Protein glutathionylation increases in the liver of patients with non-alcoholic fatty liver disease

Background and Aim:  Oxidative stress is an important pathophysiological mechanism in non-alcoholic steatohepatitis, where hepatocyte apoptosis is significantly increased correlating with disease severity. Protein glutathionylation occurs as a response to oxidative stress, where an increased concentration of oxidized glutathione modifies post-translational proteins by thiol disulfide exchange. In this study, we analyzed the protein glutathionylation in non-alcoholic fatty liver disease (NAFLD) and evaluated a potential association between glutathionylation, fibrosis, and vitamin E treatment.
Methods:  Protein glutathionylation was studied in the livers of 36 children (mean age 12.5 years, range 4–16 years) subdivided into three groups according to their NAFLD activity score (NAS) by Western blot analysis and immunohistochemistry, using a specific monoclonal antibody. In addition, we identified the hepatocyte ultrastructures involved in glutathionylation by immunogold electron microscopy.
Results:  Our findings showed that protein glutathionylation increases in the livers of patients with NAFLD and it is correlated with steatohepatitis and liver fibrosis. Its increase appears mainly in nuclei and cytosol of hepatocytes, and it is reversed by antioxidant therapy with reduced fibrosis.
Conclusion:  Protein glutathionylation significantly increases in livers with NAFLD, strongly suggesting that oxidative injury plays a crucial role in this disease. Furthermore, the marked increase of protein glutathionylation, in correlation with collagen VI immunoreactivity, suggests a link between the redox status of hepatic protein thiols and fibrosis.     

Gut microbiota dysbiosis in patients with non-alcoholic fatty liver disease

BACKGROUND: Gut microbiota plays a significant role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). This study aimed to assess the contribution of gut microbiota dysbiosis to the pathogenesis of NAFLD. METHODS: Forty-seven human feces samples (25 NAFLD patients and 22 healthy subjects) were collected and 16S rDNA amplicon sequencing was conducted on Hiseq 2000 platform. Discrepancy of species composition between controls and NAFLD group was defined by Metastats analysis under P value<0.01. RESULTS: NAFLD patients harbored lower gut microbiota diversity than healthy subjects did. In comparison to the control group, the Proteobacteria (13.50%) and Fusobacteria (2.76%) phyla were more abundant in NAFLD patients. Ad-ditionally, the Lachnospiraceae (21.90%), Enterobacteriaceae (12.02%), Erysipelotrichaceae (3.83%), and Streptococcaceae (1.39%) families, as well as the Escherichia_Shigella (10.84%), Lachnospiraceae_Incertae_Sedis (7.79%), and Blautia (4.95%) genera were enriched in the NAFLD group. However, there was a lower abundance of Prevotella in the NAFLD group than that in the control group (5.83% vs 27.56%, P<0.01). The phylum Bacteroidetes (44.63%) also tended to be more abundant in healthy subjects, and the families Prevotellaceae (28.66%) and Ruminococcaceae (26.44%) followed the same trend. Compared to those without non-alcoholic steatohepa-titis (NASH), patients with NASH had higher abundance of genus Blautia (5.82% vs 2.25%; P=0.01) and the correspond-ing Lachnospiraceae family (24.33% vs 14.21%; P<0.01). Patients with significant fibrosis had a higher abundance of genus Escherichia_Shigella (12.53% vs 1.97%; P<0.01) and the corresponding Enterobacteriaceae family (13.92% vs 2.07%;P<0.01) compared to those with F0/F1 fibrosis. CONCLUSIONS: NAFLD patients and healthy subjects harbor varying gut microbiota. In contrast to the results of previous research on children, decreased levels of Prevotella might be detrimental for adults with NAFLD. The increased level of the genus Blautia, the family Lachnospiraceae, the genus Escherichia_Shigella, and the family Enterobacteriaceae may be a primary contributor to NAFLD progression.     

Experimental models of non-alcoholic fatty liver disease in rats

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the Western world, and it persists at a high prevalence. NAFLD is characterised by the accumulation of triglycerides in the liver and includes a spectrum of histopathological findings, ranging from simple fatty liver through non-alcoholic steatohepatitis (NASH) to fibrosis and ultimately cirrhosis, which may progress to hepatocellular carcinoma. The pathogenesis of NAFLD is closely related to the metabolic syndrome and insulin resistance. Understanding the pathophysiology and treatment of NAFLD in humans has currently been limited by the lack of satisfactory animal models. The ideal animal model for NAFLD should reflect all aspects of the intricate etiopathogenesis of human NAFLD and the typical histological findings of its different stages. Within the past several years, great emphasis has been placed on the development of an appropriate model for human NASH. This paper reviews the widely used experimental models of NAFLD in rats. We discuss nutritional, genetic and combined models of NAFLD and their pros and cons. The choice of a suitable animal model for this disease while respecting its limitations may help to improve the understanding of its complex pathogenesis and to discover appropriate therapeutic strategies. Considering the legislative, ethical, economical and health factors of NAFLD, animal models are essential tools for the research of this disease.     

Non-alcoholic fatty liver disease: an overview

Non-alcoholic fatty liver disease (NAFL) includes a spectrum of clinicopathological conditions with increasing prevalence in the developed world. Although steatosis alone seems to have a benign course, those patients with the diagnosis of non-alcoholic steatohepatitis (NASH) can have a progressive course. Additionally, there is now evolving, indirect evidence that some of the patients with cryptogenic cirrhosis may be the result of 'burned-out' NASH. Although NAFL and NASH are associated with insulin-resistance syndrome, some patients with NAFL may have no obvious risk factors. Despite preliminary data from a number of pilot studies, no established therapies can be offered to patients with NASH. Over the next few years, a number of exciting research projects dealing with the epidemiology as well as the pathogenesis of NAFL are expected to be completed. It is anticipated that, through a better understanding of NAFL, more effective treatment protocols can be developed targeting only those patients with NASH that are at the highest risk for progression to cirrhosis and liver failure.     

Influence of inducible nitric oxide synthase polymorphisms in Japanese patients with non-alcoholic fatty liver disease

Aim:  Genetic factors as well as environmental factors play an important role in the development of non-alcoholic fatty liver disease (NAFLD). Recently, inducible nitric oxide synthase (iNOS) was significantly higher in the severest form of non-alcoholic steatohepatitis (NASH), and nitric oxide (NO) has been determined to play an important role in the process of fibrosis in NASH. In this study, we investigated iNOS gene polymorphisms for associations with NAFLD.
Methods:  A total of 115 NAFLD patients, consisting of 65 patients with NASH and 50 patients with simple steatosis, in whom a positive diagnosis had been made by liver biopsy, and 435 healthy control subjects, were recruited into this study.
Results:  We investigated 10 single nucleotide polymorphisms (SNP) of the iNOS gene, one of which, rs1060822, had the lowest P -value in the allele frequency model ( P  = 0.00078) with an odds ratio (95% confidence interval) of 0.49 (0.32–0.75). Four SNP, rs2297510, rs2297511, rs2797512 and rs1060822, were significantly associated with NAFLD, even when the most conservative Bonferroni's correction was applied. Linkage disequilibrium analysis revealed that SNP rs1060822 and three other SNP, rs2297510, rs2297511 and rs2797512, were in the same block. We also investigated associations between rs1060822 genotypes and the fibrosis index, and the results of the analysis revealed an additive increase in the fibrosis index and intrahepatic iNOS mRNA expression in the patients with the T allele of rs1060822.
Conclusion:  This is the first study to identify genetic variations in iNOS that may influence the risk of NAFLD and liver fibrosis in NAFLD.     

Mean platelet volume in biopsy-proven non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is increasingly recognized as the most common cause of chronic liver disease worldwide. It has been shown that NAFLD has a strong association with metabolic syndrome and its component like insulin resistance (IR). Cardiovascular disease has a relation with NAFLD. Platelet volume is an indicator of platelet function and activation. Mean platelet volume (MPV) has been reported as a risk factor for atherothrombosis. In our study, we aimed to investigate the relation of MPV with NAFLD and IR in the NAFLD patients. A total of 54 patients with histologically proven NAFLD and 41 healthy age-matched control subject were enrolled in this study. The NAFLD subjects were divided into two subgroups: 42 patients in the insulin resistant group (median age 39.5, females 22 [52%]) and 12 patients in the insulin sensitive group (median age 38, females 5 [41.7%]). MPV were significantly higher in the NAFLD group in univariate analysis (p?<?0.05). In the NAFLD patients, we did not find any relation between steatosis grade, lobular inflammation, hepatocellular ballooning, NAFLD activity score and fibrosis with MPV value. Among the insulin resistant and sensitive groups in the NAFLD patients MPV values were similar. The results of this study showed that MPV, an indicator of platelet activation, increased in biopsy proven NAFLD patients but MPV is not correlated with the increase of IR in NAFLD patients. MPV is not related with inflammation and steatosis degree, hepatocellular ballooning and fibrosis in NAFLD patients.     

Trends in hepatocellular carcinoma due to non-alcoholic fatty liver disease

Introduction: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide and is associated with hepatocellular carcinoma (HCC), the most frequent malignant liver tumor. The increasing prevalence of obesity and diabetes is influencing the epidemiology of HCC with the most dramatic increases in NAFLD-related HCC seen in Western countries. Although cirrhosis is the major risk factor for HCC in NAFLD, there is increasing recognition that NAFLD-HCC occurs in the absence of cirrhosis.

Areas covered: The epidemiology of NAFLD related HCC and its impact on changing the incidence of HCC globally. We overview risk factors for NAFLD-HCC in the presence and absence of cirrhosis and examine trends in liver transplantation (LT) related to NAFLD-HCC.

Expert commentary: The incidence of NAFLD-related cirrhosis will continue to rise globally in parallel with risk factors of obesity and diabetes. Consequently, NAFLD-related HCC will become an increasingly important cause of liver-related morbidity and mortality and a common indication for LT worldwide. Further identification of risk factors for NAFLD-HCC and effective treatments for NAFLD are required to reduce this future burden of disease.     

Metformin in the treatment of non-alcoholic fatty liver disease: safety,efficacy and mechanism

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease etiology worldwide. It encompasses a spectrum ranging from simple steatosis to non-alcoholic steatohepatitis. Although the physiopathology of NAFLD is partly known. Insulin-resistance plays a central role in the development and progression of NAFLD. Several studies have indicated that metformin, as an insulin sensitizer, effectively improves NAFLD and its related metabolic status. Metformin was effective in reducing enzyme levels in the short period, but very limited and controversial information are available on liver histology. Larger randomized controlled trials of sufficient duration using clearly defined histological endpoints are needed to fully assess the efficacy of this drug in modifying the natural history of NAFLD.     

Role of hepatic iron in non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) includes a spectrum of clinical entities ranging from simple steatosis to non-alcoholic steatohepatitis (NASH) with possible evolution to cirrhosis and hepatocellular carcinoma. Iron is considered a putative element that interacts with oxygen radicals in inducing liver damage and fibrosis. The role of hepatic iron in the progression of NASH remains controversial, but in some patients, iron may have a role in the pathogenesis of NASH. Though genetic factors, insulin resistance, dysregulation of iron-regulatory molecules, erythrophagocytosis by Kupffer cells may be responsible for hepatic iron accumulation in NASH, exact mechanisms involved in iron overload remain to be clarified. Iron reduction therapy such as phlebotomy or dietary iron restriction may be promising in patients with NASH/NAFLD to reduce insulin resistance as well as serum transaminase activities.     

The effect of ursodeoxycholic acid on liver regeneration after partial hepatectomy in rats with non-alcoholic fatty liver disease

Aim:  To investigate the effect of ursodeoxycholic acid (UDCA) on liver regeneration following partial hepatectomy in rats with non-alcoholic fatty liver disease (NAFLD).
Methods:  UDCA was administered to seven rats (group 1) and physiological saline was administered both to seven rats (group 2) with NAFLD and to seven rats with normal livers (group 3). All rats underwent two-thirds hepatectomy and the remnant liver tissues were removed 48 h later. Mitotic index (MI) and levels of proliferating cell nuclear antigen (PCNA), glutathione (GSH) and malondialdehyde (MDA) were assayed.
Results:  MI and PCNA levels in group 2 were significantly lower than in groups 1 and 3, but the values in groups 1 and 3 were similar. The GSH levels of group 2 were significantly lower than those of group 3 in the hepatectomy tissues, and lower than those of groups 1 and 3 in the remnant tissues. The differences between GSH levels in groups 1 and 3 were not significant. MDA levels in hepatectomy and remnant tissues were significantly higher in group 2 compared to groups 1 and 3; values in groups 1 and 3 were similar.
Conclusion:  UDCA increases regeneration after partial hepatectomy in rats with NAFLD, possibly due to an attenuating effect on oxidative stress.     

Placebo-controlled,randomised clinical trial: high-dose resveratrol treatment for non-alcoholic fatty liver disease

Objective “The obesity epidemic” has led to an increase in obesity-related conditions including non-alcoholic fatty liver disease (NAFLD), for which effective treatments are in demand. The polyphenol resveratrol prevents the development of experimental NAFLD through modulation of cellular pathways involved in calorie restriction. We aimed to test the hypothesis that resveratrol alleviates NAFLD in a randomised, clinical trial. Materials and methods A total of 28 overweight patients with transaminasemia and histological NAFLD were randomised 1:1 to placebo or resveratrol 1.5 g daily for 6 months. Twenty-six participants completed the trial and underwent repeated clinical investigation, blood work, MR spectroscopy; and 19 participants agreed to a repeat liver biopsy. Results Resveratrol treatment was generally not superior to placebo in improving plasma markers of liver injury (primary outcome: alanine transaminase, p?=?0.51). Resveratrol-treated patients showed a 3.8% decrease in liver lipid content (p?=?0.03), with no difference between the two treatment arms (p?=?0.38) and no improvement of histological features. Resveratrol treatment was not associated with improvements in insulin sensitivity or markers of the metabolic syndrome, except for a transient decrease in systolic BP. Microarray analysis and qRT-PCR revealed no major changes in expression profile. Also, we report a serious adverse event in a patient who developed fever and bicytopenia. Conclusions In this placebo-controlled, high-dose and long-term study, resveratrol treatment had no consistent therapeutic effect in alleviating clinical or histological NAFLD, though there may be a small ameliorating effect on liver function tests and liver fat accumulation.     

复方甘枣宁预防大鼠非酒精性脂肪肝的实验研究

[目的]研究复方甘枣宁对高脂饮食诱发的大鼠脂肪肝的预防作用.[方法]采用高脂饲料喂饲制作大鼠非酒精性脂肪肝模型,同时以复方甘枣宁灌胃,12周后检测血清中总胆固醇(TC)、三酰甘油(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、丙氨酸氨基转移酶(ALT)、天冬氨酸转氨酶(AST)水平,并作病理组织学观察,同时称体重、肝脏重,计算肝指数.[结果]复方甘枣宁明显减轻肝组织内脂肪变性,降低血清TC与AST水平.[结论]复方甘枣宁对高脂饮食诱发的大鼠非酒精性脂肪肝具有一定的预防作用.     

非酒精性脂肪性肝病的研究进展

非酒精性脂肪性肝病经历由非酒精性单纯性脂肪肝发展至非酒精性脂肪性肝炎和肝硬化或原发性肝癌的缓慢过程,并与心血管病的发生关系密切.本文针对非酒精性脂肪性肝病的流行病学、发病机制、诊断和治疗等的研究进展进行综述.     

活血化痰方对非酒精性脂肪肝大鼠炎性因子的影响

[目的]观察活血化痰方对非酒精性脂肪肝（NAFLD）大鼠炎性因子的影响。[方法]将30只大鼠随机分为正常对照组、模型组和活血化痰方组。检测各组大鼠血清中白细胞介素（IL）-6、IL-10及肿瘤坏死因子-α（TNF-α）的水平。[结果]与正常对照组比较,模型组血清中IL-6、TNF-α水平增高,血清中IL-10含量降低;与模型组相比,活血化痰方组血清中IL-6、TNF-α水平显著降低,血清中IL-10含量显著升高。[结论]活血化痰方可能通过降低血清中IL-6、TNF-α水平,提高血清中IL-10含量,调节体内炎性因子的变化,从而使NAFLD渐愈。     

姜黄素对大鼠非酒精性脂肪肝的干预作用

[目的]研究姜黄素对大鼠非酒精性脂肪肝的保护作用。[方法]30只Wismr大鼠随机分为对照组、非酒精性脂肪肝模型组和姜黄素干预组。对照组以普通饲料饲养,模型组和干预组给予高脂饲料饲养,干预组每日予50mg／kg姜黄素灌胃,共计12周。实验结束处死大鼠,收集血清和肝组织。检测血清丙氨酸氨基转移酶活性、天冬氨酸氨基转移酶活性和血清总胆固醇含量,以及肝组织γ-谷氨酰转肽酶活性、三酰甘油含量、超氧化物歧化酶活性和谷胱甘肽活性。肝组织行苏木精-伊红染色和油红O染色检测肝脏病理改变。[结果]与模型组比较,干预组能显著降低丙氨酸氨基转移酶、天冬氨酸氨基转移酶和肝组织γ-谷氨酰转肽酶活性,减少血清总胆固醇和肝组织三酰甘油含量;显著升高肝组织超氧化物歧化酶和谷胱甘肽活性;明显减轻大鼠肝内脂肪沉积,改善肝细胞的脂肪性病理改变。[结论]姜黄素通过抗氧化作用,对大鼠非酒精性脂肪肝具有良好的干预作用。     

柴苓调肝颗粒剂治疗大鼠非酒精性脂肪性肝病的实验研究

[目的]探讨柴苓调肝颗粒剂治疗大鼠非酒精性脂肪性肝病(NAFLD)的作用机制。[方法]采用高脂饲料的方法制备大鼠NAFLD模型,造模成功后,随机分为7组,柴苓调肝颗粒剂高、中、低剂量(高、中、低剂量)组,甘乐对照(甘乐)组、三七脂肝丸对照(三七脂肝丸)组、模型对照(模型)组和饮食控制组,另设正常对照(正常)组未予造模。各组分别给予相应的处理,4周后检测各组血清丙氨酸氨基转移酶(ALT)、天冬氨酸转氨酶(AST)、总胆固醇(TC)、三酰甘油(TG)、肿瘤坏死因子α(TNF-α)和瘦素(LP)以及肝组织中超氧化物歧化酶(SOD)、丙二醛(MDA)、体重及肝重指数的变化。[结果]高剂量组各项指标与模型组和饮食控制组比较差异均有统计学意义(P0.05,0.01),与正常组比较(P0.05)。[结论]柴苓调肝颗粒剂可有效治疗实验性NAFLD。