慢性铅暴露大鼠海马脑区自噬相关蛋白表达的研究

Effect of chronic lead exposure on expression of autophagy-associated proteins in rat hippocampus

目的:观察慢性铅暴露后子代大鼠海马脑区铅含量变化,采用自噬/溶酶体途径关联蛋白标记物探讨慢性铅暴露对海马脑区自噬/溶酶体途径关联信号蛋白表达的影响。方法:以慢性铅暴露子代大鼠为研究对象,随机编号分组为对照组、低剂量铅暴露组(0.5 g/L)和高剂量铅暴露组(2.0 g/L)。断乳期仔鼠分笼饲养,仔鼠饮用低、高含量的醋酸铅饮用水直至60天成熟期。分别取血样与海马脑组织样本使用石墨炉原子吸收分光光度法进行铅含量测定,免疫印迹及免疫荧光组化方法分别检测慢性铅暴露后海马脑区自噬相关蛋白Beclin 1、LC3、LAMP2及cathepsin B的表达变化情况。结果:与对照组比较,模型组大鼠血铅含量显著升高(P<0.01)。同时,低剂量与高剂量铅暴露模型组大鼠海马脑铅含量显著升高,分别为(1.26±0.31)μg/g(低剂量组)与(1.83±0.18)μg/g(高剂量组)vs对照组(0.52±0.13)μg/g。此外,免疫印迹结果显示,高剂量铅暴露组大鼠海马脑区的早期自噬标志物Beclin 1及LC3-Ⅱ/LC3-I比值较对照组显著增加(P<0.05或P<0.001)。激光共聚焦显微镜结果显示,慢性铅暴露高剂量组较正常对照组的自噬相关蛋白cathepsin B在海马锥体神经元的阳性表达明显增加。结论:慢性铅暴露诱导大鼠海马脑区发生自噬/溶酶体途径关联信号蛋白表达变化,可能与海马脑区铅含量的增加存在内在关联性。

Objective： To investigate the effects of chronic lead exposure on expression of autophagy-associated proteins in rat hippocampus. Methods： SD rats were randomly divided into three groups： control group was given distilled water,lead-exposed groups were given 0.5 g/L（low-dose） or 2.0 g/L（high-dose） lead acetate solution in drinking water.The rat pups started to drink the lead content water until 60 d maturity.The lead contents in blood and brain samples were analyzed by graphite furnace atomic absorption spectrophotometry.The expressions of Beclin 1,LC3,LAMP2 and cathepsin B proteins were detected by Western blot and immunohistochemistry. Results： Compared with control group,the contents of lead were significantly higher in blood and hippocampus samples in chronic lead-exposed rats（P 〈0.01）.Western blot showed that the expression of Beclin 1 and LC3-II/LC3-I increased significantly in high dose lead-exposed group compared with control group（P 〈0.05 or P 〈0.001）.The confocal laser immunostaining results demonstrated that increased immunofluorescence staining of cathepsin B in hippocampal neurons compared with control animals. Conclusion： The disturbance of autophagy-lysosome signaling molecules might be partially contribute to neurotoxicity of chronic lead exposure.