Claudia Pinto Marques Souza de Oliveira

Combination of N‐acetylcysteine and metformin improves histological steatosis and fibrosis in patients with non‐alcoholic steatohepatitis

Aim:u2002 There is no proven medical therapy for the treatment of non‐alcoholic steatohepatitis (NASH). Oxidative stress and insulin resistance are the mechanisms that seem to be mostly involved in its pathogenesis. The aim of our study was to evaluate the efficacy of N‐acetylcysteine (NAC) in combination with metformin (MTF) in improving the aminotransferases and histological parameters (steatosis, inflammation, hepatocellular ballooning, and fibrosis) after 12u2003months of treatment.

Association of polymorphisms of glutamate‐cystein ligase and microsomal triglyceride transfer protein genes in non‐alcoholic fatty liver disease

Background and Aims:u2002 Although the metabolic risk factors for non‐alcoholic fatty liver disease (NAFLD) progression have been recognized, the role of genetic susceptibility remains a field to be explored. The aim of this study was to examine the frequency of two polymorphisms in Brazilian patients with biopsy‐proven simple steatosis or non‐alcoholic steatohepatitis (NASH): −493u2003G/T in the MTP gene, which codes the protein responsible for transferring triglycerides to nascent apolipoprotein B, and −129u2003C/T in the GCLC gene, which codes the catalytic subunit of glutamate‐cystein ligase in the formation of glutathione.

Strategies, models and biomarkers in experimental non-alcoholic fatty liver disease research

Non-alcoholic fatty liver disease encompasses a spectrum of liver diseases, including simple steatosis, steatohepatitis, liver fibrosis and cirrhosis and hepatocellular carcinoma. Non-alcoholic fatty liver disease is currently the most dominant chronic liver disease in Western countries due to the fact that hepatic steatosis is associated with insulin resistance, type 2 diabetes mellitus, obesity, metabolic syndrome and drug-induced injury. A variety of chemicals, mainly drugs, and diets is known to cause hepatic steatosis in humans and rodents. Experimental non-alcoholic fatty liver disease models rely on the application of a diet or the administration of drugs to laboratory animals or the exposure of hepatic cell lines to these drugs. More recently, genetically modified rodents or zebrafish have been introduced as non-alcoholic fatty liver disease models. Considerable interest now lies in the discovery and development of novel non-invasive biomarkers of non-alcoholic fatty liver disease, with specific focus on hepatic steatosis. Experimental diagnostic biomarkers of non-alcoholic fatty liver disease, such as (epi)genetic parameters and -omics-based read-outs are still in their infancy, but show great promise. In this paper, the array of tools and models for the study of liver steatosis is discussed. Furthermore, the current state-of-art regarding experimental biomarkers such as epigenetic, genetic, transcriptomic, proteomic and metabonomic biomarkers will be reviewed.

Plasmatic higher levels of homocysteine in non-alcoholic fatty liver disease (NAFLD).

BackgroundNon-alcoholic fatty liver disease (NAFLD) is a chronic liver disease, which includes a spectrum of hepatic pathology such as simple steatosis, steatohepatitis, fibrosis and cirrhosis. The increased serum levels of homocysteine (Hcy) may be associated with hepatic fat accumulation. Genetic mutations in the folate route may only mildly impair Hcy metabolism. The aim of this study was to investigate the relation between liver steatosis with plasma homocysteine level and MTHFR C677T and A1298C polymorphisms in Brazilian patients with NAFLD.MethodsThirty-five patients diagnosed with NAFLD by liver biopsy and forty-five healthy controls neither age nor sex matched were genotyped for C677T and A1298C MTHFR polymorphisms using PCR-RFLP and PCR-ASA, respectively, and Hcy was determined by HPLC. All patients were negative for markers of Wilson’s, hemochromatosis and autoimmune diseases. Their daily alcohol intake was less than 100xa0g/week. A set of metabolic and serum lipid markers were also measured at the time of liver biopsies.ResultsThe plasma Hcy level was higher in NAFLD patients compared to the control group (pu2009=u20090.0341). No statistical difference for genotypes 677C/T (pu2009=u20090.110) and 1298A/C (pu2009=u20090.343) in patients with NAFLD and control subjects was observed. The genotypes distribution was in Hardy-Weinberg equilibrium (677C/T pu2009=u20090.694 and 1298 A/C pu2009=u20090.188). The group of patients and controls showed a statistically significant difference (pu2009<u20090.001) for BMI and HOMA_IR, similarly to HDL cholesterol levels (pu2009<u20090,006), AST, ALT, γGT, AP and triglycerides levels (pu2009<u20090.001). A negative correlation was observed between levels of vitamin B12 and Hcy concentration (pu2009=u20090.005).ConclusionOur results indicate that plasma Hcy was higher in NAFLD than controls. The MTHFR C677T and A1298C polymorphisms did not differ significantly between groups, despite the 677TT homozygous frequency was higher in patients (17.14%) than in controls (677TTu2009=u20094.44%) (pu2009>u20090.05). The suggested genetic susceptibility to the MTHFR C677T and A1298C should be confirmed in large population based studies.

Omega-3 polyunsaturated fatty acids in treating non-alcoholic steatohepatitis: A randomized, double-blind, placebo-controlled trial

BACKGROUNDn& aims: Few clinical trials have addressed the potential benefits of omega-3 polyunsaturated fatty acids (PUFAs) on non-alcoholic steatohepatitis (NASH). We evaluated the effects of supplementation with omega-3 PUFAs from flaxseed and fish oils in patients with biopsy-proven NASH.nnnMETHODSnPatients received three capsules daily, each containing 0.315xa0g of omega-3 PUFAs (64% alpha-linolenic [ALA], 16% eicosapentaenoic [EPA], and 21% docosahexaenoic [DHA] acids; n-3 group, nxa0=xa027) or mineral oil (placebo group, nxa0=xa023). Liver biopsies were evaluated histopathologically by the NASH activity score (NAS). Plasma levels of omega-3 PUFAs were assessed as a marker of intake at baseline and after 6 months of treatment. Secondary endpoints included changes in plasma biochemical markers of lipid metabolism, inflammation, and liver function at baseline and after 3 and 6 months of treatment.nnnRESULTSnAt baseline, NAS was comparable between the groups (pxa0=xa00.98). After intervention with omega-3 PUFAs, plasma ALA and EPA levels increased (pxa0≤xa00.05). However in the placebo group, we also observed increased EPA and DHA (pxa0≤xa00.05), suggesting an off-protocol intake of PUFAs. NAS improvement/stabilization was correlated with increased ALA in the n-3 group (pxa0=xa00.02) and with increased EPA (pxa0=xa00.04) and DHA (pxa0=xa00.05) in the placebo group. Triglycerides were reduced after 3 months in the n-3 group compared to baseline (pxa0=xa00.01).nnnCONCLUSIONSnIn NASH patients, the supplementation of omega-3 PUFA from flaxseed and fish oils significantly impacts on plasma lipid profile of patients with NASH. Plasma increase of these PUFAs was associated with better liver histology. (ID 01992809).

Experimental models of liver fibrosis

Hepatic fibrosis is a wound healing response to insults and as such affects the entire world population. In industrialized countries, the main causes of liver fibrosis include alcohol abuse, chronic hepatitis virus infection and non-alcoholic steatohepatitis. A central event in liver fibrosis is the activation of hepatic stellate cells, which is triggered by a plethora of signaling pathways. Liver fibrosis can progress into more severe stages, known as cirrhosis, when liver acini are substituted by nodules, and further to hepatocellular carcinoma. Considerable efforts are currently devoted to liver fibrosis research, not only with the goal of further elucidating the molecular mechanisms that drive this disease, but equally in view of establishing effective diagnostic and therapeutic strategies. The present paper provides a state-of-the-art overview of in vivo and in vitro models used in the field of experimental liver fibrosis research.

Hypocaloric high-protein diet improves clinical and biochemical markers in patients with nonalcoholic fatty liver disease (NAFLD).

OBJECTIVEnTo investigate the role of hypocaloric highprotein diet, a prospective clinical study was conducted in NAFLD patients.nnnRESEARCH METHODS AND PROCEDURESnPre-versus post-interventional data were analyzed in 48 stable NAFLD patients (submitted to a hypocaloric high-protein diet during 75 days. Variables included anthropometrics (body mass index/ BMI and waist circumference/WC), whole-body and segmental bioimpedance analysis and biochemical tests. Diet compliance was assessed by interviews every two weeks.nnnRESULTSnBMI, WC and body fat mass remained relatively stable (-1.3%, -1.8% and -2.5% respectively, no significance). HDL- cholesterol increased (P < 0.05) whereas total, LDL and VLDL cholesterol, triglycerides, aspartate aminotransferase/ AST, gamma glutamyltransferase/GGT, alkaline phosphatase/ AP, fasting blood glucose and glycated hemoglobin/ HbA1c decreased (P < 0.05). When patients were stratified according to increase (22/48, 45.8%) and decrease (21/48, 43.8%) of BMI, association between weight decrease and liver benefit could be elicited in such circumstances for ALT, AP and AST/ALT ratio. No change could be demonstrated in patients who gained weight. Multivariate assessment confirmed that waist circumference, ferritin, triacylglycerol, and markers of glucose homeostasis were the most relevant associated with liver enzymes.nnnDISCUSSIONnOurs results are consistent with the literature of calorie restriction in the management of NAFLD. Changes in lifestyle and weight loss are recommended for NAFLD patients. European guidelines also support this recommendation.nnnCONCLUSIONnThis is the first study that demonstrated that a high protein, hypocaloric diet were associated with improvement of lipid profile, glucose homeostasis and liver enzymes in NAFLD independent on BMI decrease or body fat mass reduction.

Yo Jyo Hen Shi Ko, a novel Chinese herbal, prevents nonalcoholic steatohepatitis in ob/ob mice fed a high fat or methionine–choline-deficient diet

Background: Oxidative stress plays a role in the pathogenesis of nonalcoholic steatohepatitis (NASH). Yo Jyo Hen Shi Ko (YHK) is a complex compound purported to reduce reactive oxygen species (ROS) by blocking the propagation of radical‐induced reactions. The aim of this study was to evaluate the role of the effect of YHK in experimental NASH.

Nonalcoholic Steatohepatitis (NASH) in OB/OB Mice Treated with Yo Jyo Hen Shi Ko (YHK): Effects on Peroxisome Proliferator-Activated Receptors (PPARs) and Microsomal Triglyceride Transfer Protein (MTP)

YHK has antioxidant properties, has a hypoglycemic effect, and may reduce plasma lipid levels. In this study, we examined the hepatic expression of PPAR-α and -γ and MTP in ob/ob mice receiving or not receiving YHK. Ob/ob mice were assigned to receive oral YHK (20 mg/kg/day) fed solution (methionine/choline-deficient [MCD] dietxa0+xa0YHK group) or vehicle (MCD group) by gavage for 4 weeks. Liver fragments were collected for histologic examination and mRNA isolation. PPAR-α and -γ and MTP gene expression was examined by RT-qPCR. YHK treatment was associated with NASH prevention, weight loss, and reduction of visceral fat and of serum concentrations of aminotransferases in comparison to the MCD group. YHK promoted an increment in PPAR-α and MTP and a decrement in PPAR-γ mRNA contents. These findings suggest that modulation of PPAR-α and -γ and MTP RNA expression may be implicated in the protective effect of YHK in experimental NASH, limiting hepatocyte lipid accumulation.

Primary Biliary Acids Inhibit Hepatitis D Virus (HDV) Entry into Human Hepatoma Cells Expressing the Sodium-Taurocholate Cotransporting Polypeptide (NTCP)

Background The sodium-taurocholate cotransporting polypeptide (NTCP) is both a key bile acid (BA) transporter mediating uptake of BA into hepatocytes and an essential receptor for hepatitis B virus (HBV) and hepatitis D virus (HDV). In this study we aimed to characterize to what extent and through what mechanism BA affect HDV cell entry. Methods HuH-7 cells stably expressing NTCP (HuH-7/NTCP) and primary human hepatocytes (PHH) were infected with in vitro generated HDV particles. Infectivity in the absence or presence of compounds was assessed using immunofluorescence staining for HDV antigen, standard 50% tissue culture infectious dose (TCID50) assays and quantitative PCR. Results Addition of primary conjugated and unconjugated BA resulted in a dose dependent reduction in the number of infected cells while secondary, tertiary and synthetic BA had a lesser effect. This effect was observed both in HuH-7/NTCP and in PHH. Other replication cycle steps such as replication and particle assembly and release were unaffected. Moreover, inhibitory BA competed with a fragment from the large HBV envelope protein for binding to NTCP-expressing cells. Conversely, the sodium/BA-cotransporter function of NTCP seemed not to be required for HDV infection since infection was similar in the presence or absence of a sodium gradient across the plasma membrane. When chenodeoxycolic acid (15 mg per kg body weight) was administered to three chronically HDV infected individuals over a period of up to 16 days there was no change in serum HDV RNA. Conclusions Primary BA inhibit NTCP-mediated HDV entry into hepatocytes suggesting that modulation of the BA pool may affect HDV infection of hepatocytes.