Background and aims Defective autophagy has 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 target of rapamycin (mTOR)-dependent autophagy and its underlying regulatory mechanisms in the intestinal epithelium in response to inflammation and oxidative stress remain poorly understood. Methods The levels of p-mTOR, LC3B, p62 and autophagy in 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 autophagy was achieved by using AZD8055, rapamycin and 3-methyladenine. Finally, to verify the involvement of TLR4 signalling and the NF-κB pathway in cells and active ulcerative colitis (UC) patients, immunofluorescence, qRT-PCR, immunoblotting and TEM were performed to determine TLR4 signalling relevance to autophagy and 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 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 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.

β-catenin, a core component of Wnt/β-catenin signaling, has been shown to be an important regulator of cellular proliferation and differentiation. Abnormal activation of Wnt/β-catenin signaling promotes tissue fibrogenesis. In the present study, the role of β-catenin during liver fibrogenesis was analyzed and the functional effects of β-catenin gene silencing in hepatic stellate cells (HSCs) using small interfering (si)RNA were investigated. The expression of β-catenin in human hepatic fibrosis tissues of different grades and normal human hepatic tissues was examined using immunohistochemistry. To inhibit the Wnt/β-catenin signaling pathway, siRNA for β-catenin was developed and transiently transfected into HSC-T6 cells using Lipofectamine 2000. β-catenin expression was evaluated by quantitative polymerase chain reaction (qPCR) and western blot analysis. The expression of collagen types I and III was evaluated by qPCR and immunofluorescent staining. Cellular proliferation and the cell cycle were analyzed using a methyl thiazolyl tetrazolium assay. Apoptosis was assessed by Annexin V staining. A higher expression level of β-catenin was identified in the patients with high-grade hepatic fibrosis in comparison with that of the normal controls. Additionally, β-catenin siRNA molecules were successfully transfected into HSCs and induced inhibition of β-catenin expression in a time-dependent manner. β-catenin siRNA treatment also inhibited synthesis of collagen types I and I in transfected HSCs. Furthermore, compared with those of the control group, siRNA-mediated knockdown of β-catenin in HSC-T6 cells inhibited cell proliferation and resulted in cell apoptosis. This study suggests a significant functional role for β-catenin in the development of liver fibrosis and demonstrates that downregulation of the Wnt/β-catenin signaling pathway inhibits HSC activation. Thus, this study provides a novel strategy for the treatment of hepatic fibrosis.

Background & Aims: It has recently been reported that thymosin beta-4 (Tβ4) has anti-fibrogenic effects in human hepatic stellate cells (HSCs) in vitro, but the mechanisms underlying these effects remain unclear. The aim of this study was to investigate the roles of Tβ4 in the proliferation, migration, and activation of HSCs. Methods: Enzyme-linked immunosorbent assays (ELISA), immunohistochemistry, and western blot assays were utilized to determine the expression levels of Tβ4 in serum, liver tissues, and LX-2 cells. Tβ4 was depleted in LX-2 cells using small interfering RNAs (siRNAs). Cell proliferation was analyzed using cell counting kit-8 (CCK-8) viability assays, and cell migration was investigated using wound-healing and transwell migration assays. Results: The expression of Tβ4 was significantly reduced during the progression of liver fibrosis. The depletion of Tβ4 significantly promoted the proliferation and migration of LX-2 cells via the activation of the PI3K/Akt signaling pathway. The pro-migratory and pro-proliferative effects of Tβ4 depletion in LX-2 cells can be counteracted by treatment with the Akt inhibitor MK-2206. In addition, Tβ4 depletion was also associated with the activation of HSCs via the enhanced expression of α-smooth muscle actin (α-SMA) and vimentin. Conclusions: Our results suggest that Tβ4 participates in liver fibrosis by inhibiting the migration, proliferation, and activation of HSCs and that Tβ4 may be an effective target in the treatment of liver fibrosis.