Susceptibility of rat non-alcoholic fatty liver to the acute toxic effect of acetaminophen

Background and Aim: Acetaminophen overdose is the most frequent cause of acute liver failure. Non‐alcoholic fatty liver disease is the most common chronic condition of the liver. The aim was to assess whether non‐alcoholic steatosis sensitizes rat liver to acute toxic effect of acetaminophen. Methods: Male Sprague–Dawley rats were fed a standard diet (ST‐1, 10% kcal fat) and high‐fat gelled diet (HFGD, 71% kcal fat) for 6 weeks and then acetaminophen was applied in a single dose (1 g/kg body weight). Animals were killed 24, 48 and 72 h after acetaminophen administration. Serum biochemistry, activities of mitochondrial complexes, hepatic malondialdehyde, reduced and oxidized glutathione, triacylglycerol and cholesterol contents, and concentrations of serum and liver cytokines (TNF‐α, TGF‐β1) were measured and histopathological samples were prepared. Results: The degree of liver inflammation and hepatocellular necrosis were significantly higher in HFGD fed animals after acetaminophen administration. Serum markers of liver injury were elevated only in acetaminophen treated HFGD fed animals. Concentration of hepatic reduced glutathione and ratio of reduced/oxidized glutathione were decreased in both ST‐1 and HFGD groups at 24 h after acetaminophen application. Mild oxidative stress induced by acetaminophen was confirmed by measurement of malondialdehyde. Liver content of TNF‐α was not significantly altered, but hepatic TGF‐β1 was elevated in acetaminophen treated HFGD rats. We did not observe acetaminophen‐induced changes in activities of respiratory complexes I, II, and IV and activity of caspase‐3. Conclusion: Liver from rats fed HFGD is more susceptible to acute toxic effect of acetaminophen, compared to non‐steatotic liver.     

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.     

Low-level mechanical strain induces extracellular signal-regulated kinase 1/2 activation in alveolar epithelial cells

Background and objective: The pattern and the degree of mechanical stimuli may determine cellular responses, but little is known about how low magnitude stimuli are converted into biochemical signals in alveolar epithelial cells (AEC). The aim of this study was to explore whether extracellular signal‐regulated kinases 1/2 (ERK1/2) are activated by low‐level strain in A549 cells and how mechanical factors affect ERK1/2 phosphorylation. Methods: A549 cells (an AEC line) were exposed to cyclic tensile strain via a four‐point bending system, with strains of different magnitude (437, 874, 1748, 3496 µstrain), duration (5, 15, 30, 60, 120 min) and frequency (0.5, 1 Hz). Phosphorylation of ERK1/2 proteins was assessed by western blot. Results: Maximal ERK1/2 phosphorylation occurred in the 874 µstrain group (a 2.25‐fold increase, P < 0.01). In this group, the peak response occurred after 30 min of exposure and slowly decreased to baseline after 90 min. Static strain did not produce a statistically significant increase in ERK1/2 phosphorylation, whereas a frequency of 0.5 Hz produced a 4.56‐fold increase compared with the control (P < 0.05). A 10.87‐fold increase in response with frequency of 1 Hz was found. Conclusion: Low‐level strain activates ERK1/2 in A549 cells. ERK1/2 may be the key signalling molecules mediating strain‐induced cellular responses.     

Protective effects of regulatory amino acids on ischaemia-reperfusion injury in the isolated perfused rat liver

Objective. Some amino acids (AAs) display potent regulatory activities on cell metabolism, including via anti-oxidative defences. The aim of this study was to evaluate the protective effect of these AAs on warm ischaemia-reperfusion (I/R) injury in the isolated perfused rat liver.

Material and methods. Livers from fasted male Sprague-Dawley rats were isolated and perfused without (control group) or with (AP group) a mixture of regulatory AAs (glutamine, histidine, leucine, methionine, proline, phenylalanine, tryptophan and alanine). After 45 min of perfusion, warm ischaemia was induced for 45 min by clamping the portal vein catheter; thereafter, reperfusion was performed for 30 min.

Results. TNF-α production was significantly lower in the AP group during reperfusion (Control: 39±7 versus AP: 16±2 pg min^?1 g^?1, p<0.05), and lactate dehydrogenase (LDH) release decreased significantly during the last 15 min of reperfusion (Control: 0.13±0.03 versus AP: 0.04±0.02 IU min^?1 g^?1, p<0.05), despite similar levels of oxidative stress. The addition of regulatory AAs was not associated with variations in portal flow, bile flow, hepatic glucose or urea metabolism. However, significant changes in intrahepatic glutamine (Control: 1.4±0.2 versus AP: 2.6±0.5 µmol g^?1, p<0.05) together with higher glutamate release in the AP group (Control: 10.2±5.4 versus AP: 42.6±10.9 nmol min^?1 g^?1, p<0.05) indicated modifications in nitrogen metabolism.

Conclusions. Taken together, the lower TNF-α production, suggesting decreased inflammatory response, the decrease in LDH release in the AP group, demonstrating a better preservation of liver viability, and the increase in hepatic glutamine indicate that AAs play an important role in the liver's response to I/R.     

Serum soluble transferrin receptor in the diagnosis of iron deficiency in chronic liver disease

Fifty-one consecutive patients with chronic liver disease (CLD) underwent investigations of their iron status (full blood count, serum iron [Fe], total iron binding capacity [TIBC], transferrin saturation [TS], serum ferritin and serum soluble transferrin receptor [sTfR] level). Twenty-six patients were anaemic; 12 patients had iron deficiency, and 10 had iron deficiency anaemia (IDA). The median (range) sTfR in the IDA patients was 16.6 (11.2–24.8) mg/l, compared with 6.6 mg/l (11.2–24.8) in the 16 patients with anaemia due to other causes (P = 0.01). The sensitivity of sTfR for diagnosing iron deficiency in CLD was 91.6% (100% if only anaemic patients are included) and the specificity was 84.6%. Patients with haemolysis and recent blood loss may have falsely elevated sTfR levels. The results suggest that the sTfR is as useful as serum ferritin in identifying a potentially treatable cause of anaemia in CLD.     

Therapeutic effects of ezetimibe for non-alcoholic steatohepatitis in fatty liver shionogi-ob/ob mice

Aim: An effective therapy for non‐alcoholic steatohepatitis has yet to be defined. This study examined the therapeutic effects of ezetimibe, a lipid‐lowering medication, on steatosis and hepatic fibrosis in fatty liver Shionogi ob/ob (FLS‐ob) mice. Methods: Low‐dose (0.2 mg/kg body weight) and high‐dose (1.0 mg/kg body weight) of ezetimibe were administered to FLS‐ob mice orally for 12 weeks. Results: Administration of ezetimibe significantly and dose‐dependently decreased liver cholesterol content. The area of hepatic fibrosis and hepatic hydroxyproline content in the low‐ and high‐dose groups were significantly decreased compared with controls. Areas of α‐smooth muscle actin positivity and F4/80 positivity were significantly decreased in a dose‐dependent manner. Percentages of 8‐hydroxy‐2‐deoxyguanosine‐positive cells in low‐ and high‐dose groups were significantly decreased compared with those in controls, and 8‐hydroxy‐2‐deoxyguanosine DNA content and thiobarbituric acid reactive substances in the high‐dose group was also significantly decreased compared to controls. Gene expression levels of procollagen I and transforming growth factor β1 mRNA levels were lower in the low‐ and high‐dose groups than in controls. Tumor necrosis factor‐α and sterol regulatory element‐binding protein 1c mRNA levels were also lower in the low‐ and high‐dose groups than in controls. Conclusions: Ezetimibe attenuated steatosis and liver fibrosis by reducing oxidative stress and lipid peroxidation and suppressing activated hepatic stellate cells and Kupffer cells.     

Plasma reactive carbonyl species levels and risk of non-alcoholic fatty liver disease

Background and Aim: Oxidative stress plays a critical role in the pathogenesis of non‐alcoholic fatty liver disease (NAFLD). However, there is still no large cohort study to explore the direct risk role of oxidative stress for NAFLD. This study is to test the hypothesis that elevated oxidative stress is a direct risk factor for the pathogenesis of NAFLD under controlling the potential effects of covariates. Methods: The levels of serum cholesterol, serum triglyceride, fasting plasma glucose and plasma reactive carbonyl species (RCS) were measured from 1204 Chinese Han adults, and the questionnaire and physical examination were administered to those with known and suspected risk factors for NAFLD. Results: Statistically significant high levels of blood pressure, fasting plasma glucose, serum cholesterol and triglyceride, body mass index, serum alanine aminotransferase and aspartate aminotransferase, and plasma RCS were observed in NAFLD subjects compared to healthy subjects (P < 0.01). Multivariate‐adjusted odds ratio illustrated that, compared with the lowest quartile of plasma RCS levels, the highest quartile subjects had a 132% increase in the risk of developing NAFLD. Further results from multi‐interaction analysis demonstrated that the underlying mechanism of the risk of NAFLD by unhealthy physical conditions and lifestyles might be, at least in part, through the oxidative stress. Conclusions: Our findings provide credible evidence from a large population that oxidative stress, as indicated by plasma RCS levels, may be a direct risk factor for developing NAFLD.     

Increase in liver antioxidant enzyme activities in non-alcoholic fatty liver disease.

AIMS: Steatosis may increase oxidative stress, which is counteracted by cellular enzymatic (cytosolic and mitochondrial superoxide dismutases (Cu/Zn-SOD and Mn-SOD), glutathione peroxidase (GPx), catalase) and non-enzymatic antioxidant systems. We aimed to determine, in patients with non-alcoholic fatty liver disease (NAFLD), the level of antioxidant defenses (1) in liver biopsies, to demonstrate the existence of oxidative stress; (2) in erythrocytes and plasma, to determine whether their antioxidant defenses reflect liver oxidative stress. METHODS: Erythrocyte and plasma antioxidant defenses were prospectively studied in two groups of 16 patients: patients with NAFLD and controls. Liver biopsies were performed in eight NAFLD patients; liver antioxidant enzyme activities were measured and compared with those in 12 control livers used for transplantation. RESULTS: Cu/Zn-SOD, GPx and catalase activities were significantly higher in NAFLD livers than in controls whereas no significant differences were observed in Mn-SOD activity, and thiobarbituric acid-reactive substance (TBARS) concentration. No differences were observed in erythrocyte antioxidant enzyme activities (GPx, catalase, Cu/Zn-SOD), erythrocyte TBARS concentration, and plasma alpha-tocopherol concentration. CONCLUSIONS: Liver antioxidant enzyme activities were high in patients with NAFLD, reflecting an oxidative stress possibly involved in inflammation and fibrogenesis. However, erythrocyte and plasma antioxidant defenses did not reflect intrahepatic peroxidation.     

Hepatic iron overload in alcoholic end-stage liver disease is associated with iron deposition in other organs in the absence of HFE-1 hemochromatosis.

BACKGROUND: End-stage cirrhosis in the absence of hereditary hemochromatosis (HHC) can be associated with moderate to marked hepatic iron overload, especially in liver disease as a result of alcohol and/or hepatitis C. However, no published studies have addressed extrahepatic iron deposition in this setting. METHOD: A retrospective case series from three autopsied patients who died from end-stage cirrhosis associated with significant hepatic iron overload. Histology of vital organs was performed to detect extrahepatic iron deposition. HFE genotyping for the C282Y and H63D mutations was determined from archival tissue. Hepatic iron index and hepatic iron concentration (HIC) were quantified from formalin-fixed, paraffin-embedded tissue. Medical records were reviewed for possible causes of iron overload. RESULTS: Two patients were H63D heterozygous (H63D +/-) and one was wild type (C282Y -/-, H63D -/-). Histology revealed evidence of stainable iron in the heart and pancreas of all three subjects. Additionally, stainable iron was seen in the stomach in one subject and in the thyroid, pituitary, choroid plexus and testes in another subject. HIC ranged from 4354 to 6834 microg/g dry weight and HII from 1.8 to 2.2 (micromol/g/years). CONCLUSION: Iron overload secondary to end-stage liver disease can be associated with iron deposition in other organs in the absence of HFE-1 HHC.     

Melatonin reduces lipid peroxidation and nitric oxide during irradiation-induced oxidative injury in the rat liver

Radiation therapy is a popular and useful tool in the treatment of cancer. Melatonin participates in the regulation of a number of important physiological and pathological processes. Melatonin, a powerful endogenous antioxidant, plays a role in the reduction of oxidative damage. Thirty adult rats were divided into five equal groups. On the day of the experiment, groups I and II were injected with 5 or 10 mg/kg melatonin, respectively, while group III received isotonic NaCl solution. Thirty minutes later, groups I, II and III were exposed to 6.0 Gy whole body ionizing radiation in a single fraction. Group IV was injected with 5 mg/kg melatonin but was not irradiated. The final group was reserved as sham treated. Liver malondialdehyde (MDA) and nitric oxide (NO*) levels were measured in all groups. Whole body irradiation caused a significant increase in liver MDA and NO* levels. Hepatic MDA and NO* levels in irradiated rats that were pretreated with melatonin (5 or 10 mg/kg) were significantly decreased. Malondialdehyde and NO* levels were reduced in a dose-related manner by melatonin. The data show that melatonin reduces liver damage inflicted by irradiation when given prior to the exposure to ionizing radiation. The radioprotective effect of melatonin is likely achieved by its ability to function as a scavenger for free radicals generated by ionizing radiation.     

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.     

铁超载在非酒精性脂肪性肝病中的作用

铁超载是非酒精性脂肪性肝病（NAFLD）的一个研究热点.从铁超载的评估方法、铁超载与血色病基因突变的关系、铁超载在NAFLD中的发生率及铁超载与NAFLD病情进展的关系等方面归纳了铁超载与NAFLD的相关性;从铁超载的原因、铁超载与脂质代谢的关系及铁沉积类型与肝损伤的关系评述了铁超载参与NAFLD进展的机制;从铁超载作为NAFLD危险分层的新标志物及可能的治疗靶点阐述了铁超载在NAFLD诊疗中的意义.目前认为不论铁超载是NAFLD进展的原因还是结果,一旦出现都将促进NAFLD进展;铁超载作为NAFLD危险分层的新标志物及治疗靶位值得进一步研究.     

Therapeutic effects of cytokine modulator Y-40138 in the rat alcoholic liver disease model

Background and Aim: Inflammatory cytokines, such as tumor necrosis factor‐α (TNF‐α) and interferon‐gamma (IFN‐γ), induce liver injury in the rat alcoholic liver disease (ALD) model. Y‐40138 is known to suppress the pro‐inflammatory cytokines and augment the anti‐inflammatory cytokines. We investigated whether or not Y‐40138 may be effective as a novel immunotherapy in the rat ALD model. Methods: Male Wistar rats were fed Lieber‐DeCarli ethanol liquid diet. The effects of Y‐40138 treatment in the ALD models were assessed by analyzing the serum and the liver tissues. Results: The serum levels of alanine aminotransferase (ALT), TNF‐α, and IFN‐γ, and the liver levels of TNF‐α and IFN‐γ were significantly higher in the ethanol‐fed group than in the pair‐fed group. The immunohistochemistry of the liver TNF‐α and 4‐hydroxynonenal (4HNE), and the expressions of TNF‐α and IFN‐γ mRNA were increased, too. The gene expressions of interleukin‐10 (IL‐10) in the ethanol‐fed group were suppressed as compared with the pair‐fed group. The serum triglyceride (TG) and liver TG were increased, and Oil Red O and α‐smooth muscle actin (α‐SMA) staining showed greater expression by ethanol‐fed feeding. After administration of Y‐40138, enzyme linked immunosorbent assay and real‐time polymerase chain reaction of the liver showed that the increased TNF‐α and IFN‐γ were suppressed, and that IL‐10 was augmented. Moreover, ethanol‐induced lipid accumulation in the liver was suppressed by administering Y‐40138. Conclusions: Y‐40138 decreased the inflammation, fibrosis, oxidative stress, and lipid synthesis, and augmented the anti‐inflammatory cytokines of the liver. These results indicate that the multiple cytokine production modulator, Y‐40138, is a promising novel therapy for ALD.     

5-ASA and lycopene decrease the oxidative stress and inflammation induced by iron in rats with colitis

Background Supplementation of 5-aminosalicylic acid (5-ASA) and of iron are among the principal therapies in patients with inflammatory bowel disease. Therapeutic iron, as well as heme iron from chronic mucosal bleeding, can increase iron-mediated oxidative stress in colitis. This study was designed to examine the influence of iron supplementation on histological expression and oxidative status relative to 5-ASA treatment and antioxidant treatment.Methods Colitis was induced using the iodoacetamide rat model, and rats were divided into different dietary groups of 6 rats each: 1, normal chow diet (control); 2, diet supplemented with iron; 3, iron supplementation and lycopene; 4, iron and -carotene; 5, 5-ASA; 6, 5-ASA and lycopene; 7, 5-ASA and iron; 8, 5-ASA, iron, and lycopene. The animals were killed after 3 days and the weight of the ulcerated area recorded. Mucosal specimens were histologically evaluated. Myeloperoxidase (MPO) was measured to evaluate inflammatory status (U/g). Malondialdehyde (MDA) was measured in colonic tissue (µmol/g) and superoxide dismutase (SOD) in erythrocytes to assess the degree of tissue oxidative stress.Results Significantly more severe colitis, including necrosis, ulceration, and hemorrhage, was seen in colonic biopsies of rats with colitis when iron was supplemented. This pathology was attenuated when iron was given in combination with 5-ASA and/or lycopene. There was no significant benefit from adding -carotene.Conclusions Iron supplementation can amplify the inflammatory response and subsequent mucosal damage in a rat model of colitis. We suggest that the resultant oxidative stress generated by iron supplementation leads to the extension and propagation of crypt abscesses, either through direct membrane disruption by lipid peroxidation or through the generation of secondary toxic oxidants. Simultaneous treatment with 5-ASA and/or lycopene minimizes the potential hazard of iron. Therefore, we suggest giving iron supplementation with 5-ASA or lycopene or both.