白果内酯促进巨噬细胞神经保护的机制研究  

Mechanism of bilobalide promoting neuroprotection of macrophages

作　　者：陈阳阳 鞠文媛 褚果果 李晓慧 魏汝恒 王青 肖保国 马存根 CHEN Yang-yang;JU Wen-yuan;CHU Guo-guo;LI Xiao-hui;WEI Ru-heng;WANG Qing;XIAO Bao-guo;MA Cun-gen(Research Center of Neurobiology/the Key Research Laboratory of Benefiting Qi for Acting Blood Circulation Method to Treat Multiple Sclerosis of State Administration of Traditional Chinese Medicine,Shanxi University of Chinese Medicine,Jinzhong 030619,China;Institute of Neurology/Institutes of Brain Science and State Key Laboratory of Medical Neurobiology,Huashan Hospital,Fudan University,Shanghai 200025,China;Institute of Brain Science,Shanxi Datong University,Datong 037009,China)

机构地区：[1]山西中医药大学神经生物学研究中心/国家中医药管理局多发性硬化益气活血重点研究室,山西晋中030619 [2]复旦大学附属华山医院神经内科/医学神经生物学国家重点实验室,上海200025 [3]山西大同大学脑科学研究所,山西大同037009

出　　处：《中国中药杂志》2023年第15期4201-4207,共7页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81473577,81903596);中国博士后科学基金面上项目(2020M680912);山西省研究生创新项目(2020SY450);国家中医药管理局多发性硬化益气活血重点研究室2021年度研究生培育项目(2021-KF-02S)。

摘　　要：探讨白果内酯(BB)通过抑制巨噬细胞/小胶质细胞炎症反应、促进神经营养因子分泌、干预外周免疫中CD4^(+)T细胞活化分化,从而发挥神经保护作用及其机制。以质量浓度梯度为12.5、25、50、100μg·mL-1的BB干预RAW264.7与BV2细胞,采用MTT、CCK-8法检测BB的细胞毒性并选取合适的药物浓度进行后续实验。分别以脂多糖(LPS)诱导RAW264.7、BV2细胞、小鼠骨髓巨噬细胞、原代小胶质细胞建立炎性模型,并使用ELISA法检测BB对细胞增殖、炎性因子和神经营养因子分泌的影响。选取7~8周龄C57BL/6雌性小鼠分离脾脏单核细胞,无菌条件下磁珠分选CD4^(+)T细胞,用骨髓巨噬细胞模型中的条件培养基与CD3/CD28诱导分化Th17细胞,ELISA法检测上清中IL-17细胞因子含量,以测定对CD4^(+)T细胞活化分化程度的影响。另将骨髓巨噬细胞与原代小胶质细胞模型中的条件培养基,孵育PC12细胞,观察细胞数量和形态,并用LDH法测定细胞毒性,结果显示BB在12.5~100μg·mL-1对RAW264.7与BV2细胞无细胞毒性,对低炎症模型细胞的活性亦无明显影响。选取BB 50μg·mL-1进行实验,结果显示BB可抑制LPS诱导的炎症细胞模型中炎性因子的分泌。使用条件培养基进行干预的CD4^(+)T细胞模型实验,结果显示LPS诱导的骨髓巨噬细胞炎症模型条件培养基能够促进CD4^(+)T细胞的活化分化,而进行BB药物干预的实验组条件培养基降低了CD4^(+)T细胞的活化分化程度。另外,BB也可以促进骨髓巨噬细胞与原代小胶质细胞神经营养因子的释放,且BB干预后的条件培养基可显著减少PC12神经元的死亡,抑制神经元损伤,保护神经元。综上表明,BB通过抑制骨髓巨噬细胞与小胶质细胞介导的炎症反应,促进神经营养因子分泌,发挥神经保护作用。This study aims to explore the neuroprotective effect of bilobalide(BB)and the mechanisms such as inhibiting inflammatory response in macrophage/microglia,promoting neurotrophic factor secretion,and interfering with the activation and differentiation of peripheral CD4^(+)T cells.BB of different concentration(12.5,25,50,100μg·mL^(-1))was used to treat the RAW264.7 and BV2 cells for 24 h.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay and cell counting kit-8(CCK-8)were employed to detect the cytotoxicity of BB and appropriate concentration was selected for further experiment.Lipopolysaccharide(LPS)was applied to elicit inflammation in RAW264.7 and BV2 cells,mouse bone marrow-derived macrophages(BMDMs),and primary microglia,respectively.The effect of BB on cell proliferation and secretion of inflammatory cytokines and neurotrophic factors was detected by enzyme-linked immunosorbent assay(ELISA).Spleen monocytes of C57BL/6 female mice(7-8 weeks old)were isolated,and CD4^(+)T cells were separated by magnetic beads under sterile conditions.Th17 cells were induced by CD3/CD28 and the conditioned medium for eliciting the inflammation in BMDMs.The content of IL-17 cytokines in the supernatant was detected by ELISA to determine the effect on the activation and differentiation of CD4^(+)T cells.In addition,PC12 cells were incubated with the conditioned medium for eliciting inflammation in BMDMs and primary microglia and the count and morphology of cells were observed.The cytoto-xicity was determined by lactate dehydrogenase(LDH)assay.The result showed that BB with the concentration of 12.5-100μg·mL^(-1)had no toxicity to RAW264.7 and BV2 cells,and had no significant effect on the activity of cell model with low inflammation.The 50μg·mL^(-1)BB was selected for further experiment,and the results indicated that BB inhibited LPS-induced secretion of inflammatory cytokines.The experiment on CD4^(+)T cells showed that the conditioned medium for LPS-induced inflammation in BMDMs promoted the activa

关 键 词：白果内酯 巨噬细胞 小胶质细胞 炎症 神经营养因子 神经保护 

分 类 号：R285[医药卫生—中药学]