OBJECTIVE Alzheimer disease(AD) is a progressive neurodegenerative disorder involving a gradual decline in many cognitive processes and in neurons.The endoplasmic reticulum(ER) is involved in several crucial cellular ...OBJECTIVE Alzheimer disease(AD) is a progressive neurodegenerative disorder involving a gradual decline in many cognitive processes and in neurons.The endoplasmic reticulum(ER) is involved in several crucial cellular functions,eg protein folding and quality control.Massive misfolded or unfolded proteins in ER can disturb the function of ER and induce ER stress,which results in neuronal death in AD.Icariin(ICA) has a wide range of neuro protection and has been researched in AD treatment.However,whether ICA has the effect on ER stress in AD condition,and how ICA affects ER stress remains stil unclear.Therefore,the current study aimed to investigate the mechanism of ICA against cognitive impairments in AD model through ER stress pathway and apoptosis.METHODS Twelve months male APP/PS1 or wild-type(WT)mice were randomly divided into four groups:APP/PS1,and APP/PS1+ICA,WT and WT+ICA groups.The treated mice were given ICA60 mg·kg-1 per day and control mice were received the same volume distilled water for consecutive 3 months.The Morris water maze and novel object recognition were used to detect animals′ behavior.Nissl staining was used to observe the neuronal morphology in hippocampus area.The protein and(or) phosphorylation level of GRP78,p-PERK,PERK,p-IRE1,IRE1,ATF6,p-e IF2α,eIF2α,ATF4,CHOP,the level of cleaved-casepase 3,Bax and Bcl-2 were examined by Western blotting.RESULTS The behavior performance testing by Morris water maze and novel object recognition deteriorated in APP/PS1 mice compared with WT mice,however,ICA significantly improved the behavior performance when compared with APP/PS1.The neuron impairments in APP/PS1 mice also were ameliorated after ICA treatment.The protein expression of GRP78,ATF4,CHOP,and the level of p-PERK and p-eI F2α were higher in APP/PS1 mice than that in WT mice.The Bax/Bcl-2 ratio elevation and caspase 3 activation have been found in APP/PS1 mice.After treated with ICA,those above-mentioned parameters were decreased compared with APP/PS1.However,the levels of IRE1,p-IRE1 and ATF6 were not change among other groups.CONCLUSION ICA may decrease the ER stress and apoptosis in AD model,and may be through inhibiting the PERK/eI F2α pathway,not IRE and ATF6 pathways.展开更多
基金National Natural Science Foundation of China(81560594).
文摘OBJECTIVE Alzheimer disease(AD) is a progressive neurodegenerative disorder involving a gradual decline in many cognitive processes and in neurons.The endoplasmic reticulum(ER) is involved in several crucial cellular functions,eg protein folding and quality control.Massive misfolded or unfolded proteins in ER can disturb the function of ER and induce ER stress,which results in neuronal death in AD.Icariin(ICA) has a wide range of neuro protection and has been researched in AD treatment.However,whether ICA has the effect on ER stress in AD condition,and how ICA affects ER stress remains stil unclear.Therefore,the current study aimed to investigate the mechanism of ICA against cognitive impairments in AD model through ER stress pathway and apoptosis.METHODS Twelve months male APP/PS1 or wild-type(WT)mice were randomly divided into four groups:APP/PS1,and APP/PS1+ICA,WT and WT+ICA groups.The treated mice were given ICA60 mg·kg-1 per day and control mice were received the same volume distilled water for consecutive 3 months.The Morris water maze and novel object recognition were used to detect animals′ behavior.Nissl staining was used to observe the neuronal morphology in hippocampus area.The protein and(or) phosphorylation level of GRP78,p-PERK,PERK,p-IRE1,IRE1,ATF6,p-e IF2α,eIF2α,ATF4,CHOP,the level of cleaved-casepase 3,Bax and Bcl-2 were examined by Western blotting.RESULTS The behavior performance testing by Morris water maze and novel object recognition deteriorated in APP/PS1 mice compared with WT mice,however,ICA significantly improved the behavior performance when compared with APP/PS1.The neuron impairments in APP/PS1 mice also were ameliorated after ICA treatment.The protein expression of GRP78,ATF4,CHOP,and the level of p-PERK and p-eI F2α were higher in APP/PS1 mice than that in WT mice.The Bax/Bcl-2 ratio elevation and caspase 3 activation have been found in APP/PS1 mice.After treated with ICA,those above-mentioned parameters were decreased compared with APP/PS1.However,the levels of IRE1,p-IRE1 and ATF6 were not change among other groups.CONCLUSION ICA may decrease the ER stress and apoptosis in AD model,and may be through inhibiting the PERK/eI F2α pathway,not IRE and ATF6 pathways.
文摘目的:观察化瘀祛痰方(Huayu-Qutan formula,HYQT)含药血清对油酸诱导的HepG2细胞脂质损伤及内质网应激(endoplasmic reticulum stress,ERS)的影响,探讨HYQT防治脂代谢紊乱的可能机制。方法:以不同终浓度的油酸诱导HepG2细胞,CCK8法、油红O染色和ELISA法筛选油酸诱导及HYQT含药血清干预的最佳浓度及时间。将HepG2细胞分为对照(control)组、模型(model)组、HYQT组、毒胡萝卜素(thapsigargin,Tg;ERS激动剂)组和Tg+HYQT组。油红O染色观察细胞脂质沉积情况;ELISA法检测细胞甘油三酯(triglyceride,TG)和游离脂肪酸(free fatty acid,FFA)含量变化;透射电镜观察细胞内质网结构变化;RT-qPCR和Wes全自动蛋白定量分析系统检测各组细胞葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)、固醇调节元件结合蛋白1c(sterol regula⁃tory element binding protein 1c,SREBP1c)、乙酰辅酶A羧化酶1(acetyl coenzyme A carboxylase 1,ACC1)、脂肪酸合成酶(fatty acid synthase,FAS)和硬脂酰辅酶A去饱和酶l(stearoyl coenzyme A desaturase 1,SCD1)的mRNA和蛋白表达情况。结果:1000μmol/L油酸诱导HepG2细胞脂质沉积效果明显,10%含药血清作用48 h作为后续实验的相对最佳浓度和时间。与control组比较,model组胞浆中橘红色脂滴形成明显增多,内质网扩张,发生ERS,细胞核固缩;TG和FFA含量明显增多(P<0.01);GRP78、SREBP1c、ACC1、FAS和SCD1 mRNA及蛋白表达量显著增高(P<0.01);与model组比较,HYQT含药血清干预后细胞内橘红色脂滴明显减少,内质网形态基本恢复正常,TG和FFA含量明显减少(P<0.01),ERS及脂肪酸从头合成相关因子的mRNA和蛋白表达量显著降低(P<0.01);加入Tg后细胞内橘红色脂滴形成更多,内质网扩张程度更明显,内质网形态迥异,大小不一,呈空泡状改变,部分可见融合成簇现象,TG和FFA含量增多(P<0.01),ERS及脂肪酸从头合成相关因子的mRNA和蛋白表达量增高(P<0.01);与Tg组比较,Tg+HYQT组细胞内橘红色脂滴减少,内质网扩张程度减轻,空泡体积减小和数量减少;TG和FFA含量明显减少(P<0.01),ERS及脂肪酸从头合成相关因子的mRNA和蛋白表达量显著降低(P<0.01)。结论:HYQT含药血清可以减轻油酸诱导的HepG2细胞脂质损伤,其机制可能与抑制ERS介导的脂肪酸从头合成有关。
