Influence Of Hyperbaric Oxygen On Keap1-Nrf2-ARE Pathway In Brain Of Rats With Acute Carbon Monoxide Poisoning

BackgroundAcute carbon monoxide poisoning(ACOP)is a major public health event and the leading agent of death by poisoning worldwide.As the development of modern technology and the improvement of daily life,the environments exist where the risk of induce carbon monoxide poisoning can be anticipated and people avoid this kind of environment as far as possible.However,we may contact with the toxic environment that we can't avoid every day such as in outdoors with congested traffic or industrial combustion source and in indoors with unvented combustion appliances.Acute poisoning has an extremely influence on organ,especicaly on brain,which resulted in cognitive problems such as a loss in attention and memory.Even after treatment,there are still 3% to 30% of patients with delayed neuropathological sequela.The pathogenesis of delayed neuropathological sequela caused by carbon monoxide poisoning has not been fully elucidated,and there has been no effective and satisfactory treatment measure.Therefore,we need to pay more attention to the harm of carbon monoxide poisoning.Hyperbaric oxygen therapy(HBOT)has been remained one of treatment measure for carbon monoxide poisoning.Studies found that hyperbaric-oxygen therapy in patients with acute carbon monoxide poisoning could reduce the rate of cognitive sequelae 6 weeks and 12 months later.However,others studies also found that hyperbaric-oxygen therapy has the potential for inducing oxidative stress.Recently,studies also found the treatment of rats with hyperbaric oxygen attenuating ischemia-reperfusion injury through decreasing oxidative stress reaction.There is a controversial wherther the hyperbaric oxygen therepy can increase the organization oxidative stress damage.Therefore this study was designed to observe the cognitional function and the antioxidant effects of hyperbaric oxygen on brains of rats after acute carbon monoxide poisoning.Keap1-Nrf2-ARE pathway has recently been demonstrated to be an important pathway regulating oxidative stress in cells and regulated expression of various antioxidant gens.Studies found that low concentrations of carbon monoxide(CO)play an important role in the treatment of ischemic stroke,and its beneficial effect would be partially mediated by activation of Nrf2 pathway.And studies also found that hyperbaric oxygen reduced the number of apoptotic nerve cells and improved cognitional function in traumatic brain injury,which was mediated by up-regulation of the Nrf2 signaling pathway.However,the effect of hyperbaric oxygen on Keap1-Nrf2-ARE pathway in brain of rats with acute carbon monoxide poisoning is still not cleared.So we observed the Keap1 and Nrf2 protein expression in the brain tissue after acute carbon monoxide poisoning and brain tissue after hyperbaric oxygen therepy.And we combined the changes of antioxidant ability with the changes of Keap1 and Nrf2 protein expression,in order to clarify the possible molecular mechanisms of the antioxidant effects of hyperbaric oxygen on brain of rats after acute carbon monoxide poisoning.Objective 1.To observe the cognitional function of hyperbaric oxygen on brains of rats with acute carbon monoxide poisoning.2.To observe the antioxidant effects of hyperbaric oxygen on brains of rats with acute carbon monoxide poisoning.3.To observe the effect of hyperbaric oxygen on Keap1-Nrf2-ARE pathway in brain of rats with acute carbon monoxide poisoning.Methods 1.114 Sprague-Dawley(SD)rats were divided into three groups including the normal control(NC)group,the acute CO poisoning(CO)group and hyperbaric oxygen therapy(HBOT)group by random number table.2.Morris water maze experiments were used for monitoring cognitive function at 1 day(d),3 day(d),7 day(d),14 day(d)and 21 day(d).3.Antioxidant capacities were evaluated by detecting T-AOC,SOD,GPX,GR and CAT activities and MDA content in the brain at 1 d,3 d,7 d,14 d and 21 d by spectrophotometric method.4.Changes in Kelch-like ECH-associated protein 1(Keap1)and nuclear erythroid factor 2-like 2(Nrf2)of cerebral cortex tissue and brain of hippocampus from each group rats at 1 d,3 d and 7 d were monitored by immunohistochemical staining and western blot.Results 1.Hyperbaric oxygen therapy can improve the abnormal behavior of rats after acute CO poisoning1)Compared with the normal control group,the CO group(1 d,7 d,14 d,21 d)had the longer escape latency(P < 0.05)in spatial navigation learning of morris water maze experiments,the shorter swimming time in I quadrant(P < 0.05)in probe trials for spatial memory.2)After hyperbaric oxygen therapy,the HBOT group(1 d?3 d?7 d?14 d?21 d)had the shorter escape latency(P < 0.05)in spatial navigation learning of morris water maze experiments,the longer swimming time in I quadrant(P < 0.05)in probe trials for spatial memory.2.Hyperbaric oxygen therapy enhanced antioxidant capacities of brain tissues in acute CO-poisoned rats.1)Hyperbaric oxygen therapy increased the activities of antioxidant enzymes of brain tissues in acute CO-poisoned rats.Compared with the normal control group,the CO group had the lower T-AOC activity of brain tissue at 1 d,3 d,7 d,14 d and 21 d(P < 0.05),lower T-SOD and Cu-Zn SOD activity at 1 d,3 d,7 d,14 d and 21 d(P < 0.05),lower GPX activity at 1 d(P < 0.05),lower GR activity at 1 d,3 d,7 d,14 d,21 d(P < 0.05)and lower CAT activity at 1 d(P < 0.05).Compared with CO group,the HBOT group had the higher T-AOC activity of brain tissue at 3 d,7 d,14 d and 21 d(P < 0.05),higher T-SOD activity at 3 d,7 d,14 d and 21 d(P < 0.05),higher Cu-Zn SOD activity at 7 d,14 d and 21 d(P < 0.05),higher GPX activity at 1 d,3 d,7 d and 14 d(P < 0.05),higher GR activity at 1 d and 14 d(P < 0.05)and higher CAT activity at 1 d,3 d and 21 d(P < 0.05).2)Hyperbaric oxygen therapy decreased the degree of lipid peroxidation of brain tissues in acute CO-poisoned rats.Acute CO poisoned rats had the higher MDA content at 3 d,7 d and 14 d(P < 0.05).Hyperbaric oxygen therapy decreased the MDA content at 3 d,7 d and 14 d(P < 0.05).3.Hyperbaric oxygen therapy could affect the Keap1-Nrf2-ARE pathway in brain tissues of acute CO-poisoned rats.1)Hyperbaric oxygen therapy had no regular influence on the expression of Keap1 protein of brain tissues in acute CO-poisoned rats.Keap1-positive cells in the cerebral cortex tissue from rats of CO group was no significant difference with NC group at 1 day and 7 day(P>0.05),but the Keap1-positive cells at 3 day was significantly less than NC group.Keap1-positive cells in the brain of hippocampus from rats of CO group at 1 day and at 3 day was no significant difference compared with NC group(P>0.05).However the samples from CO group was significantly more than NC group at 7 day(P<0.05).After hyperbaric oxygen therapy,the Keap1-positive cells in the cerebral cortex tissue were significantly more than that in CO group at 3 day(P<0.05),but there was no alteration at 1 day and 7 day.Hyperbaric oxygen therapy decreased the degree of Keap1 expression in the brain of hippocampus at 7 day,but there was no alteration at 1 day and 3 day.In subjects from cerebral cortex tissue or brain of hippocampus of rats after CO exposure,the protein expression level of Keap1 was no significantly difference relative to subjects from either NC group or HBOT group at any time point through western blot(P>0.05).2)CO exposure leads to reduction of the expression of Nrf2.The Nrf2-positive cells in the cerebral cortex tissue from rats of CO group were significantly less than that of NC group at 1 day,3 day and 7 day(P<0.05).The samples from brain of hippocampus of rats after CO exposure was significantly less than NC group at 7 day(P<0.05),but there was no alteration at 1 day and 3 day(P>0.05).Testing the Nrf2 protein entering the nucleus through western blot,the expeimetal results showed that Nrf2 protein in the cerebral cortex tissue was no significantly difference relative to subjects from NC group at any time point(P>0.05).However,at 1 day,the Nrf2 protein in hippocampus was significantly increased than that of NC group(P<0.05).At 3 day,there was no significant difference between CO group and NC group(P>0.05).At 7 day,the Nrf2 protein in hippocampus was significantly less than that of NC group(P<0.05).3)Hyperbaric oxygen therapy increased the expression of Nrf2.The Nrf2-positive cells in the cerebral cortex tissue and hippocampus from rats of HBOT group were significantly more that of CO group at 1 day,3 day and 7day(P<0.05).Testing the Nrf2 protein entering the nucleus through western blot,the expeimetal results of western blot confirmed the results of immunohistochemical staining(P<0.05)except for at 1 day.At 1 day,the Nrf2 protein in hippocampus from rats of HBOT group was significantly less than that of CO group(P<0.05).Conclusion Our date showed that hyperbaric oxygen can improve the abnormal behavior of rats after acute CO poisoning,enhanced antioxidant capacities of brain tissues in acute CO-poisoned rats,and its beneficial effect would be partially mediated by activation of Keap1-Nrf2-ARE pathway.This study provided new experimental evidence for the elucidation of the mechanism and the protective effect of hyperbaric oxygen therapy on reducing the oxidative stress of brain after acute CO poisoning.