Herbal cake-partitioned moxibustion inhibits colonic autophagy in Crohn's disease signaling involving distinct classes of phosphatidylinositol 3-kinases.

Background: Autophagy is an evolutionarily conserved biological process in eukaryotic cells that involves lysosomal-mediated degradation and recycling of related cellular components. Recent studies have shown that autophagy plays an important role in the pathogenesis of Crohn's disease (CD). Herbal cake-partitioned moxibustion (HM) has been historically practiced to treat CD. However, the mechanism by which HM regulates colonic autophagy in CD remains unclear.

Aim: To observe whether HM can alleviate CD by regulating colonic autophagy and to elucidate the underlying mechanism.

Methods: Rats were randomly divided into a normal control (NC) group, a CD group, an HM group, an insulin + CD (I + CD) group, an insulin + HM (I + HM) group, a rapamycin + CD (RA + CD) group, and a rapamycin + HM (RA + HM) group. 2,4,6-trinitrobenzenesulfonic acid was administered to establish a CD model. The morphology of the colonic mucosa was observed by hematoxylin-eosin staining, and the formation of autophagosomes was observed by electron microscopy. The expression of autophagy marker microtubule-associated protein 1 light chain 3 beta (LC3B) was observed by immunofluorescence staining. Insulin and rapamycin were used to inhibit and activate colonic autophagy, respectively. The mRNA expression levels of phosphatidylinositol 3-kinase class I (), , , sequestosome 1 (), and mammalian target of rapamycin () were evaluated by RT-qPCR. The protein expression levels of interleukin 18 (IL-18), tumor necrosis factor-α (TNF-α), nuclear factor κB/p65 (NF-κB p65), LC3B, p62, coiled-coil myosin-like BCL2-interacting protein (Beclin-1), p-mTOR, PI3KC1, class III phosphatidylinositol 3-kinase (PI3KC3/Vps34), and p-Akt were evaluated by Western blot analysis.

Results: Compared with the NC group, the CD group showed severe damage to colon tissues and higher expression levels of IL-18 and NF-κB p65 in colon tissues ( < 0.01 for both). Compared with the CD group, the HM group showed significantly lower levels of these proteins ( < 0.01 and < 0.05). There were no significant differences in the expression of TNF-α protein in colon tissue among the rat groups. Typical autophagic vesicles were found in both the CD and HM groups. The expression of the autophagy proteins LC3B and Beclin-1 was upregulated ( < 0.01 for both) in the colon tissues of rats in the CD group compared with the NC group, while the protein expression of p62 and p-mTOR was downregulated ( < 0.01 for both). However, these expression trends were significantly reversed in the HM group compared with the CD group ( < 0.01, < 0.05, < 0.05, and < 0.05). Compared with those in the RA + CD group, the mRNA expression levels of , , , and in the RA + HM group were significantly higher ( < 0.01 and < 0.05), while those of LC3B were significantly lower ( < 0.05). Compared with the RA + CD group, the RA + HM group exhibited significantly higher PI3KC1, p-Akt1, and p-mTOR protein levels ( < 0.01, < 0.05, and < 0.01), a higher p62 protein level ( = 0.057), and significantly lower LC3B and Vps34 protein levels ( < 0.01 for both) in colon tissue.

Conclusion: HM can activate PI3KC1/Akt1/mTOR signaling while inhibiting the PI3KC3 (Vps34)-Beclin-1 protein complex in the colon tissues of CD rats, thereby inhibiting overactivated autophagy and thus exerting a therapeutic effect.