2010-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658234摘要：目前已有研究指出，當巨噬細胞受感染或是在發炎的情形下會產生內質網壓力，然而對於內質網壓力在發炎中所扮演的角色仍不清楚。近期的報導顯示出在有或無發炎的情況下，內質網壓力可能具有正向或負向調控發炎反應的作用。因此，雖然已知內質網壓力能活化NF-κB 及MAPKs 等發炎相關訊息傳遞路徑，但其負調控發炎反應之作用也有可能為病原體抵抗宿主先天性免疫的機制之一。有越來越多的研究結果顯示出未折疊蛋白反應(unfolded protein response, UPR)具有抗發炎的作用，但詳細的分子機制尚需進一步的探討。巨噬細胞是宿主先天性免疫重要的第一道防線，目前對於內質網壓力和其他發炎刺激物質在巨噬細胞中互相作用後所產生的影響還不清楚。因此，在這個研究中我們將利用老鼠巨噬細胞RAW264.7 為模式,探討內質網壓力在LPS 和 interferon 所活化的發炎反應中所扮演的角色。本研究將利用tunicamycin (TM)，thapsigargin (TG)，以及brefeldin A(BFA)為內質網壓力誘導劑，進一步探討內質網壓力調控發炎相關基因表現的分子機制及訊息傳導路徑。本研究主要的研究目標如下：(1) 了解內質網壓力誘導劑對於LPS 及interferon 所活化之發炎物質表現的影響; (2) 了解內質網壓力誘導劑其調控發炎相關蛋白表現的作用層面; (3) 了解內質網壓力誘導劑調控LPS 所活化之發炎蛋白表現的分子機制; (4) 了解內質網壓力誘導劑對於IFN 所活化之訊息傳遞路徑的影響及作用機制; (5)了解未折疊蛋白反應下游活化的分子(PERK，ATF4，XBP1，ATF6，GRP78，CHOP，IRE1，and TRAF2)其參與調控LPS 和 interferon 所活化的訊息傳遞路徑及相關基因的表現。由我們初步實驗結果得知，在老鼠巨噬細胞RAW264.7 中內質網壓力誘導劑TM 及BFA 能抑制LPS 誘導的iNOS 蛋白表現及NO 生成。此抑制作用是在iNOS 基因轉錄的層面，和細胞的生存率無關。此外，LPS 刺激3 小時後給予TM 及BFA 仍能抑制LPS 誘導的iNOS基因表現，顯示出內質網壓力誘導劑的抑制作用可能是在LPS 活化的晚期訊息傳遞路徑或是在基因轉錄的層面上。由實驗結果證實，內質網壓力誘導劑對於LPS 所活化的早期訊息傳遞路徑，例如IKK/IκB/p65 訊息傳遞以及MAPKs 的活化沒有影響，但會抑制IFNγ所活化的STAT1。LPS 活化晚期訊息傳遞路徑的分子以及其對於iNOS 基因表現的調控仍需要進一步探討。我們相信更深入的了解內質網壓力調控發炎反應的機制對於學術或是臨床上的應用非常重要。本研究不僅能更深入了解未折疊蛋白反應的抗發炎作用，更能供未來發展抗病毒或抗菌劑一個作用的標靶分子。<br> Abstract: Since macrophagic ER stress is induced in infectious and inflammatory conditions, itbecomes an interesting issue to understand the association between ER stress andinflammatory response. However, recent studies regarding the role of ER stress toinflammatory responses still remains elusive and even bi-directional modes of action inregulating inflammatory response might exist depending on the status of inflammation. In thisregard, even though ER stress response itself can induce inflammatory signals such as NF-kBand MAPKs, it may be one of ways for pathogens escaping from host innate immunity. Thereare more and more evidences indicating the anti-inflammatory effect resulting from the UPR.However, the detailed molecular mechanisms remain unclear, and additional studies will benecessary. Since macrophages are the first line host cells of innate immunity, and realoutcome upon ER stress interactive with other inflammatory stimuli has not been elucidated,in this study we attempt to use murine RAW264.7 macrophage cell model to clarify the roleplayed by ER stress in lipopolysaccharide (LPS) and interferons (IFN)-mediatedinflammatory responses. Moreover, the molecular mechanisms and signaling cascadesunderlying the regulation of inflammatory gene expression will be explored. In doing so,several ER stressors including tunicamycin (TM), thapsigargin (TG) and brefeldin A (BFA)will be used. The specific aims of this study include (1) To understand the effects of ER stressinducers on LPS/IFNγ-induced expression of inflammatory mediators; (2) To understandaction levels on the regulation of inflammatory proteins by ER stress inducers; (3) Tounderstand molecular mechanisms underlying the regulation of LPS-mediated inflammatoryprotein expression by ER stress inducers; (4) To understand the effects and actionmechanisms of ER stress inducers on IFNs-induced signaling pathways; (5) To understanddownstream components of UPR activation (PERK, ATF4, XBP1, ATF6, GRP78, CHOP,IRE1, TRAF2, and GRP78) that involve in the modulation of LPS- and IFNs-elicitedsignaling cascades and gene transcription. Our preliminary results reveal the ability of ERstressors TM and BFA to attenuate LPS-elicited iNOS gene expression and NO production inmurine RAW264.7 macrophages. This event occurs at the gene transcription level, and isunrelated to the cell viability. Moreover, post-treatment with TM or BFA at 3 h as comparedto LPS still can inhibit LPS-induced NO and iNOS, suggesting that such inhibitory actions ofER stressors occur either at the late signals evoked by LPS or at the transcriptional machinery.Confirming our suggestion, we do not observe any effects of ER stressors on the earlyupstream signals evoked by LPS, such as IKK and three MAPKs activation. However, bothTM and BFA can inhibit IFNγ induced STAT1 phosphoylation. The molecular eventsoccurring at late phase and contributing to iNOS gene transcription evoked by LPS needfurther investigation. Further investigation to determine the effects and mechanismsunderlying the regulation of inflammatory response by ER stressors is believed to discloseacademic and therapeutic merits. This study may not only provide advanced understanding ofthe anti-inflammatory effect of UPR but also provide defined targets for antiviral orantibacterial agents.