APOE-TREM2介导的蛇床子素对阿尔兹海默病体外细胞模型的抗炎机制研究

Study on Anti-inflammatory Mechanism of Osthole on in vitro Cell Model of Alzheimer’s Disease through APOE-TREM2

目的探究载脂蛋白E(APOE)-髓细胞触发受体2(TREM2)信号通路介导的蛇床子素(Osthole)对阿尔兹海默病体外细胞模型的抗炎作用机制。方法将小鼠小胶质细胞(BV2细胞)分为:空白组、溶剂组(0.1%DMSO)、模型组(Aβ_(1-42),10μmol·L^(-1))、阳性药组(地塞米松,2.5μmol·L^(-1))及蛇床子素高、中、低剂量组(25、5、1μmol·L^(-1))。给药组分别给予药物预保护4 h后,再给予Aβ_(1-42)诱导损伤24 h,建立阿尔茨海默病BV2细胞模型。采用CCK-8法检测细胞存活率;ELISA法检测细胞炎性因子白细胞介素(IL)1β、肿瘤坏死因子(TNF)α的表达;免疫荧光法检测细胞促炎表型M1型标志物CD16/32的表达;倒置显微镜观察BV2细胞培养上清对SH-SY5Y细胞形态的影响;流式细胞术检测BV2细胞培养上清对SH-SY5Y细胞凋亡的影响;采用Western Blot、q PCR法检测细胞中APOE、TREM2蛋白及基因的表达情况。结果与空白组比较,模型组BV2细胞存活率明显降低(P<0.001),TNF-α、IL^(-1)β分泌量明显增加(P<0.001),CD16/32荧光明显增强,SH-SY5Y细胞损伤明显,SH-SY5Y细胞凋亡率明显升高(P<0.001),APOE、TREM2蛋白及mRNA表达显著上调(P<0.01,P<0.001)。与模型组相比,蛇床子素组的BV2细胞存活率明显升高(P<0.01,P<0.001),TNF-α、IL^(-1)β分泌量明显减少(P<0.01,P<0.001),CD16/32荧光明显减弱,SH-SY5Y细胞形态有所改善且凋亡率明显降低(P<0.05,P<0.01,P<0.001),APOE、TREM2蛋白及基因表达明显下调(P<0.05,P<0.01,P<0.001)。结论蛇床子素对Aβ_(1-42)诱导的BV2细胞的炎性反应有较好的治疗作用,其机制可能与下调APOE、TREM2蛋白及基因表达有关。

Objective To explore the anti-inflammatory mechanism of osthole(OST) on in vitro cell model of Alzheimer’s disease through APOE(apolipo protein E,APOE)-TREM2(recombinant triggering receptor expressed on myeloid cells 2,TREM2).Methods Mouse microglia(BV2 cells)were divided into blank group,solvent group(0.1% DMSO),model group(Aβ_(1-42),10 μmol·L^(-1)),positive drug group(dexamethasone group,2.5 μmol·L^(-1)),OST high,medium,and low dose groups(25,5,1 μmol·L^(-1)).OST high,medium and low dose groups and positive drug groups were given pre-protection for 4 hours and then given β-amyloid protein 1-42(Aβ_(1-42))24 hours to damage mouse microglia for establishing Alzheimer’s disease(AD)cell model in BV2 cells.CCK-8 method was used to detect the cell survival rate of each group;the ELISA kit was used to detect the expression of inflammatory factors interleukin(IL)-1β and tumor necrosis factor(TNF)-α in the supernatant of cell culture medium of each group;immunofluorescence method was applied to detect the expression of CD16/32,marker of the M1 type proinflammatory phenotype in cells.Inverted microscope was used to observe the influence of BV2 cell culture supernatant on the morphology of SH-SY5 Y cells,and the influence of BV2 cell culture supernatant on apoptosis of SH-SY5 Y was detected by flow cytometry.Western Blot and qPCR were used to detect the protein and gene expression of APOE and TREM2 in cells.Results Compared with the blank group and the solvent group,the survival rate of BV2 cells in the model group was significantly reduced(P<0.001),the secretion of TNF-α and IL^(-1)βwas significantly increased(P<0.001),the fluorescence of CD16/32 was significantly enhanced,and the damage and apoptosis of SH-SY5 Y cells was obvious(P<0.001).The protein expression and gene mRNA levels of APOE and TREM2 were significantly increased(P<0.01,P<0.001).Compared with the model group,the cell survival rates of the OST groups were significantly increased(P<0.01,P<0.001),TNF-α,IL^(-1)β secretion was significantly reduced(P<0.01,P<0.001),CD16/32 fluorescence was significantly weakened,SH-SY5 Y cell status improved,and total apoptotic rates of cells decreased(P<0.05,P<0.01,P<0.001),the expression of APOE,TREM2 protein and gene levels were significantly reduced(P<0.05,P<0.01,P<0.001).Conclusion Osthole has a good therapeutic effect on the inflammatory response of BV2 cells induced by Aβ_(1-42).The mechanism may be related to the reduction of APOE and TREM2 protein and gene levels.