Upregulation of miR-181a impairs lipid metabolism by targeting PPARα expression in nonalcoholic fatty liver disease.

Recent studies have reported elevated expression of miR-181a in patients with non-alcoholic fatty liver disease (NAFLD), suggesting that it may play an important role in liver lipid metabolism and insulin resistance. We aimed to investigate the effect of miR-181a in lipid metabolism and find new treatments for NAFLD. The expression level of miR-181a in NAFLD patient serum and a palmitic acid (PA)-induced NAFLD cell model was examined by Q-PCR. Oil red O staining and triglyceride assays were used to assess lipid accumulation in hepatocytes. Western blotting was used to detect the protein expression levels of peroxisome proliferator-activated receptor-α (PPARα) and the fatty acid ß-oxidation-related genes. Direct interactions were validated by dual-luciferase reporter gene assays. MiR-181a expression was significantly upregulated in the serum of NAFLD patients and PA-induced hepatocytes. Inhibition of miR-181a expression resulted in the increased expression of PPARα and its downstream genes, and PA-induced lipid accumulation in hepatocytes was also inhibited. Upregulation of miR-181a resulted in the downregulation of its direct target PPARα and downstream gene expression of PPARα as well as aggravated lipid accumulation in hepatocytes. At the same time, the increased expression of PPARα can offset lipid accumulation in hepatocytes induced by miR-181a mimics. This study demonstrates that reducing the expression of miR-181a may improve lipid metabolism in NAFLD. The downregulation of miR-181a expression can be a therapeutic strategy for NAFLD by modulating its target PPARα.

Notoginsenoside R1 attenuates experimental inflammatory bowel disease via pregnane X receptor activation.

Notoginsenoside R1 (R1) is the main bioactive component in Panax notoginseng, an old herb medicine widely used in Asian countries in the treatment of microcirculatory diseases. However, little is known about the effect of R1 on inflammatory bowel disease (IBD). The present study demonstrated that R1 alleviated the severity of dextran sulfate sodium-induced colitis in mice by decreasing the activity of myeloperoxidase, the production of cytokines, the expression of proinflammatory genes, and the phosphorylation of IκB kinase, IκBα, and p65 in the colon. Further studies indicated that R1 dose-dependently activated human/mouse pregnane X receptor (PXR), a known target for decreasing inflammation in IBD, and upregulated the expression of genes involved in xenobiotic metabolism in colorectal cells and the colon. Ligand pocket-filling mutant (S247W/C284W or S247W/C284W/S208W) of the human PXR abrogated the effect of R1 on PXR activation. Time-resolved fluorescence resonance energy transfer PXR competitive binding assay confirmed R1 (ligand) binding affinity. In addition, PXR overexpression inhibited nuclear factor-κB (NF-κB)-luciferase activity, which was potentiated by R1 treatment. PXR knockdown by small interfering RNA demonstrated the necessity of PXR in R1-induced upregulation of the expression of xenobiotic-metabolizing enzymes and downregulation of NF-κB activity. Finally, the anti-inflammatory effect of R1 was confirmed in trinitrobenzene sulfonic acid-induced colitis in mice. These findings suggest that R1 attenuates experimental IBD possibly via the activation of intestinal PXR signaling.

Depletion of thymosin ß4 promotes the proliferation, migration, and activation of human hepatic stellate cells.

BACKGROUND & AIMS: It has recently been reported that thymosin beta-4 (Tß4) 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 Tß4 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 Tß4 in serum, liver tissues, and LX-2 cells. Tß4 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 Tß4 was significantly reduced during the progression of liver fibrosis. The depletion of Tß4 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 Tß4 depletion in LX-2 cells can be counteracted by treatment with the Akt inhibitor MK-2206. In addition, Tß4 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 Tß4 participates in liver fibrosis by inhibiting the migration, proliferation, and activation of HSCs and that Tß4 may be an effective target in the treatment of liver fibrosis.

Unmet needs in biomarkers for autoimmune pancreatitis diagnosis.

Autoimmune pancreatitis (AIP) is a rare chronic autoimmune disorder. The diagnosis of AIP mainly depends on histopathology, imaging and response to treatment. Serum immunoglobulin 4 (IgG4) is used only as collateral evidence in diagnostic criteria for AIP because of its moderate sensitivity. Serum IgG4 levels are normal in 15%-37% of type 1 AIP and most of type 2 AIP patients. In these patients, the indeterminate imaging and histopathology may lead to the difficulty in definitive diagnosis of AIP. Therefore, discovery of new biomarkers is important for AIP diagnosis. Here, we provide some views on the progression and challenges in identifying novel serological biomarkers in AIP diagnosis.

PI3K: a potential therapeutic target for cancer.

Phosphatidylinositol 3-kinase (PI3K), one member of lipid kinase family, has been demonstrated to play a key role in regulating cell proliferation, adhesion, survival, and motility. Recent studies indicate that PI3K related signaling pathway is one of the most commonly activated pathways in human cancers. Accordingly, pharmacological inhibition of key nodes in this signaling cascade has been a focus in developmental therapeutics. To date, Inhibitors targeting PI3K or nodes in this pathway, AKT and mTOR, are best studied and have reached clinical trials. In this review, we will focus on recent progress on understanding of PI3Ks signaling pathway and the development of PI3K inhibitors.

miR-181b promotes hepatic stellate cells proliferation by targeting p27 and is elevated in the serum of cirrhosis patients.

MicroRNAs, as a kind of negative gene regulators, were demonstrated to be involved in many types of diseases. In this study, we found that transforming growth factor-beta 1 could induce the expression of miR-181a and miR-181b, and miR-181b increased in the much higher folds than miR-181a. Because of the important role of transforming growth factor-beta 1 in HSC activation and liver cirrhosis, we investigate the effect of miR-181a and miR-181b on HSC proliferation. The results showed that miR-181b could promote HSC-T6 cell proliferation by regulating cell cycle. Further study showed p27, the cell cycle regulator, was the direct target of miR-181b in HSC-T6 cell. But miR-181a had no effects on HSC-T6 cell proliferation and cell cycle, and did not target p27. Interestingly, miR-181b is elevated significantly in serum of liver cirrhosis cases comparing to that of normal persons, whereas miR-181a expression was in the similar level with that of normal persons. These results suggested that miR-181b could be induced by TGF-ß1 and promote the growth of HSCs by directly targeting p27. The elevation of miR-181b in serum suggested that it may be potential diagnostic biomarkers for cirrhosis. As for miR-181a, it may work in TGF-ß1 pathway by a currently unknown mechanism.

Coexpression of Smad7 and UPA attenuates carbon tetrachloride-induced rat liver fibrosis.

BACKGROUND: There is a great need for developing novel therapies to treat liver fibrosis. Previous studies showed that both Smad7 and uPA were inhibitors of liver fibrosis. Therefore, we explored the therapeutic effects of combinational gene therapy with Smad7 and uPA on CCl4-induced liver fibrosis. MATERIAL/METHODS: Smad7 and uPA genes were cloned into an adenovirus vector. To observe the therapeutic effects of coexpression of Smad7 and uPA genes, the recombinant adenovirus were delivered into CCL4-induced fibrosis models. Fibrillar collagen, hydroxyproline, α-SMA, TGF-ß1, MMP-13, TIMP-1, HGF and PCNA were detected to evaluate the fibrosis and to explore the mechanisms underlying the treatment with Smad7 and uPA. RESULTS: The results showed that single Smad7 or uPA adenovirus reduced CCL4 induced liver fibrosis significantly; while combination of Smad7 and uPA had more significant therapeutic effect on CCl4 induced liver fibrosis. Then the markers underlying the therapeutic effect of combination of Smad7 and uPA were also explored. Over-expression of Smad7 and uPA inhibited the expression of α-SMA and TGF- ß1 significantly. Combinational gene therapy also enhanced extracellular matrix degradation by increasing the expression of MMP-13, inhibiting TIMP-1 expression, and promoted hepatocyte proliferation, while single Smad7 or uPA only induced part of these changes. CONCLUSIONS: These results suggest that combinational gene therapy with Smad7 and uPA inhibited CCl4-induced rat liver fibrosis by simultaneously targeting multiple pathogenic pathways.

Exosomal microRNA-618 derived from mesenchymal stem cells attenuate the progression of hepatic fibrosis by targeting Smad4.

Hepatic fibrosis (HF) is a pathological phenomenon that occurs during the process of long-term damage and repair in the liver. This condition will lead to the development of cirrhosis and even liver cancer if untreated. Previous evidence has shown that exosomes derived from mesenchymal stem cells (MSCs), carrying microRNAs (miRs), can affect the pathogenesis of HF. Therefore, the present study aimed to identify novel exosomal miRs derived from MSCs that play a critical role in the progression of HF. Next, the expression data of differentially expressed miRs (DEMs) of patients with liver cirrhosis and healthy controls were obtained from the Gene Expression Omnibus dataset. DEMs were analyzed using Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Moreover, to further confirm the function of exosomal miR-618 derived from MSCs on the pathogenesis of HF in vivo, a mouse model of HF was established. The results of the present study suggested that a close associated existed between DEMs and HF. Based on the results of the bioinformatics analysis, miR-618 was one of the main downregulated miRs involved in cirrhosis. In addition, miR-618 could be transferred from MSCs to LX-2 cells via exosomes; exosomal miR-618 derived from MSCs inhibited the viability and migration of LX-2 cells that were treated with TGF-ß. Furthermore, exosomal miR-618 derived from MSCs attenuated the progression of HF via targeting Smad4. These findings indicated that treatment of exosomal miR-618 derived from MSCs might serve as a new strategy for HF.

Fluoroscopy-guided shaped endobiliary biopsy at endoscopic retrograde cholangiography can accurately diagnose biliary neoplasia: Results from a large cohort.

Background and study aims The sensitivity of using standard endobiliary forceps biopsy to diagnose neoplastic biliary lesions remains low. We have developed a unique biopsy approach, termed fluoroscopy-guided, shaped endobiliary biopsy (FSEB), in which the biopsy forceps are modified to improve diagnostic yield. In this study, we evaluate the diagnostic characteristics of FSEB for endobiliary lesions at endoscopic retrograde cholangiography (ERC). Patients and methods Consecutive patients undergoing FSEB between 1/2001 and 12/2014 were retrospectively enrolled. The identification of neoplastic lesions with FSEB, was the primary endpoint. The gold standard of neoplasia was histopathology, cytology or surgical histopathology. The benign cases were followed up for one year. Results A total of 204 patients undergoing 250 biopsy sessions by FSEB were analyzed. Per-patient analysis was performed and FSEB showed 81.1 % sensitivity and 88.2 % accuracy. FSEB detection of proximal biliary lesions was more sensitive (91.1 % vs 73.2 %, P  < 0.01) and accurate (94.9 % vs 82.2 %, P  < 0.01) compared to distal lesions. No complications from FSEB were reported. Conclusions FSEB shows high accuracy for diagnosis of neoplasia in biliary strictures, especially for proximal lesions. Future prospective randomized controlled studies are merited to further validate the role of FSEB as the first-line sampling tool for evaluation of biliary neoplasm.

A novel anticoagulant affinity membrane for enhanced hemocompatibility and bilirubin removal.

Affinity membrane is widely employed to promote specific adsorption of toxins and reduce the blood purification therapeutic time. However, it suffers from insufficient toxin binding and low hemocompatibility. Herein, a novel anticoagulant affinity membrane (AAM) was developed to clear bilirubin from human blood in a pore-flow-through way. Firstly, a nylon net membrane with a regularly arranged pore as the matrix was coated with poly(pyrrole-3-carboxylic acid) via chemical vapor deposition (CVD) method. Then, poly(L-arginine) (PLA) as a highly specific ligand of bilirubin, was immobilized onto the surface of the composited membrane after the modification of heparin. Owing to the 3-dimensional molecular architecture of PLA, up to 86.1 % of bilirubin was efficiently cleared. Besides, the AAM exhibited effective anticoagulant activity in the measurement of clotting time, with suppressed thrombus formation, low hemolysis ratio, minimized platelet and leukocyte adhesion, and excellent biosafety. Therefore, the AAM has enormous potential in blood purification therapy for enhancing hemocompatibility and bilirubin removal.

Hepatitis B-related glomerulonephritis and optimization of treatment.

Introduction: Multiple studies have revealed a strong relationship between the development of nephropathy and hepatitis B virus (HBV) infection. The underlying pathogenesis of hepatitis B-related glomerulonephritis (HBV-GN) involves immune complexes, which can be isolated from kidney tissues. Clearance of HBV antigenemia improves renal impairment and proteinuria in HBV-GN patients.Areas covered: In this review, we present our current understanding of the epidemiology, pathogenesis, pathology, diagnosis, and treatment of HBV-GN. We discuss the advantages and disadvantages of oral nucleoside/nucleotide analogs (NAs), and the main pharmaceutical treatment for hepatis B.Expert opinion: Currently, antiviral agents are the main HBV-GN therapeutic agents. Although no randomized controlled clinical trials have compared the efficacy of interferon (IFN) and NA, we suggest IFN treatment for pediatric patients (IFN-α in patients ≥1 year; pegIFN-α in patients ≥3 years) considering treatment duration and absence of resistance. Novel NAs have brought about promising treatment options involving high efficacy viral suppression and low resistance rates. NAs with a high barrier to resistance (e.g. entecavir) are recommended as first-line therapy of HBV-GN. Immunosuppression monotherapy, such as corticosteroids, is of little benefit and potentially harmful to HBV-GN patients due to the possibility of viral reactivation.

Role of Noncoding RNA in Development of Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is increasing in prevalence globally, but little is known about its specific molecular mechanisms. During the past decade, noncoding RNAs (ncRNAs) have been linked to NAFLD initiation and progression. They are a class of RNAs that play an important role in regulating gene expression despite not encoding proteins. This review summarizes recent research on the relationship between ncRNAs and NAFLD. We discussed the potential applicability of ncRNAs as a biomarker for early NAFLD diagnosis and assessment of disease severity. With further study, ncRNAs should prove to be valuable new targets for NAFLD treatment and benefit the development of noninvasive diagnostic methods.

Transplantation of urokinase-type plasminogen activator gene-modified bone marrow-derived liver stem cells reduces liver fibrosis in rats.

BACKGROUND: Bone marrow-derived liver stem cells (BDLSCs) are very robust cells that can differentiate into liver epithelial cells. These stem cells are promising targets for gene therapy treatment of liver diseases. Liver fibrosis results from chronic liver damage characterized by an accumulation of extracellular matrix (ECM) and levels of urokinase-type plasminogen activator (uPA) play an important role in ECM degradation. In the present study, we investigated the therapeutic effects of uPA gene-modified BDLSC transplantation on carbon tetrachloride (CCl4)-induced liver fibrosis in rats. METHODS: BDLSCs were obtained from the bone marrow of cholestatic rats. These stem cells were selected and proliferated in medium containing 5% cholestatic serum. BDLSCs transfected with adenovirus-mediated human urokinase-plasminogen activator were transplanted into rats with CCl(4)-induced hepatic fibrosis. Liver function and the area of hepatic fibrosis were correlated with the development and prognosis of hepatic fibrosis. RESULTS: Hepatocyte-like colony-forming units were formed by bone marrow cells after 2 weeks in culture. In the uPA gene-modified BDLSC group, the areas of hepatic fibrosis were smaller and liver function was markedly ameliorated compared to controls. The expression of alpha-smooth muscle actin protein, transforming growth factor-beta1 protein and collagen types I and III mRNA were downregulated. By contrast, the levels of matrix metalloproteinases-2, -3 and -9 mRNA, hepatic growth factor mRNA and proliferating cell nuclear antigen protein increased. CONCLUSIONS: Transplantation of uPA gene-modified BDLSCs may suppress hepatic fibrosis and ameliorate liver function.

[Effects of gene-transfected bone marrow-derived liver stem cell transplantation on accumulation of extracellular matrix in rats with liver fibrosis].

OBJECTIVE: To explore the effects of urokinase-type plasminogen activator (uPA) gene-modified bone marrow-derived stem cell (BDLSC) transplantation on accumulation of extracellular matrix (ECM) in hepatic tissue in liver fibrosis. METHODS: BDLSCs obtained from 10 male Fisher344 rats were transfected by adenovirus-mediated human uPA (AduPA) in vitro. Twenty-seven female rats were randomly divided into 3 equal groups to undergo subcutaneous injection of carbon tetrachloride to establish liver fibrosis models and then randomly divided into 3 equal groups: model group injected with normal saline via caudal vein, BDLSC group injected with 2 x 10(6) BDLSCs via caudal vein, and BDLSC-uPA group injected with 2 x 10(6) AduPA-transfected BDLSCs. Eight weeks later the serum levels of alanine transaminase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), albumin (ALB), and ECM levels, i.e., hyaluronic acid, laminin (LN), and procollagen III (PC III), were detected. Then the rats were killed with their livers taken out. The hydroxyproline (Hyp) content of the liver was detected by alkaline hydrolysis. RT-PCR was used to examine the expression of collagen I and III (COLI and COLIII), matrix metalloproteinases-2, 3, and 9 (MMP-2, 3, and 9), and tissue inhibitor of metalloproteinase-1 and 2 (TIMP-1 and 2). RESULTS: Compared with those of the model group the levels of ALT, AST, and TBIL of the BDLSC-uPA group were all significantly lower, and the ALB level was higher (all P < 0.05). The ECM levels of BDLSC-uPA group were all significantly lower than those of the model group or BDLSC group too (all P < 0.05). Hyp content of the liver decreased dramatically. The mRNA expression levels of COLI and COLIII of the liver of the BDLSC-uPA group were significantly lower (38.9 +/- 2.7, 8.5 +/- 1.6), and the mRNA expression levels of MMP-2, -3, and MMP-9 mRNA (157.5 +/- 32.6, 105.5 +/- 14.6, 187.5 +/- 22.8) were significantly higher than those of the model group or BDLSC group (all P < 0.05), but no significant differences were observed in the mRNA expression of TIMP-1 and 2 mRNA between the 3 groups (all P > 0.05). CONCLUSION: uPA gene-modified BDLSC transplantation improves the liver function and suppresses the hepatic fibrosis in liver cirrhosis through up-regulating the expression of MMPs and promoting the degradation of ECM.

miR-192-5p regulates lipid synthesis in non-alcoholic fatty liver disease through SCD-1.

AIM: To evaluate the levels of miR-192-5p in non-alcoholic fatty liver disease (NAFLD) models and demonstrate the role of miR-192-5p in lipid accumulation. METHODS: Thirty Sprague Dawley rats were randomly divided into three groups, which were given a standard diet, a high-fat diet (HFD), and an HFD with injection of liraglutide. At the end of 16 weeks, hepatic miR-192-5p and stearoyl-CoA desaturase 1 (SCD-1) levels were measured. MiR-192-5p mimic and inhibitor and SCD-1 siRNA were transfected into Huh7 cells exposed to palmitic acid (PA). Lipid accumulation was evaluated by oil red O staining and triglyceride assays. Direct interaction was validated by dual-luciferase reporter gene assays. RESULTS: The HFD rats showed a 0.46-fold decrease and a 3.5-fold increase in hepatic miR-192-5p and SCD-1 protein levels compared with controls, respectively, which could be reversed after disease remission by liraglutide injection (P < 0.01). The Huh7 cells exposed to PA also showed down-regulation and up-regulation of miR-192-5p and SCD-1 protein levels, respectively (P < 0.01). Transfection with miR-192-5p mimic and inhibitor in Huh7 cells induced dramatic repression and promotion of SCD-1 protein levels, respectively (P < 0.01). Luciferase activity was suppressed and enhanced by miR-192-5p mimic and inhibitor, respectively, in wild-type SCD-1 (P < 0.01) but not in mutant SCD-1. MiR-192-5p overexpression reduced lipid accumulation significantly in PA-treated Huh7 cells, and SCD-1 siRNA transfection abrogated the lipid deposition aggravated by miR-192-5p inhibitor (P < 0.01). CONCLUSION: This study demonstrates that miR-192-5p has a negative regulatory role in lipid synthesis, which is mediated through its direct regulation of SCD-1.

Mechanism and clinical significance of cyclooxygenase-2 expression in gastric cancer.

AIM: To determine the correlation between methylation status of 5' CpG island of cyclooxygenase-2 (COX-2) gene and protein expression in gastric cancer tissues for distinguishing the molecular characters of gastric cancers. METHODS: Methylation status of 5' CpG island of COX-2 gene was studied by PCR amplification after HpaII and Hha I restrictive enzyme digestion; COX-2 expression was evaluated by immunohistochemical method. RESULTS: Hpa II and HhaI site were all methylated in 12 normal gastric mucosa tissues, whereas they were demethylated in 77.27% (34/44) and 84.09% (37/44) gastric cancer tissues, respectively. Expression of COX-2 was detected in 68.18% (30/44) gastric cancer tissues, but no expression was found in normal gastric mucosa tissues. In gastric cancer tissues, COX-2 expression was correlated significantly with HpaII site demethylation (29/30 vs 5/14, P<0.001 and HhaI site demethylation (28/30 vs 9/14, P<0.05). CONCLUSION: The demethylation of 5' CpG island of gene is necessary for COX-2 expression in human gastric cancer. The expression status of COX-2 may provide theoretical basis for COX-2 targeting gastric cancer treatments.

Entering the duodenal diverticulum: a method for cannulation of the intradiverticular papilla.

Successful cannulation of the common bile duct may be difficult in patients in whom the papilla is located entirely within a diverticulum. In this study, we report successful biliary cannulation in three patients following intubation of the distal tip of the duodenoscope into the duodenal diverticulum and locating the major papilla. No complications occurred during the operation or during the postoperative period. This method didn't need second incubation an endoscope and might lower the burden of patients. So this skill is useful to deal with the papilla hidden inside the large diverticulum because of its safety and convenience.