Disruption Of Glutamate Neurotransmitter Transmission Is Modulated By SNAP-25 In Benzo[a]pyrene-induced Neurotoxic Effects

PART ?EFFECTS OF SUBCHRONIC B[A]P EXPOSURE ON THE EXPRESSION OF SNAP-25 AND GLUR IN HIPPOCAMPUS OF SD RATSObjective: Through the establishment of neurotoxicity model induced by benzo?a?pyrene?B[a]P?,observe the changes of glutamate?Glu?level and the expression of SNAP-25?glutamate receptor?GluR?in the hippocampus of animal.To provide a theoretical basis for the important role of SNAP-25 and Glu in the neurotoxicity induced by B[a]P.Methods: 80 SD neonatal rats,aged 5 days,were randomly divided into four groups as follows: control group,0.02,0.2,and 2.0 mg/kg of B[a]P.B[a]P was given to the rats by intragastric administration for 7 weeks.Morris water maze was utilized to evaluate the learning and memory ability after treatment.The level of Glu in the hippocampus was detected by the 1H magnetic resonance spectroscopy?1H-MRS?.Synaptic ultrastructure,synaptic cleft width and postsynaptic density of hippocampus were observed by using transmission electron microscope.The expression of SNAP-25 and GluR were detected by immunohistochemistry and Western-blot at the time point of 1 week and 7 week,respectively.Results:?1?After 35 days treatment,the body weight of the B[a]P treatment group was significantly reduced,especially in 0.2 and 2.0 mg/kg groups [F?3,76?=3.881,P=0.008].?2?The escape latency and swimming distance of 0.02 mg/kg,0.2 mg/kg and 2 mg/kg B[a]P treated groups were all significant longer than that of control group,while the time spent in target quadrant and the number of platform crossings was significantly decreased in B[a]P-treated groups.?3?The transmission electron microscope showed an expansion of endoplasmic reticulum and cytoplasmic swelling in the hippocampus of B[a]P treated groups.In addition,the observably widened synaptic cleft was also found.?4?The 1H-MRS showed that,as compared with control group?0.996±0.236 ppm?,the significant decrease on the Glu concentration were found in B[a]P-treated groups?0.722±0.185,0.687±0.172 and 0.583±0.154 ppm for different groups,respectively?.?5?Compared with control group,the expression of SNAP-25 was even more obvious,and both GluR 1 and GluR 2 protein expressions were significantly declined.Conclusion: Subchronic exposure to B[a]P could cause learning and memory deficits in neonatal rats,which could also change the ultrastructure and widening the synaptic cleft of hippocampus.The possible mechanism of neurotoxicity induced by B[a]P maybe due to the alteration of Glu transmission by decreasing the level of Glu,reducing the expression of GluR,enhancing the level of SNAP-25.PART ?STUDY ON THE MECHANISM OF SNAP-25 AND GLUR IN THE NEUROTOXICITY INDUCED BY B[A]PObjective: To investigate the effect of B[a]P on the cell viability and apoptosis of U87 cells,and observe the possible effect of SNAP-25 on the expression of GluR.Methods: MTT method was used to observe the effect of B[a]P on the cell viability of U87 cells.AO-EB staining,Hoechst 33258 staining and annexin V-FITC/PI double staining were used to observe the apoptosis of U87 cells caused by B[a]P.The concentration of intracellular [Ca²⁺]i was measured with Fluo-3/AM probe after treatment of B[a]P.After the expression of SNAP-25 protein was inhibited by si RNA transfection,the expression of Glu R was further examined by Western-blot and real-time PCR.Results: After treated with B[a]P,the cell viability of U87 cells was significantly inhibited,which showed a dose-dependent manner.?2?The results of AO-EB staining and annexin V-FITC/PI double staining showed that the apoptosis rate in B[a]P treated group was significantly elevated as compared with control group.Hoechst 33258 staining represented a dense,bright and irregularity nuclei in B[a]P treated group.?3?After treating with B[a]P for 24 h,our results showed that there was no significant change on the proportion of cells in the G0/G1,G2/M and S phase between B[a]P treated group?G0/G1:56.04% ± 2.78%;G2/M: 12.44% ± 2.71%;S: 9.05% ± 0.79%?and control group?G0/G1:56.44%±2.44%;G2/M:11.79%±3.66%;S:8.57%±0.36%?.?4?The concentration of [Ca²⁺]i was increased significantly in response to B[a]P exposure.?5?The RT-PCR and Western-blot results showed that,down-regulated mRNA expressions of Gria 1 and Gria 2 and the decreased protein expressions of GluR1 and GluR2 in B[a]P-treated cells were remarkably ameliorated in SNAP-25 siRNA transfection.Conclusion: B[a]P could decrease the cell viability and induce apoptosis.By decreasing the expression of SNAP-25,the level of intracellular Glu R was increased,which indicated that the neurotoxic effect of B[a]P was,at least partly,due to the change of SNAP-25 expression,which further affected the Glu neurotransmitter delivery.