中国中药杂志

2020, v.45(12) 2924-2931

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四君子汤调控Aβ跨血脑屏障转运相关蛋白的作用研究
Effect of Sijunzi Decoction on regulation of Aβ-related proteins across blood-brain barrier

郭园园;郑琴;吴玲;张明霞;张科楠;肖帅;李文静;杨明;
GUO Yuan-yuan;ZHENG Qin;WU Ling;ZHANG Ming-xia;ZHANG Ke-nan;XIAO Shuai;LI Wen-jing;YANG Ming;Key Laboratory of Modern Preparation of Traditional Chinese Medicine, Ministry of Education, Jiangxi University of Traditional Chinese Medicine;

摘要(Abstract):

中医认为"脾主运化",与机体物质代谢、能量代谢密切相关。研究表明,阿尔茨海默病(Alzheimer′s disease,AD)是一种代谢性疾病,与糖脂代谢和能量代谢有关,中药健脾方剂能够改善AD动物模型的学习记忆。四君子汤来源于《太平惠民和剂局方》,具有补气、益气健脾之功效,是健脾补脾基础方。该实验采用人脑微血管内皮细胞(HBMEC)与四君子汤水提液(0.25,0.5,1 mg·L~(-1))预孵育2 h,而后加入Aβ_(25-35)寡聚体(终浓度40μmol·L~(-1))共培养22 h,考察四君子汤水提液对Aβ_(25-35)寡聚体损伤细胞活性及相关蛋白表达的影响。首先运用Q-TOF-LC-MS对四君子汤水提液进行主要成分分析;然后采用MTT法研究四君子汤水提液对HBMEC细胞增殖情况的影响;采用实时荧光定量PCR技术(RT-qPCR)检测Aβ跨血脑屏障转运相关蛋白基因葡萄糖转运体1(GLUT1)、晚期糖基化终产物受体(RAGE)、低密度脂蛋白受体相关蛋白1(LRP1)mRNA的表达,采用免疫印迹(Western blot)法测定Aβ跨血脑屏障转运相关蛋白RAGE,LRP1的表达。研究发现40μmol·L~(-1) Aβ_(25-35)寡聚体能够诱导内皮细胞损伤,使细胞存活率降低,RAGE mRNA及蛋白表达增加,GLUT1 mRNA表达减少,LRP1 mRNA及蛋白表达减少;四君子汤水提液能够减弱Aβ_(25-35)寡聚体诱导的HBMEC细胞毒性,能降低RAGE mRNA及蛋白表达,升高GLUT1 mRNA表达,升高LRP1 mRNA及蛋白表达。结果表明,四君子汤水提液对Aβ_(25-35)寡聚体诱导的HBMEC细胞损伤有改善作用,并且能够调控Aβ跨血脑屏障转运相关蛋白GLUT1,RAGE,LRP1,达到调控Aβ在血脑屏障上转运的作用。
According to traditional Chinese medicine, "spleen transport" is closely related to the metabolism of substance and energy. Studies have shown that Alzheimer′s disease(AD) is a disease related to glucose and lipid metabolism and energy metabolism. The traditional Chinese medicine Jiangpi Recipe can improve the learning ability and memory of AD animal model. Sijunzi Decoction originated from Taiping Huimin Hefang Prescription is the basic prescription for strengthening and nourishing the spleen, with the effects of nourishing Qi and strengthening the spleen. In this experiment, human brain microvascular endothelial cells(HBMEC) and Sijunzi Decoction water extract(0.25, 0.5, 1 mg·L~(-1)) were pre-incubated for 2 h, and then Aβ_(25-35) oligomers(final concentration 40 μmol·L~(-1)) was added for co-culture for 22 hours. The effect of Sijunzi Decoction on the activity of Aβ_(25-35) oligomer injured cells and the expression of related proteins were investigated. Q-TOF-LC-MS was used first for principal component analysis of Sijunzi Decoction water extract. Then MTT assay was used to investigate the effect of Sijunzi Decoction water extract on the proliferation of HBMEC cells. Real-time fluorescence quantitative PCR(RT-qPCR) was employed to detect the mRNA expression of GLUT1, RAGE, and LRP1. The expression of Aβ-related proteins across blood-brain barrier(RAGE, LRP1) was detected by Western blot. The results showed that 40 μmol·L~(-1) Aβ_(25-35) oligomers could induce endothelial cell damage, reduce cell survival, increase expression of RAGE mRNA and RAGE protein, and reduce expression of GLUT1 mRNA, LRP1 mRNA, and LRP1 protein. Sijunzi Decoction water extract could reduce the Aβ_(25-35) oligomer-induced cytotoxicity of HBMEC, decrease the expression of RAGE mRNA and RAGE protein, and increase the expression of GLUT1 mRNA, LRP1 mRNA and LRP1 protein. The results indicated that Sijunzi Decoction could reduce the injury of HBMEC cells induced by Aβ_(25-35) oligomer, and regulate the transport-related proteins GLUT1, RAGE and LRP1, which might be the mechanism of regulating Aβ_(25-35) transport across the blood-brain barrier.

关键词(KeyWords): 四君子汤;阿尔茨海默病;人脑微血管内皮细胞(HBMEC);晚期糖基化终产物受体;低密度脂蛋白受体相关蛋白1;葡萄糖转运蛋白1
Sijunzi Decoction;Alzheimer′s disease;human brain microvascular endothelial cells(HBMEC);advanced glycation end products;low density lipoprotein receptor-related protein 1;glucose transporter 1

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81860874);; 江西省教育厅科学技术研究重点项目(GJJ170699)

作者(Author): 郭园园;郑琴;吴玲;张明霞;张科楠;肖帅;李文静;杨明;
GUO Yuan-yuan;ZHENG Qin;WU Ling;ZHANG Ming-xia;ZHANG Ke-nan;XIAO Shuai;LI Wen-jing;YANG Ming;Key Laboratory of Modern Preparation of Traditional Chinese Medicine, Ministry of Education, Jiangxi University of Traditional Chinese Medicine;

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