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.

Intestinal fibrosis is one of the major serious complications of Crohn’s disease (CD). However, there are no effective antifibrotic drugs to treat intestinal fibrosis in CD. Therefore, it is important to understand the pathogenesis of fibrosis in CD. It has been reported that members of the miR-200 family are essential in the regulation of renal fibrogenesis. In this study, we analyzed the function of miR-200a and miR-200b in intestinal fibrosis, which was induced by transforming growth factor β1 (TGF-β1) in vitro. Furthermore, we detected the expression of miR-200a and miR-200b in CD specimens, which were divided into groups of fibrosis and no-fibrosis. The results of this study showed that administration of miR-200b could partially protect intestinal epithelial cells from fibrogenesis in vitro. Furthermore, we found that miR-200b was overexpressed in the serum of the fibrosis group. The results suggest that miR-200b has potential value for diagnostic and therapeutic applications for CD patients with fibrosis complications.

This study was undertaken to evaluate the efficacy of infliximab (IFX) in treatment of Crohn's disease (CD) patients. 106 CD patients were undergoing treatment with IFX from five hospitals in Shanghai, China. Clinical remission to IFX induction therapy was defined as Crohn's disease activity index (CDAI) < 150. Clinical response was assessed by a decrease in CDAI ≥ 70, and the failure as a CDAI was not significantly changed or increased. Ten weeks after therapy, 61 (57.5%) patients achieved clinical remission, 17 (16.0%) had clinical response, and the remaining 28 (26.4%) were failed. In remission group, significant changes were observed in CDAI, the Simple Endoscopic Score for Crohn's Disease (SES-CD), and serum indexes. Patients with short disease duration (22.2 ± 23.2 months) and luminal lesions showed better effects compared to those with long disease duration (71.0 ± 58.2 months) or stricturing and penetrating lesions. IFX markedly downregulated Th1/Th17-mediated immune response but promoted IL-25 production in intestinal mucosa from remission group. No serious adverse events occurred to terminate treatment. Taken together, our studies demonstrated that IFX is efficacious and safe in inducing clinical remission, promoting mucosal healing, and downregulating Th1/Th17-mediated immune response in short course CD patients with luminal lesions.

Tight junction dysfunction plays a vital role in some chronic inflammatory diseases. Pro-inflammatory cytokines, especially tumor necrosis factor alpha (TNF-α), act as important factors in intestinal epithelial tight junction dysfunction during inflammatory conditions. Autophagy has also been shown to be crucial in tight junction function and claudin-2 expression, but whether autophagy has an effect on the change of claudin-2 expression and tight junction function induced by TNF-α is still unknown. To answer this question, we examined the expression of claudin-2 protein, transepithelial electrical resistance (TER), and permeability of cell monolayers, autophagy flux change, and lysosomal pH after TNF-α with or without PP242 treatment. Our study showed that claudin-2 expression, intestinal permeability, microtubule-associated protein 1 light chain 3B II (LC3B-II) and sequestosome 1 (P62) expression largely increased while TER values decreased in TNF-α treated cell monolayers. Further research using 3-methyladenine (3-MA), bafilomycin A1, and ad-mCherry-GFP-LC3B adenovirus demonstrated that LC3B-II increase induced by TNF-α was attributed to the inhibition of autophagic degradation. Moreover, both qualitative and quantitative method confirmed the increase of lysosomal pH, and mammalian target of rapamycin (mTOR) inhibitor PP242 treatment relieved this elevation. Moreover, PP242 treatment also alleviated the change of autophagy flux, TER, and claudin-2 expression induced by TNF-α. Therefore, we conclude that increase of claudin-2 levels and intestinal epithelial tight junction dysfunction are partly caused by the inhibition of autophagic degradation in TNF-α treated cell monolayers.

This study suggests a significant fun-ctional role for HMGB1 in the development of liver fibrosis. It also demonstrates that downregulation of HMGB1 expression might be a potential strategy to treat liver fibrosis.

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