山奈酚通过抑制p38 MAPK通路减轻6-羟多巴胺(6-OHDA)诱导的PC12细胞炎症  

Kaempferol attenuates 6-OHDA-induced inflammatory damage in PC12 cells via inhibting p38 MAPK signaling pathway

作　　者：蔡美云 庄文欣 吕娥 付文玉 CAI Meiyun;ZHUANG Wenxin;LYU E;FU Wenyu(Department of Histology and Embryology,School of Basic Medical Sciences,Shangdong Provincial Key Laboratory for Neurologic Disorders and Regenerative Repair,Weifang Medical University,Weifang 261053,China;Center for Experimental Medical Research,Weifang Medical University,Weifang 261053,China)

机构地区：[1]潍坊医学院,基础医学院组织学与胚胎学教研室,山东省神经疾病与再生修复重点实验室,山东潍坊261053 [2]潍坊医学院医学研究实验中心,山东潍坊261053

出　　处：《细胞与分子免疫学杂志》2020年第7期583-589,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：山东省自然科学基金(ZR2014HL043);山东省中医药科技发展计划项目(2017-214)。

摘　　要：目的研究山奈酚(KAE)对神经毒素6-羟多巴胺(6-OHDA)诱导的PC12细胞炎性损伤的保护作用,并探讨其是否与抑制p38丝裂原活化蛋白激酶(p38 MAPK)通路有关。方法将PC12细胞分为对照组、100μmol/L 6-OHDA处理组、6-OHDA联合(20、40、60、80、100)μmol/L KAE处理组、KAE组,培养24 h。倒置显微镜下观察PC12细胞的形态变化,CCK-8法检测细胞活力;在筛选出KAE的最佳作用剂量基础上,应用免疫细胞化学染色法检测环加氧酶2(COX2)、诱导型一氧化氮合酶(iNOS)、核因子κB(NF-κB)的表达,Western blot法检测COX2、iNOS、NF-κB蛋白水平。为探讨其作用机制,在PC12细胞的6-OHDA培养体系中分别给予KAE和p38 MAPK通路抑制剂SB203580,Western blot法检测iNOS、COX2、NF-κB、p38 MAPK、磷酸化的p38 MAPK(p-p38 MAPK)的蛋白水平。结果与对照组比较,6-OHDA组细胞数量显著减少,大部分细胞胞体皱缩,聚集成团;与6-OHDA组相比,6-OHDA联合(20、40、60、80、100)μmol/L KAE处理组细胞数量增多,形态明显改善,且KAE浓度为80μmol/L时,细胞形态最好、数量最多。6-OHDA处理组细胞COX2、iNOS、NF-κB表达明显增高;与6-OHDA组比较,6-OHDA联合80μmol/L KAE处理组上述分子的表达显著下降。6-OHDA组细胞的COX2、iNOS、NF-κB、p38 MAPK、p-p38 MAPK的蛋白水平明显升高,而在6-OHDA联合KAE组和6-OHDA联合SB203580组,以上蛋白表达水平较6-OHDA组显著降低。结论KAE抑制p38 MAPK通路减轻6-OHDA所致的PC12细胞损伤。Objective To investigate the protective effects of kaempferol(KAE)on PC12 cells against neurotoxin 6-hydroxydopamine(6-OHDA)-induced inflammatory damage and explore whether it is related to the inhibition of the p38 mitogen-activated protein kinase(p38 MAPK)signaling pathway.Methods After being cultured for 24 hours,PC12 cells were divided into 4 groups:control,6-OHDA(100μmol/L),6-OHDA combined with(20,40,60,80,100)μmol/L KAE and KAE groups.The morphological features of PC12 cells were observed under an inverted microscope,and the cell viability was detected by CCK-8 assay.In the case of the optimal effective dose of KAE,the expression of cyclooxygenase-2(COX2),inducible nitric oxide synthase(iNOS),nuclear factors(NF)-κB were evaluated by immunocytochemical staining and Western blot analysis.To explore the mechanism underlying the protective role of KAE,KAE and SB203580(p38 MAPK pathway inhibitor)(10μmol/L)were separately added into 6-OHDA culture medium of PC12 cells.The protein levels of iNOS,COX2,NF-κB,p38 MAPK and p-p38 MAPK were tested by Western blotting.Results Compared with the control group,the number of cells in the 6-OHDA group was significantly reduced,with most cell bodies shrunk and aggregated into clumps.Compared with the 6-OHDA group,the number of cells increased and the morphology was effectively improved in the 6-OHDA combined with KAE(20,40,60,80,and 100μmol/L)group.KAE at 80μmol/L was demonstrated the best protective effects in the present work.In the 6-OHDA group,the expression of COX2,iNOS,NF-κB significantly increased.Compared with the 6-OHDA group,the expression of the above molecules decreased markedly in the 6-OHDA combined with 80μmol/L KAE group.The protein expression levels of COX2,iNOS,NF-κB,p38 MAPK and p-p38 MAPK went up significantly in the 6-OHDA group as compared with the control group,while they were down-regulated obviously by both KAE and SB203580.Conclusion KAE exerts neuroprotective effects on PC12 cells against the damage induced by 6-OHDA probably through the p3

关 键 词：山奈酚(KAE) 帕金森病 PC12细胞 p38丝裂原活化蛋白激酶(p38 MAPK) 神经炎症 

分 类 号：R285.5[医药卫生—中药学] Q291[医药卫生—中医学]