Boosting mTOR-dependent autophagy via upstream TLR4-MyD88-MAPK signalling and downstream NF-κB pathway quenches intestinal inflammation and oxidative stress injury
2018
Abstract Background and aims Defective
autophagyhas been proposed as an important event in a growing number of autoimmune and inflammatory diseases such as rheumatoid arthritis and lupus. However, the precise role of
mechanistic targetof
rapamycin(mTOR)-dependent
autophagyand its underlying regulatory mechanisms in the
intestinal epitheliumin response to inflammation and oxidative stress remain poorly understood. Methods The levels of p-mTOR, LC3B, p62 and
autophagyin mice and LPS-treated cells were examined by immunoblotting, immunohistochemistry, confocal microscopy and transmission electron microscopy (TEM). We evaluated the expression of IL-1β, IL-8, TNF-α, MDA, SOD and T-AOC by quantitative real time-polymerase chain reaction (qRT-PCR) and commercially available kits after silencing of mTOR and
ATG5. In vivo modulation of mTOR and
autophagywas achieved by using AZD8055, rapamycin and 3-methyladenine. Finally, to verify the involvement of
TLR4signalling and the NF-κB pathway in cells and active ulcerative colitis (UC) patients, immunofluorescence, qRT-PCR, immunoblotting and TEM were performed to determine
TLR4signalling relevance to
autophagyand inflammation. Results The mTOR-dependent autophagic flux impairment in a murine model of colitis, human intestinal epithelial cells and active UC patients is probably regulated by
TLR4-MyD88-MAPK signalling and the NF-κB pathway. Silencing mTOR remarkably attenuated, whereas inhibiting
ATG5aggravated, LPS-induced inflammation and oxidative injury. Pharmacological administration of mTOR inhibitors and
autophagystimulators markedly ameliorated experimental colitis and oxidative stress in vivo. Conclusions Our findings not only shed light on the regulatory mechanism of mTOR-dependent
autophagy, but also provided potential therapeutic targets for intestinal inflammatory diseases such as refractory inflammatory bowel disease.
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