亚慢性染毒苯并[a]芘对大鼠海马热休克蛋白70表达的影响

Effects of sub-chronically exposure to benzo [a] pyrene on heat shock protein 70 expression level in rat hippocampus

目的研究亚慢性染毒苯并[a]芘（B[a]P）对大鼠海马热休克蛋白70（Hsp70）的影响。方法选择48只健康雄性SD大鼠，饲养1周后将动物随机分为空白对照组、溶剂对照组及0．5、1．5、4．5、10．0mg／kg B[a]P染毒组，隔日腹腔注射染毒90d，Morris水迷宫试验进行行为学测试。免疫印迹（Western—blot）法和反转录-聚合酶链反应（RT—PCR）法分别检测大鼠海马Hsp70蛋白和Hsp70 mRNA的表达水平，免疫组织化学法观察大鼠海马各区Hsp70表达情况，图像分析系统测定Hsp70平均灰度值。结果Morris水迷宫结果显示，4．5、10．0mg／kgB[a]P组大鼠平均潜伏期和平均总路程明显高于空白对照组、溶剂对照组、0．5、1．5mg／kgB[a]P组，差异有统计学意义（P〈0．05）；10．0mg／kgB[a]P组平台象限滞留时问明显低于空白对照组、溶剂对照组和0．5mg／kgB[a]P组，差异有统计学意义（P〈0．05）。B[a]P染毒组大鼠海马hsp70蛋白和hsp70 mRNA的表达水平均随染毒剂量增高而下降。其中，4．5、10．0mg／kgB[a]P组Hsp70蛋白表达水平明显低于空白对照组、溶剂对照组和0．5mg／kgB[a]P组，差异有统计学意义（P〈0．01，P〈0．05）；0．5、4．5、10．0mg／kgB[a]P组hsp70 mRNA表达水平明显低于空白对照组、溶剂对照组，差异有统计学意义（P〈0．01），10．0mg／kgB[a]P组hsp70mRNA表达水平明显低于其他染毒组。免疫组化结果显示，海马CA1、CA2、CA3、DG区Hsp70表达平均灰度值均随着B[a]P染毒剂量的增高而呈下降趋势。Pearson相关性分析结果显示，大鼠海马Hsp70表达水平与平均潜伏期呈负相关（r=-0．428，P〈0．05）；大鼠海马hsp70mRNA表达水平与平均潜伏期、平均总路程呈负相关（r值分别为-0．677、-0．594，P〈0．01），与平台象限滞留时间呈明显正相关（r=-0．597，P〈0．01）。结论亚慢性染毒B[a]P可抑制大鼠海马Hsp70的表达，与大鼠学习记忆和空间探索能力损害相关，该抑制作用无区域特异性。

Objective To study the influence of sub-chronically exposure to benzo[a] pyrene （B[a]P） on heat shock protein 70 （Hsp70） expression in rat hippocampus. Methods Forty eight healthy aduh male SD rats were randonly divided into 6 groups： blank control, olive control and four B [a]P-treated groups, which were exposed intra-peritoneally to B[a]P at the doses of 0.5, 1.5, 4.5 and 10.0 mg/kg for 90 days. The learning and memory function was measured by Morris water maze. The expression level of Hsp70 and hspT0 mRNA in rat hippocampus were detected by Western-blot and RT-PCR, respectively. Immunohistochemistry was used to examine the Hsp70 expression levels in hippocampus CA 1, CA2, CA3, DG regions, which were reflected by average gray values of Hsp70 immunoreactive products measured by image analysis system. Results The results of Mort＇is water maze test showed the escape latencies and total distances of navigation in 4.5 and 10.0 mg/kg B[a]P groups were significantly higher than those in two control groups and 0.5 or 1.5 mg/kg B[a]P groups （P〈0.05）. The duration of the third quadrant of the space search experiment in 10.0 mg/kg B[a]P group was significantly lower than those in two control groups and 0.5 mg/kg B[a]P group （P〈0.05）. The expression levels of Hsp70 and hsp70 mRNA in rat hippocampus decreased with the B[a]P dose. The expression levels of Hsp70 in 4.5 and 10.0 mg/kg B[a]P groups were significantly lower than those of two control groups and 0.5 mg/kg B[a]P group （P〈0.05 or P〈0.01 ）. The expression levels of hsp70 mRNA in 0.5, 4.5 and 10.0 mg/kg B[a]P groups were significantly lower than those in two control groups （P〈0.01） and the expression level of hsp70 mRNA in 10.0 mg/kg B[a]P group was significantly lower than those in other B[a]P groups. The results of immunohistochemistry examination indicated that the average gray values of Hsp70 immunoreactive products in CA1, CA2, CA3, DG regions decreased with dose of B[a]P. Pearson correlation analysis showed that there was a negative correlation between the expression of Hsp70 protein in hippocampus and the average escape latency （r=-0.428, P〈0.05）. There was a negative correlation between the expression of hsp70 mRNA in hippocampus and the average escape latency, the total distance of the third quadrant （r=-0.677 and r=-0.594, P〈0.01 ）. There was a positive correlation between the expression of hsp70 mRNA in hippocampus and the average total duration of the third quadrant （r= 0.597, P〈0.01）. Conclusion Sub-chronic exposure to B[a]P could inhibit the expression of Hsp70 in rat hippocampus, which was related to the ability of learning and rnemory of rats and the inhibition effects did not have the region-specificity.