發炎反應是一系列經由活化的巨噬細胞產生 cytokines、growth factors、NO 所調控的複雜過程。巨噬細胞經由 Toll like receptors (TLRs) 辨識多樣病原來達成免疫方面的功能；隨著對於細胞表面受器的分子
結構、受體辨識、訊息傳遞及基因剔除動物實驗等的廣泛研究，TLRs
在生物體免疫系統的重要性也是現代分子生物學上的焦點之一。LPS
這個源自Gram negative bacteria 細胞壁的病原，在經由人體血清中的 LBP 結合後，在 CD14 的輔助之下形成 LPS-LBP-CD14 三體複合體，
接著經由巨噬細胞上TLR4 的辨識，啟動一系別包括 NF-κB
pathway、MAPK pathway 在內的訊息傳遞，同時也會活化 Src family kinases (SFKs)，接著在轉錄因子 (NF-κB、AP-1)被活化之後，產生 pro-inflammatory agents (iNOS，IL-6，TNF-α)導致發炎反應 (41)。
由於 LPS 的 receptor 並不具有 intrinsic tyrosine kinase 的活性，
因此在巨噬細胞的活化過程中，令人猜想可能有一些
membrane-associated tyrosine kinases 參與其中，使得 signal
transduction 得以被啟動。早期研究懷疑 SFKs 可能參與巨噬細胞活 化的文獻中，多以Lyn、Fgr、Hck 為研究對象，認為這三者對於巨 噬細胞在受到剌激活化之後的migration、cytokines production 扮演 著重要的角色，但是這些研究多半看的是Lyn、Fgr、Hck 三者在短
時間內的活性顯現 (42；43；44；45)。而到了 2006 年由 Tzeng-Horng Leu 等人研究的結果顯示出，Src 不僅僅在 LPS 的剌激之下有立即、
短暫的表現，其持續隨著時間而增加的特性，使得我們得以解釋 Lowell C.A.等人於 1997 年使用 Lyn-/-、Fgr-/-、Hck-/- triple knockout mice 所做出的結果。而從我們以 PP2 處理巨噬細胞所得到的結果也 顯示出Src 在長時間 (48 小時)受到 LPS 剌激的情況之下的確能夠影 響巨噬細胞被活化之後的IL-6 表現，但是如果能夠使用 Src 的 knockout mice 來做實驗，或許更能直接證實 Src 對於 IL-6 表現的重
要性。雖然Src 被證實在巨噬細胞活化時扮演著重要的角色，但是
其它的SFKs 仍然有研究指出 SFK 參與了細胞在各個層面的生理表
現，令人好奇SFKs 之間究竟存在著什麼樣的關係，值得進一步探
究。
在 2008 年的一篇使用缺乏 p85α (PI3K 的 subunit) 的
Pik3rl-deficient mice 來探討 LPS 誘導巨噬細胞產生 cytokines 的文獻 中指出，PI3K-Akt pathway 會抑制 ERK1/2 的活化，並降低轉錄因子 Egr-1 的量。相較於 wild type mice 的巨噬細胞，Pik3rl-deficient mice
的巨噬細胞所產生的包括IL-6 在內的 cytokines 的量會明顯的增加 (46)，但是調節 PI3K-Akt pathway 對於 LPS 引發的 IκBα降解或 NF-κB 的 translocation 並不會有影響；然而較早之前類似的實驗指出使
用別的PI3K 抑制劑對於 NF-κB 有著調控作用 (47；48)，會產生這 樣的矛盾據推測，原因可能是抑制劑的非專一性、細胞的種類、及抑
制劑的濃度等等。我們的實驗顯示出，在LPS 剌激之下，MAPK
pathway 能夠調控 IL-6 的表現；經由 PD98059 的抑制之後，雖然 IL-6 被抑制有到達顯著的標準，但是相較於使用PP2 或 AG 等上游抑制劑 來看，IL-6 的量仍舊偏高，會導致這樣的情況可能是 iNOS/Src pathway 的下游還有許多能夠影響IL-6 轉錄的 pathway (包括 NF-κB)，而在 巨噬細胞中iNOS/Src pathway 與 PI3K-Akt pathway 之間的關聯性目前 仍不清楚。已有證據顯示Src/PI3K/Akt dependent pathway 參與在 nNOS 產生 NO 的過程 (49)，也有研究指出在 valsartan-induced eNOS activation 中，Src/PI3K/Akt pathway 可以經由調節 eNOS 來調控 NO 的產生 (50)；而在巨噬細胞中，透過抑制 Src/PI3K/Akt pathway 可以
使得NO 及 proinflammatory agents 的產量減少，但是並沒有人了解到 Src/PI3K/Akt 與 iNOS 之間的關係 (41)。綜合上述的結果：1) PI3K/Akt pathway 能抑制 MAPK pathway 而影響 IL-6 的表現；2) Src/PI3K/Akt pathway 能調控 NO 及 cytokines 的產出；3) 從我們的研究結果得知 iNOS/Src pathway 藉由調控 MAPK pathway 去影響 IL-6 的表現 (Fig.8) 。這使得我們好奇這些 pathway 之間真正的角色關係為何？在 巨噬細胞中我們已經知道iNOS/Src 與 MAPK pathway 之間的關係，
而iNOS 會不會和 eNOS、nNOS 一樣與 Src/PI3K pathway 有著相似的 情形，則有待進一步的實驗證實。
此外，許多研究的結果認為在 LPS 的剌激之下，經由 NF-κB 的 translocation 使得 iNOS 和 IL-6 的基因同時轉錄出來，而我們的研究 結果顯示出iNOS 透過 MAPK pathway 對處於慢性發炎反應中的巨噬
細胞扮演著調控IL-6 的重要角色，顛覆了先前的看法。值得一提的
是，這些研究多半是巨噬細胞處於急性期時 (小於 2 小時)的研究，
而我們使用LPS 處理巨噬細胞長達 48 小時。已知某些細胞種類在受 到剌激活化之後，pERK 的持續時間會影響 cell signaling decisions (51)； 而 transient pERK 和 sustained pERK 與 iNOS、IL-6 之間的角
色關係在受到LPS 剌激之後會不會有可能不一樣，值得深入探索。
目前有許多研究正針對 iNOS 及 Src 的抑制作用進行，而 iNOS 本身為發炎反應的重要物質，Src 則在細胞的癌化過程中扮演著重要 的角色。本實驗揭露了「LPS 在誘發 iNOS 及 Src 之後，透過增加 ERK
的磷酸化來影響IL-6 表現」的觀點，希望未來對於癌症與發炎反應
的連結以及對抗癌、免疫製劑等的應用有所幫助。
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圖表 (Figures)
Figure.1 LPS-induced IL-6 secretion in RAW264.7 macrophage.
RAW264.7 cell line were treated with LPS (100 ng/ml) for 48 hours.
Then the condition medium were collected to determine the IL-6 expression by ELISA. Total cell lysates (80 μg) from each group were collected and soon resolved by SDS-PAGE and then probed with antibodies of iNOS, Src, and Actin.
(A)
(B)
Figure.2 The inhibitory effect of AG and ODQ on LPS-induced macrophage IL-6 expression. (A) Raw264.7 cells were pre-treated with AG (2 mM) for 30 minutes and then cells were stimulated with LPS (100 ng/ml) for 48 hours. The IL-6 expression was determined by ELISA kit ( Invitrogen Corporation). Total cell lysates (80 μg) from each group were collected and resolved by SDS-PAGE and then probed with antibodies of iNOS, Src, and Actin. ***, P < 0.001 as compared with LPS-stimulated
groups. (AG -- aminoguanidine hemisulfate, inhibitor of iNOS) (B) Raw 264.7 cells were pre-treated without or with ODQ (100 μM) for 30 minutes and then stimulated with LPS (100 ng/ml) for 48 hours. Equal amounts of cell lysates (80 μg) from each groups were resolved by SDS-PAGE immediately and probed with anti-iNOS and anti-Actin antibodies. The concentration of IL-6 in culture medium was determined by ELISA. ***, P < 0.001 as compared with LPS-stimulated groups.
(ODQ -- 1H-[1,2,4]Oxadiazole[4,3-a]guinoxalin-1-one, inhibitor of sGC)
Figure.3 The inhibitory effect of PP2 in IL-6 secretion of LPS-stimulated RAW264.7 macrophage. Raw264.7 cells were pre-treated with PP2 (10 μM) for 30 minutes and then cells were
stimulated with LPS (100 ng/ml) for 48 hours. The IL-6 expression was determined by ELISA kit (Invitrogen Corporation). Total cell lysates (80 μg) from each group were collected and resolved by SDS-PAGE and then probed with antibodies of iNOS, Src, and Actin. ***, P < 0.001 as
compared with LPS-stimulated groups.
Figure.4 LPS-induced IL-6 expression in wild type and iNOS knockout mice peritoneal macrophage ( PEM ). PEM isolated from wild type ( WT ) and iNOS knockout ( iNOS-/- ) C57BL/6 mice were stimulated with LPS (100 ng/ml) for 48 hours, then the concentration of IL-6 in culture medium was determined by ELISA. ***, P < 0.001 as compared with wild type LPS-stimulated group.
Figure.5 IL-6 expression was increased by adding SNAP and cGMP in both wild type and iNOS knockout mice PEM. PEM isolated from wild type (WT) and iNOS knockout (iNOS-/-) C57BL/6 mice were treated with or without SNAP (100 μM) and 8-br-cGMP (cGMP, 100 μM) for 48 hours, then the concentration of IL-6 in culture medium was determined by ELISA. ***, P<0.001 as compared with untreated groups. (SNAP – S-nitroso-N-acetyl-DL-penicillamine, NO donor ; 8-br-cGMP –
8-bromoguanosine 3’, 5’-cyclic monophosphate sodium salt, homolog of cGMP.)
Figure.6 The inhibitory effect of PD98059 on LPS-induced
macrophage IL-6 expression. Raw264.7 cells were pre-treated with PD98059 (10 μM) for 30 minutes and then cells were stimulated with LPS (100 ng/ml) for 48 hours. The IL-6 expression was determined by
macrophage IL-6 expression. Raw264.7 cells were pre-treated with PD98059 (10 μM) for 30 minutes and then cells were stimulated with LPS (100 ng/ml) for 48 hours. The IL-6 expression was determined by