The Role Of Nf-?b Signaling Pathway In Brain Injury Induced By Acrylonitrile In Rats

Objectives To explore the effects of oxidative stress and inflammatory responses induced by acrylonitrile(ACN)on NF-?B signaling pathway,as well as the regulation of apoptosis related genes after NF-?B activation.The basis for further study of ACN neurotoxicity mechanism was provided.Methods Sixty healthy SPF adult male SD rats were randomly divided into five groups according to body weight(200-220g): control group(corn oil),ACN groups(11.5,23.0 and 46.0 mg/kg),NAC group(46 mg/kg ACN+300 mg/kg NAC).NAC group treated with 300.0 mg/kg before exposed to 46.0 mg/kg ACN.All of the rats were treated with a 5.0 ml/kg dose for gavage.Administration was conducted 6 days per week for 28 days.All rats were anesthetized with diethyl ether before collected blood from the heart and then killed by cervical dislocation.(1)Before and after ACN exposed,the open-field test was performed to evaluate the autonomous activity level of rats.(2)Weighed the brain and calculated the organ coefficient.(3)The hematoxylin and eosin(HE)staining was peformed to observe the brain lesion.(4)Preparation of brain tissue homogenate,the level of inflammatory cytokines IL-6,IL-1?,TNF-? was determined by Enzyme-Linked Immunosorbent Assay(ELISA).The GSH,MDA content and GSH-Px,SOD,CAT activity were measured by biochemical method.(5)Real-time PCR was peformed to detect the mRNA expression levels of IKK-?,I?B,NF-?B,TLR4,caspase-9 and caspase-3 in brain tissue.(6)Western Blot was used to detect the relative protin expression level of NF-?B,IKK-?,P-IKK?/?,I?B,p-I?B,TLR4,Cytc,caspase-9,caspase-3,Bax and Bcl-2 in brain tissue.(7)TUNEL assay was uesd to detect the apoptosis of testicular cells.Results(1)During the 0-10 min and 11-20 min in the OFT,the lardist and the total distance in the high dose of ACN-treated group were higher than in the control group.Compared with the high dose of ACN-treated group,the total distance in the NAC group was decreased.(2)There was no significant difference in body weight between the treatment groups before and after ACN exposed(P>0.05).The brain coefficients of each ACN-treated group was no signif icant difference(P>0.05).(3)In the middle and high dose of ACN-treated groups,cortical pyramidal cells structure turned incomplete,where some cells axons and Nissl bodies had disappeared,nucleus were pale stained,visible satellite phenomenon.The neurons in hippocampus CA1 area were disordered,and the layers of cells were not obvious.The nucleus was pale stained,cytoplasm was fuzzy,and the cell structure was unclear.Compared with the high dose of ACN-treatment group,the cortical pyramidal cells structure in the NAC group was complete,visible nucleus,axons and Nissl bodies.The neurons in hippocampal CA1 area were order,and the cell structure was clear.(4)Compared with the control group,the MDA content in each groups of ACN-treated were increased,the GSH content in the middle and high dose of ACN-treated groups were decreased,and the CAT activity in the low dose group was decreased(P<0.05).The activity of GSH-Px in the high dose group was decreased(P<0.05).Compared with the high dose of ACN-treatment group,the SOD content in the NAC group was increased,while the MDA content and CAT activity were decreased(P<0.05).(5)Compared with the control group,the levels of IL-1? and TNF-? in the middle and high dose ACN-treated groups group were increased(P<0.05),and the level of IL-6 in the high dose group was increased(P<0.05).The levels of inflammatory cytokines IL-6,IL-1? and TNF-? in NAC group were lower than in the high dose of ACN-treated group(P<0.05).(6)Compared with the control group,the relative mRNA expression of IKK-? and NF-?B in the middle and high dose of ACN-treated group were up-regulated(P<0.05),and the TLR4,Caspase-9 and Caspase-3 mRNA expression in the high dose of ACN-treated group were up-regulated(P<0.05).Compared with the high dose of ACN-treated group,the m RNA expression of IKK-?,NF-?B,Caspase-9 and Caspase-3 in NAC group were down-regulated(P<0.05).(7)Compared with the control group,the relative protein expression of p-IKK?/?,IKK-?,NF-?B,Cyt c and Caspase-9 in each groups of ACN-treated were increased(P<0.05),and the protein expression of p-I?B? and Bax in the middle and high dose groups were increased(P<0.05).The protein expression of TLR4 in the high dose group was increased,while the protein expression Bcl-2 was decreased(P<0.05).The protein expression of p-IKK?/?,NF-?B,Cytc,Caspase-9 and Caspase-3 in NAC group was lower than in the high dose of ACN-treated group(P<0.05),while the protein expression of Bcl-2 was higher than in the high dose group.(8)Compared with the control group,the number of apoptosis cells was increased in the middle and high dose of ACN-treated groups.Conclusions(1)ACN caused signif icant changes in autonomous activity in rats,and injured the normal brain architecture,which is related to oxidative stress mediated via ROS.(2)Inflammatory response,activation of NF-?B signaling pathway and mitochondrial apoptosis pathway mediated via ROS may be one of the mechanisms of brain damage induced by ACN.