Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Oct 21, 2020; 26(39): 5997-6014
Published online Oct 21, 2020. doi: 10.3748/wjg.v26.i39.5997
Herbal cake-partitioned moxibustion inhibits colonic autophagy in Crohn’s disease via signaling involving distinct classes of phosphatidylinositol 3-kinases
Shi-Yuan Wang, Ji-Meng Zhao, Ci-Li Zhou, Han-Dan Zheng, Yan Huang, Min Zhao, Zhi-Ying Zhang, Lu-Yi Wu, Huan-Gan Wu, Hui-Rong Liu
Shi-Yuan Wang, Ji-Meng Zhao, Ci-Li Zhou, Han-Dan Zheng, Yan Huang, Min Zhao, Zhi-Ying Zhang, Lu-Yi Wu, Huan-Gan Wu, Hui-Rong Liu, Key Laboratory of Acupuncture-Moxibustion and Immunology, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
Shi-Yuan Wang, Ji-Meng Zhao, Ci-Li Zhou, Han-Dan Zheng, Yan Huang, Min Zhao, Zhi-Ying Zhang, Huan-Gan Wu, Hui-Rong Liu, Shanghai Research Institute of Acupuncture and Meridian, Shanghai 200030, China
Author contributions: Wang SY, Huang Y, Wu HG, and Liu HR designed the research; Wang SY, Zhao JM, Zhou CL, and Zheng HD performed the experiments; Wang SY, Zhao M, Zhang ZY, and Wu LY collected and analyzed the data; Wang SY wrote the manuscript; all authors reviewed the manuscript prior to its submission, and read and approved the final manuscript.
Supported by the Program of Shanghai Academic Research Leader, No. 17XD1403400; National Natural Sciences Foundation of China, No. 81574079 and No. 81873374; Three-year Action Plan Project of Shanghai Traditional Chinese Medicine Development, No. ZY(2018-2020)-CCCX-2004-01; Chinese Medicine Inheritance and Innovation "100 Million" Talent Project, Qi Huang Scholar; and Shanghai Rising-Star Program, No. 16QA1403400.
Institutional review board statement: This study did not involve human subjects.
Institutional animal care and use committee statement: All animal experiments in this study were performed under guidelines approved by the Animal Ethics Committee of the Shanghai University of Traditional Chinese Medicine (No. PZSHUTCM200403009).
Conflict-of-interest statement: The authors declare no conflicts of interest.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Hui-Rong Liu, MD, PhD, Doctor, Professor, Research Fellow, Key Laboratory of Acupuncture-Moxibustion and Immunology, Shanghai University of Traditional Chinese Medicine, No. 650 South Wanping Road, Xuhui District, Shanghai 201203, China.
Received: April 16, 2020
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.

Keywords: Crohn’s disease, Colon, Moxibustion, Macroautophagy, Immunity, Phosphatidylinositol 3-kinase signaling

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.