苦参碱对LPS激活的小胶质细胞炎症因子释放和CX3CR1蛋白表达的影响  

Effects of Matrine on Inflammatory Factors Release and CX3CR1 Protein Expression in LPS Activated Microglia

作　　者：王业丰 姚遥[2] 杨欣河 邹彬 李佳 李娟[3,4] WANG Yefeng;YAO Yao;YANG Xinhe;ZOU Bin;LI Jia;LI Juan(School of Public Health and Management,Ningxia Medical University,Yinchuan 750004,China;School of Basic Medical Sciences,Ningxia Medical University,Yinchuan 750004,China;School of Pharmacy,Ningxia Medical University,Yinchuan 750004,China;Key Laboratory of Ningxia Minority Medicine Modernization,Ministry of Education,Yinchuan 750004,China)

机构地区：[1]宁夏医科大学公共卫生与管理学院,银川750004 [2]宁夏医科大学基础医学院,银川750004 [3]宁夏医科大学药学院,银川750004 [4]宁夏少数民族医药现代化教育部重点实验室,银川750004

出　　处：《宁夏医科大学学报》2022年第11期1081-1087,共7页Journal of Ningxia Medical University

基　　金：国家自然科学基金项目(82160759,82060792);宁夏重点研发计划重点项目(2018BFG02005);宁夏自然科学基金项目(2020AAC03133,2021AAC03143)。

摘　　要：目的探讨苦参碱对脂多糖(lipopolysaccharide,LPS)激活的小胶质细胞炎症因子释放和CX3CR1(CX3C chemokine receptor 1)蛋白表达的影响。方法选择小胶质N9细胞系,将细胞分为Control组、苦参碱(10μmol·L^(-1))组、LPS(300 ng·mL^(-1))组、LPS+苦参碱(0.1、0.3、1.0、3.0、10.0μmol·L^(-1))组以及LPS+米诺环素(minocycline,MINO)(30μmol·L^(-1))组。MTT法检测各组细胞的细胞存活率;ELISA法测定活性氧(ROS)和炎症因子白细胞介素-6(IL-6)、前列腺素E2(PGE2)的表达;免疫荧光法测定M1/M2极化标记物CD32与CD206的表达;Western blot与免疫荧光检测CX3CR1的表达。结果MTT实验结果显示:0~100μmol·L^(-1)的苦参碱以及0~10μg·mL^(-1)的LPS对N9细胞活力无影响(P=0.9828,P=0.7107);LPS刺激亦未降低经苦参碱预处理的N9细胞的细胞存活率(P=0.7445)。ELISA实验结果显示:与Control组相比,LPS组的ROS、IL-6、PGE2水平均升高(P均<0.01);与LPS组相比,LPS+苦参碱组ROS、IL-6、PGE2水平均下降(P均<0.01)。免疫荧光结果显示:与Control组相比,LPS组CD32的相对荧光强度升高,CD206相对荧光强度降低(P均<0.01);与LPS组相比,LPS+苦参碱组CD32相对荧光强度下降,CD206相对荧光强度升高(P均<0.001)。Western blot与免疫荧光检测结果显示:与Control组相比,LPS组的CX3CR1水平降低(P<0.05);与LPS组相比,LPS+苦参碱组CX3CR1水平升高(P<0.05)。结论苦参碱能够抑制LPS诱导的小胶质细胞炎症因子释放,抑制小胶质细胞的M1型极化,促进小胶质细胞向M2表型转化,并上调CX3CR1的表达。Objective To investigate the effects of matrine on inflammatory cytokines release and CX3CR1 protein expression in LPS-activated microglia.Methods Microglia N9 cell line was selected,and the cells were divided into the Control group,matrine(10μmol·L^(-1))group,LPS group(300 ng·mL^(-1)),LPS+matrine(0.1,0.3,1.0,3.0,10.0μmol·L^(-1))group and LPS+MINO(30μmol·L^(-1))group.MTT assay was used to detect the cell viability of each group.The expression of ROS and inflammatory factors interleukin-6(IL-6)and prostaglandinE2(PGE2)were determined by ELISA.The expression of M1/M2 polarization markers CD32 and CD206 was determined by immunofluorescence assay.The expression of CX3CR1(CX3C chemokine receptor 1)was detected by Western blot and immunofluorescence.Results The results of the MTT assay showed that 0-100μmol·L^(-1) matrine and 0-10μg·mL^(-1) LPS had no significant effects on the viability of N9 cells(P=0.9828,P=0.7107).LPS stimulation did not reduce the cell survival rate of matrine pretreated N9 cells(P=0.7445).ELISA results showed that ROS,IL-6,and PGE2 levels in the LPS group were significantly higher than those in the Control group(P all<0.01);Compared with the LPS group,ROS,IL-6,and PGE2 levels in the LPS+matrine group were significantly decreased(P all<0.01).Immunofluorescence results showed that the relative fluorescence intensity of CD32 in the LPS group was significantly higher than that in the Control group,while the relative fluorescence intensity of CD206 decreased significantly(P all<0.01);Compared with the LPS group,the relative fluorescence intensity of CD32 in LPS+matrine group decreased significantly,while the relative fluorescence intensity of CD206 increased significantly(P all<0.01).Western blot and immunofluorescence showed that CX3CR1 levels in the LPS group were significantly lower than those in the Control group(P<0.05);Compared with the LPS group,CX3CR1 level in the LPS+matrine group was significantly increased(P<0.05).Conclusion Matrine can significantly inhibit LPS-induced release o

关 键 词：苦参碱 小胶质细胞 脂多糖 CX3CR1 经典活化/替代活化 炎症 

分 类 号：R322.81[医药卫生—人体解剖和组织胚胎学]