cytochrome-c-t and Liver-Diseases--Alcoholic

Topics: Anthocyanins; Antioxidants; Apoptosis; Caspase 3; Caspase 9; Cytochromes c; Hep G2 Cells; Humans; Liver Diseases, Alcoholic; Spectroscopy, Fourier Transform Infrared

Hepatocyte apoptosis is frequently observed in alcohol-related liver disease (ARLD), which ranks among the 30 leading causes of death worldwide. In the current study, we explored the impact of S-allyl-l-cysteine (SAC), an organosulfur component of garlic, on hepatocyte apoptosis induced by alcohol. Rat liver (BRL-3A) cells were challenged by ethanol with or without SAC treatment. Cell death/viability, reactive oxygen species (ROS) generation, mitochondrial Cytochrome C release, and caspase 3 activity were then examined. We found that ethanol remarkably induced apoptosis of hepatocytes, while SAC treatment rescued ethanol-induced hepatocyte injury, as demonstrated by cell counting kit-8 (CCK8) assay, TUNEL assay, and annexin V/PI staining assay. Ethanol evoked ROS generation in BRL-3A cells, and this was abated by SAC pretreatment, as indicated by 2',7'-dichlorofluorescin diacetate (DCFDA) staining assay. Moreover, ethanol suppressed cellular anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) expression, increased pro-apoptotic protein Bcl-2-associated X protein (Bax) expression, induced mitochondrial Cytochrome C release, and activated the caspase 3-dependent apoptosis pathway in BRL-3A cells. SAC was sufficient to abolish all these changes induced by ethanol, thereby revealing the molecular mechanisms underlying its protective effects. In conclusion, SAC protects hepatocytes from ethanol-induced apoptosis and may be suitable for use as a novel anti-apoptotic agent for treating ARLD.

Topics: Animals; Antioxidants; Apoptosis; Caspase 3; Cell Death; Cell Line; Cell Survival; Cysteine; Cytochromes c; Ethanol; Hepatocytes; Liver; Liver Diseases, Alcoholic; Mitochondria; Oxidative Stress; Protective Agents; Rats; Reactive Oxygen Species; Signal Transduction

Mitochondria play an important role in the initiation and development of alcoholic liver disease (ALD). Our previous studies found lychee pulp phenolic extract (LPPE) exerted protective effect against ALD partly by inhibiting fatty acid β-oxidation, and phenolic-rich lychee pulp extract improved restraint stress-induced liver injury by inhibiting mitochondrial dysfunction. The aim of this study was to investigate whether LPPE exerted protective effect against ALD via modulating mitochondrial function. The mice were treated with an ethanol-containing liquid diet alone or in combination with LPPE for 8 weeks. LPPE supplementation significantly alleviated hepatic steatosis, suppressed serum aspartate aminotransferase activity, and decreased triglyceride levels in serum and liver. On the basis of lipid peroxidation and antioxidant enzyme analyses, LPPE supplementation inhibited serum and hepatic oxidative stress. Moreover, LPPE supplementation significantly suppressed mitochondrial 8-hydroxy-2'-deoxyguanosine level, and increased mitochondrial membrane potential, mitochondrial DNA content, activities of mitochondrial complexes I and IV, and hepatic ATP level. Furthermore, LPPE supplementation significantly inhibited cytoplasmic cytochrome c level and caspase-3 activity, repressed Bax expression and Bax/Bcl-2 ratio, and increased Bcl-2 expression in liver. In summary, LPPE exerts beneficial effects against alcoholic liver injury by alleviating mitochondrial dysfunction.

Topics: Animals; Caspase 3; Cytochromes c; Glutathione; Humans; Litchi; Liver Diseases, Alcoholic; Male; Mice; Mice, Inbred C57BL; Mitochondria; Oxidative Stress; Phenols; Plant Extracts; Protective Agents

This study evaluated the protective effects of Aplysin against ethanol-induced hepatic injury in rats and analyzed the associated mechanisms. Rats were administered orally with ethanol 8-12 ml/kg bw excluding the rats in the control group at 1h after rats were administered by gavage doses of Aplysin (50, 100, and 150 mg/kg bw) every day. After 6 weeks, rats were sacrificed, and liver injury was evaluated by biochemical and pathological examination. Hepatocyte apoptosis was analyzed by annexin V-FITC/PI staining. Ethanol metabolic enzymes, oxidative stress, mitochondrial function, and Bcl-2, Bax, cytochrome c and cleaved caspase-3 expressions were evaluated by western blot analysis. These results demonstrated that Aplysin exhibited a significant hepatoprotective effect. In the ethanol-treated group, cytochrome P4502E1 and alcohol dehydrogenase were increased significantly in liver tissue. Moreover, Aplysin not only significantly reversed the ratio of NAD(+)/NADH and mitochondrial glutathione depletion, but also reversed the decreased activity of mitochondrial respiratory chain complexes I, III and IV. Overexpression of cytoplasmic cytochrome c and caspase-3 activation was suppressed by Aplysin. These results suggest that Aplysin alleviates hepatocyte apoptosis by modulating the ethanol-metabolizing pathway, attenuating oxidative stress, ameliorating mitochondrial function, inhibiting mitochondrial damage-mediated apoptosis, which ultimately prevent and repair alcoholic liver injury.

Topics: Alanine Transaminase; Alcohol Dehydrogenase; Animals; Apoptosis; Aspartate Aminotransferases; Caspase 3; Cytochrome P-450 CYP2E1; Cytochromes c; Ethanol; Glutathione; Hydrocarbons, Brominated; Lipid Peroxidation; Liver; Liver Diseases, Alcoholic; Male; Mitochondria; Oxidative Stress; Protective Agents; Rats, Wistar; Sesquiterpenes

Ethanol-induced hepatic steatosis may induce the progression of alcoholic liver disease. The involvement of autophagic clearance of damaged mitochondria (mitophagy) and lipid droplets (LDs) (lipophagy) in chronic ethanol-induced hepatic steatosis is not clearly understood. Adult Wistar rats were fed either 5 % ethanol in Lieber-DeCarli liquid diet or an isocaloric control diet for 10 weeks. Light microscopy showed marked steatosis in hepatocytes of ethanol-treated rats (ETRs), which was further revealed by transmission electron microscopy (TEM), where significant numbers of large LDs and damaged mitochondria were detected in steatotic hepatocytes. Moreover, TEM demonstrated that hepatocyte steatosis was associated with greatly enhanced autophagic vacuole (AV) formation compared to control hepatocytes. Mitochondria and LDs were the predominant contents of AVs in steatotic hepatocytes. Immunohistochemistry of LC3, a specific marker of early AVs (autophagosomes), demonstrated an extensive punctate pattern in hepatocytes of ETRs, while LC3 puncta were much less frequent in control hepatocytes. This was confirmed by immunoelectron microscopy (IEM), which showed localization of LC3 to autophagosomes sequestering damaged mitochondria and LDs. In addition, IEM revealed that PINK1 (a sensor of mitochondrial damage and marker of mitophagy) was overexpressed in mitochondria of ETRs. Enhanced autophagic lysosomal activity was evidenced by increased immunolabeling of LAMP-2, a marker of late AVs (autolysosomes) in hepatocytes of ETRs and colocalization of LC3 and lysosomal cathepsins using double immunofluorescence labeling. Increased AVs in hepatocytes of ETRs reflect ethanol toxicity and could represent a possible protective mechanism via stimulation of mitophagy and lipophagy.

Topics: Animals; Autophagy; Cathepsins; Cytochromes c; Ethanol; Fatty Liver; Hepatocytes; Immunohistochemistry; Lipids; Liver; Liver Diseases, Alcoholic; Lysosomal-Associated Membrane Protein 2; Lysosomes; Male; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Mitochondria; Mitophagy; Phagosomes; Protein Kinases; Rats; Rats, Wistar; Ultrasonography

Alcoholic liver disease is associated with nutritional deficiency and it may aggravate within the context of fatty liver. We investigated the relationship between alcohol intake (whiskey binge drinking) and a choline-deficient diet (CD) and assessed whether stellate cells could contribute to liver injury in this model.. Rats fed the CD diet plus whiskey showed increased liver damage compared to rats fed the CD diet, as demonstrated by H&E staining, elevated transaminases, steatosis, TNF-alpha levels, enhanced CYP2E1 activity, impaired antioxidant defense, elevated lipid peroxidation, and protein carbonyls. The combined treatment triggered an apoptotic response as determined by elevated Bax, caspase-3 activity, cytochrome-c release, and decreased Bcl-2 and Bcl-XL. Stellate cells were activated as increased expression of alpha-Sma was observed over that by the CD diet alone. The combined treatment shifted extracellular matrix remodeling towards a pro-fibrogenic response due to up-regulation of collagen I, TIMP1, and Hsp47 proteins, along with down-regulation of MMP13, MMP2, and MMP9 expression, proteases which degrade collagen I. These events were accompanied by increased phosphorylation of p38, a kinase that elevates collagen I.. Repeated alcohol binges in the context of mild steatosis may promote activation of stellate cells and contribute to liver injury.

Topics: Alcoholic Beverages; Animals; Antioxidants; Apoptosis Regulatory Proteins; Choline; Choline Deficiency; Collagenases; Cytochrome P-450 CYP2E1; Cytochromes c; Diet; Lipid Peroxidation; Liver; Liver Diseases, Alcoholic; Male; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Rats; Rats, Inbred Lew; Tumor Necrosis Factor-alpha

The present study investigates the hepatoprotective effect of fenugreek seed polyphenolic extract (FPEt) against ethanol-induced hepatic injury and apoptosis in rats. Chronic ethanol administration (6 g/kg/day x 60 days) caused liver damage that was manifested by the elevation of markers of liver dysfunction--aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), bilirubin and gamma-glutamyl transferase (GGT) in plasma and reduction in liver glycogen. The effects on alcohol metabolizing enzymes such as alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) were studied and found to be altered in the alcohol-treated group. Ethanol administration resulted in adaptive induction of the activities of cytochrome p450 (cyt-p-450) and cytochrome-b5 (cyt-b5) and reduction in cytochrome-c-reductase (cyt-c-red) and glutathione-S-tranferase (GST), a phase II enzyme. Further, ethanol reduced the viability of isolated hepatocytes (ex vivo) as assessed by the trypan blue exclusion test and increased hepatocyte apoptosis as assessed by propidium iodide staining (PI). Treatment with FPEt restored the levels of markers of liver injury and mitigated the alterations in alcohol metabolizing and detoxification enzymes and the electron transport component cytochrome-c reductase. Increased hepatocyte viability and reduced apoptotic nuclei were observed in FPEt-treated rats. These findings demonstrate that FPEt acts as a protective agent against ethanol-induced abnormalities in the liver. The effects of FPEt are comparable with those of a known hepatoprotective agent, silymarin.

Topics: Animals; Apoptosis; Cell Survival; Central Nervous System Depressants; Coloring Agents; Cytochromes c; Electron Transport; Ethanol; Flavonoids; Glutathione Transferase; Hemeproteins; Hepatocytes; Inactivation, Metabolic; Liver; Liver Diseases, Alcoholic; Male; Mixed Function Oxygenases; Phenols; Plant Extracts; Polyphenols; Propidium; Rats; Rats, Wistar; Seeds; Trigonella; Trypan Blue