[Comparative study on intervention effects of electroacupuncture at single point "Zusanli" (ST36), "Sanyinjiao" (SP6) and combined points on irritable bowel syndrome mice].

OBJECTIVES: To compare the differences in interventional effects among three stimulation modalities:electroacupuncture (EA) at single acupoint "Zusanli" (ST36), single acupoint "Sanyinjiao" (SP6), and their combination (ST36 + SP6), on behavioral and histological outcomes in a mouse model of irritable bowel syndrome (IBS), and to examine the differential neuronal responses in the peripheral and central nervous systems under these modalities, so as to provide experimental evidence for exploring the neural mechanisms underlying acupoint combination stimulation in IBS treatment.

METHODS: An IBS model was established in male C57BL/6J mice by intrarectal instillation of zymosan. The mice were randomly divided into blank control group, model group, ST36 group, SP6 group, and acupoint combination group (n=20 per group except n=18 for the model group). After 7 consecutive days of EA stimulation (2 Hz/100 Hz, 1 mA, 15 min per session, once daily), abdominal mechanical hyperalgesia (referred pain) was assessed using von Frey filaments, motor function by rotarod test, and 3D spontaneous fine behavioral characteristics by an AI-based unsupervised 3D behavioral analysis system. Colon tissue structure and inflammatory levels were observed by HE staining, and colonic inflammatory cytokine expressions were detected by Western blot, to evaluate the symptom-relieving effects of single versus combined acupoint stimulation. For neural tracing, retrograde viral tracers were injected into ST36 and SP6 to label dorsal root ganglion (DRG) neurons innervating these acupoints in C57BL/6J mice. In vivo two-photon calcium imaging was performed to record DRG neuronal responses to different EA modalities (1 mA, 1 min) in Thy1-GCaMP6s mice, and activity-dependent neuronal labeling was applied using transgenic TRAP2;Ai14 mice to identify central neurons activated by EA (2 Hz/100 Hz, 1 mA, 15 min per session), aiming to reveal peripheral-central neuronal response differences between single and combined acupoint stimulation.

RESULTS: Compared with the blank control group, the model group showed increased abdominal mechanical withdrawal frequency and area under the curve (P<0.001, P<0.05), decreased motor function (latency to fall and rotarod speed at fall, P<0.01), aggravated colonic tissue damage and inflammatory responses (P<0.001), elevated expressions of interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α, P<0.001), and altered spontaneous behavioral patterns (P<0.001, P<0.01, P<0.05). All three EA modalities significantly alleviated abdominal mechanical hyperalgesia in IBS mice (P<0.001, P<0.05, P<0.01), with the combined stimulation showing a larger effective interventional window than either single acupoint. Compared with single-acupoint stimulation, combined stimulation also more effectively restored colonic tissue damage, reduced TNF-α expression (P<0.05), and shortened the duration of spontaneous arching episodes and altered the transition patterns among different spontaneous behavioral actions (P<0.01, P<0.05). DRG neurons exhibited highly heterogeneous responses to the three stimulation modalities, which could be classified into 3 major categories and 7 subcategories based on their firing characteristics. Furthermore, combined stimulation activated a greater number of DRG neurons than single acupoint stimulation (P<0.05), and the neuronal calcium signal intensity was also higher (P<0.05). Finally, significant differences were observed in the number of responsive neurons across different brain regions under the three modalities. The activated neuronal populations evoked by ST36, SP6, and combined stimulation showed maximal overlap in the paraventricular nucleus of the hypothalamus (PVN), whereas the overlap was minimal in the insular cortex (IC), suggesting that IC neuronal activation requires the synergistic effect of combined acupoint stimulation.

CONCLUSIONS: Combined stimulation at ST36 and SP6 produces superior therapeutic effects over single acupoint stimulation in ameliorating abdominal mechanical hyperalgesia, colonic structural damage, inflammatory status, and spontaneous behavioral changes induced by visceral pain in IBS model mice, and this advantage may arise from the synergistic integration of peripheral and central neuronal responses to different EA signals.