| Objective To investigate the neurobehavioral changes induced by short-term inhalation of methanol for twenty-eight days in SD rats,and the role of p25-CDK5-p53 signaling pathway in the apoptosis of rat neurons induced by methanol,and provide evidence for studies on methanol neurotoxicity mechanism.Method One hundred and four healthy SD rats[body weight(200±20)g]were randomly divided into control group(0 g/m~3),low(25.344 g/m~3),medium(50.688 g/m~3),and high(101.376 g/m~3)dose groups,with 16 rats in each group.The experimental groups were exposed to the designed dosages of methanol for 28d and two hours everyday.Then the water maze test and open field test were used for neurobehavioral evaluation.After the neurobehavioral testing,six rats in each group were fixed by 4%paraformaldehyde under heart perfusion and their brain was embedded in paraffin and then serially cut for apoptotic neurons by TUNEL.The other rats were sacrificed under anaesthetization and the cerebral cortex were taken for next examinations.The contents of malondialdehyde(MDA)and glutathione(GSH),and the activity of glutathione peroxidase(GSH-PX)and superoxide dismutase(SOD)in cerebral cortex were detected by using oxidative stress kit.The gene expressions of Calpain,p35/p25,CDK5 and p53 were detected by q-PCR.The expressions of Calpain,p25,p35,CDK5 and p53 were detected by Western-blot.Results 1.During the experimental period,rats in the experimental group had less activity,lack of gloss and poor gloss,slow reaction and dyskinesia.And lately in the experiment,individual rat had nose-bleed,and was easily irritated.2.After two weeks exposure to methanol,there was a difference in body weight gain of the four groups.The weight gain of middle dose group was significantly lower than that in the control rats(P<0.05)and the high dose group was significantly lower than that in the control group and low dose group rats(P<0.05).3.In the water maze experiment,the latency of middle dose group was significantly higher than that of control group(P<0.05),and the high dose group was significantly higher than that of control group and low dose group(P<0.05).The number of cross-platform in the experimental group was significantly less than that in the control group(P<0.05),and the high-dose group was significantly less than that of the low-dose group(P<0.05).The target quadrant distance/total distance and the target quadrant time/total time in the control group and the low dose group of rats significantly higher than the middle dose group and high dose group rats(P<0.05).The number of target quadrant/total number of the high-dose group was significantly lower than that of the control group and the low-dose group(P<0.05).4.In the open-field test,the frequency in the central area of middle-dose rats significantly less than that in the control group,and the high-dose group was significantly less than the other groups(P<0.05).The distance in the central area of middle-dose group was significantly less than the control group(P<0.05)and the high-dose group was significantly less than the control and low groups(P<0.05).The time of high dose group in the central area was significantly lower than that of the control group and the low dose group(P<0.05).The time of central area/total time and the diatance of central area/total distance of the high dose group were significantly lower than those in the control group and the low dose group(P<0.05).5.The apoptosis rate of neurons in high-dose methanol group was significantly higher than that in other groups(P<0.05),middle dose group was significantly higher than low dose group and control group(P<0.05),low dose group was significantly higher than control group(P<0.05).6.The levels of GSH in the high-dose group was significantly lower than those in the control group(P<0.05).The activity of SOD in high-dose methanol group was significantly lower than that in the control group and the low-dose group(P<0.05).The levels of MDA in the high dose group was significantly higher than the control group and low dose group(P<0.05).7.The expression of calpain gene and P35/25 gene in high dose group was significantly higher than that in control group and low dose group,and the middlle group was higher than that in control group(P<0.05).The expression of P53 gene in high dose group was significantly higher than that in control group Group(P<0.05).8.The expression of calpain protein in high-dose group was higher than that in other groups(P<0.05).The expression of P35 protein in experimental group was higher than that in control group and the high-dose group was higher than that in other groups(P<0.05).The expression level of p25 protein in middle group was higher than that in control group,and high dose group was higher than that in control group and low dose group(P<0.05).The expression of CDK5 protein in middle dose group was higher than that in control group,and the high dose group was higher than that in other groups(P<0.05).Conclusion 1.Methanol exposure could affect weight gain in SD rats.2.Methanol caused damage to the nervous system of SD rats and affects their cognitive function,motor function and emotional state.3.Methanol could lead to apoptosis of nerve cells in SD rats.4.Methanol could change the oxidative stress of rats,activate the calpain protein,and convert p35into p25-a more potent activator for CDK5,thereby leading to the accumulationof p53.The transcriptional activation of pre-progeny genes could cause nerve cell apoptosised through the classical mitochondrial apoptotic pathway... |
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