CHOP deficiency attenuates cholestasis-induced liver fibrosis by reduction of hepatocyte injury

CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is a key component in endoplasmic reticulum (ER) stress-mediated apoptosis. The goal of the study was to investigate the role of CHOP in cholestatic liver injury. Acute liver injury and liver fibrosis were assessed in wild-type (WT) and CHOP-deficient mice following bile duct ligation (BDL). In WT livers, BDL induced overexpression of CHOP and Bax, a downstream target in the CHOP-mediated ER stress pathway. Liver fibrosis was attenuated in CHOP-knockout mice. Expression levels of alpha-smooth muscle actin and transforming growth factor-beta1 were reduced, and apoptotic and necrotic hepatocyte death were both attenuated in CHOP-deficient mice. Hepatocytes were isolated from WT and CHOP-deficient mice and treated with 400 microM glycochenodeoxycholic acid (GCDCA) for 8 h to examine bile acid-induced apoptosis and necrosis. GCDCA induced overexpression of CHOP and Bax in isolated WT hepatocytes, whereas CHOP-deficient hepatocytes had reduced cleaved caspase-3 expression and a lower propidium iodide index after GCDCA treatment. In conclusion, cholestasis induces CHOP-mediated ER stress and triggers hepatocyte cell death, and CHOP deficiency attenuates this cell death and subsequent liver fibrosis. The results demonstrate an essential role of CHOP in development of liver fibrosis due to cholestatic liver damage.     

Chromium attenuates high-fat diet-induced nonalcoholic fatty liver disease in KK/HlJ mice

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease associated with insulin resistance, oxidative stress, and inflammation. Evidence indicates that chromium has a role in the regulation of glucose and lipid metabolism and may improve insulin sensitivity. In this study, we report that chromium supplementation has a beneficial effect against NAFLD. We found that KK/HlJ mice developed obesity and progressed to NAFLD after feeding with high-fat diet for 8 weeks. High-fat-fed KK/HlJ mice showed hepatocyte injury and hepatic triglyceride accumulation, which was accompanied by insulin resistance, oxidative stress, and inflammation. Chromium supplementation prevented progression of NAFLD and the beneficial effects were accompanied by reduction of hepatic triglyceride accumulation, elevation of hepatic lipid catabolic enzyme, improvement of glucose and lipid metabolism, suppression of inflammation as well as resolution of oxidative stress, probably through enhancement of insulin signaling. Our findings suggest that chromium could serve as a hepatoprotective agent against NAFLD.     

Ascorbic acid supplementation causes faster restoration of reduced glutathione content in the regression of alcohol-induced hepatotoxicity in male guinea pigs

Abstract

Alcoholic liver disease is caused mainly by free radicals. Ascorbic acid (AA) and glutathione (GSH) are the major water-soluble antioxidants in the liver. The impact of AA supplementation on GSH, AA and activities of GSH-dependent enzymes in alcoholic guinea pigs was studied and was compared with alcohol abstention. Guinea pigs were administered ethanol at a dose of 4 g/kg body weight (b.wt)/day for 90 days. After 90 days, alcohol administration was stopped and one-half of the ethanol-treated animals were supplemented with AA (25 mg/100 g b.wt) for 30 days and the other half was maintained as the abstention group. There was a significant increase in the activities of alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyl transpeptidase in the serum of the ethanol group. In addition, a significant decrease in the GSH content, activities of GSH peroxidase, GSH reductase, and increased activity of GSH-S-transferase were observed in the liver of the ethanol group. Histopathological analysis and triglycerides content in the liver of the ethanol group showed induction of steatosis. But AA supplementation and abstention altered the changes caused by ethanol. However, maximum protective effect was observed in the AA-supplemented group indicating the ameliorative effect of AA in the liver.     

Ascorbic acid attenuates endothelial permeability triggered by cell-free hemoglobin

Background

Increased endothelial permeability is central to shock and organ dysfunction in sepsis but therapeutics targeted to known mediators of increased endothelial permeability have been unsuccessful in patient studies. We previously reported that cell-free hemoglobin (CFH) is elevated in the majority of patients with sepsis and is associated with organ dysfunction, poor clinical outcomes and elevated markers of oxidant injury. Others have shown that Vitamin C (ascorbate) may have endothelial protective effects in sepsis. In this study, we tested the hypothesis that high levels of CFH, as seen in the circulation of patients with sepsis, disrupt endothelial barrier integrity.

Sialic acid attenuates puromycin aminonucleoside-induced desialylation and oxidative stress in human podocytes

Sialoglycoproteins make a significant contribution to the negative charge of the glomerular anionic glycocalyx—crucial for efficient functioning of the glomerular permselective barrier. Defects in sialylation have serious consequences on podocyte function leading to the development of proteinuria. The aim of the current study was to investigate potential mechanisms underlying puromycin aminonucleosisde (PAN)-induced desialylation and to ascertain whether they could be corrected by administration of free sialic acid.     

Biphasic Liberation of Arachidonic and Stearic Acids During Cerebral Ischemia

The mode of free fatty acid (FFA) liberation from the mouse brain during ischemia was investigated at various times after decapitation and under nizofenone treatment. Normal nonischemic brain FFAs consist mainly of palmitic acid (16:0), stearic acid (18:0), and oleic acid (18:1) with smaller amounts of arachidonic acid (20:4), docosahexaenoic acid (22:6), and others. Postdecapitative ischemia induced a rapid, biphasic release of 20:4 after a short lag of less than 30 s. The first phase showed a rapid 6.4-fold increase within 1 min of decapitation, followed by the second phase involving a slow release at less than one-fifth the rate of the first phase and lasting for at least 10 min. A similar, but not so marked, biphasic liberation was observed with 18:0. However, all of the other fatty acids (16:0, 18:1, 22:6, and others) were released only in a single phase at a slow rate. The time course for the rapid and specific liberation of 20:4 coincided with the time course for the decrease in brain ATP concentration during ischemia. Pretreatment of the animals with nizofenone resulted in a marked suppression of both FFA liberation and ATP depletion during ischemia. This suppression was particularly noteworthy with 20:4 and 18:0. The present study indicates that there is a specific and rapid liberation of 20:4 and 18:0 in a very early stage of ischemia and that this liberation seems to depend on availability of ATP in the brain. The physiological role of this transient 20:4 liberation during ischemia is discussed.     

TNFalpha is required for cholestasis-induced liver fibrosis in the mouse

TNFα, a mediator of hepatotoxicity in several animal models, is elevated in acute and chronic liver diseases. Therefore, we investigated whether hepatic injury and fibrosis due to bile duct ligation (BDL) would be reduced in TNFα knockout mice (TNFα−/−). Survival after BDL was 60% in wild-type mice (TNFα+/+) and 90% in TNFα−/− mice. Body weight loss and liver to body weight ratios were reduced in TNFα−/− mice compared to TNFα+/+ mice. Following BDL, serum alanine transaminases (ALT) levels were elevated in TNFα+/+ mice (268.6 ± 28.2 U/L) compared to TNFα−/− mice (105.9 U/L ± 24.4). TNFα−/− mice revealed lower hepatic collagen expression and less liver fibrosis in the histology. Further, α-smooth muscle actin, an indicator for activated myofibroblasts, and TGF-β mRNA, a profibrogenic cytokine, were markedly reduced in TNFα−/− mice compared to TNFα+/+ mice. Thus, our data indicate that TNFα induces hepatotoxicity and promotes fibrogenesis in the BDL model.     

Oxidative stress is a triggering factor for LPS-induced Mrp2 internalization in the cryopreserved rat and human liver slices

Cholestasis develops during inflammation and is characterized as occurring under oxidative stress. We have described the internalization of multidrug resistance-associated protein 2 (Mrp2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid or lipopolysaccharide (LPS)-induced acute oxidative stress in rat liver. However, it remains unclear whether canalicular Mrp2 internalization is observed in human liver under conditions of acute oxidative stress. In this study, we examined the effect of dimerumic acid (DMA), an antioxidant and found in traditional Chinese medicine, on endotoxin-induced Mrp2 internalization in rat and human liver slices. At 1.5 h following LPS treatment (100 μg/mL), canalicular Mrp2 localization was disrupted without changing the expression of Mrp2 protein or the integrity of filamentous actin in the rat and human liver slices. Pretreatment with DMA (10 μM) counteracted LPS-induced subcellular distribution of Mrp2. Our data clearly indicated that LPS-induced short-term rapid retrieval of Mrp2 from the canalicular surface resulted from LPS-induced oxidative stress in rat and human liver slices.     

Protective effect of docosahexaenoic acid against brain injury in ischemic rats

Evidence suggests that inactivation of cell-damaging mechanisms and/or activation of cell-survival mechanisms may provide effective preventive or therapeutic interventions to reduce cerebral ischemia/reperfusion (I/R) injuries. Docosahexaenoic acid (DHA) is an essential polyunsaturated fatty acid in the central nervous system that has been shown to possess neuroprotective effects. We examined whether different preadministrative protocols of DHA have effects on brain injury after focal cerebral I/R and investigated the potential neuroactive mechanisms involved. Sprague–Dawley rats were intraperitoneally pretreated with DHA once 1 h or 3 days being subjected to focal cerebral I/R or daily for 6 weeks before being subjected to focal cerebral I/R. Reduction of brain infarction was found in all three DHA-pretreated groups. The beneficial effect of DHA on the treatment groups was accompanied by decreases in blood–brain barrier disruption, brain edema, malondialdehyde (MDA) production, inflammatory cell infiltration, interleukin-6 (IL-6) expression and caspase-3 activity. Elevation of antioxidative capacity, as evidenced by decreased MDA level and increased superoxide dismutase activity and glutathione level, was detected only in the chronic daily-administration group. The two single-administration groups showed increased phosphorylation of extracellular-signal-regulated kinase (ERK). Elevation of Bcl-2 expression was detected in the chronic daily-administration and 3-day-administration groups. In vitro study demonstrated that DHA attenuated IL-6 production from stimulated glial cells involving nuclear factor κB inactivation. Therefore, the data suggest that the neuroprotective mechanisms of DHA pretreatment are, in part, mediated by attenuating damaging mechanisms through reduction of cytotoxic factor production and by strengthening survival mechanisms through ERK-mediated and/or Bcl-2-mediated prosurvival cascade.     

Lipoic acid ameliorates oxidative stress and renal injury in alloxan diabetic rabbits

The therapeutic potential of lipoic acid (LA) in diabetes and diabetic nephropathy treatment was elucidated. Alloxan diabetic rabbits were treated daily for three weeks with either 10 or 50 mg of LA per kg body weight (i.p.). The following parameters were measured: 1) serum glucose, urea, creatinine and hydroxyl free radical (HFR) levels; 2) blood glutathione redox state; 3) urine albumin concentration; 4) hepatic and renal HFR levels, GSH/GSSG ratios, cysteine contents and the activities of the enzymes of glutathione metabolism; and 5) the activity of renal NADPH oxidase. Histological studies of kidneys were also performed. The treatment of diabetic rabbits with 50 mg of LA resulted in lethal hypoglycaemia in 50% of animals studied. Although the low dose of LA did not change serum glucose concentration, it decreased serum urea and creatinine concentrations, attenuated diabetes-induced decline in GSH/GSSG ratio and abolished hydroxyl free radicals accumulation in serum, liver and kidney cortex. LA did not change the activities of the enzymes of glutathione metabolism, but it elevated hepatic content of cysteine, which limits the rate of glutathione biosynthesis. Moreover, LA lowered urine albumin concentration and attenuated glomerulopathy characteristic of diabetes. However, it did not affect diabetes-stimulated activity of renal NADPH oxidase. In view of these data, it is concluded that low doses of LA might be useful for the therapy of diabetes and diabetic nephropathy. Beneficial action of LA seems to result mainly from direct scavenging of HFR and restoring glutathione redox state due to elevation of intracellular cysteine levels.     

Study on injury effect of food additive citric acid on liver tissue in mice

To investigate the damaging effect and action mechanism of the food additive citric acid (CA) on mouse liver, 40 healthy male Kunming mice were randomly divided into control group (0.9 % saline), low CA dose (120 mg/kg), middle dose (240 mg/kg) and high dose groups (480 mg/kg). All experimental mice have received peritoneal injection of the corresponding reagent each week for 3 weeks. After 7 days since the third injection, morphological changes were observed by light microscope; activities of T-SOD, glutathione peroxidase (GSH-Px), caspase-3, and contents of hydrogen peroxide (H₂O₂) and malonyldialdehyde (MDA) in the liver were evaluated using the corresponding assay kits; DNA fragmentation was assayed using agarose gel electrophoresis. Microscopical detection showed a series of hispathological changes in mouse livers treated with CA, such as indiscriminate liver cell cord, blood clot in central veins, and lymphocyte infiltrating. Biochemical examination suggested the gradually but moderately reduced T-SOD activity and elevated H₂O₂ level with the increase of CA dose (P > 0.05), and the gradually reduced GSH-Px activity and increased MDA content depending on graded doses with a significant difference (P < 0.05) between the high dose group and the control group. According to cell apoptosis assays, caspase-3 activity were significantly higher in all treatment groups than in the control (P < 0.05) in a dose-dependent manner. Contrasting to the control, characteristic DNA laddering was observed when injected with any of the three graded doses. It can be concluded that certain concentrations of CA cause oxidative damage of the liver by means of the decrease of antioxidative enzyme activities, thus resulting in MDA level elevation and DNA fragmentation inducing active caspase-3.     

Efficacy of caffeic acid in preventing nickel induced oxidative damage in liver of rats

Nickel (Ni), a major environmental pollutant, is known for its wide toxic manifestations. In the present study caffeic acid (CA), one of the most commonly occurring phenolic acids in fruits, grains and dietary supplements, was evaluated for its protective effect against the Ni induced oxidative damage in liver. In this investigation, Ni (20 mg/kg body weight) was administered intraperitoneally for 20 days to induce toxicity. CA was administered orally (15, 30 and 60 mg/kg body weight) for 20 days with intraperitoneal administration of Ni. Ni induced liver damage was clearly shown by the increased activities of serum hepatic enzymes namely aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT) and lactate dehydrogenase (LDH) along with increased elevation of lipid peroxidation indices (thiobarbituric reactive acid substances (TBARS) and lipid hydroperoxides). The toxic effect of Ni was also indicated by significantly decreased levels of enzymatic (superoxide dismutase (SOD), catalase (CAT) glutathione peroxidase (GPx) and glutathione S-transferase (GST)) and non-enzymatic antioxidants (glutathione (GSH), vitamin C and vitamin E). CA administered at a dose of 60 mg/kg body weight significantly reversed the activities of hepatic marker enzymes to their near normal levels when compared with other two doses. In addition, CA significantly reduced lipid peroxidation and restored the levels of antioxidant defense in the liver. All these changes were supported by histological observations. The results indicate that CA may be beneficial in ameliorating the Ni induced oxidative damage in the liver of rats.     

Long-distance transit alters liver and skeletal muscle physiology of beef cattle

Transportation of cattle is necessary but negatively impacts animal health and production efficiency. To gain a better understanding of the physiological responses to long-distance road transit, 36 crossbred beef steers (324 ± 36 kg) were randomly assigned to treatments (n = 12 steers/treatment): no transit and ad libitum access to feed and water (CON), no transit but deprived of feed and water for 18 h (DEPR), or road transit and no access to feed or water for 18 h (1 790 km; TRANS). Blood, liver, and muscle (longissimus dorsi) samples were collected pre- and post-treatment for analysis of blood metabolites, blood leukocyte profiles, blood markers of oxidative stress, and tissue antioxidant enzyme activity. Additionally, discovery-based metabolomics and proteomics analyses were performed on tissue samples collected immediately post-treatment (d 1). Data (except for omics) were analyzed using ProcMixed of SAS 9.4 with the fixed effect of treatment and steer as the experimental unit. Omics data were analyzed using MetaboAnalyst; metabolites and proteins of interest were identified based on a fold change threshold of 1.20 and t-test P-value of 0.10. On d 1, percent of pretreatment BW and DM intake were least for TRANS steers (P ≤ 0.06). Percent of pretreatment BW remained lesser for TRANS steers on d 8 (P = 0.05). Serum haptoglobin was greatest for TRANS steers immediately post-treatment (P = 0.02). Additionally, TRANS steers exhibited the greatest increase in the neutrophil to lymphocyte ratio and serum non-esterified fatty acids during the treatment period (P < 0.01), indicating TRANS steers experienced a more robust inflammatory and neuroendocrine response. Immediately post-treatment, liver superoxide dismutase activity tended to be greatest for both DEPR and TRANS (P = 0.07) while muscle superoxide dismutase activity was only greatest for TRANS (P = 0.02), suggesting TRANS steers may have experienced more oxidative stress due to the additional physical effort required to stand and maintain balance during transit. The abundance of several proteins (alpha-2-HS-glycoprotein) and metabolites (lactate, citrate, tri-hydroxybutyric acid, and leucine) associated with energy metabolism were altered in the liver and muscle of TRANS. The differential responses for DEPR versus TRANS steers indicate muscle plays an important role in how cattle respond to and recover from transportation stress.     

Modulatory role of lipoic acid on adriamycin-induced testicular injury

The present study investigated the protective efficacy of dl-alpha-lipoic acid (LA) on adriamycin (ADR)-induced oxidative damage in rat testis. Adult male albino rats of Wistar strain were administered ADR (1 mg/kg body weight, i.v.), once a week for 10 weeks. ADR injected rats showed increased oxidative stress with a concomitant decrease in cellular thiols. The mRNA level for phospholipid hydroperoxide glutathione peroxidase (PHGPx) was also significantly decreased by ADR administration. Transmission electron microscopic (TEM) observations of testicular germ cells revealed abnormal ultrastructural changes in ADR treated rats. Treatment with lipoic acid (35 mg/kg body weight, i.p.) 1 day prior to ADR administration, effectively reverted these abnormal changes towards normalcy. These findings indicate a cytoprotective role of LA in this experimental model of testicular toxicity.     

MiR-124 attenuates doxorubicin-induced cardiac injury via inhibiting p66Shc-mediated oxidative stress

Previous studies showed that miR-124 had a protective role by reducing oxidant stress and preventing cell apoptosis and autophagy. However, its role in doxorubicin-induced cardiomyopathy was less known. In our study, we confirmed increased ROS and decreased expression of miR-124 in doxorubicin-treated heart tissues and primary cardiomyocytes. The oxidative stress and cell apoptosis were alleviated by overexpressing miR-124, characterized by decreased activity of MDA and increased activity of SOD. While inhibiting miR-124 generated opposed effects. Mechanistically, our bioinformatic prediction and luciferase assay confirmed that miR-124 inhibited the expression of p66Shc, a proapoptotic signaling pathway. Our results suggested that miR-124 was hopeful to become a therapeutic target in doxorubicin-related cardiomyopathy.     

Caffeic acid phenethyl ester (CAPE) protects brain against oxidative stress and inflammation induced by diabetes in rats

Diabetic patients reveal significant disorders, such as nephropathy, cardiomyopathy, and neuropathy. As oxidative stress and inflammation seem to be implicated in the pathogenesis of diabetic brain, we aimed to investigate the effects of caffeic acid phenethyl ester (CAPE) on oxidative stress and inflammation in diabetic rat brain. Diabetes was induced by a single dose of streptozotocin (45 mg kg⁻¹, i.p.) injection into rats. Two days after streptozotocin treatment 10 μM kg⁻¹ day⁻¹ CAPE was administrated and continued for 60 days. Here, we demonstrate that CAPE significantly decreased the levels of nitric oxide and malondialdehyde induced by diabetes, and the activities of catalase, glutathione peroxidase, and xanthine oxidase in the brain. However, glutathione levels were increased by CAPE. The mRNA expressions of tumor necrosis factor (TNF)-α and interferon (IFN)-γ, and inducible nitric oxide synthase (iNOS) were remarkably enhanced in brain by diabetes. CAPE treatments significantly suppressed these inflammatory cytokines (about 70% for TNF-α, 26% for IFN-γ) and NOS (completely). Anti-inflammatory cytokine IL-10 mRNA expression was not affected by either diabetes or CAPE treatments. In conclusion, diabetes induces oxidative stress and inflammation in the brain, and these may be contributory mechanisms involved in this disorder. CAPE treatment may reverse the diabetic-induced oxidative stress in rat brains. Moreover, CAPE reduces the mRNA expressions of TNF-α and IFN-γ in diabetic brain; suggesting CAPE suppresses inflammation as well as oxidative stress occurred in the brain of diabetic patients.     

Proteomic analysis of liver tissues subjected to early ischemia/reperfusion injury during human orthotopic liver transplantation

Knowledge of early molecular events occurring upon ischemia/reperfusion (I/R) during liver transplantation (LT) is of great importance to improve the therapeutic intervention of surgical treatment. However, nowadays, few data are available on early protein targets of I/R injury. To identify these proteins, we used a differential proteomics approach in the characterization human liver biopsies during I/R upon LT. Analyses were performed on nine donor livers during LT. By using 2-DE and MALDI-TOF MS, we identified 36 proteins which resulted significantly altered upon I/R injury. The majority of these proteins are functionally involved in lipid and energy metabolism, in different metabolic pathways, in redox signalling and in oxidative-stress response. Our data represent the first global approach in the study of I/R injury in liver.     

Protective effect of ursodeoxycholic acid on liver mitochondrial function in rats with alloxan-induced diabetes: link with oxidative stress

We investigated the effects of ursodeoxycholic acid (UDCA) on mitochondrial functions and oxidative stress and evaluated their relationships in the livers of rats with alloxan-induced diabetes. Diabetes was induced in male Wistar rats by a single alloxan injection (150 mg kg^− 1 b.w., i.p.). UDCA (40 mg kg^− 1 b.w., i.g., 30 days) was administered from the 5th day after the alloxan treatment. Mitochondrial functions were evaluated by oxygen consumption with Clark oxygen electrode using succinate, pyruvate + malate or palmitoyl carnitine as substrates and by determination of succinate dehydrogenase and NADH dehydrogenase activities. Liver mitochondria were used to measure chemiluminiscence enhanced by luminol and lucigenin, reduced liver glutathione and the end-products of lipid peroxidation. The activities of both NADH dehydrogenase and succinate dehydrogenase as well as the respiratory control (RC) value with all the substrates and the ADP/O ratio with pyruvate + malate and succinate as substrates were significantly decreased in diabetic rats. UDCA developed the beneficial effect on the mitochondrial respiration and oxidative phosphorylation parameters in alloxan-treated rats, whereas the activities of mitochondrial enzymes were increased insignificantly after the administration of UDCA. The contents of polar carbonyls and MDA as well as the chemiluminescence with luminol were elevated in liver mitochondria of diabetic rats. The treatment with UDCA normalized all the above parameters measured except the MDA content. UDCA administration prevents mitochondrial dysfunction in rats treated with alloxan and this process is closely connected with inhibition of oxidative stress by this compound.