肝豆汤改良方调控PKR/eIF2α通路改善Wilson病模型TX小鼠突触功能障碍的机制研究  

Mechanism of Modified Gandou Decoction in Improving Synaptic Dysfunction in TX Mice with Wilson's Disease by Regulating the PKR/eIF2αPathway

作　　者：刘松杨 程楠 徐陈陈 董健健 高曼莉 LIU Song-yang;CHENG Nan;XU Chen-chen;DONG Jian-jian;GAO Man-li(Graduate School of Anhui University of Chinese Medicine, Anhui Hefei 230012, China;Affiliated Hospital of Neurology Institute, Anhui University of Chinese Medicine, Anhui Hefei 230061, China)

机构地区：[1]安徽中医药大学研究生院,安徽合肥230012 [2]安徽中医药大学神经病学研究所附属医院,安徽合肥230061

出　　处：《安徽中医药大学学报》2021年第6期75-81,共7页Journal of Anhui University of Chinese Medicine

基　　金：国家自然科学基金项目(81904086)。

摘　　要：目的探究肝豆汤改良方调控双链RNA依赖蛋白激酶(double-stranded RNA-dependent protein kinase,PKR)/真核细胞起始因子2α(eukaryotic initiation factor 2α,eIF2α)通路改善Wilson病模型毒牛奶(toxic milk,TX)小鼠突触功能障碍的机制。方法实验分为正常组、正常加肝豆汤改良方组、模型组、模型加肝豆汤改良方组,每组20只小鼠;正常组和模型组每日分2次予生理盐水灌胃,每次20 mL/kg,正常加肝豆汤改良方组与模型加肝豆汤改良方组每日分2次予中药肝豆汤改良方各20 mL/kg灌胃给药,连续4周,末次给药结束后取各组小鼠海马组织进行检测;采用旷场实验、Barnes迷宫实验检测Wilson病模型小鼠认知功能;采用TUNEL染色法检测小鼠海马组织中神经细胞凋亡情况;采用免疫荧光染色检测各组小鼠海马8-羟化脱氧鸟苷(8-hydroxylated deoxyguanosine,8-OHdG)的表达水平;采用Western blot法检测海马组织PKR/eIF2α通路以及突触相关蛋白表达水平。结果模型组小鼠的认知功能显著低于正常组(P<0.05),模型加肝豆汤改良方组小鼠的认知功能显著高于模型组(P<0.05);与正常组比较,模型组小鼠海马神经细胞凋亡数目显著增加(P<0.05);与模型组比较,模型加肝豆汤改良方组小鼠海马神经细胞凋亡数量显著降低(P<0.05),8-OhdG表达水平显著降低(P<0.05);小鼠海马内突触相关蛋白突触蛋白1、突触素、突触后致密物-93、突触后致密物-95表达水平显著增加(P<0.05),小鼠海马PKR/eIF2α通路相关蛋白PKR、磷酸化eIF2α、转录激活因子4、促凋亡分子C/EBP同源蛋白表达水平显著降低(P<0.05)。结论肝豆汤改良方可通过调控PKR/eIF2α通路,促进突触相关蛋白表达,减轻高铜诱导的突触功能障碍,改善TX小鼠认知功能损伤症状。Objective To investigate the mechanism of Modified Gandou Decoction(MGDD)in improving synaptic dysfunction in TX mice with Wilson's disease by regulating the double-stranded RNA-dependent protein kinase(PKR)/eukaryotic initiation factor 2α(eIF2α)pathway.Methods The mice were divided into normal group,normal+MGDD group,model group,and model+MGDD group,with 20 mice in each group.The mice in the normal group and the model group were given 20 mL/kg normal saline by gavage twice a day,and those in the normal+MGDD group and the model+MGDD group were given 20 mL/kg MGDD by gavage twice a day,for 4 consecutive weeks.Hippocampal tissue was collected for analysis after last administration;the open field test and the Barnes maze test were used to evaluate the cognitive function of the mice with Wilson's disease;TUNEL staining was used to measure neural cell apoptosis in the hippocampus of mice;immunofluorescent staining was used to measure the expression of 8-hydroxydeoxyguanosine(8-OHDG)in the hippocampus of mice,and Western blot was used to measure the expression levels of the PKR/eIF2αpathway and synapse-associated proteins in the hippocampus.Results The model group had a significantly poorer cognitive function than the control group(P<0.05),and the model+MGDD group had a significantly better cognitive function than the model group(P<0.05).Compared with the normal group,the model group had a significant increase in the number of apoptotic neural cells in the hippocampus(P<0.05),and compared with the model group,there was a significant reduction in the number of apoptotic neural cells in the hippocampus after MGDD treatment(P<0.05).MGDD significantly reduced the expression level of 8-OHDG in the model group(P<0.05).In the TX mice with Wilson's disease,MGDD significantly increased the expression levels of synapse-associated proteins,synapsin1,synaptophysin,PSD-93,and PSD-95,in the hippocampus(P<0.05)and significantly reduced the expression levels of PKR,phosphorylated eIF2α,activating transcription factor 4,and C/EBP h

关 键 词：突触功能障碍 PKR/eIF2α通路 肝豆汤改良方 WILSON病 认知功能障碍 

分 类 号：R742.4[医药卫生—神经病学与精神病学]