Font Size: a A A

β-arrestin1/2 Negatively Regulate NOD2 Mediated Inflammatory Signaling Through The Association With TRAF6 In The Microglia

Posted on:2016-10-17Degree:MasterType:Thesis
Country:ChinaCandidate:L J KongFull Text:PDF
GTID:2284330461986058Subject:Pharmacology
Abstract/Summary:PDF Full Text Request
Background and ObjectiveStroke is a leading cause of mortality and morbidity all over the world, more than 80% of all cases are ischemic stroke. Although thrombolytic therapy restored the ischemic areas with blood and oxygenation in time, it also caused an exacerbation of tissue injury and a profound inflammatory response. Recently, increasing evidences suggest that innate immunity is involved in ischemic stroke. NOD2 (nucleotide-binding oligomerization domain protein 2), an important cytoplasmic pattern recognition receptor, is the key molecule and research hot point in innate immunity; however its role and mechanism in cerebral ischemia-reperfusion (I/R) injury hasn’t been completely elucidated.Although infiltrated immune cells contribute to inflammatory responses in the brain, the resident glial cells (mainly microglia) play important roles in the initiation and progression of inflammation following I/R insult. We previously demonstrated that NOD2 expression was significantly increased in microglia in response to the I/R insult. It indicated that NOD2 triggered inflammation in microglia may play an important role in the regulation of immune responses in cerebral I/R injury, but the detailed signaling induced by NOD2 and regulatory mechanism in microglia is not clear.p-arrestinl/2 are multifunctional proteins that mediate desensitization of G protein-coupled receptors (GPCRs) and serve as important scaffold proteins to interact with other proteins and influence intracellular signaling pathways. Accumulating evidence has proved that β-arrestinl/2 are functionally involved in the regulation of immune responses. It has recently been implicated that β-arrestinl/2 interacted with TRAF6 and negatively regulated the inflammatory reactions mediated by TLR/IL-1R. TRAF6 is also a critical factor in the NOD2-triggered signaling pathway, but whether β-arrestinl or β-arrestin2 regulated it has not been founded. Our research is based on the microglia in vitro to study the inflammatory signal pathway which induced by NOD2 deeply investigate if β-arrestinl/2 regulated the NOD2 signal pathway through the association with TRAF6 and ameliorate the role and mechanism of NOD2 triggered signal pathway in the cerebral I/R. We also want to make contributions to exploring the new targets and resolution for cerebral ischemia reperfusion.Methods1. Middle cerebral artery occlusion (MCAO) was induced in male wild-type mice and reperfusion was initiated by retracting the monofllament carefully for 2h,6h,12h, 24h and 48h after 2h of MCAO.2. BV2 cell line was recovered routinely and cultured for using when they come to an logarithmic phase.3. MDP solution was prepared in sterile condition.4. Plasmid mini mention Kit was used to extract β-arrestinl full-length vector, β-arrestin2 full-length vector,β-arrestin2-shRNA plasmid, NOD2-shRNA plasmid and their control vectors.5. Transfection was performed using Lipofectamine 2000 reagent.6. Immunoprecipitation was used to detect the interaction of TRAF6 and β-arrestinl or β-arrestin2 respectively after MDP stimulating.7. Western blot was used to detect the protein expression of NOD2, TRAF6, COX-2, NF-κB p-P65, IκBα, β-arrestinl and β-arrestin2.8. Gelatin zymography was to test the activity of MMP-9 and MMP-2 in the microglial medium.9. Statistical analysis was performed by SPSS 16.0 statistical program. A value of p< 0.05 was considered statistically significant.Results1. Both β-arrestin 1 and β-arrestin2 were upregulated by after cerebral I/R injury, the trends were similar to that of NOD2.2. MDP is specific to NOD2 receptor.3. MDP stimulation upregulated the expression of TRAP6 and COX-2 and enhanced the activation of NF-κB in the microglia time-dependently.4. MDP stimulation promoted the expression and activation of MMP-9 time-dependently, but did not have effect on MMP-2 obviously.5. MDP stimulation rapidly promoted the association of β-arrestin 1/2 and TRAF6 respectively.6. Overexpression of β-arrestinl and β-arrestin2 respectively inhibited the activation of NF-κB and the expression of COX-2 induced by MDP.7. Silence of β-arrestinl and β-arrestin2 respectively enhanced the activation of NF-κB and the expression of COX-2 induced by MDP.8. Overexpression of β-arrestinl and β-arrestin2 respectively inhibited the activity MMP-9 activition induced by MDP.ConclusionNOD2 activation induced the TRAF6/NF-κB/COX-2/MMP-9 signaling pathway in the microglia. β-arrestin1 and β-arrestin2 respectively interacted with TRAF6 after MDP stimulation, and negatively regulated the NOD2 triggered inflammatory signaling pathway in microglia.
Keywords/Search Tags:β-arrestin 1/2, NOD2, Cerebral Ischemia Reperfusion, Microglia, Innate Immunity, Inflammation
PDF Full Text Request
Related items