Therapeutic Efficacy of Acupuncture Targeting GV-1 Acupoint on Diarrhea Induced by Sennae Folium in Rabbits

Houkang Ding1, Xiao Wang1, Mingchao Liu1, Kun Li2, Ahmad R. Alhimaidi3, Ramzi A. Amran3, Aituan Ma1* and Dengshan Shiau4*

1Department of Traditional Chinese Veterinary Medicine, College of Veterinary Medicine, Hebei Agricultural University, Baoding 071001, Hebei, China

2Institute of Traditional Chinese Veterinary Medicine and MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China

3Department of Zoology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia.

4Department of Graduate Studies, Chi University, Reddick, Florida 32686, USA

ABSTRACT

Diarrhea is among one of the significant digestive disorders having significant implications related to rabbit farming. This study aimed to investigate the effects of GV-1 (Hou-hai) acupuncture on behavior, intestinal structure and immune function in rabbits having diarrhea induced by Sennae Folium. The mechanism underlying GV-1 acupuncture’s efficacy related to treatment of diarrhea was explored by measuring serum levels of various brain-gut peptides along with the expression of key protein molecules involved in 5-HT signaling pathway in colon tissue. After establishing diarrhea using Sennae Folium the rabbits were subjected to aqua-acupuncture or dry-needle acupuncture at GV-1 point for 7 days. The rabbits’ behavior, body weight and diarrhea index were observed and recorded. The therapeutic effects of GV-1 acupuncture on diarrhea were evaluated using histopathological techniques, enzyme-linked immunosorbent assay and western blotting assay. Results revealed rabbits with GV-1 acupuncture significantly improved the production performance of rabbits and effectively relieved the intestinal tract damage caused by diarrhea. Inflammatory factors including IL-2, IL-1β, TNF-α and IFN-γ were downregulated following dry-needle acupuncture and aqua-acupuncture at GV-1 points. Moreover, 5-HT, CCK, SS, SP, and VIP brain-gut peptides were downregulated while the levels of MTL, GAS, ENK and β-EP were upregulated. Results of western blotting show that compared to CG protein level of TPH-1 and 5HT3R increased significantly in MG and BCG. However, treatment by acupuncture significantly overturns the expression of TPH-1 and 5HT3R in ATG and DTG (P<0.05). SERT showed an opposite trend with the expression levels of TPH-1 and 5HT3R. It was concluded that acupuncture at GV-1 point is a favorable therapeutic option for alleviating diarrhea. Both dry-needle acupuncture and aqua-acupuncture treatments demonstrate significant efficacy in reducing FSAE-induced diarrhea with aqua-acupuncture having superior therapeutic effects compared to dry-needle acupuncture at the GV-1 point. Acupuncture targeting GV-1 relieves diarrhea by regulating various inflammatory cytokines and brain-gut peptides as brain-intestine peptides potentially serving as a crucial mediator in its mechanism of action.

Article Information

Received 11 February 2024

Revised 05 March 2024

Accepted 26 March 2024

Available online 12 August 2024

(early access)

Published 19 July 2025

Authors’ Contribution

HD, XW, ML and KL designed and conceived the study, carried out the research, analysed the data and wrote the manuscript. KL, ARA, RAA, AM and DS arranged funding and resources, supervised and investigation the study, and critically reviewed and revised the manuscript.

Key words

Acupuncture, GV-1, Diarrhea, Rabbit, Brain-gut peptide, 5-HT signaling pathway

DOI: https://dx.doi.org/10.17582/journal.pjz/20240211132542

* Corresponding author: [email protected]; [email protected]

0030-9923/2025/0005-2089 $ 9.00/00

Copyright 2025 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Diarrhea is a serious and common health concern in rabbits which leads to damage to gut mucosa and death resulting in significant losses to rabbit farming industry (Chen et al., 2017; Li et al., 2022). Diarrhea can be attributed to pathogenic infections or gastrointestinal distress induced by environmental pollutants however, antibiotics and probiotics have been widely used to cure diarrhea (Zubair et al., 2022; Pasha et al., 2022; Rehan et al., 2023). The administration of antibiotics and probiotics is associated with high costs reducing profit gain at numerous farms. Due to this reason antibiotic replacement therapy has been studied extensively. Acupuncture is an ancient therapeutic modality of traditional Chinese medicine which has been used for many years (Zhuang et al., 2012). Extensive research has demonstrated that the widespread utilization of acupuncture in managing diverse gastrointestinal (GI) disorders including diarrhea, gastric paralysis syndrome, dyspepsia and irritable bowel disease (Li et al., 2015; Rehman et al., 2022; Younas et al., 2023). The efficacy of acupuncture has been extensively verified making it a well-established therapeutic intervention however, the details related to the mechanisms regarding acupuncture therapy are still insufficient and require further investigations.

Governing vessel (GV) is called Du Mai in traditional Chinese medicine (TCM). Du means to govern or dominate, Du Mai (GV) can dominate all Yang Channels, while GV-1 (Hou-hai) is the first acupoint of the Du Mai, and it is located in the caldera of dorsomeson between archos and veutro of the tail. Acupuncture is recognized for its local efficacy and GV-1 is commonly applied in veterinary clinics to cure different ailments such as diarrhea, constipation and perianal diseases (Li et al., 2015; Long et al., 2016). However, there is diminished literature on the therapeutic effects and mechanism of GV-1 in treating diarrhea it is possible that the location of GV-1 acupoints can alter its therapeutic effects. Given the easy accessibility of GV-1 for acupuncture in veterinary medicine and investigating its effects on diarrhea would offer valuable theoretical support for utilizing acupuncture as a suitable treatment option for diarrhea.

Brain­gut peptides have crucial functions in along the digestive tract and central nervous system (Bonaz and Sabate, 2009). Gastrin and cholecystokinin are peptides which effectively regulate gastrointestinal motility. It has been reported that electroacupuncture at ST-36 promoted excretion of MTL and CCK which can reveal the mechanism of acupuncture enhancing gastrointestinal myoelectric activity (Niu et al., 2007). Crucial functions of brain gut peptides (Gastrin, Serotonin), cytokines (Tumor necrosis factor-alpha, Interleukin-1β) and signaling mediators establishing connections between the immune system and various cell types (Anderson and Delgado, 2008; Wei et al., 2020). Neuropeptides and cytokines can modulate the inflammation response by affecting phagocytosis of phagocytes, apoptosis of epithelial cells and the release of inflammatory molecules (Anderson and Delgado, 2008).

A close association has been reported between the 5-HT signaling pathway, oncogenesis of IBS and intestinal inflammation (Koopman et al., 2021). The 5-HT is synthesized by two types of cells in intestine that are enterochromaffin cells and enteric serotonergic neurons. Those cells produce tryptophan hydroxylase-1 or TpH2 to generate 5-HT. Serotonin reuptake transporter is the releasing regulator of 5-HT whereas, the functional effects of 5-HT are exerted through activation of specific 5-HT receptors (Gershon and Tack, 2007). The 5-HT3 receptor plays a crucial role in regulating inflammatory reactions (Najjar et al., 2023).

Acupuncture generates a noxious stimulus that leads to inflammatory response enhancing the therapeutic potential by activating the nerve-endocrine-immune systems and is the important medium connecting brain-gut axis (Li et al., 2019). To investigate the therapeutic effect of GV-1 acupuncture on diarrhea and to compare the efficacy of dry-needle acupuncture at GV-1 and aqua-acupuncture treatment at GV-1 diarrhea was induced in rabbits by sennae folium aqueous extract (FSAE). Moreover, bladder meridian -37 (BL-37) point was set as the acupoint control of GV-1 to verify the effectiveness and specificity of GV-1 acupoint. The 37th acupoint (BL-37, Han-gou) of the bladder meridian is located on the caudo-lateral aspect of the pelvic, 1/3 of the distance ischial tubercles to the middle of biceps femoris tendon, in the rut between the semitendinosus and musculus vastus intermedius. This acupoint is not often used, and no studies have shown that it has a therapeutic effect on diarrhea. This study was conducted to investigate the underlying mechanism of GV-1 acupuncture including rabbit behavior, pathological alterations in intestinal tissue, serum inflammatory factors, brain-gut peptides and expression levels of key proteins in the 5-HT signaling pathways. This study will highlight the results aimed at identifying brain-gut peptides and cytokines functions in diarrhea development through acupuncture treatment.

Materials and Methods

Preparation of sennae folium

Sennae folium (8kg) was obtained from Tongrentang Co., Ltd., Beijing, China. Sennae folium (100g) was soaked with 1 L of water in beaker for 30 min and placed in water bath preset to 100℃ for 25 min. The obtained filtrate was filtered with a gauze. This procedure was repeated 80 times to produce about 49 L of sennae folium aqueous extract. Subsequently, the aqueous extract was concentrated to 8L using multi-functional miniature extractor concentrator under a vacuum of 55°C that was equal to 1 g/mL of sennae folium aqueous extract (FSAE) which was then stored at 4°C for further use according to previous study.

Experimental animals

Thirty male New Zealand rabbits aged at 5 months (2.0~2.5 kg of bodyweight) were purchased from the Quanyou farm (Baoding, China). All rabbits were housed in the animal facility of Hebei Agricultural University. Each rabbit was kept in a single cage having 22±3°C temperature and 50% ~ 60% relative humidity with free access to water. Each rabbit was provided with a daily ration of 160g divided equally into two meals of 80g in the morning and evening throughout the experiment.

Model evaluation criteria

The evaluation criteria for the diarrhea model were as follows: (1) loose feces with mucous or watery consistency; (2) reduced appetite and body weight; (3) peripheral vasoconstriction, dorsal curvature, and arching posture; (4) a lack luster and soiled coat or presence of fecal matter on the coat; (5) easy fatigue and lethargy. (1) to (3) were the major symptoms, and (4) and (5) were the concurrent symptoms. The successful establishment of a rabbit diarrhea model requires the presence of at least 2 major symptoms and 2 concurrent symptoms (Li et al., 2023; Zhu et al., 2018).

Experimental design

After one week of acclimatization period the rabbits were randomly divided into five groups comprising of a control group and four model groups. The control group was administered with saline at the dosage of 8 mL/kg daily for two weeks. The rabbits in model group were fed with sennae folium aqueous extracts by gavage (8 mL/kg) daily for two weeks. The gavage was administered daily at 10 am and the gavage procedures were all completed within a one-hour time frame. To ensure the efficacy of FSAE on rabbits no medication was given to rabbits. Moreover, acute toxicity of senna in mice demonstrate that senna is a well-tolerated laxative with no significant pain (Boeira et al., 2010). The methodology was derived from preliminary experiments revealing the successful establishment of a rabbit diarrhea model by FSAE (8ml/kg) for 5 days. On the 7th day after confirming that all four model groups had successfully established diarrhea, which were randomly divided into GV-1 dry-needle acupuncture treatment group (DTG), GV-1 aqua-acupuncture treatment group (ATG), BL-37 acupoint control group (BCG) and diarrhea model group (MG). Subsequently, acupuncture protocol was initiated and applied for a duration of 7 consecutive days.

Interventions

The acupuncture treatment was performed by licensed acupuncturists. Sterile 0.25 mm disposable acupuncture needles used in this study was obtained from JingTang Herbal Co.Ltd (Baoding, China). Acupoints GV-1 and BL-37 were chosen for DTG and BCG animals. The acupoint site was disinfected and the dry-needle was inserted vertically to a depth of 1 cun. A dull needling consciousness was applied through raising, thrusting and entwisting maneuver for 20 min/day. In ATG treatment was administered daily at GV-1 acupoint using 1 mL syringe to inject a mixed solution of vitamin B12 and normal saline in a ratio of 1:3 with a dosage of 0.2 mL. Rabbits in both the control group (CG) and the model group (MG) continued to receive gavage without any other interventions. Rabbits that experienced infections or premature death were excluded from the study.

Behavioral monitoring

Starting from the 7th day of the experiment the residual food in each cage was measured every morning before feeding. The daily feed intake of each rabbit was calculated and the total feed intake of each rabbit during the 7-13 days of experiment was recorded. Moreover, the mental well-being, physical activities, appetite and body coat condition of rabbits were observed and recorded during the acupuncture treatment procedure.

Body weight and diarrhea index detection

Body weight of rabbits was measured and documented on the 1st, 4th, 7th, 10th, and 13th day. Faecal status was observed and recorded 6 h after intragastric administration after which the rate of diarrhea and diarrhea index were calculated. The degree of diarrhea was divided into 5 grades as per previous studies reported (Blake et al., 2016). Dry, hard, and dark feces were classified as grade 1 (constipation); round and uniform feces were classified as grade 2 (normal); soft and large feces that were attached to each other or with mucus are classified as grade 3 (mild diarrhea); small or unformed feces with mucus were classified as grade 4 (diarrhea); watery feces with mucus or blood were classified as grade 5 (severe diarrhea). The presence of diarrhea is defined as a stool grade of 3 or higher.

Sample collection

Experimental rabbits were euthanized by intravenous injection of pentobarbital sodium (100mg/kg) into the auricular vein. Blood samples from all rabbits were gathered from the jugular vein of animals, and the serum was obtained by centrifugation (1200 g, 4°C, 10 min) for ELISA. The colon tissues were excised, immediately immersed in liquid nitrogen and transferred to a freezer at -80 ℃ for cryopreservation. Simultaneously, duodenum, jejunum and colon samples were kept in paraformaldehyde (4%). The no of rabbits in each group was (n=6). All the obtained samples were labelled and the personnel who conducted the analyses were blinded to the grouping.

Pathomorphological examination and ELISA

The contents of serum interferon gamma, interlenkin 1β, IL-2, tumor necrosis factor α, gastrin (GAS), motilin (MLT), somatostatin, Substance P, vasoactive intestinal peptide, cholecystokinin (CCK), 5-hydroxytryptamine (5-HT), enkephalin (ENK), β- endorphins (β-EP) and acetylcholinesterase (AChE) were analyzed using ELISA kits purchased from Enzyme-linked Biotechnology (Shanghai, China). The duodenum, jejunum and colon tissues were collected, fixed and paraffin embedded. Then a 4 μm section from each sample was cut and subjected to hitopathological processing followed by staining with hematoxylin and eosin. Histopathological slide analysis was observed for pathological alterations under microscope (Olympus Corporation, Tokyo, Japan).

Western blot analysis

Protein extraction was performed through bicinchoninic acid protein assay kit (BioChain, USA). Then protein products were quantified using the BCA protein assay kit (Thermo, USA). Antibodies of β-actin, 5-HT3R, TPH-1 and SERT were purchased from CST (USA). Image Quant LAS 4000 mini (USA) was used for densitometric values analysis.

Statistical analysis

Data is presented as mean ± SD. One-way analysis of variance (ANOVA) was performed using SPSS (16.0, USA). Results were considered significant at P > 0.05.

ResultS

Behavioral performance

Rabbits in the CG exhibited vigorous activity levels, exhibited a healthy appetite, possessed well-maintained shiny fur and had regular grainy fecal pellets. While rabbits in the remaining four MG gradually exhibited signs of fatigue following intragastric administration, including activity, sluggish response, diminished appetite and loose stools. The rabbit diarrhea model was successfully established on the 7th day. Under the intervention of acupuncture negative behavior of rabbits in both the ATG and DTG groups exhibited a significant improvement. During the 7-13 days of the experiment as depicted in Figure 1A, a significant decrease in total feed intake per rabbit was observed in the MG compared to the CG. In contrast to MG both ATG and DTG groups demonstrated a substantial increase in total feed intake per rabbit.

Body weight and diarrhea index

As shown in Figure 1 body weight of CG increased significantly during the whole experiment whereas, it decreased significantly in other groups (Fig. 1B). Compared with the CG all rabbits in experimental group were significantly lower in body weight due to diarrhea at 7 days. After 7 days of acupuncture intervention GV-1 acupoint showed significant increase in the body weight of diarrheic animals (P<0.05) (Fig. 1C). In contrast with the trend of body weight change animals of CG did not show any diarrhea symptom while the diarrhea rate and its index in experimental group increased (Fig. 1D, E). Compared to CG diarrhea was induced successfully in all experimental groups and the diarrhea index increased significantly (P<0.05) after 7 days the diarrhea index in ATG and DTG decreased significantly (P<0.05 or P<0.01) (Fig. 1F).

 

Intestinal histopathology

The histomorphology changes of duodenal structures were observed by H and E staining on pathological sections from different experimental groups is shown in Figure 2. In CG the duodenal morphology and structures were normal. The surface of the duodenal mucosa appeared as dense with increased number of intestinal villi. The villous surface epithelial cells were normal and the goblet cells were evenly distributed among the epithelial cells (oval mark), the Lieberkühn crypts was clearly visible in the basal parts of intestinal villi (triangle mark) (Fig. 2A). The duodenal mucosa showed evidence of edema which was characterized by separation of epithelium and lamina propria to form substantial gaps In MG (rectangular mark) accompanied by marked infiltration of inflammatory cells in certain sections (arrow mark) (Fig. 2B). The degree of injury in BCG was similar to that of MG (Fig. 2C) aqua-acupuncture and dry needle treatment significantly alleviated the inflammatory injury induced by sennae folium. After 7 days treatment the duodenal villi length and morphology in both the ATG and DTG did not fully recovered to normal state. However, there was a significant reduction in duodenal mucosa edema and inflammatory cell infiltration (Fig. 2D, E).

 

The effect of acupuncture on jejunum histomorphology were shown in Figure 2. The villi of the jejunum of healthy animals were intact and there were no significantly visible signs of edema and prominent inflammatory reaction (Fig. 2F). In MG and BCG severe edema in mucosa and submucosa could be found in some local jejunum tissue (arrow mark), whereas, the villi length of jejunum significantly decreased due to diarrhea (Fig. 2G, H); The mucosal edema (arrow mark) and the decreased connective tensile tissue strength (rectangular mark) were partially enhanced in both ATG and DTG however, complete recovery was not achieved (Fig. 2I, J).

There were no significant pathological changes in CG (Fig. 2). The colon was structurally intact with clear morphology and goblet cells (arrow mark). In MG the mucous membrane was damaged, the basal layer edema was observed (rectangular contour mark), and the goblet cell apoptosis (Fig. 2L). The colon morphology of the BCG was basically consistent with that of MG whereas, the presence of hydropic degeneration in the epithelial cells within the mucosal layer was evident (Fig. 2M). The colonic injury in the ATG and DTG were significantly improved (Fig. 2N, O).

ELISA profile of inflammatory mediators

The impact of acupuncture treatment on serum inflammatory cytokines was assessed using the ELISA assay. As depicted in Figure 3 there was a significant increase in the release of TNF-α, INF-γ, IL-1β and IL-2 in MG and BCG (P<0.01). However, these effects were significantly decreased in ATG and DTG after 7 days of intervention (P<0.05) (Fig. 3).

 

Serum protein profile of brain-gut peptides

The serum samples of each group were collected and the contents of 9 different types of brain-gut peptides including 5-HT were detected. Compared to CG the serum levels of 5-HT, CCK, SP, VIP and SS were significantly increased in MG and BCG (Fig. 4). Compared to MG the expression of the above 5 types of brain-gut peptides were significantly down-regulated by aqua-acupuncture treated via dry needle (P<0.05 or P<0.01). However, CCK, SP and SS in ATG and DTG animals did not exhibit significant differences compared to those in BCG which proved that these above 3 peptides were not only affected by acupuncture at GV-1 (Fig. 4A, E). Contrary to the results of 5-HT and other peptides diarrhea significantly down-regulated the level of GAS, MTL, ENK and β-EP (P<0.01). The expression levels of the above 4 types of peptides were significantly up-regulated in ATG and DTG (P<0.05 or P<0.01) compared to GM. However, ENK and EP did not show any significant differences among BCG, ATG and DTG groups which showed that the above 2 peptides were not regulated by GV-1 point (Fig. 4F, I). Compared to CG, SP, ENK and β-EP in ATG did not exhibit significant differences. However, significant differences were observed between DTG and CG. These findings suggest that aqua-acupuncture at GV-1 point exhibited superior therapeutic efficacy compared to dry-needle acupuncture in rabbits with diarrhea.

 

Regulation effects of acupuncture treatment on 5-TH signaling pathway

Compared to CG, the protein levels of TPH-1 and 5HT3R significantly increased in MG (P<0.05) and BCG (P<0.05). However, acupuncture treatment suppressed the expression of TPH-1 and 5HT3R in ATG and DTG (P<0.05) (Fig. 5A, C). The SERT was opposite with the trend that of TPH-1 and 5HT3R (Fig. 5A, D).

 

Discussion

Acupuncture is a widely applied method in traditional medicine with high efficacy. Studies have revealed that acupuncture therapy is effective for more than one hundred diseases and symptoms including diarrhea (Wang et al., 2022; Zhang et al., 2021). Though acupuncture-related techniques were relatively commonly used in treating diarrhea. Limited knowledge is available related to the mechanisms and therapeutic effects (Li et al., 2015). In this study, we employed sennae folium to induce diarrhea in rabbits for exploring the treatment effects of aqua-acupuncture with dry needle and the potential mechanisms of acupuncture method.

Rabbit diarrhea induced by FSAW is a recognized model used to study the pathology and mechanism of diarrhea (Zhang et al., 2020). The efficacy of GV-1 as an acupuncture point in the treatment of diarrhea has been demonstrated in various clinical studies (Yang et al., 2022). Therefore, this stimulatory was selected using both aqua-acupuncture and dry-needle acupuncture in a FSAE-induced diarrhea rabbit to detect the treatment effects and explore its mechanisms. It was found that rabbits treated by aqua-acupuncture treatment and dry-needle acupuncture could ameliorate body weight loss and diarrhea index after 7 days and inhibit the severe inflammation leading to necrosis in duodenal, jejunal and colonic mucosa which was different in BCG and MG. Our results are in corroboration with previous studies (Dahlgren et al., 2022; Li et al., 2019) and provide reference for suitable treatment modalities of diarrhea.

Diarrhea is considered as a severe inflammatory disorder and inflammatory cytokines have important relationship with development of diarrhea (Dong et al., 2017). Acupuncture therapy affect immune and endocrine function in host, it was hypothesized that the treatment effect of acupuncture in diarrhea is related to its regulation of immune cells and endocrine function. The pivotal involvement of IFN-γ and α, IL-1β and 2 in immune and inflammatory responses has been widely acknowledged (Liu et al., 2021). It was found that the expression levels of inflammatory cytokines in both aqua-acupuncture and dry needle treatment significantly improves the release of four inflammatory cytokines induced by diarrhea. Our results are consistent with a previous study (Dong et al., 2017; Yang et al., 2021).

Clinical and experimental results indicated that during acupuncture treatment brain-gut peptides are related to the modulation of diarrhea (Augustyniak et al., 2021). It has been reported that 9 types of brain-gut peptides associated with diarrhea, which were screened and detected in our research. The result shows that acupuncture at GV-1 point regulated the levels of serum 5-HT, VIP, GAS and MTL in rabbits with diarrhea. However, no significant regulatory effect on these brain-gut peptides was observed with acupuncture at BL-37. Moreover, both acupuncture at GV-1 and BL-37 acupoints exhibited significant down-regulation of serum SP levels and up-regulation of serum ENK and β-EP levels in rabbits with diarrhea.

The 5-HT is mainly derived from enteroendocrine cells playing crucial roles in the gastrointestinal inflammatory response. Excessive release of 5-HT leads to diarrhea whereas, the administration of 5-HT receptor antagonists induce constipation (Yang et al., 2021). The VIP is a commonly found mammalian neuropeptide innervating gastrointestinal tract, which regulates physiological processes like gut secretion and motility (Iwasaki et al., 2019). Excessive secretion of VIP has been demonstrated to induce severe watery diarrhea in humans (Ito et al., 2012). However, the function of VIP in acute diarrhea remains imperfectly established. The VIP is recognized as a biomarker of inflammatory bowel disease as higher VIP concentrations are reported in these inflammatory diseases (Abad et al., 2012). Our findings are in agreement with the increase of VIP plasma concentrations which indicated a drastic intestinal inflammatory response induced by sennae folium (Xie et al., 2021). Compared to MG rabbits the animals in ATG and DTG exhibited significant decrease (P<0.05) of serum concentrations of 5-HT and VIP after acupuncture at GV-1 indicating that this treatment method might have effectively cured diarrhea and suppressed intestinal inflammation by regulating levels of 5-HT and VIP. Furthermore, the impact of acupuncture treatment duration on serum concentrations of 5-HT and VIP remains unclear as further research is required to explain the findings of this study.

Gastrin stimulates acid secretion and fundic mucosal growth (Heilmann et al., 2017). The content of gastrin can be affected by factors of diarrhea, hypercalcemia and use of antacids. The mechanisms of acupuncture treatment on gastrin expression and release in rabbits with diarrhea yet remains unexplored. It was observed that serum gastrin concentrations in rabbits treated with aqua-acupuncture and dry-needle acupuncture were significantly lower when compared with that in healthy rabbits, but significantly higher compared to the MG. In contrast to our findings, in dogs with chronic enteritis the serum gastrin concentrations can reach up to as high as double in concentration as compared to dogs without gastrointestinal disease (Garcia-Sancho et al., 2005). The difference of gastrin contents between chronic enteritis and acute diarrhea is due to chronic inflammation leading to gastric lesions and decreased acidity in gastric antrum. The variation trend of the gastrin seen in this study is associated with the inflammation as inflammatory cytokines affect gastrin concentrations (Hopman et al., 2003; Suzuki et al., 2001).

The primary physiological effects of MTL include increased pepsin secretion, migrating motor complex (MMC) and promoting gastrointestinal motility (Kitazawa and Kaiya, 2021). The MTL and CCK controls hunger and is related to gut-brain signaling (Tack et al., 2021). The CCK has been shown to enhance satiety and reduce food consumption. Consistent with these effects, we observed a significant increase in serum CCK levels and a notable drop in MTL levels among rabbits within the diarrhea model group leading to increased abdominal satiety and decreased feed intake. The SP is a pro-inflammatory medium which releases upon nociceptive stimulation (Mazzoni et al., 2024). Furthermore, SP is a reliable indicator of peripheral nociceptive afference in several studies (Ma et al., 2023). Compared to the diarrhea model group, both the aqua-acupuncture and the dry-needle acupuncture at GV-1 exhibited significantly decreased plasma concentrations of CCK and SP. This suggests that GV-1 effectively mitigates gastrointestinal tissue injury and inflammation induced by FSAE in rabbits with diarrhea which is consistent with the histopathological results of this study. However, the concentrations of SP were significantly reduced by acupuncture at BL-37 acupoints whereas, no significant difference was observed in CCK levels compared with GM. These findings suggest that acupuncture at BL-37 has the potential to modulate brain-gut peptide levels while its application is not favorable for animals with diarrhea.

The ENK and β-EP belonging to endogenous opioid polypeptides that contribute to ease pain, self-governing regulation of immune responses and better intestine functioning (Bodnar, 2023). This endogenous opioid system could be broken by pain and exogenous opioids (Roeckel et al., 2016). In this study, the concentrations of ENK and β-EP in the serum of rabbits with FSAE-induced diarrhea were significantly decreased compared to GM indicating a potential correlation between pain and diarrhea. However, the levels of ENK and β-EP at both GV-1 and BL37 points exhibited significant upregulation following acupuncture treatment, thereby suggesting that acupuncture exerts a universal analgesic effect. However, further investigation is required to establish the correlation between the analgesic efficacy of acupuncture at BL-37 points and its potential for alleviating diarrhea.

The 5-HT signaling pathway is involved in sustaining gut homeostasis, and the regulation of 5-HT in intestinal tissue is mediated by TPH-1 and SERT, which are key enzymes involved in the biosynthesis and reuptake of serotonin (Koopman et al., 2021). The SERT is related to the termination of 5-HT in the mucosa and low levels of SERT is associated with intestinal inflammation (Gershon, 2004). The abnormal expression of 5-HT and 5-HT3R has been extensively documented to induce inflammation in intestinal tissues. Recently, several 5-HT3 receptor antagonists have demonstrated their utility in various intestinal inflammatory diseases. These antagonists effectively inhibit the releasing of inflammatory cytokines, like IL-1β and TNF-α (Kato, 2013). Following the administration of FSAE to rabbits, a significant spike in peripheral free 5-HT levels was observed. The potential inactivation defect caused by reduced SERT expression id due to the underlying cause. Moreover, elevated 5-HT concentrations prolong its interaction with the 5-HT3R thereby augmenting the effects of 5-HT3R and subsequently leading to gastrointestinal disorders. After acupuncture at GV-1 point, the expression of SERT in colon tissue was increased whereas, the expression of 5HT3R was decreased which is consistent with the observed changes in serum 5-HT and inflammatory cytokines levels. These findings suggest that acupuncture may alleviate FSAE-induced diarrhea and inflammation by modulating the 5-HT signaling pathway in rabbits.

Conclusion

Nexus to the above it is concluded that acupuncture at GV-1 point is a favorable therapeutic option for alleviating diarrhea. Both dry-needle acupuncture and aqua-acupuncture treatments demonstrate significant efficacy in reducing FSAE-induced diarrhea, with aqua-acupuncture exhibiting superior therapeutic effects compared to dry-needle acupuncture. Acupuncture exerts regulatory effects on the 5-HT signaling pathway thereby regulating brain-gut peptides production. The antidiarrheal effect of acupuncture at G-V1 is attributed to the regulation of various inflammatory cytokines and brain-gut peptides.

Declarations

Funding

The authors sincerely acknowledge the Researcher Supporting Project number (RSP2024R232) for funding this work at King Saud University, Riyadh, Saudi Arabia.

Ethical approval

The experimental procedures were reviewed and approved by the Laboratory Animal Ethics Committee of Hebei Agricultural University (Approval No. 2021066).

Statement of conflict of interest

The authors have declared no conflict of interest.

References

Abad, C., Gomariz, R., Waschek, J., Leceta, J., Martinez, C., Juarranz, Y. and Arranz, A., 2012. VIP in inflammatory bowel disease: State of the art. Endocrine, Metab. Immune Disorders Drug Targets, 12: 316-322. https://doi.org/10.2174/187153012803832576

Anderson, P. and Delgado, M., 2008. Endogenous anti-inflammatory neuropeptides and pro-resolving lipid mediators: A new therapeutic approach for immune disorders. J. cell. mol. Med., 12: 1830–1847. https://doi.org/10.1111/j.1582-4934.2008.00387.x

Augustyniak, D., Kramarska, E., Mackiewicz, P., Orczyk-Pawiłowicz, M. and Lundy, F.T., 2021. Mammalian neuropeptides as modulators of microbial infections: Their dual role in defense versus virulence and pathogenesis. Int. J. mol. Sci., 22: 3658. https://doi.org/10.3390/ijms22073658

Blake, M.R., Raker, J.M. and Whelan, K., 2016. Validity and reliability of the Bristol Stool Form Scale in healthy adults and patients with diarrhoea-predominant irritable bowel syndrome. Aliment. Pharmacol. Ther., 44: 693-703. https://doi.org/10.1111/apt.13746

Bodnar, R.J., 2023. Endogenous opiates and behavior: 2021. Peptides, 164: 171004. https://doi.org/10.1016/j.peptides.2023.171004

Boeira, J.M., Fenner, R., Betti, A.H., Provensi, G., Lacerda, L.A., Barbosa, P.R., González, F.H., Corrêa, A.M., Driemeier, D., Dall’Alba, M.P., Pedroso, A.P., Gosmann, G., da Silva, J. and Rates, S.M., 2010. Toxicity and genotoxicity evaluation of Passiflora alata Curtis (Passifloraceae). J. Ethnopharmacol., 128: 526–532. https://doi.org/10.1016/j.jep.2009.09.037

Bonaz, B. and Sabate, J.M., 2009. Le dysfonctionnement du brain-gut (Brain-gut axis dysfunction). Gastroenterol. Clin. Biol., 33(Suppl 1): S48–S58. https://doi.org/10.1016/S0399-8320(09)71525-8

Chen, Y., Zhao, B., Wu, Y., Hu, S., Mu, L., Zhu, C., Pan, Y. and Wu, X., 2017. Impacts of diarrhea on the immune system, intestinal environment, and expression of PGRPs in New Zealand rabbits. PeerJ, 5: e4100. https://doi.org/10.7717/peerj.4100

Dahlgren, D., Rosenqvist, E., Hellström, P.M., Nygren, P., Kullenberg, F., Peters, K., Sjöblom, M. and Lennernäs, H., 2022. Evaluation and validation of chemotherapy-specific diarrhoea and histopathology in rats. Basic clin. Pharmacol. Toxicol., 131: 536-546. https://doi.org/10.1111/bcpt.13790

Dong, Y., Han, Y., Wang, Z., Qin, Z., Yang, C., Cao, J. and Chen, Y., 2017. Role of serotonin on the intestinal mucosal immune response to stress-induced diarrhea in weaning mice. BMC Gastroenterol., 17: 82. https://doi.org/10.1186/s12876-017-0634-5

García-Sancho, M., Rodríguez-Franco, F., Sainz, Á., Rodríguez, A., Silván, G. and Illera, J.C., 2005. Serum gastrin in canine chronic lymphocytic-plasmacytic enteritis. Can. Vet. J., 46: 630-634. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1168880

Gershon, M.D., 2004. Review article: Serotonin receptors and transporters roles in normal and abnormal gastrointestinal motility. Aliment. Pharmacol. Therapeut., 20 (Suppl 7): 3–14. https://doi.org/10.1111/j.1365-2036.2004.02180.x

Gershon, M.D. and Tack, J., 2007. The serotonin signaling system from basic understanding to drug development for functional GI disorders. Gastroenterology, 132: 397–414. https://doi.org/10.1053/j.gastro.2006.11.002

Heilmann, R.M., Berghoff, N., Grutzner, N., Parnell, N.K., Suchodolski, J.S. and Steiner, J.M., 2017. Effect of gastric acid-suppressive therapy and biological variation of serum gastrin concentrations in dogs with chronic enteropathies. BMC Vet. Res., 13: 321. https://doi.org/10.1186/s12917-017-1233-y

Hopman, W., Jong, D.J., Naber, A. and J, B.M.J.J., 2003. Tumour necrosis factor alpha antibody affects gastrin release in Crohn disease. Scand. J. Gastroenterol., 38: 522-525. https://doi.org/10.1080/00365520310002896

Ito, T., Igarashi, H. and Jensen, R.T., 2012. Pancreatic neuroendocrine tumors: Clinical features, diagnosis and medical treatment: Advances. Best Pract. Res. Clin. Gastroenterol., 26: 737-753. https://doi.org/10.1016/j.bpg.2012.12.003

Iwasaki, M., Akiba, Y. and Kaunitz, J.D., 2019. Recent advances in vasoactive intestinal peptide physiology and pathophysiology: focus on the gastrointestinal system. F1000 Res. Fac. Rev., 8: 1629. https://doi.org/10.12688/f1000research.18039.1

Kato, S., 2013. Role of serotonin 5-HT₃ receptors in intestinal inflammation. Biol. Pharma. Bull., 36: 1406–1409. https://doi.org/10.1248/bpb.b13-00363

Kerckhoffs, A.P., M. Ter Linde, J.J., Akkermans, A.L.M. and Samsom, M., 2008. Trypsinogen IV, serotonin transporter transcript levels and serotonin content are increased in small intestine of irritable bowel syndrome patients. Neurogastroenterol. Motil., 20: 900-907. https://doi.org/10.1111/j.1365-2982.2008.01100.x

Kitazawa, T. and Kaiya, H., 2021. Motilin comparative study: Structure, distribution, receptors, and gastrointestinal motility. Front. Endocrinol., 12: 700884. https://doi.org/10.3389/fendo.2021.700884

Koopman, N., Katsavelis, D., Hove, A.S.T., Brul, S., Jonge, W.J. and Seppen, J., 2021. The multifaceted role of serotonin in intestinal homeostasis. Int. J. mol. Sci., 22: 9487. https://doi.org/10.3390/ijms22179487

Li, D., Wang, Y., Liu, N., Chen, S., Liu, H., Wang, P., Yu, Z., Shu, G., Lin, J., Zhang, W., Peng, G., Zhao, L., Tang, H., Zhang, K., Wen, B. and Fu, H., 2022. Modified Sijunzi granule decreases post-weaning diarrhea in Rex rabbits via promoting intestinal development. Front. Vet. Sci., 9: 972326. https://doi.org/10.3389/fvets.2022.972326

Li, H., He, T., Xu, Q., Li, Z., Liu, Y., Li, F., Yang, B.F. and Liu, C.Z., 2015. Acupuncture and regulation of gastrointestinal function. World J. Gastroenterol., 21: 8304-8313. https://doi.org/10.3748/wjg.v21.i27.8304

Li, N.C., Li, M.Y., Chen, B. and Guo, Y., 2019. A new perspective of acupuncture: The interaction among three networks leads to neutralization. Evid Based Complement. Altern. Med., 2019: 2326867. https://doi.org/10.1155/2019/2326867

Li, X., Zhu, J., Wu, Y. and Tan, Z., 2023. Correlation between kidney function and intestinal biological characteristics of adenine and sennae folium induced diarrhea model in mice. Turk. J. Gastroenterol., 34: 4-12. https://doi.org/10.5152/tjg.2022.211010

Li, Y., Li, J., Liu, X., Zhang, J., Mei, X., Zheng, R., Chen, W., Zheng, Q. and Zhong, S., 2019. Antidiarrheal activity of methanol extract of Sophora tonkinensis in mice and spasmolytic effect on smooth muscle contraction of isolated jejunum in rabbits. Pharma. Biol., 57: 477-484. https://doi.org/10.1080/13880209.2019.1645701

Liu, C., Chu, D., Kalantar-Zadeh, K., George, J., Young, H.A. and Liu, G., 2021. Cytokines: From clinical significance to quantification. Adv. Sci. (Weinheim, Baden-Wurttemberg, Germany), 8: e2004433. https://doi.org/10.1002/advs.202004433

Long, Q., Li, Y., Li, J., Wen, Y., Du W, Wan, C. and Yue, C., 2016. Clinical observation on anal pendant expansion after PPH of mixed hemorrhoid treated with acupuncture at Xialiao (BL 34) and Changqiang (GV 1). Chinese Acupunct. Moxibust. 36: 603-606.

Ma, J., Nguyen, D., Madas, J., Bizanti, A. Mistareehi, A., Kwiat AM., Chen, J., Lin, M., Christie, R., Hunter, P., Heal, M., Baldwin, S., Tappan, S., Furness, J.B., Powley, T.L. and Cheng, Z.J., 2023. Organization and morphology of calcitonin gene-related peptide-immunoreactive axons in the whole mouse stomach. J. comp. Neurol., 531: 1608-1632. https://doi.org/10.1002/cne.25519

Mazzoni, M., Cabanillas, L., Costanzini, A., Caremoli, F., Million, M., Larauche, M., Clavenzani, P., De Giorgio, R. and Sternini, C., 2024. Distribution, quantification, and characterization of substance P enteric neurons in the submucosal and myenteric plexuses of the porcine colon. Cell Tissue Res., 395: 39–51. https://doi.org/10.1007/s00441-023-03842-x

Najjar, S.A., Hung, L.Y. and Margolis, K.G., 2023. Serotonergic control of gastrointestinal development, motility, and inflammation. Comprehen. Physiol., 13: 4851-4868. https://doi.org/10.1002/cphy.c220024

Niu, W.X., He, G.D., Liu, H. and Qin, X.Y., 2007. Effects and probable mechanisms of electroacupuncture at the Zusanli point on upper gastrointestinal motility in rabbits. J. Gastroenterol. Hepatol., 22: 1683-1689. https://doi.org/10.1111/j.1440-1746.2007.05049.x

Pasha, R.H., Qureshi, A.S., Ali, A., Rehman, S.U. and Khan, M.A., 2022. Exposure to arsenic induces histomorphological alterations in testis of rabbits. Asian J. Agric. Biol. 2022: 202104178.

Rehan, S., Qureshi, A.S., Kausar, R. and Saleemi, M.K., 2023. Effects of maternal undernutrition on coronary vasculature of fetuses and neonates in rabbits (Oryctolagus cuniculus). Pak. Vet. J., 43: 49-54.

Rehman, H.U., Ghafoor, A., Rabbani, M., Riaz, A., Awan, F.N. and Raza, S., 2022. Locally prepared bovine herpesvirus 1 gE deleted vaccine induced immunogenicity in rabbits. Pak. Vet. J., 42: 127-130.

Roeckel, L.A., Le Coz, G.M., Gavériaux-Ruff, C. and Simonin, F., 2016. Opioid-induced hyperalgesia: Cellular and molecular mechanisms. Neuroscience, 338: 160–182. https://doi.org/10.1016/j.neuroscience.2016.06.029

Suzuki, T., Grand, E., Bowman, C., Merchant, J.L., Todisco, A., Wang, L. and Del Valle, J., 2001. TNF-α and interleukin 1 activate gastrin gene expression via MAPK- and PKC-dependent mechanisms. Am. J. Physiol. Gastroint. Liver Physiol., 281: G1405-G1412. https://doi.org/10.1152/ajpgi.2001.281.6.G1405

Tack, J., Verbeure, W., Mori, H., Schol, J., Van den Houte, K., Huang, I.H., Balsiger, L., Broeders, B., Colomier, E., Scarpellini, E. and Carbone, F., 2021. The gastrointestinal tract in hunger and satiety signalling. United Eur. Gastroenterol. J., 9: 727–734. https://doi.org/10.1002/ueg2.12097

Wang, X., Shi, X., Lv, J., Zhang, J., Huo, Y., Zuo, G., Lu, G., Liu, C. and She, Y., 2022. Acupuncture and related therapies for the anxiety and depression in irritable bowel syndrome with diarrhea (IBS-D): A network meta-analysis of randomized controlled trials. Front. Psych., 13: 1067329. https://doi.org/10.3389/fpsyt.2022.1067329

Wei, P., Keller, C. and Li, L., 2020. Neuropeptides in gut-brain axis and their influence on host immunity and stress. Comput. Struct. Biotechnol. J., 18: 843–851. https://doi.org/10.1016/j.csbj.2020.02.018

Xie, Y., Zhan, X., Tu, J., Xu, K., Sun, X., Liu, C., Ke, C., Cao, G., Zhou, Z. and Liu, Y. 2021. Atractylodes oil alleviates diarrhea-predominant irritable bowel syndrome by regulating intestinal inflammation and intestinal barrier via SCF/c-kit and MLCK/MLC2 pathways. J. Ethnopharmacol., 272: 113925. https://doi.org/10.1016/j.jep.2021.113925

Yang, B., Yue, Y., Chen, Y., Ding, M., Li, B., Wang, L., Wang, Q., Stanton, C., Ross, R.P., Zhao, J., Zhang, H. and Chen, W., 2021. Lactobacillus plantarum CCFM1143 alleviates chronic diarrhea via inflammation regulation and gut microbiota modulation: A double-blind, randomized, placebo-controlled study. Front. Immunol. 12: 746585. https://doi.org/10.3389/fimmu.2021.746585

Yang, Y., Rao, K., Zhan, K., Shen, M., Zheng, H., Qin, S., Wu, H., Bian, Z. and Huang, S., 2022. Clinical evidence of acupuncture and moxibustion for irritable bowel syndrome: A systematic review and meta-analysis of randomized controlled trials. Front. Publ. Hlth., 24: 1022145. https://doi.org/10.3389/fpubh.2022.1022145

Younas, M., Ashraf, K., Rashid, M.I., Ijaz, M., Suleman, M., Chohan, T.A. and Sarfraz-ur-Rahman, 2023. Expression and purification of recombinant multi-epitope protein of Rhipicephalus microplus tick and its antigenicity in rabbits. Pak. Vet. J., 43: 778-784.

Yu, C.P., Peng, Y.H., Huang, C.Y., Hsieh, Y.W., Hou, Y.C. and Lin, S.P., 2021. Sennae folium increased the bioavailability of methotrexate through modulation on MRP 2 and BCRP. Pharmaceuticals, 14: 1036. https://doi.org/10.3390/ph14101036

Zhang, C., Shao, H., Li, D., Xiao, N. and Tan, Z., 2020. Role of tryptophan-metabolizing microbiota in mice diarrhea caused by Sennae Folium extracts. BMC Microbiol., 20: 185. https://doi.org/10.1186/s12866-020-01864-x

Zhang, J., Li, Z., Li, Z., Li, J., Hu, Q., Xu, J. and Yu, H., 2021. Progress of acupuncture therapy in diseases based on magnetic resonance image studies: A literature review. Front. Hum. Neurosci., 15: 694919. https://doi.org/10.3389/fnhum.2021.694919

Zhu, J., Liu, S., Guo, Y., Hou, L., Su, X., Li, Y., Han, B., Liu, D., Wang, Q., Chen, J.J. and Wei, W., 2018. A new model of diarrhea with spleen-kidney yang deficiency syndrome. Evid. Based Complement. Altern. Med. 2018: 4280343. https://doi.org/10.1155/2018/4280343

Zhuang, Y., Xing, J., Li, J., Zeng, B. and Liang, F., 2012. History of acupuncture research. Int. Rev. Neurobiol., 111: 1-23. https://doi.org/10.1016/B978-0-12-411545-3.00001-8

Zubair, M., Shafique, S., Shahbaz, M., Husain, A.M., Khalique, M.A., Saleemi, M.K., Khan, M.I. and Hameed, N., 2022. Toxicological effects of arsenic trioxide on blood, serum biochemical constituents and hormonal profile of rabbits and their amelioration with olive oil. Asian J. Agric. Biol., 2022: 202012550.