自噬抑制剂3-甲基腺嘌呤对铜过量负荷大鼠海马神经损伤的影响  

The impact of the autophagy inhibitor 3-methyladenine on the hippocampus neurodegeneration in the copper overload rats

作　　者：刘星 秦思思 秦昌宇 赵会新 王雅楠 侯炳旭 李欣 李爽 LIU Xing;QIN Si-si;QIN Chang-yu;ZHAO Hui-xin;WANG Ya-nan;HOU Bing-xu;LI Xin;LI Shuang(Affiliated Hospital,North China University of Science and Technology,Tangshan,Hebei 063000,China;School of Public Health,North China University of Science and Technology,Tangshan,Hebei 063210,China;Jitang College,North China University of Science and Technology,Tangshan,Hebei 063210,China)

机构地区：[1]华北理工大学附属医院,河北唐山063000 [2]华北理工大学公共卫生学院,河北唐山063210 [3]华北理工大学冀唐学院,河北唐山063210

出　　处：《毒理学杂志》2024年第6期430-436,共7页Journal of Toxicology

基　　金：河北省科技厅中央引导地方科技发展资金项目(236Z7705G);河北省卫健委医学科学研究课题计划(20191113,20201231)。

摘　　要：目的探讨自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)对Wilson病(Wilson’s disease,WD)大鼠神经保护作用。方法30只雄性SD大鼠分为对照组、WD组及3-MA干预组,每组10只,WD组和3-MA干预组给予含硫酸铜1 g/kg的饲料和0.185%硫酸铜的水,染毒11周之后,3-MA干预组给予腹腔注射200μl(100 nmol/μl)的3-MA,连续给药1周。记录大鼠体重及一般状态。旷场实验、新物体识别实验及Morris水迷宫实验检测大鼠神经行为学变化,电感耦合等离子体质谱法检测大鼠血清及海马铜水平,苏木素-伊红(HE)染色观察大鼠海马组织病理变化,Western blot及Real-time PCR方法检测大鼠海马组织自噬标志物LC3、Beclin-1和P62蛋白及mRNA的表达。结果与WD组比较,3-MA干预组大鼠在整个实验期间体重差异无统计学意义(P>0.05),大鼠自主行为、探究能力及学习记忆能力改善,血清和海马铜水平差异无统计学意义(P>0.05),海马神经元细胞数量增多,细胞坏死减少,排列整齐。海马组织中Beclin-1、LC3的蛋白(F=64.212,F=12.430,P<0.05)及mRNA(F=32.225,F=6.633,P<0.05)表达水平均降低,P62蛋白(F=9.856,P<0.05)及mRNA(F=8.481,P<0.05)表达水平均升高,差异均有统计学意义。结论3-MA可降低铜过量负荷大鼠海马自噬作用,缓解神经损伤作用,改善大鼠神经行为能力。Objective To examine the potential neuroprotective effects of the autophagy inhibitor 3-methyladenine(3-MA)in rats with Wilson′s disease(WD).Methods Thirty male SD rats were divided into a control group,WD group,and 3-MA intervention group,with 10 rats in each group.The rats of WD group and 3-MA intervention group rats were given with the feed containing 1 g/kg copper sulfate and 0.185% copper sulfate water.After 11 weeks of exposure,the rats in the 3-MA intervention group were given with intraperitoneal injection of 200μl(100 nmol/μl)of 3-MA,the body weight and general condition were carefully monitored.Neurobehavioral changes were assessed through the open field test,novel object recognition test and Morris water maze test.Copper levels in the serum and hippocampus were measured using inductively coupled plasma mass spectrometry.Pathological changes in the hippocampus tissue were observed through the HE staining.The expression of autophagy markers LC3,Beclin-1,and P62 protein and mRNA in the hippocampus tissue was measured using Western blot and Real-time PCR methods.Results Compared to the WD model group,there was no significant difference in body weight of the rats in the 3-MA intervention group(P>0.05).The rats exhibited significant improvement in autonomous behavior,exploratory ability,and learning and memory abilities.There was no significant difference in serum and hippocampal copper levels(P>0.05).Notably,the number of hippocampal neurons increased,while cell necrosis decreased,with a more orderly arrangement.Furthermore,the protein and mRNA expression levels of Beclin-1(F=64.212,F=32.225,P<0.05)and LC3(F=12.430,F=6.633,P<0.05)in the hippocampus tissue were significantly decreased,whereas the protein and mRNA expression levels of P62(F=9.856,F=8.481,P<0.05)were significantly increased.Conclusion 3-MA demonstrated the potential function to reduce autophagy in rats with excessive copper load in the hippocampus,thereby alleviating neurotoxicity and improving neurobehavioral abilities.

关 键 词：铜过量负荷 Wilson病(WD) 大鼠 细胞自噬 3-甲基腺嘌呤 

分 类 号：R99[医药卫生—毒理学]