Our results demonstrate that acute BEA at ST36 acupoints improved visceral pain in AA-treated IBS rats. This finding is consistent with our previous study, where EA at ST36 acupoint with a similar stimulation protocol significantly reduced visceral hypersensitivity in rats [13]. Moreover, acute applications of UEA and TEA at ST36 demonstrated effectiveness in alleviating visceral pain in IBS rats. These results suggest that TEA is as effective as direct ST36 stimulation (BEA). However, acute SNS stimulation did not reduce visceral pain in AA-treated rats. Previous studies demonstrated that acute SNS at 14 Hz, pulse width of 330 ms, and stimulation amplitude of 40% MT normalized acute restraint stress-induced visceral hypersensitivity in rats [18]. Other research demonstrated that SNS with the 5 Hz, 500 µs, 10 seconds on, 90 seconds off parameters increased vagal activity and decreased sympathetic activity in 2, 4, 6-Trinitrobenzenesulfonic acid (TNBS) induced rats [21; 27]. The variation in results could be attributed to differences in the animal model and stimulation parameters used in these studies. Furthermore, our results demonstrate that acute BEA at ST36 improved visceral pain in female rats, suggesting similar efficacy between the sexes.
The animal model we used in this study to induce IBS is well-established. These animals develop visceral hypersensitivity in adulthood, and the visceral pain response can be measured with EMG in response to CRD [28; 29]. CRD is a reproducible and reliable visceral stimulus, which is helpful in assessing visceral pain [30]. The abdominal EMG is a well-established method for assessing visceral pain in animal models that measure the electromyogram signal (reflecting abdominal muscle contractions) generated during the CRD. Acupoint ST36 stimulation is most commonly used in clinical settings to treat GI disorders, including IBS [31]. Furthermore, acupoint ST36 is a critical site that modulates sympathetic and parasympathetic nervous systems since it is in the vicinity of peroneal, sciatic, and tibial nerves. Stimulation at ST36 impacts distal gut functions through anatomical proximity and influences upper gut functions through a functional connection with the central and vagal nerve systems [32; 33; 34]. Accordingly, we chose to use AA-treated IBS model rats, EMG as a surrogate for pain measurement, and ST36 as the focus of our research study.
Most interestingly, the findings of this comparative methodological study demonstrated similar ameliorating effects between unilateral and bilateral stimulation, and between direct ST36 stimulation and transcutaneous ST36 stimulation. These findings suggest a novel therapeutic approach for pain in IBS: unilateral transcutaneous ST36 stimulation. This unilateral TEA method will have several advantages: 1) it is completely noninvasive; 2) it can be self-administered at home since it does not use needles: 3) the unilateral stimulation (preferably use of a wireless wearable stimulator) does not interfere with daily activity of the user.
IBS is more predominant in women than in men, with a female-to-male ratio of 2–2.5:1 [26]. However, its pathophysiologic mechanisms are still unclear. While both men and women with IBS experience similar symptoms, including abdominal pain or discomfort, diarrhea, and constipation, women experience more abdominal pain and constipation-related symptoms. Sex hormones are thought to play a critical role that most influences the clinical manifestation and physiologic responses in men and women with IBS. Some research suggests that women may have increased sensitivity to visceral pain compared to men. This heightened sensitivity could contribute to differences in the perception and experience of IBS symptoms between genders [26; 35]. While IBS can significantly impact the quality of life for both men and women, studies have found that women with IBS may experience more severe symptoms and more significant impairment in quality of life compared to men. Understanding these similarities and differences in the medical care environment and applying them to IBS patients can help healthcare providers tailor treatment approaches for individuals with IBS.
Alteration in the inputs from the gut, known as afferent sensitization, is thought to play a crucial role in pain sensitization in patients with IBS [2; 36]. Under pathophysiological conditions, primary visceral afferent neurons, aka vagal afferent, convey pain signals from the viscera to the NTS [37]. On the other hand, spinal visceral afferent neurons from the intestinal tract are located in different spinal segments, and this viscerosomatic cross-organ sensitization may be involved in a central mechanism of nociceptive signaling. For example, increased expression of transient receptor potential vanilloid type-1 (TRPV1) contributes to visceral hypersensitivity and pain [38]. Thus, afferent sensitization is an important factor contributing to pain in IBS. EA at ST36 significantly decreased chronic visceral hypersensitivity and colon 5-HT3 receptor levels in AA-treated rats [39]. Moreover, EA decreased rectal sensitivity by decreasing TRPV1 in both colon and dorsal root ganglions [13]. Pre-EA at acupoint EX-B2 significantly reduced intracolonic formalin-induced visceral pain by decreasing p38 phosphorylation and c-Fos expression in the spinal cord and colon [40]. Colonic biopsies from IBS patients had elevated mucosal N-methyl-D-aspartate receptor (NMDAR) levels that were positively correlated with the severity and rate of recurrence of abdominal symptoms [41]. Clinical and animal studies demonstrated that administering NMDAR antagonist dextromethorphan in IBS patients and MK801 in mice blocked somatic and visceral hypersensitivity [41; 42]. Moreover, the injection of D-2-amino-5-phosphonopentanoate (AP5) into the rostral ventromedial medulla (RVM) inhibited visceral pain [43], and locus coeruleus-RVM circuit was found to be essential for the comorbidity of colorectal visceral pain [44]. EA at ST36 and ST37 improved visceral hyperalgesia, decreased c-Fos, and NMDAR expression in the RVM in IBS model rats [45], suggesting an analgesic effect of EA, which may mediated by inhibiting NMDAR activation in the RVM. These studies have suggested that EA desensitizes visceral and sensory afferents and improves visceral pain in IBS.
Chronic, low-grade inflammation is thought to play a critical role in the pathophysiology of IBS [46; 47]. Increased levels of inflammatory cytokines, including interleukin (IL)-6, IL-1β, IL-8, and tumor necrosis factor (TNF)-α, have been reported in the blood and serum of IBS model animals and IBS patients [48; 49; 50]. A clinical study demonstrated that increased levels of serotonin (5-HT) in IBS patients contributed to abdominal pain [51]. Thus, low-grade inflammation may contribute to pain in IBS patients. EA reduced pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6, and suppressed myeloperoxidase activity in the colon via the autonomic mechanism [20]. Another research showed that EA suppressed the expression of inflammatory cytokines, such as IL-8, IFN-γ, and TNF-α and in water avoidance stress (WAS) induced IBS mice and alleviated pain by suppressing the expression of inflammatory cytokines, such as IL-8, IFN-γ, and TNF-α [52]. Results from these studies suggest that EA may improve pain in IBS by reducing inflammation.
The role of the cholinergic anti-inflammatory pathway in reducing inflammation in GI disorders is well-documented [53; 54; 55]. This pathway functions through vagal efferent fibers that link to enteric neurons and release acetylcholine [56; 57]. Disruption of this pathway can synthesize pro-inflammatory cytokines, including TNF- α and IL-1, which may lead to intestinal mucosal inflammation, thus contributing to visceral pain. Upon parasympathetic activation, enteric neurons release acetylcholine, which interacts with α7 nicotinic acetylcholine receptors (α7nAChRs) on macrophages, inhibiting pro-inflammatory cytokine production [58]. Moreover, by activating parasympathetic outflow, the cholinergic anti-inflammatory pathway inhibits macrophage activation and regulates inflammation [53]. EA at ST36 restored the impaired colonic contraction and transit induced by rectal distension by enhancing vagal activity and mediated via the cholinergic pathway [59].
The gastrointestinal epithelium acts as a barrier, preventing the penetration of harmful substances in the lumen from other tissues via the intestinal mucosa. Human and animal studies have reported increased intestinal permeability in GI disorders [60; 61]. Previous studies reported that alteration in epithelial tight junctions (TJ) proteins, such as Zonula Occludens (ZO-1), Claudins, and Occludin, led to epithelial barrier dysfunction and contributed to the pathogenesis of IBS and pain [61; 62; 63]. EA increased ZO-1 and enhanced the repair of the intestinal mucosal barrier by decreasing corticotropin-releasing factor-receptor 1 expression in the gastrointestinal mucosa [64], as well as EA improved intestinal permeability by increasing the expression of TJ proteins in IBS mice and rats [52; 65]. Thus, EA may modulate TJ, improving mucosal barrier function and ameliorating visceral hypersensitivity and pain.
Mast cells are widely distributed in the colonic mucosa and release substances like histamine, proteases, growth factors, prostaglandins, and cytokines. These mediators were reported to increase the excitability of enteric [66] and primary afferent neurons [67], leading to visceral hypersensitivity [42]. Previous studies suggested that mast cell activation correlated with the severity of abdominal pain [51; 68]. Furthermore, mast cell dysfunction compromises epithelial barrier function, which alters mucosal permeability, potentially leading to altered bowel function and pain [69]. A clinical study demonstrated that the number and activity of mucosal mast cells in IBS patients positively correlated with the degree of intestinal permeability [70]. Thus, from these preclinical and human studies, it is clear that mast cells are more likely to be activated in patients with IBS, releasing mediators known to interact with nerve endings and trigger pain. A recent research study reported that the EA at ST36 acupoint ameliorates post-inflammation rectal hypersensitivity by down-regulating mast cells activated nerve growth factor and tropomyosin receptor kinase A [13].
Whole-brain imaging techniques such as functional magnetic resonance imaging (fMRI) [71; 72] have been used to assess the mechanism of pain and suggested that changes in brain structure and functional connections (FCs) correlate with pain in IBS patients. Alteration of the serotonergic signaling in the emotional arousal circuit has been reported in both male and female IBS patients, which contributes to visceral hypersensitivity [73]. Moreover, IBS patients had alterations in grey matter in brain areas associated with cognitive and evaluative functions [74; 75]. Abnormal FCs in brain areas, including the hippocampus, occipital gyrus, and cerebellum, have been reported in IBS patients, and acupuncture treatment has improved these FCs [71; 76]. EA may exert an analgesic effect in IBS by enhancing the FC between the hippocampus and various brain regions and modulating the default mode and sensorimotor networks [72]. Thus, EA alleviates visceral pain in IBS model rats by regulating the peripheral, central, and endocrine systems, reducing inflammation, improving colon permeability, stabilizing mast cell function, and altering brain activity.
In conclusion, transcutaneous ST36 stimulation is as effective as direct ST36 stimulation and unilateral ST36 stimulation is comparable to bilateral stimulation. Development of a novel therapy using unilateral transcutaneous ST36 stimulation is warranted.