小檗碱对LPS诱导神经元损伤的作用机制研究

Mechanistic study of berberine on LPS-induced neuronal injury

为探究小檗碱(berberine,Ber)对LPS诱导的神经元损伤及腺苷一磷酸活化蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)-mTOR-p70核糖体蛋白S6激酶(ribosomaiprotein S6 kinase,S6K)信号通路的影响,MTT法筛选最佳的Ber处理HT22神经元浓度。将HT22细胞分为NC组、LPS组、Ber+LPS组和AMPK抑制剂Compound C(Com.C)+Ber+LPS组。FACS检测HT22细胞凋亡率;qRT-PCR法检测HT22细胞IL-1β、IL-6和TNF-α的mRNA相对表达量;DCFH-DA荧光探针检测HT22细胞中活性氧类(reactive oxygen species,ROS)含量;TBA法、比色法和WST-1法分别检测HT22细胞中丙二醛(malondialdehyde,MDA)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)和总超氧化物歧化酶(superoxide dismutase,SOD)的含量;Western blotting检测HT22细胞中B淋巴细胞瘤2基因(B-cell lymphoma 2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein,BAX)、脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)、原肌球蛋白受体激酶B(tropomyosin receptor kinase B,TrkB)、神经生长因子(nerve growth factor,NGF)、磷酸化AMPK(p-AMPK)、磷酸化mTOR(p-mTOR)和磷酸化p70S6K(p-p70S6K)蛋白的表达水平。结果显示,与0μmol/L Ber比较,5μmol/L和10μmol/L Ber处理对HT22细胞增殖活力的影响差异无统计学意义(均P>0.05),15μmol/L和20μmol/L Ber处理可显著降低HT22细胞的增殖活力(均P<0.05),因此选择10μmol/L Ber用于后续研究。与NC组相比,LPS处理可增加HT22细胞的凋亡率(P<0.05),上调细胞中IL-1β、IL-6和TNF-α的mRNA相对表达水平(均P<0.05),上调ROS和MDA的含量以及BAX、p-mTOR和p-p70S6K蛋白的表达水平(均P<0.05),下调细胞中GSH-Px和SOD含量以及Bcl-2、BDNF、TrkB、NGF和p-AMPK蛋白表达水平(均P<0.05)。Ber干预显著减轻了LPS诱导的HT22细胞损伤(均P<0.05),Com.C预处理部分逆转了Ber处理对LPS诱导的HT22细胞的保护作用(均P<0.05)。由此,Ber可能通过调控HT22细胞中AMPK-mTOR-p70S6K信号通路缓解LPS诱导的神经元损伤。

The goal of this study was to investigate the effects of berberine(Ber)on LPS-induced neuronal injury and on adenosine monophosphate-activated protein kinase(AMPK)-mTOR-p70 ribosomaiprotein S6 kinase(S6K)signaling pathway.For this purpose,the optimal concentration of Ber to treat HT22 neuron was selected by MTT assay.10μmol/L Ber was determined as the optimal concentration and was used in subsequent experiments.HT22 cells were divided into the NC group,LPS group,Ber+LPS group,and AMPK inhibitor Compound C(Com.C)+Ber+LPS group.Cell apoptotic rate,the mRNA expression levels of inflammatory cytokines,the quantification of reactive oxygen species(ROS),malondialdehyde(MDA),glutathione peroxidase(GSH-Px),superoxide dismutase(SOD),B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(BAX),brain-derived neurotrophic factor(BDNF),tropomyosin receptor kinase B(TrkB),nerve growth factor(NGF),p-AMPK,p-mTOR and p-p70S6K were manipulated.The results showed that compared to cells in the NC group,LPS treatment increased the apoptotic rate of HT22 cells(P<0.05),up-regulated the mRNA levels of IL-1β,IL-6 and TNF-α,the levels of ROS and MDA,the protein levels of BAX,p-mTOR and p-p70S6K(all P<0.05),down-regulated the levels of GSH-Px and SOD and the protein levels of Bcl-2,BDNF,TrkB,NGF and p-AMPK(all P<0.05).Ber treatment significantly reduced the damages induced by LPS on HT22 cells(all P<0.05).Com.C pretreatment partially reversed the protective effect of Ber on LPS-induced HT22 cells injury(all P<0.05).In conclusion,Ber may play a protective role in LPS-induced HT22 neuronal injury by regulating the AMPK-mTOR-p70S6K signaling pathway.