Mechanism study on electroacupuncture regulating IL-4/JAK1/STAT6 signaling pathway to promote M2 macrophage polarization and improve intestinal mucosal barrier injury in mice with ulcerative colitis

To observe the effect of electroacupuncture(EA) intervention on the interleukin-4 (IL-4)/Janus kinase 1 (JAK1)/signal transducer and activator of transcription 6 (STAT6) signaling pathway in colon tissue of chronic ulcerative colitis (UC) mouse model, in order to explore the potential mechanism of EA in promoting M2 macrophage polarization and improving intestinal mucosal barrier injury in UC mice.

A total of 56 eight-week-old SPF BALB/c mice (28 males and 28 females) were stratified by sex and randomly assigned to blank group (15 mice) and modeling group (41 mice). 3% DSS solution was used to induce the chronic UC model. The successfully induced mice were further divided into model, EA+11B11, and EA groups. Finally, 12 mice were included in each group. Mice in the EA+11B11 group received intraperitoneal injection of IL-4 inhibitor 11B11 (10 mg/kg) before EA, and mice in the EA group were treated with EA (10 Hz/50 Hz, 1 mA) at “Guanyuan” (CV4), “Tianshu” (ST25), “Zusanli” (ST36) and “Shangjuxu” (ST37) for 20 min. The treatments were performed once daily for 14 consecutive days. After the intervention, the following aspects were evaluated. ① Symptom improvement: including disease activity index (DAI) score, macroscopic morphology score in colon, and hematoxylin-eosin (HE) staining to observe the pathological changes in colon tissue; ②Structure and function of intestinal mucosal barrier: including the observation of colonic tight junction structure by transmission electron microscopy, detection of intestinal leakage by in vivo imaging with fluorescein isothiocyanate-4D (FITC-4D), detection of the positive expressions of tight junction protein ZO-1 and occludin in colon tissue by immunofluorescence staining; ③ Activation of IL-4/JAK1/STAT6 signaling pathway: detection of the relative expression levels of IL-4, p-JAK1/JAK1, and p-STAT6/STAT6 proteins in colon tissue by Western blot; ④ Markers of M2 macrophage polarization: detection of the CD206⁺/CD86⁺ ratio in mesenteric lymph nodes, and CD206 and arginase-1 (Arg-1) positive expressions in colon tissue by flow cytometry and immunofluorescence staining, respectively.

Compared with the blank group, mice in the model group showed significantly increased DAI score and macroscopic morphology score (P<0.05), disorganized colonic mucosal structure, severe intestinal fluorescence leakage, significantly decreased positive expressions of ZO-1 and occludin (P<0.05), reduced relative expression levels of IL-4, p-JAK1/JAK1, and p-STAT6/STAT6 proteins (P<0.05), as well as decreased CD206⁺/CD86⁺ ratio and positive expressions of CD206 and Arg-1 (P<0.05). Compared with the model group, the EA group exhibited reduced DAI and macroscopic morphology scores (P<0.05), improved colonic mucosal structure, and alleviated intestinal fluorescence leakage. The positive expressions of ZO-1 and occludin, and relative expression levels of IL-4, p-JAK1/JAK1, and p-STAT6/STAT6 proteins were significantly increased (P<0.05), along with elevated CD206⁺/CD86⁺ ratio, and CD206 and Arg-1 positive expressions (P<0.05). Compared with the EA group, the EA+11B11 group showed higher DAI and macroscopic morphology scores (P<0.05), disrupted colonic mucosal structure, obvious intestinal fluorescence leakage, decreased positive expressions of ZO-1 and occludin, and relative protein expression levels of IL-4, p-JAK1/JAK1, and p-STAT6/STAT6 (P<0.05), as well as reduced CD206⁺/CD86⁺ ratio, CD206 and Arg-1 positive expressions (P<0.05).

EA can promote the repair of colonic mucosal barrier in mice with chronic UC and alleviate symptoms such as diarrhea and hematochezia, which may be associated with the activation of IL-4/JAK1/STAT6 signaling pathway and the inducing of M2 macrophage polarization in colon tissue.