远志皂苷元缓解脂多糖诱导的小鼠小胶质细胞炎症反应的机制  

Mechanism of senegenin in improving lipopolysacchride-induced inflammatory response of BV2 microglial cell

作　　者：穆秉桃 郭敏芳[1] 于婧文[1] 曹佳蕾 杨凤君 贾思玮 苏琴 孟涛 马存根 尉杰忠[1,4] 宋丽娟 Mu Bing-Tao;Guo Min-Fang;Yu Jing-Wen;Cao Jia-Lei;Yang Feng-Jun;Jia Si-Wei;Su Qing;Meng Tao;Ma Cun-Gen;Yu Jie-Zhong;Song Li-Juan(Institute of Brain Science,Shanxi Datong University/the Key Laboratory of Molecular Cellular Immunology in Datong,Datong,Shanxi 037009,China;School of Medicine,Shanxi Datong University,Datong,Shanxi 037009,China;The Key Research Laboratory of Benefiting Qi for Acting Blood Circulation Method to Treat Multiple Sclerosis of State Administration of Traditional Chinese Medicine/Research Center of Neurobiology,Shanxi University of Chinese Medicine,Jinzhong,Shanxi 030619,China;Department of Neurology,Datong Fifth People's Hospital,Datong,Shanxi 037009,China)

机构地区：[1]山西大同大学脑科学研究所/分子细胞免疫学大同市重点实验室,山西大同037009 [2]山西大同大学医学院,山西大同037009 [3]山西中医药大学国家中医药管理局多发性硬化益气活血重点研究室/神经生物学研究中心,山西晋中030619 [4]大同市第五人民医院神经内科,山西大同037009

出　　处：《解放军医学杂志》2025年第2期188-196,共9页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(82004028);山西省中医重点实验室建设项目(zyyyjs2024027);山西省“四个一批”科技兴医创新计划项目(2022XM33);山西省卫健委中医药科研课题立项计划(2022ZYYC090);山西省中医药管理局科研课题(2023ZYYB2042)。

摘　　要：目的探究远志皂苷元(SEN)通过核转录因子E2相关因子2(Nrf2)/NOD样受体蛋白3(NLRP3)通路缓解小鼠小胶质细胞炎症反应的机制。方法将BV2小鼠小胶质细胞随机分为对照组、模型组、SEN组与MCC950组。对照组细胞不做处理,模型组细胞加入1μg/ml脂多糖(LPS),SEN组细胞加入1μg/ml LPS+4μmol/L SEN,MCC950组加入1μg/ml LPS+10μmol/L MCC950,作用24 h。采用CCK-8法检测不同浓度SEN对BV2细胞活力的影响。采用Griess法测定上清液一氧化氮(NO)释放量;实时荧光定量PCR测定NLRP3、淋巴细胞凋亡相关斑点样蛋白(ASC)、胱天蛋白酶-1(caspase-1)、白细胞介素(IL)-1β和IL-18 mRNA的表达水平;免疫荧光染色检测ASC、IL-1β、Nrf2和血红素加氧酶-1(HO-1)的表达水平;Western blotting检测NLRP3、caspase-1、ASC、IL-1β、IL-18、Nrf2、HO-1及核因子κB(NF-κB)、诱导型一氧化氮合酶(iNOS)的表达水平。结果CCK-8法检测结果显示,2~20μmol/L SEN处理的BV2细胞活力与对照组差异无统计学意义(P>0.05)。与对照组比较,模型组BV2细胞活力明显下降(P<0.05);与模型组比较,4μmol/L SEN组BV2细胞活力明显恢复(P<0.05)。Griess法检测结果显示,与对照组比较,模型组细胞NO释放量明显增加(P<0.01);实时荧光定量PCR和Western blotting检测结果显示,与对照组比较,模型组细胞NLRP3、ASC、caspase-1、IL-1β和IL-18 mRNA和蛋白表达水平均明显增高(P<0.05);免疫荧光染色结果显示,与对照组比较,模型组细胞iNOS、NF-κB蛋白表达水平增高,Nrf2和HO-1表达水平下降,差异均有统计学意义(P<0.05)。与模型组比较,SEN组和MCC950组细胞NO释放量减少,NLRP3、ASC、caspase-1、IL-1β、IL-18 mRNA和蛋白表达水平降低,差异均有统计学意义(P<0.05);SEN组iNOS和NF-κB表达水平降低,免疫荧光染色显示Nrf2易位到核内,Nrf2和HO-1蛋白表达明显增多,差异均有统计学意义(P<0.05)。结论SEN可缓解LPS诱导的小鼠小胶质细胞炎症反应,抑制NLRObjective To investigate the mechanism by which Senegenin(SEN)alleviates microglial inflammatory response through the nuclear factor erythroid 2-related factor 2(Nrf2)/NOD-like receptor protein 3(NLRP3)pathway.Methods BV2 mouse microglia cells were randomly divided into control group,model group,SEN group and MCC950 group.Cells in control group were not treated,and cells in model group were added with 1μg/ml lipopolysaccharide(LPS);Cells in SEN group were added with 1μg/ml LPS+4μmol/L SEN,and cells in MCC950 group were added with 1μg/ml LPS+10μmol/L MCC950 for 24 hours.CCK-8 method was used to detect the effect of different concentrations of SEN on the viability of BV2 cells.Griess method was used to determine the release amount of nitric oxide(NO)in the supernatant.Real-time fluorescent quantitative PCR was used to determine the mRNA expression levels of NLRP3,lymphocyte apoptosis-associated spect-like protein containing a CARD(ASC),caspase-1,interleukin(IL)-1βand IL-18 mRNA.Immunofluorescence staining was used to detect the expression levels of ASC,IL-1β,Nrf2 and heme oxygenase-1(HO-1).Western blotting was used to detect the expression levels of NLRP3,caspase-1,ASC,IL-1β,IL-18,Nrf2,HO-1,nuclear factor kappa B(NF-κB)and inducible nitric oxide synthase(iNOS).Results The results of CCK-8 method showed that there was no significant difference in the viability of BV2 cells treated with 2~20μmol/L SEN compared with control group(P>0.05).Compared with control group,the viability of BV2 cells in model group decreased significantly(P<0.05).Compared with model group,the viability of BV2 cells in 4μmol/L SEN group was significantly restored(P<0.05).Compared with control group,the results of Griess method showed that the release amount of NO in cells of model group increased significantly(P<0.05);the results of realtime PCR showed that the expression levels of NLRP3,ASC,caspase-1,IL-1βand IL-18 mRNA in cells of model group increased significantly(P<0.05);the results of Western blotting showed that the protein e

关 键 词：远志皂苷元 脂多糖 小胶质细胞 NLRP3炎性小体 炎症反应 

分 类 号：R742[医药卫生—神经病学与精神病学] R285.5[医药卫生—临床医学]