Published online Oct 21, 2020. doi: 10.3748/wjg.v26.i39.5997
Peer-review started: April 16, 2020
First decision: May 1, 2020
Revised: July 14, 2020
Accepted: September 4, 2020
Article in press: September 4, 2020
Published online: October 21, 2020
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.
To observe whether HM can alleviate CD by regulating colonic autophagy and to elucidate the underlying mechanism.
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 (PI3KC1), Akt1, LC3B, sequestosome 1 (p62), and mammalian target of rapamycin (mTOR) 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.
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 (P < 0.01 for both). Compared with the CD group, the HM group showed significantly lower levels of these proteins (PIL-18 < 0.01 and Pp65 < 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 (P < 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 (P < 0.01 for both). However, these expression trends were significantly reversed in the HM group compared with the CD group (PLC3B < 0.01, PBeclin-1 < 0.05, Pp62 < 0.05, and Pm-TOR < 0.05). Compared with those in the RA + CD group, the mRNA expression levels of PI3KC1, Akt1, mTOR, and p62 in the RA + HM group were significantly higher (PPI3KC1 < 0.01 and PAkt1, mTOR, and p62 < 0.05), while those of LC3B were significantly lower (P < 0.05). Compared with the RA + CD group, the RA + HM group exhibited significantly higher PI3KC1, p-Akt1, and p-mTOR protein levels (PPI3KC1 < 0.01, Pp-Akt1 < 0.05, and Pp-mTOR < 0.01), a higher p62 protein level (P = 0.057), and significantly lower LC3B and Vps34 protein levels (P < 0.01 for both) in colon tissue.
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.
Core Tip: Here, we demonstrated that overactivation of colonic autophagy can be observed in a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced rat Crohn’s Disease (CD) model. Herbal cake-partitioned moxibustion ameliorated TNBS-induced inflammation and colon damage, facilitated the repair of colonic epithelial cells, and inhibited colonic overactivated autophagy by activating the phosphatidylinositol 3-kinase class I/protein kinase B akt-1/mammalian target of rapamycin signaling pathway while inhibiting the class III phosphatidylinositol 3-kinase -coiled-coil myosin-like BCL2-interacting protein (Beclin-1) complex in CD rats.