Remote ischemic perconditioning prevents liver transplantation-induced ischemia/reperfusion injury in rats: Role of ROS/RNS and e NOS

AIM To investigate the underlying mechanisms of the protective role of remote ischemic perconditioning(RIPerC) in rat liver transplantation. METHODS Sprague-Dawley rats were subjected to sham, orthotopic liver transplantation(OLT), ischemic postconditioning(IPostC) or RIPerC. After 3 h reperfusion, blood samples were taken for measurement of alanine aminotransferase, aspartate aminotransferase, creatinine(Cr) and creatinine kinase-myocardial band(CK-MB). The liver lobes were harvested for the following measurements: reactive oxygen species(ROS), H2O2, mitochondrial membrane potential(ΔΨm) and total nitric oxide(NO). These measurements were determined using an ROS/H2O2, JC1 and Total NOx Assay Kit, respectively. Endothelial NO synthase(e NOS) was analyzed by reverse transcription-polymerase chain reaction(RTPCR) and western blotting, and peroxynitrite was semiquantified by western blotting of 3-nitrotyrosine. RESULTS Compared with the OLT group, the grafts subjected to RIPerC showed significantly improved liver and remote organ functions(P < 0.05). ROS(P < 0.001) including H2O2(P < 0.05) were largely elevated in the OLT group as compared with the sham group, and RIPerC(P < 0.05) reversed this trend. The collapse of ΔΨm induced by OLT ischemia/reperfusion(I/R) injury was significantly attenuated in the RIPerC group(P < 0.001). A marked increase of NO content and phosphoserine eN OS, both in protein and mR NA levels, was observed in liver graft of the RIPer C group as compared with the OLT group(P < 0.05). I/R-induced 3-nitrotyrosine content was significantly reduced in the RIPerC group as compared with the OLT group(P < 0.05). There were no significant differences between the RIPerC and IPostC groups for all the results except Cr. The Cr level was lower in the RIPerC group than in the IPostC group(P < 0.01).CONCLUSION Liver graft protection by RIPerC is similar to or better than that of IPostC, and involves inhibition of oxidative stress and up-regulation of the PI3K/Akt/e NOS/NO pathway.

Control of oxidative stress in hepatocellular carcinoma: Helpful or harmful?

Oxidative stress is becoming recognized as a key factor in the progression of chronic liver disease(CLD) and hepatocarcinogenesis. The metabolically important liver is a major reservoir of mitochondria that serve as sources of reactive oxygen species, which are apparently responsible for the initiation of necroinflammation. As a result, CLD could be a major inducer of oxidative stress. Chronic hepatitis C is a powerful generator of oxidative stress, causing a high rate of hepatocarcinogenesis among patients with cirrhosis. Non-alcoholic steatohepatitis is also associated with oxidative stress although its hepatocarcinogenic potential is lower than that of chronic hepatitis C. Analyses of serum markers and histological findings have shown that hepatocellular carcinoma correlates with oxidative stress and experimental data indicate that oxidative stress increases the likelihood of developing hepatocarcinogenesis. However, the results of antioxidant therapy have not been favorable. Physiological oxidative stress is a necessary biological response, and thus adequate control of oxidative stress and a balance between oxidative and anti-oxidative responses is important. Several agents including metformin and L-carnitine can reportedly control mechanistic oxidative stress. This study reviews the importance of oxidative stress in hepatocarcinogenesis and of control strategies for the optimal survival of patients with CLD and hepatocellular carcinoma.

Autophagic cell death induced by reactive oxygen species is involved in hyperthermic sensitization to ionizing radiation in human hepatocellular carcinoma cells

AIM To investigate whether autophagic cell death is involved in hyperthermic sensitization to ionizing radiation in human hepatocellular carcinoma cells, and to explore the underlying mechanism.METHODS Human hepatocellular carcinoma cells were treated with hyperthermia and ionizing radiation. MTT and clonogenic assays were performed to determine cell survival. Cell autophagy was detected using acridine orange staining and flow cytometric analysis, and the expression of autophagy-associated proteins, LC3 and p62, was determined by Western blot analysis. Intracellular reactive oxygen species(ROS) were quantified using the fluorescent probe DCFH-DA.RESULTS Treatment with hyperthermia and ionizing radiation significantly decreased cell viability and surviving fraction as compared with hyperthermia or ionizing radiation alone. Cell autophagy was significantly increased after ionizing radiation combined with hyperthermia treatment, as evidenced by increased formation of acidic vesicular organelles, increased expression of LC3 II and decreased expression of p62. Intracellular ROS were also increased after combined treatment with hyperthermia and ionizing radiation. Pretreatment with N-acetylcysteine, an ROS scavenger, markedly inhibited the cytotoxicity and cell autophagy induced by hyperthermia and ionizing radiation.CONCLUSION Autophagic cell death is involved in hyperthermic sensitization of cancer cells to ionizing radiation, and its induction may be due to the increased intracellular ROS.

Anti-apoptotic effect of banhasasim-tang on chronic acid reflux esophagitis

AIM To evaluate the anti-apoptotic effect of banhasasimtang(BHSST) on chronic acid reflux esophagitis(CARE) using a rat model.METHODS A surgically-induced CARE model was established in Sprague-Dawley rats. The modeled rats were divided into a treatment group or untreated group, and given BHSST(1 g/kg body weight per day) or water, respectively, for 15 consecutive days(n = 7 each group). Changes in expression of proteins related to nicotinamide adenine dinucleotide phosphate(NADPH) oxidase and apoptosis were assessed by western blotting. Changes in esophageal pathology were analyzed by gross and histological examinations.RESULTS The CARE exposure modeled rats showed increased levels of the NADPH oxidase subunit, NOX4 and p47 phox in the esophagus. The BHSST treatment completely resolved these CARE-related increases. The CARE rats also showed markers of cytokine stress, including elevated levels of TNF-α and reactive oxygen species as well as of the consequent increase in JNK activation, and subsequent decrease in pro-survival gene expression, such as of Bcl-2. BHSST treatment resolved the CARE-related changes. BHSST also exerted an antiapoptotic effect, as evidenced by altered expression of the apoptosis-related genes for bax, cytochrome c,and caspase 3. Finally, the BHSST treatment markedly ameliorated the CARE-related esophageal mucosal ulcerations. CONCLUSION In the rat model of CARE, BHSST can suppress development of esophageal mucosal ulceration via regulation of reactive oxygen species-dependent apoptosis.

NADPH oxidase-1 deficiency offers little protection in Salmonella typhimurium-induced typhlitis in mice

AIM To test whether Nox1 plays a role in typhlitis induced by Salmonella enterica serovar Typhimurium(S. Tm) in a mouse model.METHODS Eight-week-old male wild-type(WT) and Nox1 knockout(KO) C57BL6/J(B6) mice were administered metronidazole water for 4 d to make them susceptible to S. Tm infection by the oral route. The mice were given plain water and administered with 4 different doses of S. Tm by oral gavage. The mice were followed for another 4 d. From the time of the metronidazole application, the mice were observed twice daily and weighed daily. The ileum, cecum and colon were removed for sampling at the fourth day post-inoculation. Portions of all three tissues were fixed for histology and placed in RNAlater for m RNA/c DNA preparation and quantitative real-time PCR. The contents of the cecum were recovered for estimation of S. Tm CFU.RESULTS We found Nox1-knockout(Nox1-KO) mice were not more sensitive to S. Tm colonization and infection than WT B6 mice. This conclusion is based on the following observations:(1) S. Tm-infection induced similar weight loss in Nox1-KO mice compared to WT mice;(2) the same S. Tm CFU was recovered from the cecal content of Nox1-KO and WT mice regardless of the inoculation dose, except the lowest inoculation dose(2 × 106 CFU) for which the Nox1-KO had one-log lower CFU than WT mice;(3) there is no difference in cecal pathology between WT and Nox1-KO groups; and(4) there are no S. Tm infection-induced changes in gene expression levels(IL-1b, TNF-α, and Duox2) between WT and Nox1-KO groups. The Alpi gene expression was more suppressed by S. Tm treatment in WT than the Nox1-KO cecum. CONCLUSION Nox1 does not protect mice from S. Tm colonization. Nox1-KO provides a very minor protective effect against S. Tm infection. Using NOX1-specific inhibitors for colitis therapy should not increase risks in bacterial infection.

Genetic polymorphisms of MAFK, encoding a small Maf protein, are associated with susceptibility to ulcerative colitis in Japan

AIM To investigate whether single nucleotide polymorphisms in maf protein K(MAFK), which encodes the MAFK, lead to increased susceptibility to ulcerative colitis in the Japanese population. METHODS This case control study examined the associations between MAFK single nucleotide polymorphisms(rs4268033 G>A, rs3735656 T>C and rs10226620 C>T) and ulcerative colitis susceptibility in 174 patients with ulcerative colitis(UC) cases, and 748 subjects without no lower abdominal symptoms, diarrhea or hematochezia(controls). In addition, as the second controls, we set 360 subjects, who have an irregular bowel movement without abnormal lower endoscopic findings(IBM controls).RESULTS The genotype frequency of rs4268033 AA and allelic frequency of the rs4268033 A allele were significantly higher in the UC cases than in both controls(P = 0.0005 and < 0.0001, P = 0.015 and 0.0027 vs controls and IBM controls, respectively). Logistic regression analysis after adjustment for age and gender showed that the rs4268033 AA and rs3735656 CC genotypes were significantly associated with susceptibility to UC development(OR = 2.63, 95%CI: 1.61-4.30, P = 0.0001 and OR = 1.81; 95%CI: 1.12-2.94, P = 0.015, respectively). Similar findings were observed by the comparison with IBM controls. In addition, the rs4268033 AA genotype was significantly associated with all phenotypes of UC except early onset. There was no significant association between rs10226620 and ulcerative colitis. CONCLUSION Our results provide the first evidence that MAFK genetic polymorphisms are significantly associated with susceptibility to UC development. In particular, rs4268033 is closely associated with an increased risk for the development of UC.

Interaction of hyperlipidemia and reactive oxygen species:Insights from the lipid-raft platform

Reactive oxygen species(ROS) and oxidative stress are closely associated with the development of atherosclerosis,and the most important regulator of ROS production in endothelial cells is NADPH oxidase.Activation of NADPH oxidase requires the assembly of multiple subunits into lipid rafts,which include specific lipid components,including free cholesterol and specific proteins.Disorders of lipid metabolism such as hyperlipidemia affect the cellular lipid components included in rafts,resulting in modification of cellular reactions that produce ROS.In the similar manner,several pathways associating ROS production are affected by the presence of lipid disorder through raft compartments.In this manuscript,we review the pathophysiological implications of hyperlipidemia and lipid rafts in the production of ROS.

Response to temperature stress of reactive oxygen species scavenging enzymes in the cross-tolerance of barley seed germination

A number of studies have shown the existence of cross-tolerance in plants,but the physiological mechanism is poorly understood.In this study,we used the germination of barley seeds as a system to investigate the cross-tolerance of low-temperature pretreatment to high-temperature stress and the possible involvement of reactive oxygen species (ROS) scavenging enzymes in the cross-tolerance.After pretreatment at 0 °C for different periods of time,barley seeds were germinated at 35 °C,and the content of malondialdehyde (MDA) and the activities of ROS scavenging enzymes were measured by a spectrophotometer analysis.The results showed that barley seed germinated very poorly at 35 °C,and this inhibitive effect could be overcome by pretreatment at 0 °C.The MDA content varied,depending on the temperature at which seeds germinated,while barley seeds pretreated at 0 °C did not change the MDA content.Compared with seeds germinated directly at 35 °C,the seeds pretreated first at 0 °C and then germinated at 35 °C had markedly increased activities of superoxide dismutase (SOD),ascorbate peroxidase (APX),catalase (CAT),and glutathione reductase (GR).The SOD and APX activities of seeds germinated at 35 °C after 0 °C-pretreatment were even substantially higher than those at 25 °C,and GR activity was similar to that at 25 °C,at which the highest germination performance of barley seeds was achieved.These results indicate that low-temperature pretreatment can markedly increase the tolerance of barley seed to high temperature during germination,this being related to the increase in ROS scavenging enzyme activity.This may provide a new method for increasing seed germination under stress environments,and may be an excellent model system for the study of cross-tolerance.