Ethanol binging enhances hepatic microvascular responses to acetaminophen in mice

OBJECTIVE: Chronic alcoholism has been considered to be a risk for acetaminophen (APAP) hepatotoxicity, but little is known about the effect of binge alcohol drinking on APAP-induced liver injury. The present study was conducted to examine the effect of ethanol binging on APAP-induced hepatic microcirculatory dysfunction. METHODS: Male C57Bl/6 mice received 3 weekly ethanol binges (4 g/kg every 12 h x 5 doses/ week) or water binges. At 12 h after the last gavage, APAP (300 mg/kg) was given by oral gavage. In one group of mice, gadolinium chloride (GdCl3, 10 mg/kg) was intraperitoneally administered 2 and 1 days before the start of each weekly ethanol binge. RESULTS: Ethanol binging enhanced APAP-induced liver injury as indicated by ALT levels. Intravital microscopic study showed that APAP further increased the area occupied by infiltrated erythrocytes into the extrasinusoidal space as well as Kupffer cell phagocytic activity in ethanol-binged mice when compared with water-binged mice, while no significant differences in sinusoidal perfusion and leukocyte adhesion were observed. ALT levels after APAP were exacerbated in ethanol-binged mice treated with GdCl3, but APAP-induced hepatic microcirculatory dysfunction was not changed significantly. CONCLUSIONS: These results suggest that ethanol binging increases APAP-induced liver injury by exacerbating infiltration of the Disse space with blood cells. Kupffer cells exert a protective role in the liver against APAP intoxication following ethanol binging.     

Early hepatic microvascular injury in response to acetaminophen toxicity

OBJECTIVE: The hepatic toxic response to acetaminophen (APAP) is characterized by centrilobular (CL) necrosis preceded by hepatic microvascular injury and congestion. The present study was conducted to examine changes in liver microcirculation after APAP dosing. METHODS: Male C57Bl/6 mice were treated with APAP (600 mg/kg body weight) by oral gavage. The livers of anesthetized mice were examined using established in vivo microscopic methods at 0, 0.5, 1, 2, 4, 6, 12 hours after APAP. RESULTS: The levels of hepatic transaminases (i.e., alanine aminotransferase [ALT] and aspartate transaminase) increased minimally for up to 2 hours. Thereafter, their levels were significantly and progressively increased. The numbers of swollen sinusoidal endothelial cells (SECs) in periportal regions were increased (3.5-fold) from 0.5 to 6 hours, and those in CL regions were increased (4.0-fold) at 0.5 and 1 hour. The intensity of in vivo staining for formaldehyde-treated serum albumin, which is a specific ligand for SECs, was reduced from 2 to 12 hours. Erythrocytes infiltrated into the space of Disse as early as 2 hours, and the area occupied by these cells was markedly increased at 6 hours. Sinusoidal perfusion was reduced from 1 through 12 hours, with a nadir (35% decrease) at 4 and 6 hours. Phagocytic Kupffer cell activity was significantly elevated from 0.5 through 12 hours. Although gadolinium chloride minimized the changes in sinusoidal blood flow and reduced ALT levels 6 hours after APAP, it failed to inhibit endothelial swelling, extravasation of erythrocytes, and CL parenchymal necrosis. CONCLUSIONS: These results confirm that APAP-induced SEC injury precedes hepatocellular injury, supporting the hypothesis that SECs are an early and direct target for APAP toxicity. These findings also suggest that reduced sinusoidal perfusion and increased Kupffer cell activity contribute to the development of APAP-induced liver injury.     

Silymarin protects against acute ethanol-induced hepatotoxicity in mice

BACKGROUND: Accumulated evidence has demonstrated that both oxidative stress and abnormal cytokine production, especially tumor necrosis factor-alpha (TNF), play important etiological roles in the pathogenesis of alcoholic liver disease (ALD). Agents that have both antioxidant and anti-inflammation properties, particularly anti-TNF production, represent promising therapeutic interventions for ALD. We investigated the effects and the possible mechanism(s) of silymarin on liver injury induced by acute ethanol (EtOH) administration. METHODS: Nine-week-old mice were divided into 4 groups, control, silymarin treatment, EtOH treatment, and silymarin/EtOH treatment, with 6 mice in each group. Because control and silymarin values were virtually identical, only control treatment is shown for ease of viewing. Ethanol-treated mice received EtOH [5 g/kg body weight (BW)] by gavage every 12 hours for a total of 3 doses. Control mice received an isocalorical maltose solution. In the silymarin/EtOH group, silymarin was dissolved in the EtOH and gavaged simultaneously with EtOH at a dose of 200 mg/kg BW. At 4 hours after the last dosing, the mice were anesthetized and subsequent serum alanine aminotransferase (ALT) level, hepatic lipid peroxidation, enzymatic activity of hepatic cytochrome P450 2E1, hepatic TNF-alpha, and glutathione (GSH) levels were measured. Histopathological change was assessed by hematoxylin and eosin staining. RESULTS: Acute EtOH administration caused prominent hepatic microvesicular steatosis with mild necrosis and an elevation of serum ALT activity, induced a significant decrease in hepatic GSH in conjunction with enhanced lipid peroxidation, and increased hepatic TNF production. Supplementation with a standardized silymarin attenuated these adverse changes induced by acute EtOH administration. CONCLUSIONS: Silymarin protects against the liver injury caused by acute EtOH administration. In view of its nontoxic nature, it may be developed as an effective therapeutic agent for alcohol-induced liver disease by its antioxidative stress and anti-inflammatory features.     

Sampling variability of liver biopsy in nonalcoholic fatty liver disease

BACKGROUND & AIMS: In nonalcoholic fatty liver disease (NAFLD), the distinction between steatosis and steatohepatitis (NASH) and the assessment of the severity of the disease rely on liver histology alone. The aim of this study was to assess the sampling error of liver biopsy and its impact on the diagnosis and staging of NASH. METHODS: Fifty-one patients with NAFLD underwent percutaneous liver biopsy with 2 samples collected. The agreement between paired biopsy specimens was assessed by the percentage of discordant results and by the kappa reliability test. RESULTS: No features displayed high agreement; substantial agreement was only seen for steatosis grade; moderate agreement for hepatocyte ballooning and perisinusoidal fibrosis; fair agreement for Mallory bodies; acidophilic bodies and lobular inflammation displayed only slight agreement. Overall, the discordance rate for the presence of hepatocyte ballooning was 18%, and ballooning would have been missed in 24% of patients had only 1 biopsy been performed. The negative predictive value of a single biopsy for the diagnosis of NASH was at best 0.74. Discordance of 1 stage or more was 41%. Six of 17 patients with bridging fibrosis (35%) on 1 sample had only mild or no fibrosis on the other and therefore could have been under staged with only 1 biopsy. Intraobserver variability was systematically lower than sampling variability and therefore could not account for most of the sampling error. CONCLUSIONS: Histologic lesions of NASH are unevenly distributed throughout the liver parenchyma; therefore, sampling error of liver biopsy can result in substantial misdiagnosis and staging inaccuracies.     

Voglibose potentiates the hepatotoxicity of carbon tetrachloride and acetaminophen by inducing CYP2E1 in rats.

BACKGROUND:: Voglibose is an alpha-glucosidase inhibitor used to decrease postprandial hyperglycemia in diabetic patients. Although clinical concern has not yet been raised, hepatic dysfunction has been reported in a few patients taking this drug. METHOD:: In the present study, we studied the effects of voglibose on the hepatotoxicity of carbon tetrachloride (CCl(4)) and acetaminophen (APAP) in rats, since both of these agents exert their effects through isoforms of cytochrome P450. Male Sprague-Dawley rats were given a daily ration (20g) of powdered chow diet containing 0, 2.5, 5.0 or 10.0mg/100g of voglibose. Three weeks later, the rats were challenged with either 0.50g/kg CCl(4) orally or 0.75g/kg APAP intraperitoneally for biochemical examinations or killed for an in vivo metabolism study. RESULTS:: Voglibose at these three experimental doses potentiated CCl(4) and APAP hepatotoxicity, as evidenced by significantly increased levels of both plasma asparate transaminase (AST) and alanine transaminae (ALT). The glutathione (GSH) content was decreased while malondialdehyde (MDA) increased in the liver after CCl(4) or APAP administration. Hepatic cytochrome P450 2E1 (CYP2E1) concentration was increased at doses of 5.0 and 10.0mg/100g of voglibose and its activity increased in the three voglibose dosage groups, while hepatic cytochrome P450 3A (CYP3A) and cytochrome P450 1A2 (CYP1A2) were only slightly changed at any dose. CONCLUSION:: Our study demonstrated that voglibose can potentiate CCl(4) and APAP hepatotoxicity in rats by inducing hepatic CYP2E1.     

CYP2E1与非酒精性脂肪肝

非酒精性脂肪性肝病(nonalcoholie fatty liver disease,NAFLD)是一种最常见的肝脏疾病,它的高发病率越来越受到广泛重视。细胞色素氧化酶P450-2E1亚型(CYP2E1)是参与氧化应激和脂质过氧化过程中的关键酶。"二次打击"学说为医学界普遍接受的非酒精性脂肪肝的发病机制。在非酒精性脂肪肝的"二次打击学说"中,CYP2E1介导的脂质过氧化亦发挥重要的作用。通过对CYP2E1介导的脂质过氧化在脂肪性肝病发病机制中作用的进一步深入研究,有可能探索出有效治疗脂肪性肝病的新途径。     

Endocannabinoid receptor CB2 in nonalcoholic fatty liver disease.

BACKGROUND AND AIM: Fatty infiltration and fibrosis are major issues in chronic liver disease. Recent reports suggest a role for the endocannabinoid system in these processes. AIM: To characterize localization and expression of CB2 in normal liver and nonalcoholic fatty liver. METHODS: We studied 64 liver biopsies: eight were considered normal; 56 had a diagnosis of nonalcoholic fatty liver disease (NAFLD); 32 with nonalcoholic steatosis and 24 nonalcoholic steatohepatitis (NASH). CB2 immunolocalization was studied in 38 samples in paraffin blocks using immunohistochemistry, and a computerized semiquantitative analysis was carried out. CB2 mRNA expression was assessed through RT-PCR in 26 frozen liver samples and the ratio CB2/beta-actin was used to evaluate differences between groups. Statistical analysis was performed with central tendency measures and the Mann-Whitney U-test. We considered as significant differences those with a P-value <0.05. RESULTS: Neither parenchymal nor nonparenchymal cells in normal liver tissue react towards anti-CB2 antibodies. All the samples from patients with steatosis and nonalcoholic steatohepatitis showed hepatocellular immunoreactivity. Cholangiocytes were positive only in the NAFLD group. Normal liver tissue showed a normalized CB2/beta-actin ratio of 0.001+/-0.01, steatosis 6.52+/-17.3 (P=0.05 vs normal) and NASH 6.49+/-12.2 (P=0.06 vs normal and P=0.6 vs steatosis). CONCLUSION: CB2 receptors are expressed by hepatocytes in nonalcoholic fatty liver disease but not in normal liver.     

壳脂胶囊对小鼠非酒精性脂肪性肝炎氧化应激的影响

目的 观察壳脂胶囊对小鼠非酒精性脂肪性肝炎(NASH)肝组织氧化应激及脂质过氧化反应的影响,探讨其对NASH的防治作用及机制.方法 健康雄性C57BL/6J小鼠18只,随机分为三组,每组6只.采用高脂、胆碱-蛋氨酸缺乏(MCD)饮食4周建立小鼠NASH模型,应用壳脂胶囊进行干预实验,并以胆碱-蛋氨酸充足饮食设立对照组....     

乙醇对大鼠肝窦内皮细胞窗孔的影响

目的 研究慢性乙醇摄取对大鼠肝窦内皮细胞(LSEC)窗孔的影响。方法 用乙醇直接灌胃的方法建立大鼠酒精性肝病动物模型，并于开始灌胃4周末、8周末、12周末，以及停止灌酒后(用平衡饲料继续喂养)12周末，分别处死实验组及对照组动物，经心脏灌流后取肝脏组织行HE染色和透射电镜观察LSEC窗孔的动态变化。结果 正常的LSEC扁平，胞核及细胞器排列规则，远侧胞质呈薄片状，有许多窗孔，内皮下缺乏基底膜(BM)；乙醇喂养4周末，可见部分LSEC远侧胞窗孔数减少，但BM未形成；8周末，窗孔数明显减少或消失，内皮下开始有不完全的BM形成，同时有功能活跃的纤维母细胞形成；12周末进一步加重，甚至可见完整的BM形成，但这种改变也多限于单个或邻近的窦状隙内，极少有广泛的纤维化形成；停药12周末，失窗孔及内皮下BM形成明显减轻。结论 随着乙醇的慢性刺激LSEC的去窗孔化和BM的形成也逐渐发生，严重时可形成肝窦毛细血管化和肝纤维化；这种早期即有局限性的去窗孔化改变和毛细血管化的生成可能是形成酒精性周围型纤维化的基础；去除病因后这种肝纤维化是可逆的。     

Influence of clinicopathological variables on CYP protein expression in human liver

Drug metabolism is usually impaired in malnourished patients with decompensated cirrhosis, but the separate influence of clinicopathological variables, including nutritional status, on the expression of hepatic cytochrome P450 proteins has not been well characterized. We determined the hepatic content of CYP1A2, CYP2C8/10, CYP2E1 and CYP3A proteins in 71 subjects, 21 with histologically normal livers and 50 with chronic liver disease, and then tested for potential relationships between patient variables and individual CYP proteins by multivariate linear regression analysis. Variables analysed included nutritional status (determined by experienced clinicians), serum albumin and bilirubin concentrations, prothrombin time, the grade of ascites and hepatic encephalopathy, and the Child-Pugh score. Impaired nutrition and cachexia were associated with reductions of CYP2C8/10 levels of approximately 19 and 39%, respectively, relative to cases in which nutrition was replete. Similarly, CYP2E1 protein was reduced by approximately 13 and 26%, according to the apparent severity of nutritional impairment. In contrast, nutritional status did not contribute to variability in expression of CYP1A2 or CYP3A proteins. Of the clinicopathological variables analysed, only serum bilirubin was shown to have an independent influence on CYP protein content. Thus, elevated serum bilirubin concentrations were associated with significant declines in the contents of CYP1A2 and CYP2C8/10 but not CYP3A or CYP2E1. The mechanisms for the effects of nutritional status and serum bilirubin concentration on the levels of CYP proteins are unclear, but could be mediated by factors such as cytokines, dietary composition and alterations in the level of serum bile acids. Knowledge of the influence of clinicopathological factors and nutritional status on CYP expression should lead to more rational drug prescribing in patients with hepatic disease.     

Mild hypothermia attenuates liver injury and improves survival in mice with acetaminophen toxicity

BACKGROUND & AIMS: Body temperature may critically affect mechanisms of liver injury in acetaminophen (APAP) hepatotoxicity. In addition, mild hypothermia is used to treat intracranial hypertension in human liver failure without detailed information on its effects on the injured liver itself. Therefore, we investigated the effects of body temperature on the progression of APAP-induced liver injury in mice. METHODS: Male C57BL6 mice treated with saline or APAP (300 mg/kg intraperitoneally) were maintained at normothermia (35.5-37.5 degrees C) by external warming or were allowed to develop mild hypothermia (32.0-35.0 degrees C) after 2 hours from APAP administration. RESULTS: Mild hypothermia resulted in improved survival after APAP intoxication. Liver damage was reduced, as assessed histologically and by plasma alanine aminotransferase levels. Early effects of hypothermia included a reduction of hepatic congestion and improved recovery of glycogen stores. At later time points (8-12 hours), APAP-treated mice that were maintained at normothermia manifested increased hepatocyte apoptosis, as assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining and cleavage of poly(adenosine diphosphate-ribose) polymerase. Mild hypothermia did not affect the formation of APAP-protein adducts or the depletion of glutathione, nor did it abrogate hepatocyte DNA synthesis. CONCLUSIONS: Mild hypothermia improved survival and attenuated liver injury and apoptosis in APAP-treated mice by reducing hepatic congestion and improving glycogen recovery without affecting hepatic regeneration. Results of the study underscore the need for a strict control of body temperature in animal models of liver failure and suggest that the benefits of mild hypothermia in liver failure may extend beyond those related to reduced cerebral complications.     

Nonalcoholic fatty liver disease from a primary care perspective

Nonalcoholic fatty liver disease (NAFLD) affects up to one-third of the US population. Approximately one-fifth of patients with NAFLD have nonalcoholic steatohepatitis (NASH), characterized by hepatocyte damage and inflammation with or without fibrosis. NASH leads to greater risk of liver-related complications and liver-related mortality, with the poorest outcomes seen in patients with advanced fibrosis. NASH is also associated with other metabolic comorbidities and conveys an increased risk of adverse cardiovascular outcomes and extrahepatic cancers. Despite its high prevalence, NAFLD is frequently underdiagnosed. This is a significant concern, given that early diagnosis of NAFLD is a key step in preventing progression to NASH. In this review, we describe the clinical impact of NASH from the perspective of both the clinician and the patient. In addition, we provide practical guidance on the diagnosis and management of NASH for primary care providers, who play a pivotal role in the frontline care of patients with NASH, and we use case studies to illustrate real-world scenarios encountered in the primary care setting.     

氧化应激与核因子E2相关因子2在非酒精性脂肪性肝病中的作用

非酒精性脂肪肝病(NAFLD)是全球最流行的慢性肝病,发病率逐年上升。氧化应激是非酒精性脂肪肝病经典“二次打击”发病机制的第二次打击,是目前公认的NAFLD发病机制之一。核因子E2相关因子2(Nrf2)是保护肝细胞免受氧化应激的一组正向调节因子,是细胞抗氧化应激的关键因子,也是拮抗肝脏氧化应激的关键转录因子,在NAFLD发生发展中起重要作用,Nrf2可能是改善NAFLD的潜在治疗靶点。对氧化应激及Nrf2通路在NAFLD中的发病机制进行了综述。