The Time-Effect Relationship Of Central Nervous System Damages Induced By 1-Bromopropane In The Rats

Objective1-bromopropane(1-BP),a colorless,transparent and irritating organic solvent,has gradually become an alternative for ozone depleting substance from the 1990s due to its characteristics such as volatile,non-flammable,small damage to the ozone layer and short half-life(about 20 days in the air).The increase of production and consumption has led to a gradual expansion populations who exposed 1-BP in China.The epidemiological data and laboratory studies have demonstrated that 1-BP could be toxic to the central nervous system(CNS)and peripheral nervous systems(PNS),which earlier damages in CNS than that in PNS.1-BP occupational exposure poisoning has been reported continuously,since 1999 Sclar reported the 1st case of 1-BP occupational exposure poisoning.In recent years,the case of 1-BP nerve poisoning has been reported in China.At present,the mechanism of 1-BP neurotoxicity is not clear yet,and there is no special effective antidote for clinic treatment.Our previous research showed that 1-BP exposure could lead to the obvious activation of microglia in the brains of experimental animals,activation of the NF-κB signaling pathway,increase of the inflammatory factors and oxidative stress,and the loss of neurons,which resulted in the changes of neurological behavior in experimental animals.Although previous studies have confirmed that significant neuroinflammation in the brain,the experimental data came from the animals exposed 1-BP for a relatively long time.It is unable to make sure whether the neuroinlflammation was the early event in the neurotoxicity of 1-BP,and its etiological association with the CNS damages induced by 1-BP.In addition,it is whether there is a relationshipin between the 1-BP exposures chronic degenerative diseases such as Alzheimer’s disease(AD).In present study,firstly,we treated male Wistar rats with 1-BP last for different time(Id,3d,5d,7d,12d),observed changes in animal neurobehavior,detected the oxidative damage and neuroinflammation in the brains of experimental animals.On the basis of the above experiments,the rats were co-treated 1-BP with pyrrolidine dithiocarbamate(PDTC),which is a special NF-κB inhibitor.Then the reversal effects of PDTC on neurological impairment and pathological changes in 1-BP induced in rats were observed,to further verify the role of 1-BP-induced inflammatory response in 1-BP neurotoxicity.Methods1 Time-effect relationship of 1-BP induced brain damages of the ratsAfter acclimation for 5d,72 SPF male Wistar rats weighted 220-240g were randomly divided into control group,1-BP exposed Id,3d,5d,7d and 12 d goups with 12 in each(i.e.con,Id,3d,5d,7d and 12d).The animals in the 12d,7d,5d,3d and 1d groups were treated 1-BP started from 1st day,6th day,8th day,10th day and 12th day respectively.1-BP is diluted with corn oil and oral administreaed to rats at dosage of 800mg/kg.bw.The control rats and other rats in 1-BP-free time were given equivalent volume corn oil.Since the experiment 13th day,the Morris water maze was used to evaluate the learning and memory ability of rats with continuous 5 days of cruise experiment and 1 day of space exploration experiments.The following day after the end of the Morris water maze experiment,8 rat were randomly selected in each group and sacrified.The the brains were harvested immediately,and freezed in liquid nitrogen,then transferred into the-80℃ refrigerator to be subsequent experiments.The 4 remaining rats in each group were fixed by heart perfusion with 4%paraformaldehyde,and the frozen slices were prepared for pathological morphological observation.During the experiment,animals were free to feed and drink water.2 Reversal effect of PDTC on the damages of rat brain induced by 1-BPAfter acclimation for 5d,50 SPF class male Wistar rats weighted 220-240g were randomly divided into control group,1-BP exposed 7d group,1-BP exposed 12d group,1-BP exposed 7d plus PDTC group,and 1-BP exposed 12d and PDTC 12d with 10 rats in each group(i.e con,7d,12d,7d+PDTC and 12d+PDTC group).1-BP is diluted with corn oil and oral administreaed to rats at dosage of 800mg/kg.bw.PDTC were intraperitoneal injected to rat at dosage of 100mg/kg.bw.Since the experiment 13th day,the Morris water maze was used to evaluate the learning and memory ability of rats with continuous 5 days of cruise experiment and 1 day of space exploration experiments.The following day after the end of the Morris water maze experiment,8 rat were randomly selected in each group and sacrified.The the brains were harvested immediately,and freezed in liquid nitrogen,then transferred into the-80℃ refrigerator to be subsequent experiments.The 4 remaining rats in each group were fixed by heart perfusion with 4%paraformaldehyde,and the frozen slices were prepared for pathological morphological observation.During the experiment,animals were free to feed and drink water.Results1 Time-effect relationship of 1-BP induced neuroinflammation in brain of the rats1.1 Effects of 1-BP poisoning on neurobehavioral behavior of the ratsMorris water maze test results showed that the total swimming distance and escape incubation period of rats increased gradually with the prolongation of 1-BP poisoning time,compared with the control group,the total swimming distance and escape incubation period of rats of 1-BP exposed 7d,12d group was a statistical difference(P<0.05).The number of crossing platforms and the target quadranttime/the total time decreased gradually with the prolongation of 1-BP,and compared with the control group 12d,the number of crossing platformsand the target quadrant time/the total time decreased statistically(P<0.05),which excluded the mixed interference caused by the inconvenient mobility of rats.1.2 Effects of 1-BP poisoning on activation of glial cells and formation of NLRP3 inflammasome in brainCompared with the control group,microglia in the rat brain were activated once exposed 1-BP,and 5d,7d and 12d group could observe the "amoeba like" microglia in the prefrontal cortex of the rats brain,iNOS,The expression of iNOS(the specific markers of M1 type microglia)has significantly increased with the prolongation of 1-BP poisoning time(P<0.05),the expression of Argl(the specific markers of M2 type microglia)has a slight increased(P<0.05).Astrocytes were activated in the brain of rats after 1-BP exposure,and the activation of astrocytes was obvious with the prolongation of 1-BP poisoning time,and astrocyte plaques could be observed.Our detection inflammasomes found that caspase 1,IL-1β and NLRP3 in NLRP3 inflammasomes increased with the prolongation of 1-BP poisoning time(P<0.05)。The above results suggest that 1-BP exposure can stimulate the polarization of small glial cells in the direction of M1,M1/M2 polarization out of balance,produce inflammasomes,and aggravate the inflammatory response in the brain.1.3 The effects 1-BP poisoning on the oxidative/nitrosative stress of rat brainCompared with the control group,and the content of ROS in the prefrontal cortex and hippocampus of rats in 1-BP poisoning group increased significantly with the prolongation of the time of poisoning,which showed time-dependent,The content of ROS in the prefrontal cortex and hippocampus of rats in 7d and 12d was significantly higher than that in the control group(P<0.05),and the content of NOin the prefrontal cortex and hippocampus of rats in the 1-BP group was significantly increased with the increase of toxicity time compared with the control group(P<0.05),Time dependent,of which the decrease of NO content in the prefrontal cortex and hippocampus of rats in 7d and 12d group was statistically significant(P<0.05).Compared with the control group,the expression of oxidative modified protein MDA in the hippocampus and prefrontal cortex of the 1-BP group increased significantly(P<0.05).The above results suggest that 1-BP exposure can lead to oxidation and nitriding stress in the body,and increase the expression of oxidized modified protein and nitro protein in the relevant region of the brain,and aggravate the imbalance of oxidation nitriding.1.4 The 1-BP poisoning on brain A20 and NFκB activationCompared with control group,the content of NFκB in the prefrontal cortex of the brain was increased and the nuclear transposition was enhanced in 1-BP exposed 7d,12d group(P<0.05),NFκB signal pathway inhibited protein A20 expressed significantly reduced(P<0.05),the content of inflammatory factor IL-1(3 also increased significantly(P<0.05),the expression of NO increased(P<0.05),iNOS activity increased(P<0.05).It is suggested that 1-BP can aggravate oxidative imbalance and nerve inflammation in the brain by weakening the inhibitory effect of zinc finger protein A20 on NF-κB signaling pathway.1.5 Effects of 1-BP on the damages of brain neurons with different times exposureNeurons nuclear protein(neuronal nuclei,NeuN)is a neuron specific nuclear protein,using anti-NeuN antibody immunohistochemical staining showed that,compared with the control group,1-BP can lead to the reduction of neurons in the prefrontal cortex of the brain after poisoning,As the time of infection increases,the more obvious the loss of neurons.The results of Western Blot showed that the expression of NeuN in cerebral cortex began to decrease from 3d,and the decrease of NeuN protein increased with the prolongation of poisoning,among which the decrease of NeuN expression was statistically significant compared with that in the control group of 1-BP and 5d,7d and 12d group(P<0.05,P<0.01),which is consistent with the change of morphological test results.2 Reversal effect of PDTC on the damages of rat brain induced by 1-BP2.1 PDTC on the activation of microglia,astrocyte and neuronal injury induced by 1-BPCompared with the control group,the microglia and astrocyte in the prefrontal cortex of the brain were significantly activated after 1-BP exposure of 7d and 12d,The number of neurons decreased significantly(P<0.05).After PDTC intervention,both groups of small glial cells activated the marker protein ion calcium joint protein molecule 1(ionized calcium-binding adapter molecule l,Ibal)Ibal expression was significantly reduced(P<0.05),The activation of astrocytes was obviously inhibited,the secretion of inflammatory factors decreased,and the number of neurons increased(P<0.05).It 1s suggested that 1-BP can lead to inflammatory reactions in the brain and lead to loss of neurons,and blocking NF-κB activation and inhibiting inflammatory reactions in the brain can effectively reverse the expression of NFκB inhibition protein A20 and protect 1-BP damage to the brain.2.2 The reversal effect of PDTC on the degradation of beta amyloid in the brainCompared with the control group,the expression of Aβ in 1-BP exposed 12d group was significantly increased(P<0.05).Compared with 12d group,the Ap expression ofl2d+PDTC groupwas decreased(P<0.05).Compared with the control group,the expression of IDEin 1-BP exposed 12d group was significantlydecreased(P<0.05).Compared with 12d group,he expression of IDEin the 12d+PDTC group was significantly increased(P<0.05),indicating that PDTC intervention could significantly reverse Apaccumulation and IDE reduction,reducing Aβgeneration increases the degradation of Aβby the IDE.2.3 The effects of PDTC on the A20 and NF-κB in rat brainCompared with the control group,the expression of NF-κB inhibition protein A20were significantly reduced in 1-BP exposed 7d,12d group(P<0.05).After PDTC intervention,compared with 1-BP poisoning group,A20expression was increased(P<0.05).The results showed that PDTC intervention could obviously increasethe expressionof NFκB inhibition protein A20,inhibit NFκB activation,and weaken the inflammatory reaction in the brain to a certain extent.Conclusion1.1-BP exposure could damage the learning and memory ability of rats,with the time dependence.2.1-BP could lead to activation of NF-κB signaling pathway in rat brain,activation of microglia and astrocytes,oxidative/introsative stress in the brain,resulting in inflammatory damage of brain.3.1-BP exposure could lead to the Aβ accumulation in the brain of rats,which is related to the exposure time of 1-BP.4.Inhibition of NF-κB could relieve neuroinflammation,decrease the Aβ,protect the neurons,and improve the learning and memory ability of rats.