细胞外囊泡在肝纤维化诊治中的研究进展

细胞外囊泡(extracellular vesicles, EVs)介导细胞间的物质信号交流参与肝纤维化的发生发展,肝星状细胞(hepatic stellate cells, HSCs)和肝实质细胞分泌的EVs携带microRNA能激活邻近的HSCs,上调TGF-β信号通路,导致结缔组织生长因子表达增加,最终导致α-平滑肌肌动蛋白和胶原蛋白分泌增加,从而介导肝纤维化.另一方面,健康群体和间充质干细胞分泌的EVs对于肝纤维化起到一定的治疗作用.本文就EVs的结构、来源、功能及介导肝纤维化的发生发展与诊治的机制作一综述.

Signal transduction mechanism of TRB3 in rats with non-alcoholic fatty liver disease

AIM:To evaluate the possible role of Tribble 3(TRB3) in a rat model of non-alcoholic fatty liver disease(NAFLD) and its signal transduction mechanism.METHODS:Thirty Sprague-Dawley rats were randomized into three groups:normal control group,non-alcoholic fatty liver group A(fed on a high-fat diet for 8 wk) and group B(fed on a highfat diet for 16 wk) .To determine the degree of hepatic steatosis in rats of each group,livers were stained with hematoxylin and eosin,and evaluated;realtime fluorescent quantitative reverse transcriptase-polymerase chain reaction was performed to measure the expression levels of TRB3 mRNA;and Western blotting analysis was done to determine the expression levels of protein kinase B(Akt) and phosphorylated protein kinase B(p-Akt-Thr308,p-Akt-Ser473) .RESULTS:Hepatic steatosis was evident in both NAFLD groups:mild to moderate hepatic steatosis occurred in group A,mainly as mild steatosis.Moderate to severe hepatic steatosis occurred in group B,mainly as severe steatosis.The expression level of TRB3 mRNA in group B was signifi cantly higher than in the control group(122.28 ± 95.37 vs 3.06 ± 2.33,P = 0.001) and group A(122.28 ± 95.37 vs 5.77 ± 4.20,P = 0.001) .There was no signif icant difference in the expression levels of Akt(1.03 ± 0.53 vs 1.12 ± 0.77,P = 0.729) and p-Akt-Thr308(0.82 ± 0.45 vs 0.92 ± 0.38,P = 0.592) between group A and the control group.The expression level of Akt and p-Akt-Thr308 in group B was signifi cantly lower than in group A(Akt 0.41 ± 0.16 vs 1.12 ± 0.77,P = 0.008;p-Akt-Thr308 0.47 ± 0.19 vs 0.82 ± 0.45,P = 0.036) and the control group(Akt 0.41 ± 0.16 vs 1.03 ± 0.53,P = 0.018;p-Akt-Thr308 0.47 ± 0.19 vs 0.92 ± 0.38,P = 0.010) .The expression level of p-Akt-Ser473 in group A was signif icantly higher than in group B(1.48 ± 0.50 vs 0.81 ± 0.39,P = 0.041) as well as the control group(1.48 ± 0.50 vs 0.45 ± 0.26,P = 0.003).CONCLUSION:TRB3 blocks insulin signaling by inhibiting Akt activation,which contributes to insulin resistance.It may be an important factor in the occurrence and development of NAFLD.

Givinostat inhibition of hepatic stellate cell proliferation and protein acetylation

AIM: To explore the effect of the histone deacetylase inhibitor givinostat on proteins related to regulation of hepatic stellate cell proliferation.METHODS: The cell counting kit-8 assay and flow cytometry were used to observe changes in proliferation, apoptosis, and cell cycle in hepatic stellate cells treated with givinostat. Western blot was used to observe expression changes in p21, p57, CDK4, CDK6, cyclin D1, caspase-3, and caspase-9 in hepatic stellate cells exposed to givinostat. The scratch assay was used to analyze the effect of givinostat on cell migration. Effects of givinostat on the reactive oxygen species profile, mitochondrial membrane potential, and mitochondrial permeability transition pore opening in JS-1 cells were observed by laser confocal microscopy.RESULTS: Givinostat significantly inhibited JS-1 cell proliferation and promoted cell apoptosis, leading to cell cycle arrest in G0/G1 phases. Treatment with givinostat downregulated protein expression of CDK4, CDK6, and cyclin D1, whereas expression of p21 and p57 was significantly increased. The givinostat-induced apoptosis of hepatic stellate cells was mainly mediatedthrough p38 and extracellular signal-regulated kinase 1/2. Givinostat treatment increased intracellular reactive oxygen species production, decreased mitochondrial membrane potential, and promoted mitochondrial permeability transition pore opening. Acetylation of superoxide dismutase(acetyl K68) and nuclear factor-κB p65(acetyl K310) was upregulated, while there was no change in protein expression. Moreover, the notable beneficial effect of givinostat on liver fibrosis was also confirmed in the mouse models.CONCLUSION: Givinostat has antifibrotic activities via regulating the acetylation of nuclear factor-κB and superoxide dismutase 2, thus inhibiting hepatic stellate cell proliferation and inducing apoptosis.

Glucose deprivation induces chemoresistance in colorectal cancer cells by increasing ATF4 expression

AIM: To investigate the role of activating transcription factor 4(ATF4) in glucose deprivation(GD) induced colorectal cancer(CRC) drug resistance and the mechanism involved.METHODS: Chemosensitivity and apoptosis were measured under the GD condition. Inhibition of ATF4 using short hairpin RNA in CRC cells under the GD condition and in ATF4-overexpressing CRC cells was performed to identify the role of ATF4 in the GD induced chemoresistance. Quantitative real-time RTPCR and Western blot were used to detect the mR NA and protein expression of drug resistance gene 1(MDR1), respectively.RESULTS: GD protected CRC cells from drug-induced apoptosis(oxaliplatin and 5-fluorouracil) and induced the expression of ATF4, a key gene of the unfolded protein response. Depletion of ATF4 in CRC cells under the GD condition can induce apoptosis and drug resensitization. Similarly, inhibition of ATF4 in the ATF4-overexpressing CRC cells reintroduced therapeutic sensitivity and apoptosis. In addition, increased MDR1 expression was observed in GD-treated CRC cells. CONCLUSION: These data indicate that GD promotes chemoresistance in CRC cells through up-regulating ATF4 expression.

Mechanisms of trichostatin A inhibiting AGS proliferation and identification of lysine-acetylated proteins

AIM: To explore the effect of lysine acetylation in related proteins on regulation of the proliferation of gastric cancer cells, and determine the lysine-acetylated proteins and the acetylated modified sites in AGS gastric cancer cells. METHODS: The CCK-8 experiment and flow cytometry were used to observe the changes in proliferation and cycle of AGS cells treated with trichostatin A (TSA). Real time polymerase chain reaction and Western blotting were used to observe expression changes in p21, p53, Bax, Bcl-2, CDK2, and CyclinD1 in gastric cancer cells exposed to TSA. Cytoplasmic proteins in gastric cancer cells before and after TSA treatment were immunoprecipitated with anti-acetylated lysine antibodies, separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and silver-stained to detect the proteins by mass spectrometry after removal of the gel. The acetylated proteins in AGS cells were enriched with lysine-acetylated antibodies, and a high-resolution mass spectrometer was used to detect the acetylated proteins and modified sites. RESULTS: TSA significantly inhibited AGS cell proliferation, and promoted cell apoptosis, leading to AGS cell cycle arrest in G0/G1 and G2/M phases, especially G0/G1 phase. p21, p53 and Bax gene expression levels in AGS cells were increased with TSA treatment duration; Bcl-2, CDK2, and CyclinD1 gene expression levels were decreased with TSA treatment duration. Two unknown protein bands, 72 kDa (before exposure to TSA) and 28 kDa (after exposure to TSA), were identified by silver-staining after immunoprecipitation of AGS cells with the lysine-acetylated monoclonal antibodies. Mass spectrometry showed that the 72 kDa protein band may be PKM2 and the 28 kDa protein band may be ATP5O. The acetylated proteins and modified sites in AGS cells were determined. CONCLUSION: TSA can inhibit gastric cancer cell proliferation, which possibly activated signaling pathways in a variety of tumor-associated factors. ATP5O was obviously acetylated in AGS cells following TSA treatment.

Gut microbiota-derived metabolites as key mucosal barrier modulators in obesity

A significant breakthrough in the field of obesity research was the demonstration that an obese phenotype could be manipulated by modulating the gut microbiota.An important next step is to elucidate a human-relevant"map"of microbiota-host interactions that regulate the metabolic health of the host.An improved understanding of this crosstalk is a prerequisite for optimizing therapeutic strategies to combat obesity.Intestinal mucosal barrier dysfunction is an important contributor to metabolic diseases and has also been found to be involved in a variety of other chronic inflammatory conditions,including cancer,neurodegeneration,and aging.The mechanistic basis for intestinal barrier dysfunction accompanying metabolic disorders remains poorly understood.Understanding the molecular and cellular modulators of intestinal barrier function will help devise improved strategies to counteract the detrimental systemic consequences of gut barrier breakage.Changes in the composition and function of the gut microbiota,i.e.,dysbiosis,are thought to drive obesity-related pathogenesis and may be one of the most important drivers of mucosal barrier dysfunction.Many effects of the microbiota on the host are mediated by microbiota-derived metabolites.In this review,we focus on several relatively well-studied microbial metabolites that can influence intestinal mucosal homeostasis and discuss how they might affect metabolic diseases.The design and use of microbes and their metabolites that are locally active in the gut without systemic side effects are promising novel and safe therapeutic modalities for metabolic diseases.