Hepatoprotective effect of a curcumin/absinthium compound in experimental severe liver injury

OBJECTIVE: A preliminary in vitro study with hepatocyte culture showed that concentrations as low as 10 µg/mL of PN‐M001 are able to significantly mitigate CCl4 hepatocyte damage (P < 0.05) comparable to 100 µg/mL silymarin, and 100 µg/mL proved to be more protective than either silymarin 100 µg/mL or glycyrrhizin 10 µg/mL (P < 0.05). METHODS: Wistar rats were allocated into three groups: (A) 0.1 mL/100 g body weight (BW) mixture of CCl₄ in olive oil (1 : 1 v/v) subcutaneous injection twice daily for 4 weeks; (B) as A, plus oral administration of 50 mg/kg of K‐17.22 dissolved in 5% glucose; (C) as B but with PN‐M001 given 1 week after the first injection of CCl₄. Rats were killed at the end of the study and blood and liver samples were obtained. RESULTS: When compared with a control, group A showed a significant decrease of glutathione (GSH;>45%, P < 0.001) and oxidized GSH (GSSG; P < 0.01) liver content, a lower liver wet weight (P < 0.01) together with an increase of both transaminases (>15‐fold, P < 0.001) whereas groups B and C both showed only a mild increase in transaminases (<4‐fold, P < 0.05). Group A showed a significant decrease of Y‐protein fraction and of GST activity, as tested by both substrates (P < 0.01 vs control). However, both these parameters were reverted to normal by PN‐M001 (P < 0.05 vs A). CONCLUSIONS: These preliminary data suggest that PN‐M001 exerts a highly protective and prolonged effect (either preventive or therapeutic) on GSH depletion in CCl₄‐induced liver injury, which suggests its potential use in the clinical setting.     

Cellular endo‐lysosomal dysfunction in the pathogenesis of non‐alcoholic fatty liver disease

Non‐alcoholic fatty liver disease (NAFLD), an increasingly devastating human disorder, is characterized by intrahepatic fat accumulation. Although important progress has been made in understanding NAFLD, the fundamental mechanisms involved in the pathogenesis of NAFLD have not been fully explained. The endo‐lysosomal trafficking network is central to lipid metabolism, protein degradation and signal transduction, which are involved in a variety of diseases. In recent years, many genes and pathways in the endo‐lysosomal trafficking network and involved in lysosomal biogenesis have been associated with the development and progression of NAFLD. Mutations of these genes and impaired signalling lead to dysfunction in multiple steps of the endo‐lysosomal network (endocytic trafficking, membrane fusion and lysosomal degradation), resulting in the accumulation of pathogenic proteins. In this review, we will focus on how alterations in these genes and pathways affect endo‐lysosomal trafficking as well as the pathophysiology of NAFLD.     

Protective effect of Kalpaamruthaa in combating the oxidative stress posed by aflatoxin B1‐induced hepatocellular carcinoma with special reference to flavonoid structure–activity relationship

Aims/Background: Aflatoxin B₁ (AFB₁) is a potent hepatotoxic and hepatocarcinogenic mycotoxin. It has been postulated to play a major role in the aetiology of primary human liver cancer. Lipid peroxidation (LPO) is one of the main manifestations of oxidative damage and has been found to play an important role in the toxicity and carcinogenesis of many carcinogens. The present investigation aimed at assessing the effect of Kalpaamruthaa (KA), a modified Siddha preparation, on AFB₁‐mediated hepatocellular carcinoma (HCC). Methods: The drug was administered orally (300 mg/kg body weight/day) for 28 days to HCC‐bearing rats. The level of lipid peroxides, antioxidant enzymes, glutathione and glutathione‐metabolizing enzyme activity were determined in the plasma, haemolysate and liver homogenate of control and experimental rats. Results: Rats subjected to AFB₁ showed a decline in the thiol capacity of the cell, accompanied by high malondialdehyde levels along with lowered activities of enzymic and non‐enzymic antioxidant and glutathione‐metabolizing enzyme levels. KA treatment restored the deranged LPO and enzyme activities almost to control levels, thereby suggesting hepatoprotection. Conclusion: This study highlighted the beneficial effect of KA in reversing the damage posed by AFB₁ and thereby bringing about an improvement in the antioxidant status to combat the oxidative stress.     

Protective effect of melatonin against Opisthorchis viverrini‐induced oxidative and nitrosative DNA damage and liver injury in hamsters

Abstract: The liver fluke, Opisthorchis viverrini, is the risk factor of cholangiocarcinoma, which is a major health problem in northeastern Thailand. Production of reactive oxygen and nitrogen species during the host’s response leads to oxidative and nitrosative stress contributing to carcinogenesis. We investigated the protective effect of melatonin against O. viverrini‐induced oxidative and nitrosative stress and liver injury. Hamsters were infected with O. viverrini followed by oral administration of various doses of melatonin (5, 10, and 20 mg/kg body weight) for 30 days. Uninfected hamsters served as controls. Compared to the levels in O. viverrini‐infected hamsters without melatonin treatment, the indoleamine decreased the formation of oxidative and nitrosative DNA lesions, 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine and 8‐nitroguanine, in the nucleus of bile duct epithelium and inflammatory cells, in parallel with a reduction in 3‐nitrotyrosine. Melatonin also reduced the expression of heme oxygenase‐1 and cytokeratin 19, nitrate/nitrite levels, and bile duct proliferation in the liver. Alanine transaminase activity and the levels of 8‐isoprostane and vitamin E were also dose dependently decreased in the plasma of melatonin‐treated hamsters. Melatonin reduced the mRNA expression of oxidant‐generating genes [inducible nitric oxide synthase, nuclear factor‐kappa B (NF‐κB), and cyclooxygenase‐2] and proinflammatory cytokines (TNF‐α and IL‐1β), accompanied by an increase in the expression of antioxidant genes [nuclear erythroid 2‐related factor 2 (Nrf2) and manganese superoxide dismutase]. Thus, melatonin may be an effective chemopreventive agent against O. viverrini‐induced cholangiocarcinoma by reducing oxidative and nitrosative DNA damage via induction of Nrf2 and inhibition of NF‐κB‐mediated pathways.     

Protective effects of melatonin in reducing oxidative stress and in preserving the fluidity of biological membranes: a review

Free radicals generated within subcellular compartments damage macromolecules which lead to severe structural changes and functional alterations of cellular organelles. A manifestation of free radical injury to biological membranes is the process of lipid peroxidation, an autooxidative chain reaction in which polyunsaturated fatty acids in the membrane are the substrate. There is considerable evidence that damage to polyunsaturated fatty acids tends to reduce membrane fluidity. However, adequate levels of fluidity are essential for the proper functioning of biological membranes. Thus, there is considerable interest in antioxidant molecules which are able to stabilize membranes because of their protective effects against lipid peroxidation. Melatonin is an indoleamine that modulates a wide variety of endocrine, neural and immune functions. Over the last two decades, intensive research has proven this molecule, as well as its metabolites, to possess substantial antioxidant activity. In addition to their ability to scavenge several reactive oxygen and nitrogen species, melatonin increases the activity of the glutathione redox enzymes, that is, glutathione peroxidase and reductase, as well as other antioxidant enzymes. These beneficial effects of melatonin are more significant because of its small molecular size and its amphipathic behaviour, which facilitates ease of melatonin penetration into every subcellular compartment. In the present work, we review the current information related to the beneficial effects of melatonin in maintaining the fluidity of biological membranes against free radical attack, and further, we discuss its implications for ageing and disease.     

Antioxidant deficit and enhanced susceptibility to oxidative damage in individuals with different forms of alpha-thalassaemia

alpha-Thalassaemia is a common red cell disorder in Taiwan, affecting 6-8% of Taiwanese. Previous studies have shown that reactive oxygen species are generated in increased amounts in thalassaemic red cells. This implies the possible alteration of redox status in thalassaemic patients, which may adversely affect their health. In the present study, the redox status of patients with alpha-thalassaemia trait and haemoglobin H (Hb H) disease was investigated. Lipid peroxidation, as measured by the level of plasma thiobarbituric acid reactive substances (TBARS), was increased in alpha-thalassaemic patients, with the highest level of TBARS in Hb H disease patient. The plasma levels of vitamin A, C, and E were significantly lower in alpha-thalassaemic patients than in controls. The overall antioxidant capacity in plasma was inversely correlated with the severity of alpha-globin gene defect: the more severe the form of alpha-thalassaemia, the lower the overall antioxidant capacity in plasma. Erythrocytes isolated from alpha-thalassaemia patients had lower levels of vitamin E, glutathione, catalase and superoxide dismutase. In addition, these alpha-thalassaemic red cells were more susceptible to hydrogen peroxide-induced lipid peroxidation and decrease in deformability. All these data suggest that the alpha-thalassaemic patients suffer from increased oxidative stress and antioxidant deficit, which may complicate the pathophysiology of alpha-thalassaemia.     

Identification of a novel biomarker for oxidative stress induced by hydrogen peroxide in primary human hepatocytes using the 2‐nitrobenzenesulfenyl chloride isotope labeling method

Aim: Oxidative stress is involved in the progression of non‐alcoholic steatohepatitis (NASH). However, there are few biomarkers that are easily measured and accurately reflect the disease states. The aim of this study was to identify novel oxidative stress markers using the 2‐nitrobenzenesulfenyl (NBS) stable isotope labeling method and to examine the clinical utility of these diagnostic markers for NASH. Methods: Proteins extracted from phosphate buffered saline‐ and hydrogen peroxide‐loaded human primary hepatocyte were labeled with the [¹²C]‐ and [¹³C]‐NBS reagents, respectively. Pairs of peaks with 6‐Da differences in which the [¹³C]‐NBS labeling was more intense than the [¹²C]‐NBS labeling were detected by MALDI‐TOF/MS and identified by MS/MS ion searching. Results: Four pairs of peaks, m/z 1705–1711, m/z 1783–1789, m/z 1902–1908 and m/z 2790–2796, were identified as cytochrome c oxidase VIb (COX6B), liver carboxylesterase 1 (CES1), carbamoyl‐phosphate synthase 1 (CPS1) and superoxide dismutase (MnSOD), respectively. Furthermore, serum MnSOD protein levels were significantly higher in NASH patients than in simple steatosis (SS) patients. The serum MnSOD levels tended to increase in parallel with the stage of fibrosis. Conclusion: The NBS labeling technique was useful to identify biomarkers. Serum MnSOD may be a useful biomarker that can distinguish between SS and NASH.     

Glucagon‐like peptide‐1 receptor activation stimulates hepatic lipid oxidation and restores hepatic signalling alteration induced by a high‐fat diet in nonalcoholic steatohepatitis

Background/Aims: High‐fat dietary intake and low physical activity lead to insulin resistance, nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Recent studies have shown an effect of glucagon‐like peptide‐1 (GLP‐1) on hepatic glucose metabolism, although GLP‐1 receptors (GLP‐1r) have not been found in human livers. The aim of this study was to investigate the presence of hepatic GLP‐1r and the effect of exenatide, a GLP‐1 analogue, on hepatic signalling. Methods: The expression of GLP‐1r was evaluated in human liver biopsies and in the livers of high‐fat diet‐treated rats. The effect of exenatide (100 nM) was evaluated in hepatic cells of rats fed 3 months with the high‐fat diet. Results: GLP‐1r is expressed in human hepatocytes, although reduced in patients with NASH. Similarly, in rats with NASH resulted from 3 months of the high‐fat diet, we found a decreased expression of GLP‐1r and peroxisome proliferator‐activated receptor γ (PPARγ), and reduced peroxisome proliferator‐activated receptor α (PPARα) activity. Incubation of hepatocytes with exenatide increased PPARγ expression, which also exerted an insulin‐sensitizing action by reducing JNK phosphorylation. Moreover, exenatide increased protein kinase A (PKA) activity, Akt and AMPK phosphorylation and determined a PKA‐dependent increase of PPARα activity. Conclusions: GLP‐1 has a direct effect on hepatocytes, by activating genes involved in fatty acid β‐oxidation and insulin sensitivity. GLP‐1 analogues could be a promising treatment approach to improve hepatic insulin resistance in patients with NAFLD/NASH.     

Oxidative stress and hippocampus in a low‐grade hepatic encephalopathy model: protective effects of curcumin

Aim: The present study was performed on prehepatic portal hypertensive rats, a model of low‐grade hepatic encephalopathy, designed to evaluate whether oxidative stress was a possible pathway implicated in hippocampal damage and if so, the effect of an anti‐oxidant to prevent it. Methods: Prehepatic portal hypertension was induced by a regulated portal vein stricture. Oxidative stress was investigated by assessing related biochemical parameters in rat hippocampus. The effect of the anti‐oxidant curcumin, administered in a single i.p. dose of 100 mg/kg on the seventh, ninth and eleventh days after surgery, was evaluated. Results: Oxidative stress in the rat hippocampal area was documented. Curcumin significantly decreased tissue malondialdehyde levels and significantly increased glutathione peroxidase, catalase and superoxide dismutase activities in the hippocampal tissue of portal hypertensive rats. Conclusion: Oxidative stress was found to be implicated in the hippocampal damage and curcumin protected against this oxidative stress in low‐grade hepatic encephalopathic rats. These protective effects may be attributed to its anti‐oxidant properties.     

Melatonin protects against paraquat‐induced damage during in vitro maturation of bovine oocytes

Paraquat (PQ), a broad‐spectrum agricultural pesticide, causes cellular toxicity by increasing oxidative stress levels in various biological systems, including the reproductive system. PQ exposure causes embryotoxicity and reduces the developmental abilities of embryos. However, there is little information regarding the toxic effects of PQ on oocyte maturation. In this study, we studied the toxic effects of PQ exposure and the effects of melatonin on PQ‐induced damage in bovine oocytes. PQ exposure disrupted nuclear and cytoplasmic maturation, which was manifested as decreased cumulus cell expansion, reduced first polar body extrusion, and abnormal distribution patterns of cortical granules and mitochondria. In addition, PQ treatment severely disrupted the ability of the resulted in vitro‐produced embryos to develop to the blastocyst stage. Moreover, PQ exposure significantly increased the intracellular reactive oxygen species (ROS) level and early apoptotic rate, and decreased the glutathione (GSH) level, antioxidative CAT and GPx4 mRNA, and apoptotic‐related Bcl-2/Bax mRNA ratio. These results indicated that PQ causes reproductive toxicity in bovine oocytes. Melatonin application resulted in significant protection against the toxic effects of PQ in PQ‐exposed oocytes. The mechanisms underlying the role of melatonin included the inhibition of PQ‐induced p38 mitogen‐activated protein kinase (MAPK) activation, and restoration of abnormal trimethyl‐histone H3 lysine 4 (H3K4me3) and trimethyl‐histone H3 lysine 9 (H3K9me3) levels. These results reveal that melatonin serves as a powerful agent against experimental PQ‐induced toxicity during bovine oocyte maturation and could form a basis for further studies to develop therapeutic strategies against PQ poisoning.     

Fluorographic study on the oxidative stress in the process of gastric mucosal injury: Attenuating effect of Vitamin E

Abstract In vivo oxidative change was visualized in the gastric mucosa of rats and the alteration was analysed by using a fluorescence microscope equipped with a digital imaging processor during the development of mucosal damage. Dichlorofluorescein (DCF)-associated fluorescence increased after the repeated electrical stimulation on the gastric artery (irritation), suggesting the occurrence of lipid peroxidation. The increase was enhanced in the mid-zone of two adjacent collecting venules. Allopurinol attenuated the oxidative stress in mucosa, showing the involvement of xanthine oxidase. Luminol-dependent chemiluminescence value in the blood taken from gastric vein was elevated by the irritation, suggesting that leucocyte-generated oxygen radicals also participate in this oxidative process. α-Tocopherol attenuated both the DCF activation and the increase in chemiluminescence value and prevented gastric mucosal injury. The present results suggest that α-tocopherol may be useful for the prevention of oxidative alteration in gastric mucosa.