[Study on the mechanism of moxibustion in improving autophagy of intestinal epithelial cells in rats with diarrhea-predominant irritable bowel syndrome based on PI3K/AKT/mTOR signaling pathway].

Objectives: To observe the effect of moxibustion on visceral hypersensitivity and the phosphatidylinositol-3 kinase (PI3K) /protein kinase B (AKT) /mammalian target of rapamycin (mTOR) signaling pathway in rats with diarrhea-predominant irritable bowel syndrome (IBS-D), so as to explore its mechanisms underlying amelioration of pain reaction of IBS-D by regulating the autophagy level of intestinal epithelial cells and protecting the intestinal mucosal barrier.

Methods: Forty male SD rats were randomly divided into control, model, moxibustion and moxibustion+LY294002 groups, with 10 rats in each group. The IBS-D model was established by acetic acid enema + chronic binding method. In the moxibustion group, the rats were given suspended moxibustion on bilateral "Tianshu" (ST25) and "Shangjuxu" (ST37) for 20 minutes. Rats of the moxibustion+LY294002 group received intraperitoneal injection of PI3K inhibitor LY294002 (0.75 mg/kg) 30 min before each moxibustion intervention. Both groups were treated once a day for 7 days. The minimum colon-rectal water injection volume (minimum water-injection volume) for achieving a score of 3 points of the abdominal withdrawal reflex (AWR) was measured to assess the rats' visceral pain reaction before and after modeling, and after the intervention. H.E. staining was used to observe inflammatory damage of the colonic mucosa. A transmission electron microscopy was employed to examine the mitochondrial structure and the number of autophagosomes in the colon mucosa. The expressions of Occludin, ZO-1, Claudin-2, p62 proteins, and the ratios of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR and LC3B-Ⅱ/Ⅰ were detected by Western blot. The mRNA expression levels of PI3K, AKT, mTOR, Occludin, ZO-1 and Claudin-2 in the colon tissues were detected by qPCR.

Results: Compared with the control group, the minimum water injection volume, and the expression levels of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR ratios, Occludin, ZO-1 and p62 proteins and the mRNA expression of PI3K, AKT, mTOR, Occludin and ZO-1 were significantly decreased (P<0.01), while the protein and mRNA expression level of Claudin-2 and the ratio of LC3B-Ⅱ/Ⅰ were strikingly increased in the model group (P<0.01). H.E. staining showed disordered arrangement of the intestinal epithelial cells, and infiltration of local inflammatory cells, and electron microscopy observation showed multiple autophagosomes in the model group. In comparison with the model group, the minimum water injection volume, the p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR ratios, the protein expression levels of Occludin, ZO-1 and p62 and the mRNA expressions of PI3K, AKT, mTOR, Occludin and ZO-1 in the colon tissue were considerably increased (P<0.05, P<0.01), while the protein and mRNA expression of Claudin-2 and the ratio of LC3B-Ⅱ/Ⅰ were apparently decreased in the moxibustion group (P<0.01). After administration of PI3K inhibitor, the above effects of moxibustion were eliminated (P<0.01, P<0.05). In the moxibustion group, the intestinal mucosal structure was relatively intact, inflammatory cell infiltration was decreased, and the quantity of autophagosomes under the electron microscope was reduced, while in the moxibustion+LY294002 group, the intestinal mucosal structure was relatively disordered, the inflammatory cell infiltration was increased, and the quantity of autophagosomes was increased.

Conclusions: Moxibustion can relieve visceral pain of IBS-D rats, which may be related to its functions in alleviating intestinal epithelial damage and protecting the intestinal mucosal barrier by activating the PI3K/AKT/mTOR signaling pathway and reducing the level of autophagy of the intestinal epithelial cells.