[Electroacupuncture improves colonic mucosal barrier damage by regulating NLRP3/Caspase-1/GSDMD signaling pathway and inhibiting pyroptosis in ulcerative colitis mice]

Abstract

Objectives: To observe the effect of electroacupuncture (EA) at "Tianshu" (ST25) and "Zusanli" (ST36) on NOD-like receptor protein 3(NLRP3)/cysteine aspartate specific protease 1 (Caspase-1)/gasdermin D (GSDMD) pyroptosis signaling pathway in ulcerative colitis (UC) mice, so as to explore its mechanism in improving UC by protecting intestinal barrier.

Methods: Fifty male C57BL/6 mice were randomly divided into normal, model, EA, sham acupuncture and medication groups, with 10 mice in each group. The UC mouse model was established by 3% DSS solution free drinking for 7 consecutive days. The mice in the EA group received EA (2 Hz/10 Hz, 0.2 mA) at bilateral ST25 and ST36 for 20 min, while the mice in the sham acupuncture group received only sham acupuncture (light and shallow acupunture at ST25 and ST36). Mice in the medication group were orally administered mesalazine (33.4 g/kg) solution. All the interventions were performed once daily for a total of 7 days. The changes of body weight, stool shape and hematochezia of mice, the disease activity index (DAI) score, and the length of colon were recorded. The intestinal mucosal permeability was observed by in vivo small animal imaging system. HE staining was used to observe the pathological changes of colon tissue. TUNEL staining was used to observe the apoptosis of colon cells. The contents of interleukin (IL)-18 and IL-1β in serum were detected by ELISA. The average fluorescence intensity of tumor necrosis factor-α (TNF-α) and IL-6 in colon tissue was detected by immunofluorescence. The positive expression of IL-18 and IL-1β in colon tissue was detected by immunohistochemistry. The relative expression levels of zonula occludens-1 (ZO-1), Occludin, NLRP3, Caspase-1 and GSDMD in colon tissue were detected by Western blot. The mRNA expressions of NLRP3, Caspase-1 and GSDMD in colon tissue were detected by qPCR.

Results: After modeling, the DAI, inflammatory infiltration, number of apoptotic cells, fluorescence intensity of TNF-α, IL-6 and FITC, positive expression rate of IL-18 and IL-1β, mRNA and protein expression levels of NLRP3, Caspase-1 and GSDMD, serum IL-18 and IL-1β contents were significantly increased (P<0.01) in the model group relevant to the normal group. At the same time, the colon length, ZO-1 and Occludin protein expression were significantly reduced (P<0.01). The increased DAI, inflammatory infiltration, number of apoptotic cells, fluorescence intensity of TNF-α, IL-6 and FITC, positive rate of IL-18 and IL-1β, mRNA and protein expression levels of NLRP3, Caspase-1 and GSDMD, serum IL-18 and IL-1β contents, and the decreased colon length, ZO-1 and Occludin protein expression were all reversed after the EA and medication interventions compared with the model group and in the EA group than in the sham acupuncture group (P<0.01, P<0.05).

Conclusions: EA can protect the intestinal mucosal permeability in UC mice, which may be related to its functions in regulating the NLRP3/Caspase-1/GSDMD axis and inhibiting pyroptosis, thereby alleviating the inflammatory injury of intestinal mucosa.

Keywords: Electroacupuncture; Intestinal barrier; Pyroptosis; Ulcerative colitis.