摘要
目的 研究大黄酚(chrysophanol,CHR)对Wilson病(WD)铜负荷大鼠模型的神经保护作用,并探讨其作用机制。方法 SD大鼠随机分为4组:对照组(Control组)、模型组(Model组)、CHR低剂量组(CHR-L组)、CHR高剂量组(CHR-H组),每组10只。通过含铜(1 g/kg)饲料、含铜(0.185%)水喂养12周构建WD铜负荷大鼠模型,铜离子(Cu)测定试剂盒检测肝铜、24 h尿铜水平;Y迷宫实验评估大鼠空间探索和记忆能力;HE染色法观察海马组织CA1区神经元形态学改变;透射电镜观察海马组织CA1区超微结构;免疫荧光法检测自噬标志蛋白LC3A/B的表达情况;Western Blot检测自噬相关蛋白LC3A/B、Beclin-1、p62以及p-AMPK、p-mTOR的表达量。结果 与Control组比较,Model组中肝铜及24 h尿铜水平增加(P<0.01);空间探索和记忆能力明显减弱(P<0.01);CA1区海马神经元出现损伤;自噬小体数量增多;LC3A/B荧光表达增高;LC3Ⅱ/Ⅰ、Beclin-1及p-AMPK表达量升高(P<0.01),p62、p-mTOR表达量降低(P<0.01)。经CHR不同剂量干预后,进一步降低肝铜水平、提高24 h尿铜水平(P<0.01);改善空间探索和记忆能力(P<0.01,P<0.05);缓解海马神经元损伤情况;减少自噬小体数量;降低LC3A/B荧光表达;下调LC3Ⅱ/Ⅰ、Beclin-1及p-AMPK蛋白表达量(P<0.01,P<0.05),上调p62、p-mTOR蛋白表达量(P<0.01,P<0.05)。结论 CHR对WD铜负荷大鼠模型具有神经保护作用,其机制可能与调控AMPK/mTOR信号通路抑制自噬的过度激活有关。
Objective To study the neuroprotective effect of chrysophanol(CHR)on copper-laden rats with Wilson disease(WD),and explore its possible mechanism.Methods SD rats were randomly divided into 4 groups:control group(Control group),model group(Model group),low dose CHR group(CHR-L group),high dose CHRl group(CHR-H group),with 10 rats in each group.The model of copper-laden rats with WD were established by feeding copper containing(1 g/kg)feed and copper containing(0.185%)water for 12 weeks.The copper level in liver and 24 h urine was detected by copper ion determination kit.The spatial exploration and memory ability of rats were evaluated by Y maze test.HE staining was used to observe the morphological changes of CA1 region neurons in hippocampus.The ultrastructure of CA1 region in hippocampus was observed by transmission electron microscope.The expression of LC3A/B was detected by immunofluorescence,the expression of autophagy related proteins LC3A/B,Beclin-1,p62,p-AMPK and p-mTOR were detected by Western Blot.Results Compared with the Control group,the liver copper level and 24 h urinary copper level in the Model group were significantly increased(P<0.01),the ability of space exploration and memory was obviously weakened(P<0.01),the hippocampal neurons in CA1 area were damaged,the number of autophagosomes increased,LC3A/Bfluorescence expression increased,the expression of LC3Ⅱ/Ⅰ,Beclin-1 and p-AMPK increased(P<0.01),while the expression of p62 and p-mTOR decreased(P<0.01).After the intervention of different doses of CHR,it could further reduce the copper level in liver and increase the copper level in 24 h urine(P<0.01),improve the ability of space exploration and memory(P<0.01,P<0.05),alleviate the damage of hippocampal neurons,reduce the number of autophagosomes,lower the expression of LC3A/B fluorescence,down regulate the expression of LC3Ⅱ/Ⅰ,Beclin-1 and p-AMPK(P<0.01,P<0.05),and up regulate the expression of p62 and p-mTOR(P<0.01,P<0.05).Conclusion CHR has neuroprotective effect on copper-laden rats with WD,and its mechanism may be related to the regulation of AMPK/mTOR signaling pathway to inhibit the over activation of autophagy.
作者
张笑颜
王谢
王杰
蔡标
谢道俊
ZHANG Xiao-yan;WANG Xie;WANG Jie;CAI Biao;XIE Dao-jun(School of Integrated Chinese and Western Medicine,Anhui University of Chinese Medicine,Hefei,230012;The First Clinical Medical College,Anhui University of Chinese Medicine,Hefei,230031;School of Nursing,Anhui University of Chinese Medicine,Hefei,230012;Encephalopathy Center,The First Affiliated Hospital of Anhui University of Chinese Medicine,Hefei,230031)
出处
《中国中西医结合杂志》
CAS
CSCD
北大核心
2023年第5期578-584,共7页
Chinese Journal of Integrated Traditional and Western Medicine
基金
国家自然科学基金资助项目(No.81874389)。
