Studies On Molecular Mechanism Of N-butylphthalide Against Brain Damage In Rats With Acute Carbon Monoxide

PART 1 Role of neuronal apoptosis in brain damage induced by acute CO poisoning in rats and the mechanism of intervention with N-butylphthalideBackground and ObjectiveNeuronal apoptosis,which is generally accompanied by the destruction of mitochondria,is an important mechanism in the injury of central nervous system.Therefore,therapeutic strategies of anti-apoptotic and mitochondrial-protective has been extensively studied in CO poisoning.N-Butylphthalide(NBP)play an active role in reducing neurological deficits by alleviating the brain damage caused by apoptosis in a variety of animal models such as cerebral ischemia and protecting the relative normality of mitochondrial structure and function.So in this part,a rat model of acute CO poisoning was established to observe the dynamic changes of apoptotic cells,apoptotic genes and mitochondrial function before and after CO poisoning.Meanwhile,the role of apoptosis mechanism in brain damage of rats with CO poisoning and the feasibility of NBP in the treatment of acute CO poisoning brain injury were investigated.MethodsA total of 165 healthy adult male Sprague-Dawley rats were randomly selected as normal control group(NC group),CO poisoning group(CO group)and NBP treatment group(NBP group),with 55 rats in each group.Rats in the CO group and NBP group were used to established animal models of acute CO poisoning with hyperbaric chamber method,and all rats received hyperbaric oxygen therapy.Rats in NBP group were administered orally NBP 60 mg/kg twice a day at 2 hours after poisoning until death.The rats in NC group and CO group were treated with equal amount of pure olive oil.TUNEL was used to detect the cell apoptosis at day 1,day 3 and day 7;RT-PCR was used to detect the apoptosis-related gene expressions(Bcl-2 and BaxmRNA)at 6 hour,day 1,day 3,day 7;The mitochondrial membrane potential(MMP)of neurons in brain tissue was detected by JC-1 method after CO poisoning in each group at 6 hour,day 1,day 3,day 7.The results were analyzed using SPSS 19.0 softwere.Data were expressed as means±SEM(x±s),One-way ANOVA was used to compare difference between multiple groups.And LSD was used to compare difference between two groups,P<0.05 means statistically significant difference.Results1.There were few apoptosis cells in the NC group The number of apoptosis cell in the CO group and the NBP group was significantly increased as compared with that in the NC group(P<0.05);And as we expected,the amout of apoptosis cells in NBP group was relatively fewer than that in the CO group with significant differences(P<0.05).2.Low levels of Bcl-2 mRNA and Bax-mRNA expression were detected in NC group,while CO group and NC group showed significantly higher expression(P<0.05).Comparing with CO group at the same time,expression of Bcl-2 mRNA in NBP group was significantly increased,while Bax mRNA was significantly decreased(P<0.05).3.The MMP was normal and RFP value kept at a high level in NC group.Comparing with NC group,the mitochondrial RFP value of CO group was rapidly decreased after exposure at all time point,and could remain at a low level at days 7(P<0.05).The RFP value of NBP group was lower than NC group(P<0.05),while significantly higher CO group(P<0.05).Conclusion1.Acute CO poisoning can lead to the destruction of mitochondrial membrane potential,induce neuronal apoptosis,and accordingly cause neurological damage.2.N-Butylphthalide can play a therapeutic role in acute CO poisoning brain damage by reducing the number of apoptotic cells,increasing the expression of anti-apoptotic gene Bcl-2,decreasing the expression of proapoptotic gene Bax,reducing damage of MMP in CO poisoning rats.PART 2 Role of ZO-1 and Claudin-5 protein activity changes in brain damage induced by acute CO poisoning in rats and the mechanism of intervention with N-butylphthalide.Background and ObjectiveThe blood-brain barrier(BBB)is an important component of vascular nerve units.Brain damage caused by CO poisoning has many characteristics in common with hypoxic-ischemic diseases.Structure and function of BBB could be changed after ischemia and hypoxia,and therefore,CO poisoning can also cause damage to BBB and pathological opening.ZO-1 and Claudin-5 are two important proteins in tight junction of blood-brain barrier,and their expression is closely related to BBB function.It has been well recognized that the therapeutic effect of N-butylphthalide on BBB injury of cerebrovascular disease,however,it's still uncertain and worth further investigating of its therapeutic effect on BBB damage caused by CO poisoning.The purpose of this study was to explore the mechanism of ZO-1 and Claudin-5 on BBB injury and NBP intervention in rats after CO poisoning by investigating the changes of BBB ultrastructure,and the expression of ZO-1 and Claudin-5.Accordingly,the feasibility of NBP in the treatment of BBB damage caused by acute CO poisoning is elucidated.MethodsA total of 135 adult healthy male Sprague-Dawley(SD)rats were randomly divided into normal control(NC)group,CO poisoning(CO)group,and NBP treatment(NBP)group,with 45 rats in each group.The acute CO poisoning model was reproduced in hyperbaric oxygen chamber,and all model rats were given hyperbaric oxygen therapy once daily.The rats in the normal control group were free to breathe fresh air.The rats in NBP treatment group were administered orally NBP 60 mg/kg twice a day at 2 hours after poisoning until death.The rats in normal control group and CO poisoning group were treated with equal amount of pure olive oil.Five rats were sacrificed from each group at 1 day,3 days,7 days after model reproducing,respectively.The changes in ultrastructure of BBB were observed under transmission electron microscope.The expressions of ZO-1 and Claudin-5 proteins were determined by immunofluorescence staining and Western Blot.The localization of the two target proteins was observed by immunofluorescence double staining.Statistical methods were the same as the previous part.Results1.The structure of BBB in NC group was intact from TEM observation.In CO group,cerebral edema and BBB dysfunction were observed 1 day and 3 days after CO poisoning,and the structural integrity of the vessel wall was severely damaged.After NBP intervention,the ultrastructural damage of cortical microvessels in rats was significantly alleviated all time..2.Some ZO-1 and a large number of Claudin-5 positive cells were observed in NC group.But the positive cells and the expressions of two proteins were significantly lower in CO group than those of NC group(P<0.05)at each time point.By the treatment of NBP,the amount of ZO-1 and claudin-5 positive cells,as well as the expressions of two proteins were significantly increased,compairing with CO group from 1 day,3 days to 7 days(P<0.05),but lower than NC group.3.It's found that,in the same field of view,almost all Claudin-5 positive cells showed positive immunoreactivity of ZO-1,while not all ZO-1 positive cells showed Claudin-5 immunogenicity,which indicated that these two proteins can be present both in the same and different cells.ZO-1 positive cells those do not exhibit Claudin-5 immunogenicity may be other types of cells associated with non-vascular barriers,such as microvascular endothelial cells.Conclusion1.Acute CO poisoning can impair the ultrastructure and function of the blood-brain barrier in rats.2.ZO-1 and Claudin-5 proteins are important targets for structural and functional damage of BBB after acute CO poisoning.3.NBP can maintain the relative integrity of BBB structure and function by up-regulating the expression of ZO-1 and Claudin-5 protein,thereby significantly reducing brain damage caused by acute CO poisoning.NBP will be a new strategy for clinical treatment of acute CO poisoningPART3 The role of Nogo/NgR signaling pathway-related molecules in brain damage induced by CO monoxide poisoning in rats and the mechanism of intervention with N-butylphthalide.Background and ObjectiveThe presence of axonal growth inhibitory factor in the microenvironment of the central nervous system is the key reason why the central nervous system is difficult to regenerate after injury.Nogo/NgR pathway-related molecules are the most important inhibitors of axon growth,and their activation inhibits nerve regeneration and remodeling.In addition,there is extensive demyelinating pathological changes in the central nervous system of patients with CO-induced delayed encephalopathy.The parietal cortex,lateral paraventricular white matter and globus pallidus are common areas of demyelination,and deep white matter and nucleus damage are also becoming more and more serious with increased time and level,which is likely to lead to delayed encephalopathy.Nogo/NgR pathway-related molecules may be closely related to cellular immunity and inflammatory demyelination.N-Butylphthalide has the equivalent of increasing cerebral blood flow in the ischemic area and reconfiguring the microcirculation of the ischemic area,improving energy metabolism after global cerebral ischemia,inhibiting platelet aggregation,protecting mitochondrial function,reducing apoptosis damage and reducing neurological damage.This study intends to observe the role of Nogo/NgR pathway-related molecules in brain damage after CO poisoning through animal experiments.It's also investigated whether NBP can play a neuroprotective role by regulating Nogo/NgR pathway,and further provide a new treatment pathways of acute CO poisoning and delayed encephalopathy.MethodsA total of 45 rats were randomly divided into normal control(NC)group,CO poisoning(CO)group,and NBP treatment(NBP)group,with 15 rats in each group.The rat poisoning model and administration method are the same as the previous part.Experiments below were carried out on different time(1 day,7 day,28 day)to investigate the influence of NBP.Immunohistochemistry was used to detect the expression of MAG protein.Immunofluorescence and immunofluorescence double labeling were used to detect the expression of Nogo and NgRl proteins and their localization relationship,respectively.Statistical methods were the same as the previous part.Results1.MAG-positive cells were rare in NC group by high-magnification microscope.A small amount of lightly stained MAG-positive cells could be observed in the early stage of CO poisoning(day 1).With the increasing time of poisoning,the mumber of MAG-positive cells of CO group gradually increased on the day 7,and decreased on week 4,comparing with NC group(P<0.05).The number of MAG-positive cells in NBP group was significantly increased comparing with NC group(P<0.05),while comparing with CO group at the same time,the number was slightly decreased,but the difference was not statistically significant(P>0.05).2.It could observed by fluorescence microscope that few Nogo-positive cells were stained green in NC group,while few NgR1-positive cells were red-colored and unevenly distributed in various regions of the brain tissue.With increased time of poisoning,the number of Nogo and NgR1 positive cells in CO group increased gradually from the 1st day to the 7th day,and many Nogo and NgR1 positive cells could still by observed on the 4th week,which is showing a significant difference(P<0.05)comparing with NC group all time.After NBP treatment,the number of Nogo and NgRl positive cells decreased significantly,comparing with CO group at the same time(P<0.05),which suggests that NBP can effectively inhibit the expression of Nogo and NgRl after CO poisoning.Conclusion1.CO poisoning can increase the expression levels of NgRl,Nogo and MAG proteins in rat brain,which makes it difficult to repair nerve damage.These three proteins continue to be highly expressed after poisoning,and can still be observed 4 weeks after poisoning.This time point is consistent with the time of onset of encephalopathy,therefore,these three proteins may be associated with extensive demyelination damage after CO poisoning.2.N-Butylphthalide can show neuroprotective effects by reducing the expression of NgRl and Nogo proteins(not MAG)and thus regulating the activity of the Nogo/NgR signaling pathway.Therefore,NBP may become an effective treatment for acute brain injury and delayed encephalopathy caused by CO poisoning.