非瑟酮通过上调GPR35抑制吗啡诱导的小鼠小胶质细胞BV2活化和炎症反应  

Fisetin inhibits activation and inflammatory response of mouse microglia BV2 cells induced by morphine via up-regulating GPR35

作　　者：关森 王慧淼 王莉萍 孙艳斌[1] GUAN Sen;WANG Huimiao;WANG Liping;SUN Yanbin(Department of Anesthesiology,Chengde Central Hospital,Chengde 067000,China;Department of Laboratory,Chengde Central Hospital,Chengde 067000,China)

机构地区：[1]承德市中心医院麻醉科,河北承德067000 [2]承德市中心医院检验科,河北承德067000

出　　处：《中国病理生理杂志》2023年第7期1218-1224,共7页Chinese Journal of Pathophysiology

基　　金：承德市科学技术研究与发展计划项目(No.202102A009)。

摘　　要：目的:探讨非瑟酮抑制吗啡诱导的小鼠小胶质细胞BV2活化和炎症的机制。方法:通过吗啡干预BV2细胞构建细胞模型,将细胞随机分为6组:对照组、吗啡组、吗啡+2μmol/L非瑟酮组、吗啡+4μmol/L非瑟酮组、吗啡+8μmol/L非瑟酮组和吗啡+10μmol/L米诺环素组,实验重复3次。CCK-8法检测细胞活力;ELISA试剂盒检测细胞上清液中炎症因子水平;Western blot检测炎症相关蛋白及BV2细胞活化标志物表达水平;RT-qPCR和Western blot检测G蛋白偶联受体35(GPR35)的表达;BV2细胞转染GPR35干扰质粒验证非瑟酮是否通过调控GPR35抑制吗啡诱导的BV2细胞活化和炎症反应。结果:与对照组相比,不同剂量非瑟酮单独处理后BV2细胞活力均无显著差异;吗啡组细胞活力升高,炎症因子[肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)和IL-6]、环加氧酶2(Cox-2)和诱导型一氧化氮合酶(iNOS)蛋白表达增加,BV2细胞活化标志物离子钙结合接头分子1(Iba-1)和CD11b表达上调(P<0.05)。与吗啡组相比,吗啡+非瑟酮组和吗啡+米诺环素组细胞活力降低,炎症因子(TNF-α、IL-1β和IL-6)、Cox-2和iNOS蛋白表达减少,BV2细胞活化标志物Iba-1和CD11b表达下调(P<0.05)。RT-qPCR和Western blot结果显示,非瑟酮剂量依赖性地增加吗啡诱导的BV2细胞中GPR35的表达(P<0.05)。干扰GPR35逆转非瑟酮对吗啡诱导的BV2细胞活化和炎症反应的抑制作用。结论:非瑟酮可能通过上调GPR35抑制吗啡诱导的小鼠小胶质细胞BV2活化和炎症。AIM:To investigate the impacts of fisetin on morphine-induced activation and inflammation of mouse microglia BV2 cells,and to discuss its mechanism.METHODS:Initially,morphine was applied for the induction of BV2 cells.The cells were divided into 6 groups:control group,morphine group,morphine+2μmol/L fisetin group,morphine+4μmol/L fisetin group,morphine+8μmol/L fisetin group and morphine+10μmol/L minocycline group.The cell viability was detected by CCK-8 assay,and ELISA kits were used to detect the levels of inflammatory factors in cell supernatants.The expression levels of inflammation-related proteins and BV2 cell activation markers were measured by Western blot.The mRNA and protein expression levels of G protein-coupled receptor 35(GPR35)were evaluated by RTqPCR and Western blot.TheBV2 cells were transfected with GPR35 interfering plasmid to verify whether fisetin inhibited morphine-induced BV2 cell activation and inflammatory response by regulating GPR35.RESULTS:Compared with control group,fisetin had no significant effect on the viability of BV2 cells.Morphine increased the viability of BV2 cells,and elevated the expression levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6,cyclooxygenase-2(Cox-2),inducible nitric oxide synthase(iNOS),ionized calcium-binding adapter molecule-1(Iba-1)and CD11b(P<0.05).The BV2 cell viability and the expression levels of TNF-α,IL-1β,IL-6,Cox-2,iNOS,Iba-1 and CD11b in morphine-induced BV2 cells were reduced after fisetin or minocycline treatment(P<0.05).Fisetin increased the mRNA and protein expression levels of GPR35 in morphine-induced BV2 cells in a dose-dependent manner(P<0.05).The rescue experiments confirmed that GPR35 knockdown reversed the inhibitory effects of fisetin on morphine-induced BV2 cell activation and inflammatory response.CONCLUSION:Fisetin inhibits morphine-induced BV2 cell activation and inflammation by up-regulating GPR35.

关 键 词：非瑟酮 G蛋白偶联受体35 小胶质细胞 炎症 

分 类 号：R741[医药卫生—神经病学与精神病学] R363.2[医药卫生—临床医学]