加减薯蓣丸通过Akt/GSK3β/Nrf2通路改善APP/PS1小鼠神经元凋亡

Modified Shuyuwan Ameliorate Neuronal Apoptosis of APP/PS1 Mice Via Akt/GSK3β/Nrf2 Pathway

目的:探讨加减薯蓣丸对APP/PS1模型小鼠神经保护的效应和作用机制。方法:5月龄雄性APP/PS1小鼠20只和野生型小鼠10只,分为空白组、模型组、加减薯蓣丸组(14 g·kg-1·d-1),灌胃28 d,空白组、模型组给予等体积生理盐水;APP/PS1背景和野生型背景原代神经元,分为空白组、模型组、加减薯蓣丸组、衣霉素组和磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(Akt)抑制剂组,空白组、模型组给予10%空白血清,加减薯蓣丸组给予5%加减薯蓣丸含药血清干预,衣霉素组和PI3K/Akt抑制剂组分别在加减薯蓣丸基础上加用2 mg·L-1的衣霉素和10μmol·L-1的LY294002。采用Morris水迷宫检测小鼠学习记忆能力;采用蛋白免疫印迹法(Western blot)检测海马核因子E2相关因子2(Nrf2)蛋白表达;Western blot检测内质网应激相关蛋白糖调节蛋白78(GRP78),蛋白激酶样内质网激酶(PERK),磷酸化(p-) PERK,凋亡通路蛋白真核生物的翻译起始因子2的α亚基(e IF2α),p-eIF2α,增强子结合蛋白同源蛋白(CHOP),半胱氨酸蛋白酶-3(Caspase-3)和p-Akt,Akt,糖原激酶3β(GSK3β),Nrf2蛋白表达。结果:体内实验中,与空白组比较,模型组APP/PS1小鼠学习记忆能力显著下降(P <0. 01),海马Nrf2表达显著下调(P <0. 01);与模型组比较,加减薯蓣丸干预4周后,加减薯蓣丸组APP/PS1小鼠学习记忆能力明显提升,海马Nrf2表达水平显著增加(P <0. 01)。体外实验中,与空白组比较,模型组GRP78,p-PERK/PERK,p-eIF2α/e IF2α,CHOP,cleaved Caspase-3蛋白表达显著增加(P <0. 01);与模型组比较,加减薯蓣丸含药血清明显降低GRP78,p-PERK/PERK,p-eIF2α/e IF2α,CHOP,cleaved Caspase-3蛋白表达(P <0. 05,P <0. 01);而衣霉素抑制加减薯蓣丸对内质网应激诱导凋亡的保护效应(P <0. 05);与空白组比较,模型组p-Akt/Akt,Nrf2蛋白表达显著下降,GSK-3β表达显著增加(P <0. 01);与模型组比较,加减薯蓣丸含药血清干预后p-Akt/Akt,Nrf2蛋白表达显著增加,GSK-3β表达明显降低(P <0. 05,P <0. 01);LY294002抑制加减薯蓣丸对p-Akt/Akt,Nrf2和GSK3β蛋白表达的影响(P <0. 05,P <0. 01)。结论:加减薯蓣丸通过PI3K/Akt/GSK3β信号通路上调Nrf2蛋白表达,减轻内质网应激诱导的神经元凋亡,改善APP/PS1模型小鼠学习记忆能力。

Objective:To explore the effect and mechanism of modified Shuyuwan neuroprotection in APP/PS1 model mice.Method:Selecting 20 male APP/PS1 mice of 5 months old and 10 wild type mice.The mice were divided into blank group,model group and modified Shuyuwan group(14 g·kg-1·d-1),drug delivery for 28 days,and blank group and model group were given the same amount of normal saline,APP/PS1 background primary neuron model and wild type primary neurons were divided into blank group,model group and modified Shuyuwan group,tunicamycin group,phosphatidylinositol 3-kinase(PI3 K)/protein kinase B(Akt)inhibitor group.The blank group and model group were given 10%blank serum,the modified Shuyuwan group was given5%modified Shuyuwan-containing serum,the tunicamycin group and the PI3 K/Akt inhibitor group were respectively added with 2 mg·L-1 tunicamycin and 10μmol·L-1 LY294002 on the basis of 5%modified Shuyuwancontaining serum.The spatial learning and memory ability of mice was measured by Morris water maze,and Western blot was used to detect nuclear factor erythroid-2 related factor 2(Nrf2)protein expression in hippocampus.Western blot was used to detect the protein expression of endoplasmic reticulum stress related proteins glucose regulatory protein 78(GRP78),protein kinase-like endoplasmic reticulum kinase(PERK),phosphorylation(p-)PERK and apoptosis expression of the pathway proteins eukaryotic translation initiation factor2α(eIF2α),p-eIF2α,enhancer binding protein homologous protein(CHOP),and cysteinyl aspartate apecific proteinase 3(Caspase-3),p-Akt,Akt,Glycogen Synthase kinase-3β(GSK3β),Nrf2.Result:In vivo experiment,compared with blank group,the learning and memory ability of APP/PS1 mice in the model group was impaired(P<0.01),and the expression level of Nrf2 in the hippocampus was decreased(P<0.01).Compared with model group,after 4 weeks of modified Shuyuwan intervention,the learning and memory ability of APP/PS1 mice in the modified Shuyuwan group was improved,and the expression level of Nrf2 in the hippocampus was significantly increased(P<0.01).In vitro experiment,Western blot analysis showed that compared with the blank group,the expression of GRP78,p-PERK/PERK,p-eIF2α/eIF2α,CHOP,and cleaved Caspase-3 proteins was increased in the model group(P<0.01).Compared with model group,modified Shuyuwancontaining serum intervention significantly reduced the expression of GRP78,p-PERK/PERK,p-eIF2α/e IF2α,CHOP,and cleaved Caspase-3 proteins(P<0.05,P<0.01).Whereas tunicamycin inhibited the protection effect of SHhuyuwan on the endoplasmic reticulum and ERS-induced apoptosis(P<0.05).Western blot results showed that compared with blank group,the expression of p-Akt/Akt and Nrf2 protein was significantly decreased and the expression of GSK-3βwas increased in the model group(P<0.01).Compared with model group,the expression of p-Akt/Akt and Nrf2 protein was significantly increased and the expression of GSK-3βwas decreased after modified Shuyuwan-containing serum intervention(P<0.05,P<0.01).The effect of modified Shuyuwan on the expression of p-Akt,Nrf2 and GSK3βprotein was inhibited by LY294002.(P<0.05,P<0.01).Conclusion:Modified Shuyuwan can increase Nrf2 protein expression through PI3 K/Akt/GSK3βsignaling pathway,reduce neuronal apoptosis induced by endoplasmic reticulum stress,improve the learning and memory ability of APP/PS1 model mice.