Celecoxib-mediated attenuation of non-alcoholic steatohepatitis is potentially relevant to redistributing the expression of adiponectin receptors in rats.

Pharmacological inhibition of cyclooxygenase-2 (COX-2) activity ameliorated the severity of non-alcoholic steatohepatitis (NASH) rats. It is not completely understood that the role of COX-2 inhibitor celecoxib on adiponectin receptors (Adipo-R1/R2) expression in different tissues in NASH rats. Sprague-Dawley male NASH rats induced by a high-fat diet (HFD) were administrated with or without celecoxib for 8 weeks. Biochemical parameters of liver function, glucose, and lipid metabolism, and the levels of adiponectin, tumor necrosis factor-alpha (TNF-α), prostaglandin E2 (PGE2) in the serum or liver were collected according to the standard protocols. The mRNA and protein levels of Adipo-R1, Adipo-R2, and COX-2 in the liver, muscle, and visceral fat were performed by quantitative real-time polymerase chain reaction (q-PCR) and Western blot analysis, respectively. The results showed that celecoxib ameliorated the various clinical indicators and pathological characteristics in the NASH rats, including body weight, liver function, liver index, and redox activities in serum and hepatic samples. The serum concentrations of adiponectin and TNF-α and PGE2 were negatively correlated. As expected, these ameliorative effects of celecoxib were associated with the gene and protein levels up-regulation of Adipo-R1, Adipo-R2 in the liver and visceral fat tissues, and seeming to be compensatory down-regulation expression in muscle tissues (P <0.05). Additionally, COX-2 protein expression was negatively correlated with serum adiponectin levels, protein expression of adiponectin receptors from the liver and visceral fat, conversely, positively correlated with those from the muscle. Our current study demonstrate that celecoxib might effectively alleviate NASH rats in a unique manner closely relevant to redistributing the expression of adiponectin receptors in the liver, visceral fat, and muscle. However, the precise molecular mechanism needs further study.

Analysis of Risk Factors and Establishment of a Prediction Model for Endoscopic Primary Bile Reflux: A Single-Center Retrospective Study.

Background: Endoscopic primary bile reflux is one of the main diagnostic criteria for bile reflux gastritis (BRG). Presently, the risk factors and prediction models of endoscopic primary bile reflux (EPBR) in gastropathy patients who cannot or will not undergo endoscopy due to contraindications are not clear. Thus, this study aimed to evaluate the risk factors of EPBR and to establish and verify a prediction model. Methods: A series of 844 patients (564 subjects with EPBR and 280 control subjects) were retrospectively selected for this study and divided into a training set (n = 591) and a validation set (n = 253) according to the usual ratio of 70:30% for the subsequent internal validation of the logistic regression model for EPBR. Fifteen parameters that might affect the occurrence of EPBR were collected. Subsequently, univariate and stepwise logistic regression analyses were introduced to reveal the risk factors and the multivariate prediction model. An R package was dedicated to the corresponding internal validation of the EPBR model. Results: The univariate analysis showed that gender, age, smoking, Helicobacter pylori (H. pylori) infection status, metabolic syndrome (MS), non-steroidal anti-inflammatory drugs (NSAIDs) use history, and previous medical histories of chronic liver diseases, cholelithiasis, and erosive gastritis were statistically significant between the two groups (P < 0.05). Multivariate regression described that being a male [OR (95%confidence interval (CI)) = 2.29 (1.50-3.50), P < 0.001], age≥45 years old [OR (95% CI) = 4.24 (2.59-6.96), P < 0.001], H. pylori infection status [OR (95% CI) = 2.34 (1.37-4.01), P = 0.002], MS [OR (95% CI) = 3.14 (1.77-5.54), P < 0.001], NSAIDs use history [OR (95% CI) = 1.87 (1.03-3.40), P = 0.04], cholelithiasis history [OR (95% CI) = 3.95 (2.18-7.18), P < 0.001] and erosive gastritis history [OR (95% CI) = 6.77 (3.73-12.29), P < 0.001] were the risk factors for the occurrence of EPBR. Based on the results of these risk factors, an EPBR prediction model with an adequate calibration and excellent discrimination was established [area under the curve (AUC): 0.839, 95% CI = 0.806-0.872]. Conclusions: Being a male, age ≥ 45 years old, H. pylori infection, histories of MS, NSAIDs use, cholelithiasis, and erosive gastritis appear to be the risk factors for EPBR, and our favorable prediction model might be an option for the prediction of EPBR.

LncRNA GAS6-AS2 facilitates tumor growth and metastasis of hepatocellular carcinoma by activating the PI3K/AKT/FoxO3a signaling pathway.

The morbidity and mortality of hepatocellular carcinoma (HCC) are growing yearly. Several reports emphasize the importance of long non-coding RNAs (lncRNAs) in HCC. This paper provides a molecular mechanism for the function of GAS6-AS2 in HCC. The expressions of GAS6-AS2, miR-493-5p and OTUB1 were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation, apoptosis, migration, and invasion were measured by cell counting kit-8 (CCK-8), flow cytometry and transwell assay, respectively. The interaction of miR-493-5p and GAS6-AS2 or OTU domain-containing Ubiquitin Aldehyde-binding Protein 1 (OTUB1) was analyzed by starBase v2.0 and verified by luciferase reporter assay. The protein level of OTUB1 as well as PI3K, p-PI3K, AKT, p-AKT, FoxO3a, p-FoxO3a and ß-actin protein levels were distinguished by western blot. GAS6-AS2 was up-regulated in HCC tissues and cells. GAS6-AS2 knockdown inhibited proliferation, migration, and invasion but promoted apoptosis. MiR-493-5p, a target of GAS6-AS2, was down-regulated in HCC tissues and cells. Inhibition of miR-493-5p reversed the effects of GAS6-AS2 knockdown on HCC cells. OTUB1, a target of miR-493-5p, was up-regulated in HCC cells and its expression was modulated by miR-493-5p. Overexpression of OTUB1 recovered the positive effects of miR-493-5p enrichment or GAS6-AS2 knockdown on HCC cells. GAS6-AS2 knockdown impeded the activation of PI3K/AKT/FoxO3a signaling pathway, while this activation was reversed by miR-493-5p inhibition or OTUB1 overexpression. In conclusion, GAS6-AS2 knockdown suppressed proliferation, migration, and invasion but promoted apoptosis of HCC cells by impeding PI3K/AKT/FoxO3a signaling pathway through regulating the GAS6-AS2/miR-493-5p/OTUB1 axis.

Prolyl oligopeptidase attenuates hepatic stellate cell activation through induction of Smad7 and PPAR-γ.

Prolyl oligopeptidase (POP) is a serine endopeptidase widely distributed in vivo with high activity in the liver. However, its biological functions in the liver have remained largely elusive. A previous study by our group has shown that POP produced N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) and thereby exerted an anti-fibrogenic effect on hepatic stellate cells (HSCs) in vitro. It was therefore hypothesized that POP may affect the activation state of HSCs and has an important role in liver fibrosis. The HSC-T6 immortalized rat liver stellate cell line was treated with the POP inhibitor S17092 or transfected with recombinant lentivirus to overexpress POP. Cell proliferation and apoptosis were determined using a Cell Counting Kit-8 and flow cytometry, respectively. The activation status of HSCs was determined by examination of the expression of α-smooth muscle actin (α-SMA), collagen I, monocyte chemoattractant protein-1 (MCP-1), transforming growth factor (TGF)-ß-Smad signaling and peroxisome proliferator activated receptor-γ (PPAR-γ). Inhibition by S17092 decreased, whereas lentiviral expression increased the activity of POP and cell proliferation, while neither of the treatments affected cell apoptosis. Of note, S17092 significantly increased, whereas POP overexpression decreased the expression of α-SMA and MCP-1 without affecting the expression of collagen I and TGF-ß1. Furthermore, S17092 caused a reduction, whereas POP overexpression caused an upregulation of Smad7 protein and PPAR-γ, but not phosphorylated-Smad2/3 expression. In conclusion, POP attenuated the activation of HSCs through inhibition of TGF-ß signaling and induction of PPAR-γ, which may have therapeutic potential in liver fibrosis.

Galanin receptor 2 mediates antifibrogenic effects of galanin on hepatic stellate cells.

Galanin is an endogenous factor involved in the negative regulation of the biological effects of leptin in bioenergetic metabolism. Leptin promotes fibrogenic effects in hepatic stellate cells (HSCs), however, little is known about the effects of galanin on HSCs. In the present study, the biological functions of galanin and its receptors (GalRs) in HSCs were investigated using cell culture in vitro. It was found that galanin and GalR3 mRNA are expressed in quiescent and activated HSCs. GalR2 expression was undetectable in quiescent HSCs but was markedly induced in activated HSCs. In the HSC-T6 cell line, which is an activated rat HSC cell line, treatment with 100 nmol/l galanin significantly inhibited cell proliferation. It also inhibited transforming growth factor (TGF)-ß1 and α-smooth muscle actin (SMA) expression and upregulated peroxisome proliferator-activated receptor (PPAR)-γ expression. Following the knockdown of GalR2 by specific small interfering RNA, the activation of GalR3 by galanin does not influence these effects of galanin on HSCs. However, activation of GalR2 alone by galanin following the knockdown of GalR3 inhibits HSC proliferation and TGF-ß1 and α-SMA expression, in addition to inducing PPAR-γ expression. These data suggest that galanin inhibits HSC activation and suppresses the profibrogenic features of these cells, and these effects might be mediated by GalR2. Thus, galanin is a potential endogenous factor in the inhibition of liver fibrosis.

Prolyl Oligopeptidase Inhibition Attenuates Steatosis in the L02 Human Liver Cell Line.

BACKGROUND: Prolyl oligopeptidase (POP) is a serine endopeptidase that is widely distributed in vivo, particularly in the liver. Significant changes in functional mitochondrial proteins involved with mitochondrial oxidoreductases/transporters and nucleic acid binding proteins were observed after POP inhibition in the liver, which suggested a role of POP in regulating liver energy metabolism. Steatosis in nonalcoholic fatty liver disease (NAFLD) is associated with disturbances in lipid and energy metabolism in hepatocytes. Here, we aimed to study the effect of POP on hepatocyte steatosis. METHODS: The human liver cell line L02 was used to investigate the biological effects of POP. An in vitro cell model of steatosis was successfully induced with oleic acid and palmitic acid. L02 cells were also subjected to S17092 (a POP inhibitor) at different concentrations for 24 or 48 h. Ac-SDKP levels and POP activity were measured to assess the rate of inhibition of POP by S17092. The POP gene and protein expression levels were detected using real-time PCR and Western blots, respectively. Oil red O staining was performed and the triglyceride levels in the L02 cells were also measured. Cell proliferation and apoptosis were detected using CCK-8 and flow cytometry, respectively. The expression of genes involved in lipid metabolism was detected using real-time PCR. The effects of POP inhibition on LC3B II were detected by Western blot. RESULTS: Compared with the control, the POP mRNA levels increased by approximately 30%, and the POP protein levels increased by almost 60% in the steatotic L02 cells. After S17092 (0.026~130 µM) incubation for 24 or 48 h, cell proliferation was significantly decreased in the free fatty acid (FFA)-treated cells at 26-130 µM; however, S17092 did not affect the proliferation of L02 cells after 24 h of incubation with S17092 at 0.026-65 µM without FFA treatment. S17092 treatment (13 and 26 µM) also elicited no significant effect on apoptosis in normal L02 cells, but FFA treatment increased cell apoptosis, which was attenuated by S17092 incubation. S17092 treatment inhibited intracellular POP activity and decreased the AcSDKP level at the concentration of 0.026-26 µM. After treatment with FFA for 24 h, oil red O staining revealed significant lipid accumulation in the cells in the model group compared with the controls; however, lipid accumulation was suppressed after the administration of S17092 (13 and 26 µM). Accordingly, the triglyceride levels in the FFA-treated cells were approximately 5-fold greater than those of the controls and were decreased by approximately 25% and 45% after the administration of S17092 at 13 and 26 µM, respectively. The mRNA levels of FASN, PPAR-γ, and SREBP-1c were higher in the FFA-treated cells than in the normal controls, and all of these levels were significantly inhibited in the presence of S17092 at both 13 and 26 µM. S17092 treatment did not affect LC3B II in the FFA-treated cells compared with FFA treatment alone. CONCLUSION: The expression of POP increases with hepatocyte steatosis, and POP inhibitors can significantly reduce intracellular lipid accumulation, which might be related to the inhibition of genes involved in lipid synthesis.