Boosting mTOR-Dependent Autophagy Via Upstream TLR4-MyD88-MAPK Signaling and Downstream NF-κB Pathway Quenches Intestinal Inflammation and Oxidative Stress Injury

Abstract

Background and Aims: Defective autophagy has been perceived as a significant event in a growing number of autoimmune and inflammatory diseases such as rheumatoid arthritis and lupus. However, the precise role of Mechanistic target of rapamycin (mTOR)-dependent autophagy and its underlying regulatory mechanisms in intestinal epithelium in response to inflammation and oxidative stress remain poorly understood. Methods: The levels of p-mTOR, LC3B, p62 and autophagy activity in mice and LPS-treated cells were examined by immunobloting, immunohischemistry, confocal microscopy and transmission electron microscope (TEM). We evaluated expression of IL-1β, IL-8, TNF-α, MDA, SOD and T-AOC after silencing mTOR and ATG5 by quantitative real time-polymerase chain reaction (qRT-PCR) and commercially available kits. In vivo modulation of mTOR and autophagy was achieved by using AZD8055, rapamycin and 3-Methyladenine. Finally, to testify the involvement of TLR4 signaling and NF-κB pathway in cells and active Ulcerative Colitis (UC) patients, immunofluorescence, qRT-PCR, immunoblotting and TEM were performed to access its relevance to autophagy and inflammation. Results: The mTOR-dependent autophagic flux impairment in murine model of colitis, human epithelial cells and active UC patients may probably be regulated by TLR4-MyD88-MAPK signaling and NF-κB pathway. Silencing mTOR remarkably attenuated, whereas inhibiting ATG5 aggravated, LPS-induced inflammation and oxidative injury. Pharmacological administration of mTOR inhibitors and autophagy stimulators markedly ameliorated experimental colitis and oxidative stress in vivo. Conclusions: Our findings not only shed novel insights into the regulatory mechanism for mTOR-dependent autophagy but also provided potential therapeutic targets for intestinal inflammatory diseases like refractory inflammatory bowel disease. Funding: This work was supported by National Natural Science Foundation of China (Grant no. 81370485). Conflicts of Interest: The authors declare no conflicts of interest. Ethics Approval Statement: The study was approved by Research and Ethics committee of Xinhua Hospital. Written informed consent was also obtained from all subjects before the study protocol.