The Role Of INOS/p38 MAPK Signaling Pathway In Acrylonitrile-induced Brain Tissue Damage In Rats

Objective: To investigate the effects of acrylonitrile(ACN)on oxidative damage and iNOS/p38 MAPK signaling pathway in rat brain,and to provide evidence for the further study on the mechanism of ACN neurotoxicity.Methods: Fifty specific pathogen free(SPF)healthy adult male SD rats were selected and fed in the SPF laboratory of Gansu University of Traditional Chinese Medicine.According to body weight,all the rats were randomly divided into five groups,which included control group,low,middle,high dose group and N-acetylcysteine(NAC)group,each group had ten rats.After adaptively feeding in one week,rats in the exposed groups were respectively treated to 12.5,25.0and 50.0 mg/kg of ACN via gavage,and rats in the control group were given corn oil.After being treated with 300.0 mg/kg NAC 30 minutes later,rats in NAC group were treated with 50.0 mg/kg ACN.All the rats were gavaged for one time per day,six days per week,which lasted for 13 weeks.After the next day of last exposure,being weighed the body weight,all rats were sacrificed.Firstly,we separated the brain tissues,then calculated the relative organ weights.Secondly,the biochemical indexes of oxidative stress in the rats brains were detected by visible light spectrophotometry and microplate reader,which contained the activities of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and catalase(CAT),and the contents of glutathione(GSH)and malondialdehyde(MDA).Thirdly,using the instruments of visible light spectrophotometry and microplate reader,we measured the biochemical indexes of the contents of nitric oxide(NO)and the activities of total nitric oxide synthase(tNOS)in the rats brains.Fourthly,we also measured the expression levels of inducible or immunological nitric oxide synthase(iNOS),endothelial nitric oxide synthase(eNOS)and neuronal nitric oxide synthase(nNOS)by Western blot.Lastly,the expression levels of p38 mRNA in rat brains were detected by Real Time PCR,and the expression levels of phospho-p38 and p38 protein were measured by Western blot.Results:(1)The relative organ weights of the rat brain tissues in the low,middle and high dose group were significantly lower than that in the control group,but no significant differencewas showed between NAC group and the high dose group.(2)With the increase of ACN exposure,the activities of SOD and CAT decreased,while the contents of MDA increased.The results of one-way ANOVA showed in the high dose group,the activities of GSH-Px and SOD were obviously lower than that in the control group,and the contents of MDA were obviously higher than that in the control group.Compared with the control group,GSH in the middle dose group significantly decreased,and the activities of CAT in the low group also significantly decreased.There were no significant difference in the changes of GSH,SOD,GSH-Px,CAT and MDA between NAC group and the high dose group.(3)With the increase of ACN exposure,the contents of NO and the activities of tNOS in the rats brains also increased.Compared with the control group,the contents of NO and the activities of tNOS in the high group significantly increased.There was no significant difference in the changes of NO and tNOS between NAC group and the high dose group.(4)The results of Western blot showed that compared with the control group,the levels of iNOS and nNOS proteins in the high group significantly increased,and eNOS significantly decreased.Compared with NAC group,the levels of iNOS protein in the high dose group obviously increased.(5)The levels of p38 mRNA in the high dose group were significantly higher than that in the control by Real Time PCR,as well as NAC group.The results of Western blot showed that levels of phospho-p38 protein and the ratios of phospho-p38 protein and p38 protein in the high dose group were significantly higher than that in the control and NAC group.Conclusions: The chronic exposure of ACN could induce oxidative damage in rat brain tissue,resulting the activation of iNOS/p38 MAPK signaling pathway,which may be one of the mechanisms of brain tissue damage induced by ACN in rat.