Simvastatin Reduces Hepatic Oxidative Stress and Endoplasmic Reticulum Stress in Nonalcoholic Steatohepatitis Experimental Model.

OBJECTIVE: In this study, we evaluated the efficacy of simvastatin in the treatment of nonalcoholic steatohepatitis induced by methionine and choline-deficient diet in mice and its possible effect on factors involved in the pathogenesis of the disease including oxidative stress and endoplasmic reticulum stress. METHOD: Male C57BL6 mice were fed either a normal diet (control) or a methionine and choline-deficient diet for four weeks and then treated orally with simvastatin (4 mg/kg once a day) for two final weeks. At the end of the experimental period, liver integrity, biochemical analysis, hepatic lipids, histology, DNA damage, biomarkers of oxidative stress, and endoplasmic reticulum stress were assessed. RESULTS: Simvastatin treatment was able to significantly reduce hepatic damage enzymes and hepatic lipids and lower the degree of hepatocellular ballooning, without showing genotoxic effects. Simvastatin caused significant decreases in lipid peroxidation, with some changes in antioxidant enzymes superoxide dismutase and glutathione peroxidase. Simvastatin activates antioxidant enzymes via Nrf2 and inhibits endoplasmic reticulum stress in the liver. CONCLUSIONS: In summary, the results provide evidence that in mice with experimental nonalcoholic steatohepatitis induced by a methionine and choline-deficient diet, the reduction of liver damage by simvastatin is associated with attenuated oxidative and endoplasmic reticulum stress.

Ductular reaction, cytokeratin 7 positivity, and gamma-glutamyl transferase in multistage hepatocarcinogenesis in rats.

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver and is characterized by multistage formation. The presence of ductular reaction, cytokeratin 7 positivity (PCK7), and increased levels of gamma glutamyltransferase (γGT) has been observed during liver carcinogenesis and contribute to tumor progression. Our goal was to evaluate the ductular reaction in multistage carcinogenesis and to correlate PCK7 and γGT levels with tumor incidence, histological characteristics, liver DNA damage index, and the expression of oxidative stress proteins. HCC was induced in 24 male Wistar rats weighing 145-150 g by chronic and intermittent exposure to 50 or 100 mg/kg diethylnitrosamine (DEN). Six control animals received only vehicle. Blood was collected to determine hepatic enzyme levels. Animals were divided into three groups: control (CO), precancerous lesions (PL), and advanced HCC. Liver samples were obtained for immunohistochemical analyses and the measurement of protein expression. Statistical analyses included Tukey's test and Pearson's correlation analyses. We observed an extensive ductular reaction in advanced HCC and a strong correlation between PCK7 and levels of γGT and the poor prognosis and aggressiveness of HCC. The extent of PCK7 and high γGT levels were associated with overexpression of inducible nitric oxide synthase (iNOS) and heat shock factor protein 1 (HSF-1). However, PCK7 and γGT levels were negatively correlated with protein expression of nuclear factor erythroid 2-related factor 2 (NRF2) and inducible heat shock protein 70 (iHSP70). These findings suggest that ductular reaction is involved in the progression of multistage hepatocarcinogenesis.

Melatonin Activates Endoplasmic Reticulum Stress and Apoptosis in Rats with Diethylnitrosamine-Induced Hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is one of the most lethal human cancers worldwide because of its high incidence, its metastatic potential and the low efficacy of conventional treatment. Inactivation of apoptosis is implicated in tumour progression and chemotherapy resistance, and has been linked to the presence of endoplasmic reticulum stress. Melatonin, the main product of the pineal gland, exerts anti-proliferative, pro-apoptotic and anti-angiogenic effects in HCC cells, but these effects still need to be confirmed in animal models. Male Wistar rats in treatment groups received diethylnitrosamine (DEN) 50 mg/kg intraperitoneally twice/once a week for 18 weeks. Melatonin was given in drinking water at 1 mg/kg/d, beginning 5 or 12 weeks after the start of DEN administration. Melatonin improved survival rates and successfully attenuated liver injury, as shown by histopathology, decreased levels of serum transaminases and reduced expression of placental glutathione S-transferase. Furthermore, melatonin treatment resulted in a significant increase of caspase 3, 8 and 9 activities, polyadenosine diphosphate (ADP) ribose polymerase (PARP) cleavage, and Bcl-associated X protein (Bax)/Bcl-2 ratio. Cytochrome c, p53 and Fas-L protein concentration were also significantly enhanced by melatonin. Melatonin induced an increased expression of activating transcription factor 6 (ATF6), C/EBP-homologous protein (CHOP) and immunoglobulin heavy chain-binding protein (BiP), while cyclooxygenase (COX)-2 expression decreased. Data obtained suggest that induction of apoptosis and ER stress contribute to the beneficial effects of melatonin in rats with DEN-induced HCC.

Diethylnitrosamine-induced cirrhosis in Wistar rats: an experimental feasibility study.

The experimental models of the development of cirrhosis in rats require a long time. Many studies in animals have demonstrated similarities in histological pattern with human cirrhosis. Just like the relation between cirrhosis and increased lipid peroxidation (LPO), which contributes to the worsening of the disease. However, few studies have focused on the reduction of time to establish cirrhosis and evaluated the expression of heat-shock protein 70 (HSP70) in cirrhotic livers of rodents. The present study proposes the adaptation of an experimental cirrhosis model using diethylnitrosamine (DEN). Twenty-six male Wistar rats, weighing ±270 g, divided into two groups: (i) CO-control and (ii) DEN-diethylnitrosamine. The DEN group received 50 mg/kg of DEN twice a week intraperitoneally for 7 weeks. The model developed cirrhosis in 7 weeks. The liver function tests showed that the animals with DEN-induced cirrhosis had increased levels when compared to control. The histological examination showed changes in the liver architecture, with severe ductal proliferation, signs of chronic damage, cholestasis, lymphocytic infiltrate, steatosis, and extensive parenchymal loss. We also found nodular formations with homogeneous pattern, increased LPO, increased expression of iNOS, TGF beta, α-SMA, and NQO1. However, the HSP70 expression was reduced in cirrhotic animals. This study showed signs of cirrhosis in liver based on biochemical, histological, and molecular analysis. The reduced expression of HSP70 appears to be associated with increased oxidative stress, contributing to the worsening of the disease.

Nitric oxide and repair of skeletal muscle injury.

The muscle wound healing occurs in three overlapping phases: (1) degeneration and inflammation, (2) muscle regeneration, and (3) fibrosis. Simultaneously to injury cellular infiltration by neutrophils and macrophages occur, as well as cellular 'respiratory burst' via activation of the enzyme NADPH oxidase. When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the cell cycle, divide, differentiate and fuse with muscle fibers to repair damaged regions and to enhance hypertrophy of muscle fibers. This process depends on nitric oxide (NO) production, metalloproteinase (MMP) activation and release of hepatocyte growth factor (HGF) from the extracellular matrix. Generation of a fibrotic scar tissue, with partial loss of function, can also occur, and seems to be dependent, at least in part, on local TGF-beta expression, which can be downregulated by NO. Hence, regeneration the muscle depends on the type and severity of the injury, the appropriate inflammatory response and on the balance of the processes of remodeling and fibrosis. It appears that in all these phases NO exerts a significant role. Better comprehension of this role, as well as of the participation of other important mediators, may lead to development of new treatment strategies trying to tip the balance in favor of greater regeneration over fibrosis, resulting in better functional recovery.

Effects of low-level laser therapy (LLLT) on the nuclear factor (NF)-kappaB signaling pathway in traumatized muscle.

BACKGROUND AND OBJECTIVE: To investigate the effects of low-level laser therapy (LLLT) on nuclear factor kappa B (NF-kappaB) activation and inducible nitric oxide synthase (iNOS) expression in an experimental model of muscle trauma. STUDY DESIGN/MATERIALS AND METHODS: Injury to the gastrocnemius muscle in the rat was produced by a single impact blunt trauma. A low-level galium arsenide (Ga-As) laser (904 nm, 45 mW, and 5 J/cm2) was applied for 35 seconds duration, continuously. RESULTS: Histological abnormalities with increase in collagen concentration, and oxidative stress were observed after trauma. This was accompanied by activation of NF-kappaB and upregulation of iNOS expression, whereas protein concentration of I kappa B alpha decreased. These effects were blocked by LLLT. CONCLUSION: LLLT reduced the inflammatory response induced by trauma and was able to block the effects of reactive oxygen species (ROS) release and the activation of NF-kappaB. The associated reduction of iNOS overexpression and collagen production suggest that the NF-kappaB pathway may be a signaling route involved in the pathogenesis of muscle trauma.

Croton cajucara Benth. leaf extract scavenges the stable free radical DPPH and protects against oxidative stress induced by paraquat.

Antioxidant effects of extracts from Croton cajucara BENTH. leaves was investigated in different in vitro and in vivo models. Extracts showed inhibitory radical scavenging activity against the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) (75%, 43% and 25% of the standard trolox at 1, 10 and 100 mg/ml, respectively; IC50 218 mg/ml). Percentage survival of Saccharomyces cerevisiae cells treated with 10 mM paraquat increased by 21% and 55%, when 1 mg/ml and 10 mg/ml concentrations of the extract, respectively, were added. The cytosolic concentration of TBARS increased in animals treated with paraquat (+283%), while values did not significantly differ from the controls in rats additionally receiving the leaf extract. Paraquat administration also induced a significant increase in hydroperoxide-initiated chemiluminiscence (+76%), that was partially prevented by the leaf extract (+31%). Liver SOD activity was a 158% higher in animals receiving paraquat as compared to the controls. This effect was abolished by administration of the leaf extract. Paraquat administration did not significantly modify the activity of GPx or catalase. Croton cajucara extract increased GPx and catalase activities in paraquat treated-animals by 342% and 70%, respectively. Our results confirm that Croton cajucara leaf extract present radical scavenging activity and reduce oxidative stress induced by paraquat, suggesting the beneficial use as a potential source of antioxidant agents of natural origin.

Low-level laser therapy (LLLT) prevents oxidative stress and reduces fibrosis in rat traumatized Achilles tendon.

BACKGROUND AND OBJECTIVES: The present study investigated the effects of low-level laser therapy (LLLT) on oxidative stress and fibrosis in an experimental model of Achilles tendon injury induced by a single impact trauma. STUDY DESIGN/MATERIALS AND METHODS: Male Wistar rats were randomly divided into four groups (n = 8): control, trauma, trauma+LLLT for 14 days, and trauma+LLLT for 21 days. Achilles tendon traumatism was produced by dropping down a load with an impact kinetic energy of 0.544 J. A low level Ga-As laser was applied with a 904 nm wavelength, 45 mW average power, 5 J/cm(2) dosage, for 35 seconds duration, continuously. Studies were carried out at day 21. RESULTS: Histology showed a loss of normal architecture, with inflammatory reaction, angiogenesis, vasodilatation, and extracellular matrix formation after trauma. This was accompanied by a significant increase in collagen concentration when compared the control group. Oxidative stress, measured by the concentration of thiobarbituric acid reactive substances and hydroperoxyde-initiated chemiluminiscence, was also significantly increased in the trauma group. Administration of LLLT for 14 or 21 days markedly alleviated histological abnormalities reduced collagen concentration and prevented oxidative stress. Superoxide dismutase activity was significantly increased by LLLT treatment over control values. CONCLUSION: LLLT by Ga-As laser reduces histological abnormalities, collagen concentration, and oxidative stress in an experimental model of Achilles tendon injury. Reduction of fibrosis could be mediated by the beneficial effects on the oxidant/antioxidant balance.