Effects Of Sub-chronically Benzo[a]pyrene On Rat Neurobehavior And Hippocampal Heat Shock Protein 70 Expression

Objective:Observation of effects of sub-chronically benzo[a]pyrene on rat neurobehavior,detect hippocampal Hsp70 expression, Investigate benzo[a]pyrene neurotoxicity and its mechanism of neurotoxicity.Methods:PartⅠ:Forty male SDrats, adult and healthy,were randomly divided into 4 groups:blank control group,trained group,swimming control group,heat shock group.The Morris water maze test was performed to test the learning and memory abilities of rat.After the experiment,18-24 hours, the rats were decapitated, detect hippocampal Hsp70 expression by immunohistochemistry, detect hippocampal Hsp70 expression by Western blot.PartⅡ:Sixty male SD rats,adult and healthy,were randomly divided into 6 groups:blank control, olive control, B[a]P0.5mg/kg, B[a]P1.5mg/kg, B[a]P4.5mg/kg, B[a]P10mg/kg. After exposure by benzo[a]pyrene intraperitioneal injection,90d, the Morris water maze test was performed to test the learning and memory abilities of rat. After the experiment, after weigh body weight,the rats were decapitated, take the brain, liver, lung, kidney, heart, spleen, testis, calculate Organ coefficient; Brain tissue for pathological examination andmorphological changes were observed by optical microscope; The apoptosis rate of hippocampus cells was measured by method of fluorescent analysis; Detect hippocampal Hsp70 and Hsp70mRNA expression by Western blot and.QRT-PCR; detect hippocampal Hsp70 expression by immunohistochemistry.Results:PartⅠ:Experimental short-term learning and memory in rats:The Immunohistochemistry showed that the control group's every part ot Hippocampus's Hsp70 expressed sporadicly.The swimming group's Hsp70 expressed difussed in Hippocampal pyramidal cells in plasma.It expressed fewer in CA3 area.The Learning and Memory Group's Hsp70 expressed a lot in CA3 area,it also expressed in cytoplasm and synapse.The heated treatment group's Hsp70 mainly expressed in the Hippocampal pyramidal nucleus. The Western-blot test results shows that the learning and memory group's and heated treatment group's Hsp70 expression values are 0.55±0.07 and 0.84±0.14,the differences are Statistically significant (P< 0.05, P< 0.01)PartⅡ:Intraperitoneal rat subchronic exposure B[a]P experiment:The rat's body weights all grew, The weight of rats in all groups the growth rate is less than the control and solvent control group.From the first month on,Weight in all groups were lower than the control group.When the exposure ended, In all groups compared with the control group,the results are stattistically significant (P< 0.05), The B[a]P4.5mg/kg group and B[a]P10mg/kg group's results are statistically significant compared with the solvent control group's.Navigation test:B[a]P1.5mg/kg,4.5mg/kg and 10mg/kg group's escape latencies and total distances are 32.5S,49.19S,69.74 S and 313.57 cm,422.81 cm,632.60 cm, and they are significantly higher than the control group's (17.17S and 252.71 cm) and the solvent control group's (20.33S和197.08 cm),the differences are Statistically significant (P＜0.05) The space search experiments:B[a]P0.5mg/kg group,1.5mg/kg group,4.5 mg/kg group,10 mg/kg group's durations of the third quadrant and numbers of cross platform are 7.17S,7.93S,6.01 S,5.30 S and1.75times,1.25times,1.20times,0.50times, they are lower than the control group's (13.31S and 4.20times), the differences are statistically significant (P＜0.05);The 4.5 mg/kg group and10 mg/kg group are statistically lower than the solvent control group (9.97S and3.0times) the difference is Statistically significant (P＜0.05)The expression of hippocampal Hsp70mRNA was detected using the RT-PCR. Compared with the control group.The expression of hippocampal Hsp70mRNA in B[a]P0.5 mg/kg group,4.5mg/kg group and 10mg/kg group was Significantly decreased and the difference was significant (P<0.01).Compared with solvent control the expression of mRNA was Significantly decreased and the difference was significant (P<0.05).The expression of hippocampal Hsp70protein was detected using the western-blot. Compared with the control group and solvent control The expression in B[a]P4.5mg/kg group and 10mg/kg group was Significantly decreased and the difference was significant (P<0.01).By immunohistochemistry we found that there have a large number of immunoreactive nerve cells stained in brown in Hippocampal pyramidal cell layer from the control group and solvent control.All groups with BaP wo found th expression of the Hsp70 in Nucleus. In B[a]P0.5 mg/kg group we found the immunoreactive expression was increased.But B[a]P4.5mg/kg group and 1 Omg/kg group the immunoreactive expression was decreased.Correlation analysis showed that there have a correlation between the early apoptosis rate and the total distance, the average escape latency, the number of cross platform and the Correlation coefficient r= 0.527,0.529,-0.401 (P<0.05). And there have a correlation between the expression of hippocampal Hsp70mRNA and the total distance, average escape latency, the duration of the third quadrant, the number of related cross platform, and the Correlation coefficient r=-0.594,-0.677,0.597,0.625, (P<0.05).Conclusion:In the short-term learning and memory rat hippocampus expression of Hsp70 increased, Hsp70 may be involved in learning and memory process.Subchronic exposure to benzo [a]pyrene can lead to rats learning and memory damage.The ability of learning and memory in rats with Neural cell apoptosis,expression of Hsp70 mHsp70 are significant correlated. Subchronic exposure to benzo[a]pyrene in rat hippocampus induced hippocampus pathological changes, neuronal apoptosis increased and the expression of Hsp70 and mHSP70 decreased may be related with benzo [a]pyrene neurotoxicity.