探讨BV2细胞活化中是否存在p38MAPK及JAK2-STAT通路的磷酸化激活
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摘要
目的:探索CD200R在LPS诱导的小胶质细胞炎症模型中的作用以及是否有pho-p38MAPK及pho JAK2-STAT通路的激活。方法:采用小胶质细胞进行研究,将细胞用CD200R抗体及LPS处理,ELISA检测TNF-α及IL-1β的分泌。Western blot检验p38MAPK、JAK2、pho-p38MAPK及pho-JAK2-STAT表达。为证实这两条通路是炎症的下游通路,分别应用两者的阻断剂处理细胞后再次检测炎症因子的分泌。结果:在小胶质细胞表面有CD200R的表达;应用CD200R的阻断性抗体后,TNF-α及IL-1β分泌均增多,较对照组及LPS组比较差异具有统计学意义(P<0.05),并且有pho-p38MAPK及pho-JAK2-STAT的激活;阻断剂SB203580及AG490均能抑制TNF-α及IL-1β的分泌。结论:pho-p38MAPK及pho-JAK2-STAT两条通路是LPS及CD200R抗体诱导的小胶质细胞炎症反应的下游通路。
Objective: To explore the important role of CD200 R in the microglia inflammation model induced by LPS as well as whether there is the activation of the pho-p38 MAPK and pho-JAK2-STAT pathway or not. Methods: Microglia was used for this study.Immunofluorescence confirms there was the expression of CD200 R in microglia. Enzyme-linked immunosorbent assay detected the secretory content of TNF-α and IL-1β after using CD200R-blocking antibody and LPS handle the microglia. Western blot disclose whether p38 MAPK and JAK2-STAT pathway can be phosphorylated or not. Finally,we detect the secretion of TNF-α,IL-1β after applicating different doses of channel blockers. Results: We observed that CD200 R expresses on the surface of microglia. To determine the relationship of CD200 R and inflammatory cytokines,we tested whether blocking CD200 R promotes the release of inflammatory cytokines or not. After using administrating antibodies of CD200 R,TNF-α and IL-1β secreted by microglia was significantly increased and phop38 MAPK and pho-JAK2-STAT were activated,which let us predict that pho-p38 MAPK and pho JAK2-STAT participate in the microglia activation of LPS induction. To further confirm the pho-p38 MAPK and pho-JAK2-STAT pathway were involved in microglia activation,we apply SB203580 and AG490 to inhibit both pathways respectively,and then we found that the number of TNF-α and IL-1β was significantly decreased,and the number of TNF-α and IL-1β was negatively correlation with the concentration of AG490 and SB203580. Conclusion: Not only pho-p38 MAPK but also pho-JAK2-STAT are involved in microglia activation induced by LPS and CD200 R antibody response.
Objective: To explore the important role of CD200 R in the microglia inflammation model induced by LPS as well as whether there is the activation of the pho-p38 MAPK and pho-JAK2-STAT pathway or not. Methods: Microglia was used for this study.Immunofluorescence confirms there was the expression of CD200 R in microglia. Enzyme-linked immunosorbent assay detected the secretory content of TNF-α and IL-1β after using CD200R-blocking antibody and LPS handle the microglia. Western blot disclose whether p38 MAPK and JAK2-STAT pathway can be phosphorylated or not. Finally,we detect the secretion of TNF-α,IL-1β after applicating different doses of channel blockers. Results: We observed that CD200 R expresses on the surface of microglia. To determine the relationship of CD200 R and inflammatory cytokines,we tested whether blocking CD200 R promotes the release of inflammatory cytokines or not. After using administrating antibodies of CD200 R,TNF-α and IL-1β secreted by microglia was significantly increased and phop38 MAPK and pho-JAK2-STAT were activated,which let us predict that pho-p38 MAPK and pho JAK2-STAT participate in the microglia activation of LPS induction. To further confirm the pho-p38 MAPK and pho-JAK2-STAT pathway were involved in microglia activation,we apply SB203580 and AG490 to inhibit both pathways respectively,and then we found that the number of TNF-α and IL-1β was significantly decreased,and the number of TNF-α and IL-1β was negatively correlation with the concentration of AG490 and SB203580. Conclusion: Not only pho-p38 MAPK but also pho-JAK2-STAT are involved in microglia activation induced by LPS and CD200 R antibody response.
引文
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[11]Linlin Yin,Yongyan Chen,Zhao Qu.Involvement of JAK/STAT signal-Ing in the effect of cornel iridoid glycoside on experimental autoimmune encephalomyelitis amelioration in rats[J].J Neuroimmunol,2014,274(6):28-37.
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[14]Wright GJ,Cherwinski H,Foster-Cuevas M.Characterization of the CD200R family in mice and humans and their interactions with CD200[J].J Immunol,2003,171(6):3034-3036.
[15]Barclay AN,Wright GJ,Brooke G.CD200 and membrane protein interactions in the control of myeloid cells[J].Trends Immunol,2002,23(6):285-290.
[16]Luo XG,Zhang JJ,Zhang CD,et al.Altered regulation of CD200receptor in monocyte-derived macrophages from individuals with parkinson's disease[J].Neurochem Res,2010,35(4):540-547.
[2]Le W,Rowe D,Xie W.Microglial activation and dopaminergic cell injury:an in vitro model relevant to Parkinson's disease[J].J Neurosicience,2001,21(21):8447-8455.
[3]Masocha W.Systemic lipopolysaccharide(LPS)-induced microglial activatoion results in different temporal reduction of CD200 and CD200 receptor gene expression in the brain[J].J Neuroimmunol,2009,214(1-2):78-82.
[4]Aedín M minogue,James PBarrett,Marina A Lynch.LPS-induced release of IL-6 from glia modulates production of IL-1βin a JAK2-dependent manner[J].J Neuroinflammation,2012,126(9):1186-1742.
[5]Dutta G,Zhang P.The lipopolysaccharide Parkinson's disease animal model:mechanistic studies and drug discovery[J].Fundamental Clin Pharmacol,2008,22(5):453-464.
[6]Rosenblum MD.Expression of CD200 on epithelial cells of the murine hair follicle:a role in tissue-specific immune tolerance?[J].Invest Dermatol,2004,123(5):880-887.
[7]Barclay AN,Wright GJ,Brooke G.CD200 and membrane protein interactions in the control of myeloid cells[J].Trends Immunol,2002,23(6):285-290.
[8]Cox FF,Camey D,Miller AM,et al.CD200 fusion protein decreases microglial activation in the hippocampus of aged rats[J].Brain Behav Immun,2012,26(5):789-796.
[9]Koning N,Swaab DF,Hoek RM,et al.Distribution of the immune inhibitory molecules CD200 and CD200R in the normal central nervous system and multiple sclerosis lesions suggest neuronglia and glia-glia interaction[J].Neuropathol Exp Neurol,2009,68(2):159-167.
[10]Hoek RM,Ruuls SR,Murphy CA.Down-regulation of the macrophage lineage through interaction with OX2(CD200)[J].Science,2000,290(5497):1768-1771.
[11]Linlin Yin,Yongyan Chen,Zhao Qu.Involvement of JAK/STAT signal-Ing in the effect of cornel iridoid glycoside on experimental autoimmune encephalomyelitis amelioration in rats[J].J Neuroimmunol,2014,274(6):28-37.
[12]Meuth SG,Simon OJ,Grimm A.CNS inflammation and neuronal degeneration is agravattedby impaired CD200-CD200R mediated macrophage silencing[J].Neuroimmunology,2008,194(1-2):62-69.
[13]Walker DG,Dalsing Hernandez JE,Campbell NA.Decreased expression of CD200 and CD200R in Alzheimer's disease:A potential mechanism for chronic inflammation[J].Exp Neurol,2009,215(1):5-19.
[14]Wright GJ,Cherwinski H,Foster-Cuevas M.Characterization of the CD200R family in mice and humans and their interactions with CD200[J].J Immunol,2003,171(6):3034-3036.
[15]Barclay AN,Wright GJ,Brooke G.CD200 and membrane protein interactions in the control of myeloid cells[J].Trends Immunol,2002,23(6):285-290.
[16]Luo XG,Zhang JJ,Zhang CD,et al.Altered regulation of CD200receptor in monocyte-derived macrophages from individuals with parkinson's disease[J].Neurochem Res,2010,35(4):540-547.