Tellimagrandin II 對於巨噬細胞在酯多醣體
誘導後所產生的一氧化氮及前列腺素E2的抑制

當身體受到感染，且免疫系統過度反應時，容易引發敗血症。大部分敗血症的起因來自於革蘭氏陰性菌上的內毒素-Lipopolysaccharide (LPS)；而在LPS所引起的發炎反應中，一氧化氮(Nitric oxide) 之產生，扮演著重要的角色，如調控血管的擴張、消除病菌等功能；但LPS 的刺激可能會導致Nitric Oxide Synthase II (NOS2)大量表現在許多細胞及組織中，如巨噬細胞跟噬中性球，尤其是巨噬細胞在大量表現NOS2時，同時也會移動到組織中，並釋放大量的細胞激素，因此會造成血壓過低、組織傷害，甚至是器官衰竭等嚴重結果。我們的研究主要是利用天然純化物- Tellimagrandin II (TGII)-為水解性的單寧 (tannin)，來降低由LPS在巨噬細胞中所誘導的一氧化氮產量，以降低發炎反應。一開始我們以不同濃度的TGII來測試細胞的存活率，之後用一氧化氮產量測定，來看一氧化氮(NO)的產量是否會受到抑制，發現TGII 25 uM 就能有效抑制一氧化氮的產生；下一步則以反轉錄聚合酶連鎖反應 (RT-PCR)和西方點墨法 (Western blot)來確定TGII對於調控一氧化氮上游基因的影響，主要是針對MAPKs及NF-?羠的調控；且在給予細胞LPS後，將核蛋白質萃取出來。初步的結果發現，調控第二型一氧化氮合成酶 (Nitric oxide synthase II) promoter的轉錄因子 (transcription factor)-NF-κB的子單位(subunit)-p65，從細胞質移動到細胞核內的情況有受到抑制，且有可能是經由p38 kinase的路徑；而且不管是在一氧化氮或其上游的信使核醣核酸 (mRNA)及蛋白質(protein)都隨TGII的劑量多寡而有不同程度的抑制。另一方面，我們探討發炎反應有重要調控地位的第二型環氧酵素(COX-2)。我們的實驗結果發現COX-2的蛋白質表現量隨著TGII 的濃度遞減，在COX-2的產物, 前列腺素E2 (prostaglandin E2)也有被TGII抑制表現的情況，但在COX-2 mRNA的表現上，卻發現並不受TGII的調控；所以TGII調控COX-2主要應該是在蛋白質的裂解及後轉譯修飾的部份。我們進一步測試TGII對於巨噬細胞型態、移動及吞噬能力之影響，初步發現在TGII 50 uM 的劑量下，有明顯抑制巨噬細胞移動的能力，且細胞在給予TGII 5 uM　TGII對於E. coli.的吞噬能力也有下降，因此預期此天然藥物會降低由LPS所誘導的發炎反應，來減緩免疫細胞的過度發炎。

關鍵字

酯多醣體；一氧化氮；前列腺素E2

並列摘要

Sepsis occurs from a serious infection that causes the body's immune system to go into overdrive. The major microorganisms that induce sepsis of bodies are gram-negative bacteria with lipopolysaccharide (LPS) in their cell wall. Nitric oxide is one kind of elements that involved in pro-inflammatory regulation. Therefore, reduction of nitric oxide might be a reasonable pathway to decrease the symptoms of patients with sepsis. Tellimagrandin II (TGII), a nature compound that extracted from Trapa bispinosa has been reported with anti-inflammatory ability. Our study were invesitigated the molecular mechanisms of anti-inflammatory effects of TGII. Murine macrophage RAW264.7 has about 90% cell viability when we treated 50 uM TGII. We found TGII could significantly reduce lipopolysaccharide-induced NO production at 25 uM. From Western blot and RT-PCR, we also found TGII reduced both nitric oxide saynthase II (NOS2) protein and mRNA expression at 25 uM. As we have known this compound regulated NO expression, we expected the transcription factor NF-kB involved in compound regulation. From western blot analysis, NF-kB active subunit, phospho-p65 translocated from cytoplasm to the nucleus has been reduced by TGII. MAPKs are also critical to NF-kB stimulation, and we found expression of phospho-p38 has been blocked at 25uM of TGII in 10 minutes. TGII efficiently reduces lipopolysaccharide-induced NO production and its upstream regulation factors. Prostaglandin (PG), which is synthesized by cyclooxygenases (COX), is another important mediator of inflammation. Two isoforms of COX are known, COX-1 and COX-2. Our studies were to be aimed at COX-2 that induced by several pro-inflammatory stimuli, such as growth factor, cytokines, and endotoxin. From our results, we found that TGII reduced COX-2 protein expression after 6 hours LPS induction in dose dependent, and COX-2 downstream product, prostaglandin E2 , was also reduced by TGII. But the interesting part is that mRNA expression of COX-2 not affected by TGII. Maybe TGII regulated COX-2 at protein degradation and post-translational part. We further observed TGII’s effects on morphology and functions of macrophages. From cell migration assay, we knew that TGII 50 uM blocked macrophage migration apparently after MCP-1 induction and also reduced the phagocytosis ability under 5 uM. From previous results, we expect that TGII will be a useful compound to reduce the inflammation response induced by LPS.

並列關鍵字

Lipopolysaccharide ； Nitric Oxide ； Prostaglandin E2

參考文獻

Fabián J. A literature review of the epidemiology of sepsis in Latin America. Revista Panamericana de Salud Pública (2005), 18:163–171

Google Scholar

Pryor W.A., Squadrito G.L. The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide. American Journal of Physiology (1995), 268: 699-722

Google Scholar

Goldstein S., Squadrito G.L., Pryor W.A., Czapski G. Direct and indirect oxidations by peroxynitrite, neither involving the hydroxyl radical. Free Radical Biology & Medicine (1996), 21: 965-974

Google Scholar

Middleton SJ., Shorthouse M., Hunter JO. Increased nitric oxide synthesis in ulcerative colitis. Lancet (1993),341:465-466

Google Scholar

Kolb-Bacliofen V., Fehsel K., Michel G., Ruzicka T. Epidermal keratinocyte expression of inducible nitric oxide synthase in skin lesions of psoriasis vulgaris. Lancet (1996), 344:139

Google Scholar

國際替代計量

Tellimagrandin II 對於巨噬細胞在酯多醣體誘導後所產生的一氧化氮及前列腺素E2的抑制

未授權

主題瀏覽