| 摘要: | 研究背景：憂鬱症的病因並非單一因素造成。由於干擾素治療會誘發憂鬱症，其次憂鬱症病人被發現體內細胞激素的含量較高，此外經由發炎物質刺激的動物會產生類似憂鬱症的行為，暗示發炎反應可能是造成憂鬱症的重要因素。Omega-3多元不飽和脂肪酸具有抗發炎的效果，且在臨床上表現出抗憂鬱劑的效果，加上流行病學調查及臨床研究的報告，Omega-3多元不飽和脂肪酸似乎在憂鬱症治療上扮演重要角色。二十二碳六烯酸（Docosahexaenoic acid, DHA）是大腦中最重要的Omega-3多元不飽和脂肪酸，然而目前關於DHA調控發炎反應的機轉並未釐清，本論文將探討DHA如何調控微膠細胞（microglia）的發炎反應，以及其中所牽涉的訊息傳遞途徑，並推測Omega-3多元不飽和脂肪酸在憂鬱症治療應用上的細胞機轉。研究方法：利用即時聚合?連鎖反應（real-time polymerase chain reaction）測量細胞激素（cytokine）、inducible isoform of nitric oxide synthase（iNOS）、cyclooxygenase-2（COX-2）、血紅素氧化?-1（Heme oxygenase-1, HO-1）的mRNA表現量，使用西方點墨法測定iNOS、COX-2、血紅素氧化?-1及相關路徑之蛋白質濃度、利用螢光染色及核質分離方法萃取細胞核蛋白並觀察轉錄因子NF-kB translocation的現象。研究結果：IFN-g誘發iNOS及COX-2蛋白和mRNA的表現。IFN-g也可造成前發炎細胞激素，如tumor necrosis factor-alpha（TNF-a和interlukin-6（IL-6）的增加，亦活化STAT蛋白。此外當加入DHA與IFN-g同處理細胞時，發現會抑制iNOS、COX-2 mRNA及蛋白的表現以及TNF-a和IL-6的增加，DHA亦可以誘發血紅素氧化?-1蛋白及mRNA的表現。此外NO的表現會因ZnPP IX（血紅素氧化?-1抑制劑）的介入而又有增加的趨勢，且DHA不抑制由IFN-g誘發的STAT蛋白活化。當給予LY294002（PI3k抑制劑）和PD98059（MEK抑制劑）時，可以發現兩者皆明顯抑制血紅素氧化?-1的表現。此外，給予DHA則可以造成下游p85、p65、IKKa/b、pIkBa產的磷酸化及IkBa的降解。NF-kB的抑制劑（PDTC和BAY）亦會抑制DHA所誘導血紅素氧化?-1的表現，證實了NF-kB確實調控DHA誘發血紅素氧化?-1之路徑。最後給予DHA前，先處理LY294002、wortmannin、PD98059、U0126在BV-2細胞上，會抑制p65、IKKa/b及pIkBa磷酸化及IkBa降解研究結論：綜合以上結果，證實了DHA增加血紅素氧化?-1的表現確實經由PI3 kinase/AKT及MEK/ERK pathway，進而啟動下游transcription factor NF-kB的活化。
Background: There are many evidences showing that inflammation may play an important role in depression. First, about 30 % of patients with hepatitis C virus infection experience major depression after receiving interferon (IFN)-a therapy. Second, patients with depression have been found to have higher serum levels of proinflammatory cytokines including interlukin-1b, interleukin (IL)-6, tumor necrosis factor(TNF)-a etc.. Final, the results from animal studies showed that the inflammatory stimulation could induce depression-like behaviors. Omega-3 PUFAs are anti-inflammatory and have antidepressant effects. Docosahexaenoic acid (DHA) is the major Omega-3 PUFAs in the brain. In this study, we investigated how DHA modulated inflammation in cellular levels. Methods: Western blotting, real-time Polymerase Chain Reaction (real-time PCR) and immunochemical methods was applied in BV-2 microglia. BV-2 was treated with DHA and IFN-g for indicated times and doses. The expression of cytokines, including inducible isoform of nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and heme oxygenase-1 (HO-1), were detected in both qPCR and western blotting. We used western blotting for measuring PI3 kinase/AKT, MEK/ERK, p65, p50, IKKa/b, IkBa and pIKba protein expression. We also applied immunochemical methods and nuclear/cytosolic isolation to assess transcription factor NF-kB translocation. Results: DHA attenuated IFN-g-induced iNOS, COX-2, tumor necrosis factor (TNF)-a, interleukin (IL)-6 and nitric oxide (NO) synthase, but not STAT protein phosphorylation. Besides, DHA also up regulated HO-1 and Interleukin (IL)-4 and Interleukin (IL)-10 expressions. Pre-treatment with HO-1 inhibitor zinc protoporphyrin IX diminished DHA’s inhibitory effect on NO production. In addition, DHA caused AKT and ERK activation in time-dependent manner and PI3 kinase/AKT and MEK/ERK inhibitors were abolished DHA-induced HO-1 expressions. DHA also increased IKKa/b, IkBa, p65 phosphorylation, and IkBa degradation. Moreover, the activation of NF-kB pathway was blocked by PI3 kinase/AKT and MEK/ERK inhibitors. Conclusion: In this study, we found that the expression of HO-1 was in response to DHA stimulation. DHA induce HO-1 expression by activate PI3 kinase/AKT and MEK/ERK pathway and increase NF-kB translocation. In connecting with inflammation hypothesis in depression and the potential antidepressant effects of Omega-3 PUFAs, our findings provide a novel implication of the antidepressant mechanisms of DHA. |
