ENS) plays a pivotal role in maintaining this inflammatory balance [9,10]: it exerts regulatory effects on inflammation to ensure responses are sufficient for tissue defense while preventing excessive damage. The sympathetic nervous system (SNS) exhibits dual pro-inflammatory and anti-inflammatory properties [11,12]. Its neurotransmitters, such as norepinephrine and adenosine, can elicit strikingly opposing effects depending on their concentration. Additionally, peptidergic neurons—characterized by neurotransmitters including substance P (SP) and nerve growth factors—have emerged as major focuses in neuroimmunomodulation research related to UC [13,14]. For these reasons, strategies targeting the intestinal SNS, including EA, have been employed in both clinical practice and animal models.

Previous studies have established the vagus nerve’s (parasympathetic nerve) anti-inflammatory role in colitis [15]. However, substantial evidence supports sympathetic anti-inflammatory functions, demonstrating that the SNS exerts anti-inflammatory effects in UC, with evidence showing that loss of sympathetic nerve activity contributes to disease pathogenesis [12,16]. One plausible explanation for such differential regulatory effects of the SNS may lie in the tissue-specific actions of different organs as well as the specific characteristics under distinct disease conditions, such as UC. Therefore, exploring these differences is conducive to gaining novel insights into the role of the sympathetic nerve in the pathogenesis of UC. Furthermore, EA stimulation at ST36 (Zusanli) activates sympathetic pathways in a somatotopic and intensity-dependent manner, and can dampen inflammatory responses by triggering the vagal-adrenal axis [17,18]. These findings suggest that EA may exert protective effects against UC pathogenesis through sympathetic-mediated anti-inflammatory mechanisms.

In the present study, we aimed to clarify the effects of different frequencies of EA on acute colitis induced by dextran sulfate sodium (DSS) and further confirm the protective effects of EA at ST36 through histological analysis. Moreover, we explored whether sympathetic degeneration is involved in the EA-induced amelioration of DSS-induced colitis using whole-tissue immunolabeling and optical clearing techniques targeting the ENS. This study demonstrated the protective effects of EA at ST36 with both lower and higher intensities in the DSS-induced UC model and identified the sympathetic protective role in this inflammatory model.

2 Materials and methods

2.1 Animals

Thirty-two male C57BL/6 mice (6–8 weeks old) were purchased from Jihui Animal Biotechnology Co., Ltd. (Certification No. 20270012020873). The mice were housed in the Animal Care Facility of Shanghai Jiao Tong University School of Medicine (Shanghai, China), and all experimental procedures were conducted in strict accordance with the Guidelines for the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research. Briefly, the mice were maintained under standard housing conditions (temperature: 22 °C; light-dark cycle: 12 h/12 h) with free access to standard chow and drinking water. All animals were allowed to acclimate to the environment for 2 weeks prior to the initiation of the formal experiment.

2.2 Chemical agents and antibodies

The following antibodies were purchased, with their respective catalog numbers provided: anti-mouse TCRβ (BV510, BioLegend); anti-mouse TCRβ (BV605, BioLegend); anti-mouse CD45.2 (AF700, Thermo Fisher Scientific); anti-mouse KLRG1 (APC, BioLegend); Fixable Viability Stain (AF700, BD Biosciences); anti-mouse CD8α (AF647, BioLegend); anti-mouse CD4 (PE, BioLegend); anti-tyrosine hydroxylase antibody (Sigma-Aldrich, Cat#: AB152); and purified anti-SNAP-25 antibody (BioLegend, Cat#: 836304). All these assays were performed in accordance with the manufacturers’ protocols. Other information of reagents was shown in Table S1.

2.3 DSS-induced mouse model and evaluation

For the induction of DSS-induced colitis, DSS (MP Biomedicals, Cat#: 160110) was dissolved in double-distilled water (ddH₂O) to a final concentration of 2.5%. The solution was filtered through a 0.22-μm membrane filter and provided to mice as drinking water ad libitum. The body weight of each mouse was monitored daily both before and after DSS administration.

To assess the pathogenesis of UC, the disease activity index (DAI) was recorded for each mouse and scored according to established criteria [19]. The DAI was calculated by combining and averaging scores for three parameters: weight loss (0 = none; 1 = < 5%; 2 = 5%–10%; 3 = 10%–15%; 4 = > 15%), stool consistency (0 = normal; 2 = loose; 4 = diarrhea), and rectal bleeding (0 = none; 2 = mild bleeding; 3 = bleeding for > 1 day; 4 = bleeding for > 2 days). This scoring system effectively reflects the progression of UC and the general health status of the mice.

2.4 Experimental protocols

All mice were randomly assigned to four groups (n = 8 per group) and all interventions were performed without anaesthetization to mimic the condition in clinical practice. Previous studies have shown that the frequencies of either 100 Hz [20] or 10 Hz at the ST36 acupoint could ameliorate colonic inflammation in mice and preferentially the 10 Hz is selected, which causes potential less harmful responses to the mice. Moreover, the other parameters (intensity, time) of EA and the location of bilateral ST36 (2 mm below the fibular head) were selected based on previous studies that published [20]. The mice were grouped as the following: Group A (Control, Con): mice received saline treatment and sham EA (Sham-EA, i.e., acupuncture without electrical stimulation);Group B (DSS + Sham-EA): Mice were administered 2.5% DSS solution and subjected to sham EA; Group C (DSS + Low-intensity EA, EAL): Mice with DSS-induced colitis were treated with low-intensity EA (0.5 mA, 10 Hz, 15 min); Group D (DSS + High-intensity EA, EAH): Mice with DSS-induced colitis were treated with high-intensity EA (1 mA, 10 Hz, 15 min).

2.5 Histological analysis

Colonic tissues from six mice in each group were harvested, fixed, and embedded in paraffin, with sections prepared at three different angles: transverse, longitudinal, and vertical. To confirm histological findings, colonic segments were presented as sections in a “duodenal roll” perspective. Histological scoring was performed according to the criteria for DSS-induced colitis described previously [21], with scores ranging from 0 to 4.

2.6 EA intervention

The EA intervention was performed using a Huatuo SDZ-IIB nerve and muscle stimulator (Cat#: 20172270710, Suzhou Medical Appliance Factory), which was purchased and used for this study. Based on a previously published paper [17], the bilateral ST36 acupoints were selected; these acupoints are located on the posterolateral aspect of the knees, approximately 2 mm inferior to the fibular head. For groups receiving actual EA treatment, pairs of stainless-steel needles (0.16 mm × 7 mm) were inserted to a depth of 2–3 mm into the bilateral hindlimb acupoints (Fig. S1B).

2.7 Hematoxylin-eosin staining

On Day 7, the mice were euthanized. Colonic tissues from 6 mice per group were fixed in 4% paraformaldehyde (PFA), routinely sectioned into 5 μm slices, and stained with hematoxylin and eosin (HE). The HE-stained sections were imaged under a light microscope at appropriate magnifications (5×, 10×, and 20×), and the images were subsequently analyzed.

2.8 Flow cytometry analysis of lamina propria lymphocytes

Colonic lamina propria mononuclear cells (LPMCs) from 3 mice per group were subjected to flow cytometry analysis. Briefly, after cell counting, LPMCs were incubated with the following fluorescently conjugated antibodies for 1 h at room temperature: anti-mouse F4/80-PE (BD Biosciences, New Jersey, USA), anti-mouse CD16/32-PerCP/Cy5.5 (BD Biosciences, New Jersey, USA), and anti-mouse CD206-Alexa Fluor 647 (BD Biosciences, New Jersey, USA). Cells were washed twice with phosphate-buffered saline (PBS) and then analyzed using a BD LSRFortessa X-20 flow cytometer. Data were processed with FlowJo v10 software.

2.9 Whole-tissue immunolabeling and optical clearing of ENS

Colonic tissues were harvested to assess the ENS in mice, nonhuman primates, and humans using advanced 3D imaging, as previously described [22]. Briefly, colonic tissues were placed in a 10-cm dish containing 10 mL of pre-cooled PBS. Mesentery and adipose tissue adhering to the intestinal wall were removed, and the tissues were rinsed with cold PBS. The colon was cut longitudinally, and 1-cm segments were excised. These tissue segments were placed in a 48-well plate, and 200 μL of 1% PFA was added; they were then incubated for 6 h with gentle rotation. The tissues were washed twice with PBS at room temperature (5 min per wash) before being blocked with 200 μL of M.O.M. (Mouse on Mouse) Blocking Reagent solution overnight at 4 °C. Subsequently, the tissues were washed three times with PBS at room temperature (5 min per wash), followed by incubation with 120 μL of blocking buffer containing the primary antibody overnight. All incubation steps were performed with gentle rotation and protected from light.

Next, the tissues were transferred to a 6-cm dish and washed with PBS three times (30 min per wash), then transferred to a new 48-well plate containing 120 μL of secondary antibody solution for overnight incubation, protected from light. Subsequently, the tissues were transferred to a 6-cm dish and washed with 3 mL of PBS overnight, followed by two additional washes with PBS (1 h per wash). The tissues were then blotted dry with paper towels, placed into an electroless plating tube, and incubated with 600 μL of RapiClear solution at room temperature for 40 min until completely transparent. Finally, the hydrated tissues (with the lumen facing upward) were placed on a cover glass, fixed using an iSpacer, and then imaged.

2.10 Statistical analysis

All data are expressed as the mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) was used to compare differences among the groups. A P value < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS 21.0 software (IBM, USA).

3 Results

3.1 EA at ST36 exerts protective effect in DSS-induced UC mice

Our research strategy for EA intervention is illustrated in Fig. S1A. The selection of acupoint ST36, mouse fixation, and EA intervention procedures are depicted in Fig. S1B–S1D. Although numerous studies have reported that EA at ST36 alleviates DSS-induced UC in mice, slight discrepancies exist among these findings, likely due to differences in stimulation intensity and duration. We therefore investigated whether EA at ST36 with low intensity (0.5 mA, 10 Hz, 15 min) and high intensity (1 mA, 10 Hz, 15 min) could ameliorate UC. Following EA intervention at ST36 using the aforementioned parameters, the weight loss in DSS-induced mice (Fig. 1A and 1B) were measured. Moreover, the DAI were measured and quantified (Fig. 1C) and colonic tissues from each group were harvested and their lengths were measured and quantified (Fig. 1D). After EA intervention at ST36 with the above parameters, both low-intensity EA (EAL; 0.5 mA, 10 Hz, 15 min) and high-intensity EA (EAH; 1 mA, 10 Hz, 15 min) alleviated histological deterioration. Specifically, the mucosal tissues in the EAL and EAH groups were intact, with epithelial cells arranged in an orderly manner and glands remaining intact.

3.2 EA at ST36 alleviates destruction of intestinal tissue morphology in DSS-induced UC mice

Next, we further confirmed that EA at ST36 alleviates the phenotypic characteristics of UC through histological analysis using HE staining. Given the complexity of UC pathogenesis and the potential influence of different slicing methods and angles, colonic tissues from DSS-induced mice were sectioned into rolled sections (Fig. 2A), transverse sections (Fig. 2B), vertical sections (Fig. 2C), and longitudinal sections (Fig. 2E). To further confirm our findings, colonic histological scores were measured, quantified, and statistical analyses were performed between Con and DSS, ESL, ESH independently (Fig. 2D). Taken together, these analyses revealed that DSS intervention induces local inflammation and structural damage to the colon in DSS-treated mice. However, EA administered at the ST36 acupoint exerts a protective effect in mice with DSS-induced UC, and notably, it can alleviate intestinal inflammatory damage.

3.3 ES at ST36 exerts no significant regulatory effect on the numbers of immune cells in DSS-induced mice

Alterations in inflammatory cells and immune regulation contribute significantly to the pathological changes in DSS-induced mice. We therefore further investigated the effects of EA at ST36 on immune cells using flow cytometry (FACS, BD FACSVerse, USA) for analysis. As shown in Fig. S2, there was a significant increase in the number of intestinal immune-related inflammatory cells in DSS-induced mice, including neutrophils (Fig. S2A), monocytes (Fig. S2B and S2C), macrophages (Fig. S2D), and eosinophils (Fig. S2G). However, EA at the ST36 acupoint did not exert a significant regulatory effect on immune cell counts in DSS-induced mice. Specifically, this intervention fails to produce any notable changes in the numbers of immune cells within the context of DSS-induced conditions.

3.4 EA at ST36 exerts protective effect in DSS by increasing the density of TH positive neurons in colon

Numerous studies have demonstrated that sympathetic nerves in the ENS play key roles in regulating the development of UC. Conversely, degeneration of sympathetic nerves in the ENS exacerbates the inflammatory process in the ENS and promotes the progression of UC. Furthermore, EA at ST36 has been reported to exert protective effects in inflammatory mouse models. In the present study, EA was administered at ST36, and 5 cm colonic segments were collected. Whole-tissue immunolabeling and optical clearing of the ENS were performed as previously reported, with results presented in 3D-projection images. The results showed that DSS intervention induced structural and histological damage to both sympathetic nerves (tyrosine hydroxylase-positive, TH⁺) and total nerves (SNAP25⁺) in colonic tissues (Fig. 3A), which mimics the pathological features of clinical UC. Notably, the reduction in TH⁺ sympathetic nerves was more prominent compared to that of SNAP25⁺ non-sympathetic nerves in DSS-treated mice, indicating that sympathetic nerves play a critical role in UC pathogenesis. Moreover, EA at ST36 significantly increased the density of TH⁺ sympathetic nerves relative to SNAP25⁺ total nerves (Fig. 3B). These results were confirmed by whole-tissue immunolabeling of 300 μm sections (Fig. S3) and 3D animations of 100 μm segments (Fig. S4). Taken together, these findings indicate that EA at ST36 primarily alleviates the deterioration of sympathetic nerves in the ENS, thereby ameliorating the development of UC.

4 Discussion

Our findings demonstrated that 2.5% DSS induces pathological morphological changes in the colon, as well as increases the DAI. The DAI, which integrates the severity of weight loss, stool consistency, and rectal bleeding, reflects the overall condition of the mice. Additionally, optical clearing techniques were used to investigate sympathetic nerves in the ENS in the DSS-induced UC model. Furthermore, EA at ST36 was administered to intervene in the DSS-induced mouse model. Mechanistically, EA at ST36 alleviates the degeneration of sympathetic nerves in DSS-induced UC mice and mitigates the pathological progression of DSS-induced UC, primarily by attenuating sympathetic nerve degeneration in the ENS. These findings indicate that EA at ST36 primarily alleviates the deterioration of sympathetic nerves in the ENS, thereby ameliorating the development of UC.

Notably, the interplay between the autonomic nervous system and intestinal inflammation has long been a focus of research, with the parasympathetic nervous system—particularly the vagus nerve—having been extensively studied for its anti-inflammatory role in colitis. For instance, vagal stimulation has been shown to ameliorate colitis via SUMOylation-mediated pathways [15], and EA itself can drive the vagal-adrenal axis to exert anti-inflammatory effects, laying a clear neuroanatomical foundation for such interventions. These findings confirm the critical role of the parasympathetic branch in regulating intestinal inflammatory homeostasis, yet they also highlight a relative gap in understanding the SNS in this context. In contrast to the well-documented parasympathetic effects, reports on the SNS in colitis remain scarcer, but accumulating evidence supports its distinct anti-inflammatory function. Studies on liver metabolism have demonstrated that sympathetic nerves release NE to act on CD11b⁺F4/80⁺ macrophages, reducing TNF-α release and mitigating inflammation [23], while research on lung innate immunity further confirms that local sympathetic innervation can suppress inflammatory responses [24]. Extrapolating from these findings, our observation that DSS-induced colitis leads to a significant reduction in TH⁺ sympathetic nerves in the ENS strongly suggests that intestinal SNS dysfunction contributes to inflammatory progression—and that restoring SNS integrity may represent a key therapeutic target.

Regarding the mechanism underlying SNS reduction in colitis, the aforementioned [23] study provides critical clues: macrophage-derived TNF, a key pro-inflammatory cytokine elevated in DSS-induced colitis, can directly induce sympathetic neuropathy and reduce sympathetic nerve density in the liver. Given that DSS challenge in our model similarly upregulates intestinal inflammatory cytokines, it is highly plausible that a parallel mechanism operates in the colon—i.e., inflammation-driven, cytokine-mediated damage to sympathetic axons leads to sympathetic degeneration in the ENS. Our finding that EA at ST36 reverses this TH⁺ nerve reduction further implies that EA may interrupt this vicious cycle, potentially by modulating cytokine levels or directly protecting sympathetic nerve integrity.

Comparing the two autonomic branches, the vagal-adrenal axis primarily exerts systemic anti-inflammatory effects, while our data suggest the intestinal SNS mediates more localized regulation of colonic tissue inflammation. This complementary role highlights the complexity of neuroimmune modulation in UC and positions EA at ST36 as a versatile intervention that may engage multiple autonomic pathways—strengthening both parasympathetic and sympathetic anti-inflammatory defenses. Together, these insights not only fill the gap in understanding intestinal SNS function in colitis but also reinforce the clinical potential of EA by identifying its ability to target understudied sympathetic mechanisms.

Future studies will further explore the regulatory effects of EA at ST36 on parasympathetic nerves and other components of the ENS, aiming to clarify the neuroimmune mechanisms in UC mouse models. Systemic and diffuse inflammation contributes to the pathogenesis of UC, and currently marketed drugs are used to reduce the inflammatory process and alleviate disease progression. However, the economic burden and recurrent nature of UC necessitate the development of a cost-effective and convenient approach for UC management. EA has long been utilized as effective, simple, and convenient therapy in TCM [25–27]. Numerous studies have elucidated its mechanisms, including anti-inflammatory effects and neuroimmune interactions. A notable advantage of EA in UC treatment is its rapid and reliable analgesic effect [28], which takes effect within several minutes and persists for several days. Beyond its local effects on colonic tissues, EA reduces systemic and diffuse inflammation via the vagal-adrenal axis [17–18], thereby improving the overall health status of patients. EA at ST36, which is located on the Stomach Meridian of Foot-Yangming according to the meridian-collateral theory in TCM [6,29], has been demonstrated to significantly regulate GI functions, including modulation of the SNS. It has been reported that EA at ST36 reduces inflammation by activating the vagal-adrenal axis [17–18]. Additionally, studies have indicated that EA exerts regulatory effects on the stimulation and modulation of intestinal sympathetic nerves [30–31].

EA at ST36 showed no statistically significant difference in the number of immune cells in the enteritis animal model. This may be attributed to the potential anti-inflammatory effect of EA being primarily mediated by stimulating the function of inflammatory cells, with no obvious impact on immune cell counts [32–33]. Therefore, EA at ST36 exerted no significant effect on the number of immune cells. However, ongoing studies will further quantify intestinal immune and inflammatory cells by increasing the number of animals and optimizing the intervention timing and intensity, aiming to elaborate on the detailed regulatory mechanisms involving intestinal immune cells. Furthermore, these analyses demonstrated that neuroimmune interactions in UC and the gut microbiota are more complex than previously thought [34], and additional experimental evidence is required to clarify the anti-inflammatory effects of EA at ST36.

However, the present study has several limitations. First, we have not yet elucidated the detailed mechanism underlying the anti-inflammatory effect of EA in DSS-induced UC, particularly regarding its downstream targets, and we lack a parallel antagonist experimental group. Second, time-dependent intervention studies of EA at ST36 in the DSS-induced colitis have not been conducted. Therefore, additional studies are required to investigate the underlying mechanisms and characteristics of EA in TCM. Ongoing research will further explore sympathetic nerve-mediated anti-inflammatory effects in other immunological diseases.

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