| Objective: This research aims to establish acute rat model of delayed encephalopathy after acute carbon monoxide poisoning(DEACMP)and use tert-butylhydroquinone(tBHQ)to interfere the model,and then conduct behavioral pathological observation and explore the expression of nuclear factor E2-related factor 2(Nrf2)and NAD(P)H:quinone oxidoreductase 1(NQO1)in hippocampus of brain tissue and to analyze their correlation,and finally explore the potential pathophysiological mechanisms of these two proteins for the study of the pathogenesis of DEACMP and disease treatment and providing experimental basis and reference.Methods: 1.180 healthy normal male SD(Sprague-Dawley)rats were selected randomly and 120 were given DEACMP model with the first dose plus additional intraperitoneal injections of carbon monoxide(CO).Ways of injection of air are same as the air control group(AC group).DEACMP rats were randomly divided into simple carbon monoxide poisoning group(CO group),carbon monoxide poisoning + tBHQ group(TC group),60 rats in each group were exposed to 1,3,7,14,21,28 days after treatment as monitoring points,divided into 6 subgroups,10 in each subgroup.In TC group,intraperitoneal injection of tBHQ was given to the rats in AC group and no treatment in AC group and CO group.The rats were fed with drinking water freely during the experiment.2.The HbCO concentration in the femoral artery and Morris water maze(MWM)test were used to test the learning and memory cognitive function of the rats before and after the exposure,to verify whether the model was established successfully and to observe the rat cognition The changes of function were observed;The pathological changes of hippocampal CA1 area were observed by HE staining;The apoptosis of hippocampal CA1 area was observed by Tunel staining;The expression of Nrf2 and NQO1 in hippocampal CA1 area was detected by immunohistochemical method.NQO1 expression in the hippocampus was detected by Western Blot.3.mean standard deviation of experimental data,then SPSS17.0 statistical software analysis of data.Results: 1.The rat DEACMP model was successfully established:(1)The rats in CO group and TC group were accelerated in breathing shortly after poisoning,their activities were decreased,their limbs were weak and their spirit was sluggish.With the increase of exposure times,some rats suffered from incontinence,Limbs tonic convulsions,and even loss of consciousness,death,survival of poisoning rats showed activity reduction,restlessness,loss of appetite and other phenomena,AC rats no significant change;(2)HbCO concentration monitoring: CO group,TC group rats femoral artery blood HbCO concentration>50%,and the maintenance time>16h;(3)MWM test: before and 7d after exposure,the average escape latency,platform quadrant activity time/total time percentage difference between groups were not statistically significant(P>0.05).Compared with AC group,the mean escape latency and the quadrant activity time/total time percentage in CO group and TC group were longer than those in AC group at 14 d,21d and 28d(P<0.05);Compared with CO group,the above changes of TC group were more obvious at 21 d and 28 d after exposure(P<0.05);(4)HE staining: There was no obvious abnormality of pyramidal cells in hippocampal CA1 area in AC group,Rat hippocampal CA1 area cone Cells were different degrees of pathological changes occur,cell disorder,the number of cells is reduced,the gap increases,cell swelling,nuclear condensation,fragmentation,degeneration and necrosis.2.Tunel staining: There was no obvious apoptosis in AC group(P>0.05).Compared with AC group,apoptosis index of hippocampal CA1 area in CO group and TC group was significantly increased Compared with CO group,the apoptosis of TC group decreased(P<0.05)on day 1,3 and 7 after exposure(P<0.05)At 21 d,28d,the apoptosis in TC group increased(P<0.05).3.The expression of Nrf2 and NQO1 protein:(1)Immunohistochemical method: The expression of Nrf2 and NQO1 in hippocampal CA1 area of AC group were very few,there was no significant difference among the monitoring points(P>0.05);Compared with AC group,The expression of Nrf2 and NQO1 in the hippocampus CA1 of CO group and TC group were all increased(P<0.05),and increased in the CO group and the TC group(P<0.05).(2)The expression trend of Western Blot was basically the same as that of immunohistochemistry.The expression of NQO1 in hippocampus of AC group was less and there was no significant difference among the monitoring points(P>0.05),Compared with control group,the relative density of NQO1/β-actin in hippocampus of CO group and TC group were all increased(P<0.05),and increased from the first day to the peak(3d)TC group were higher than CO group(P<0.05).Conclusions: 1.the first dose + additional intraperitoneal injection of CO method is an ideal method to create DEACMP model,with simple operation,routine instruments,CO less and easy to control,safe,no pollution,no waste and so on.2.By comparing the change of memory and cognitive function in rats before and after exposure to the virus by MWM,monitoring the changes of Hb CO concentration in the arterial blood of the poisoned rats,and combining the pathological and behavioral changes of the hippocampus cells of the poisoned rats,whether the DEACMP is successfully established Rat models and rats with advanced cognitive impairment.3.In rat model of DEACMP,t BHQ can promote the expression of Nrf2 nuclear translocation and up-regulate the expression of downstream target protein NQO1,and play an antioxidant and anti-apoptotic role in the early stage after CO poisoning(≤3d),at 14 d,21d,28 d,sustained overexpression of both continued to promote delayed apoptosis of rat hippocampal cells,demonstrated that Nrf2/NQO1 pathway may participate in the occurrence of DEACMP. |
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