15型脂氧酶在發炎性關節炎中調控MMP-2及PLGF之探討

Abstract

類二十碳脂酸是花生四烯酸經由多種酵素代謝而來的，在發炎反應中扮演著重要的角色。在這些酵素當中，第15型脂肪氧化酶(15-LOX)在很多種疾病中都佔有重要角色。除了脂肪氧化酶之外，花生四烯酸也可以被環氧化酶(COX)代謝而產生一些具生理活性的類二十碳脂酸。雖然COX的下游產物(例如前列腺素E2)已經是廣為人知的發炎性刺激物，脂肪氧化脢的產物在關節炎當中的角色則還不那麼明確。本研究主要是想探討15型脂肪氧化酶對於第二型基質金屬蛋白酶(MMP-2)的表現以及在發炎性關節炎中扮演的角色為何。我們發現給予15-LOX的下游產物：15-（S）-羥基二十碳四烯酸(15-(S)-HETE)時，可以刺激源自於病人的類風濕性關節炎滑膜纖維母細胞(RASF)表現MMP-2的信使核糖核酸以及蛋白質。而15-(S)-HETE的刺激作用可以經由給予LY294002 (PI3K 抑制劑) 以及 PDTC (NF-κB 抑制劑)而將其抑制。同時，給予15-(S)-HETE時可以增加AKT的磷酸化、p65在細胞核內的轉移、以及IκBα的分解。作為關節炎中主要的致炎性細胞激素，TNF-α 和IL-1β也可以刺激RASF產生MMP-2，而其作用也可經由給予15-LOX的抑制劑以及將15-LOX基因減弱而抑制；同時，這兩種細胞激素也可以刺激RASF產生15-LOX。此外，糖皮質激素可以抑制經15-(S)-HETE所誘導產生的MMP-2，而非類固醇消炎止痛藥則沒有此作用。相較於野生型的小鼠，在15-LOX基因剔除的小鼠當中，輔助劑所誘導產生的關節炎及增加的MMP-2都有減少的現象。這些結果顯示15-LOX在關節炎的病程中扮演著重要的角色，而且可能參與TNF-α 以及 IL-1β所誘導的發炎現象。因此，15-LOX在治療發炎性關節炎當中，或許可作為一個發展藥物的好標的。 胎盤生長因子(PLGF)是血管內皮生長因子(VEGF)成員之一，其在正常生理狀態的作用遠不如其在病理狀態時的作用為多。在類風濕性關節炎的病人身上，其滑膜液中PLGF的表現量會增加。動物實驗當中，當PLGF被抑制後，關節炎的病程也同時會被抑制。在本實驗中我們主要是探討15-LOX在RASF細胞調控PLGF的作用。在本實驗中，我們發現15LOX的下游產物，15-(S)-HETE可以增加PLGF的產生量；而PLGF在類風濕性關節炎的發展中又佔有重要的角色。15-(S)-HETE以時間依賴性及劑量依賴性的方式在人類的RASF增加PLGF的表現，而其作用包含經過PI3K-Akt,以及NF-kB的訊息傳遞路徑。此外，15-(S)-HETE也可以刺激COX-2的產生，而此作用也參與在15-(S)-HETE誘導增加PLGF產生的作用當中，此一作用也經由給予COX-2的小髮夾信使核糖核酸以及其抑制劑的作用而確認。此外，我們發現給予COX-2的主要下游產物：前列腺素E2之後，可以增加PLGF的產生；而EP1、EP2、EP3、EP4這幾個受體刺激劑也可以刺激PLGF產生。在動物實驗當中，我們發現在15-LOX基因剔除的小鼠，給予輔助劑所誘導發炎而產生的PLGF和COX-2都被抑制了。這些結果顯示15-LOX下游產物會刺激PLGF表現，這作用可能有參與關節炎的發展過程。 在類風濕性關節炎的發展過程中，TNF-α是極其重要的細胞激素之一，在花生四烯酸的代謝過程中也有所參與。在動物實驗中，我們將TNF-α局部注射到野生型或15-LOX基因剔除型的小鼠腳掌中，以探討脂肪氧化酶在TNF-α所造成的發炎現象中所扮演的角色。實驗結果顯示TNF-α所造成的發炎現象會因為15-LOX基因剔除而被抑制。此外，TNF-α所造成的腳掌腫脹現象也會因為給予5-LOX的抑制劑而受抑制。這些結果顯示了TNF-α所造成的發炎中，15-LOX及5-LOX均可能參與其中，在類風濕性關節炎中，脂肪氧化脢或許可作為新穎的治療標的。

The eicosanoids produced from arachidonic acids (AA) play the pivotal roles in inflammatory arthritis, which are metabolized by a variety of enzymes. Of these, 15-Lipoxygenase (15-LOX) is involved in many pathological processes. In addition to LOX, AA could also be metabolized by cyclooxygenase (COX) to produce the bioactive eicosanoids. Although the downstream products of COX including prostaglandin E2 are well-known inflammatory stimulators, the role of LOX products in inflammatory arthritis is still unclear. The aim of this study is to examine the role of 15-LOX in the matrix metalloproteinase (MMP) expression and inflammatory arthritis. Here we found that treatment of 15-LOX downstream product, 15-(S)-HETE (15-S-hydroxyeicosatetraenoic acid), increased the mRNA and protein levels of MMP-2 in rheumatoid arthritis synovial fibroblast (RASF) derived from rheumatoid arthritis patients. The enhancement effect of 15-(S)-HETE was antagonized by the addition of LY294002 (PI3K inhibitor) and PDTC (NF-κB inhibitor). Treatment of 15-(S)-HETE increased the phosphorylation of AKT, nuclear translocation of p65 and the breakdown of IκBα. TNF-α and IL-1β are the key cytokines involved in arthritis and also increase the activity of MMP-2 in RASF, which was antagonized by pretreatment with 15-LOX inhibitor PD146176 or knockdown of 15-LOX. It was also found that these two cytokines increased the expression of 15-LOX in RASF. Treatment of glucocorticoid but not NSAIDs inhibited 15-(S)-HETE-induced expression of MMP-2. In comparison with wild-type mice, adjuvant-induced arthritis and MMP-2 expression in synovial fibroblasts were markedly inhibited in 15-LOX knockout (KO) mice. These results indicate that 15-LOX plays an important role in the disease progression of arthritis and may be involved in the inflammatory action induced by TNF-α and IL-1β. 15-LOX is thus a good target for developing drugs in the treatment of inflammatory arthritis. PLGF (placenta growth factor) is a member of vascular endothelial growth factor (VEGF) family, which is redundant in physiological state but is more important in pathological condition. PLGF is increased in RA synovial fluid, and deficiency of PLGF suppresses the progression of arthritis in animal model. In this study, we examined the role of 15-LOX in the regulation of PLGF in RASF. Here we found that 15-(S)-HETE can enhance the expression of PLGF, which is recently considered to play an important role in rheumatoid arthritis. 15-(S)-HETE increased the expression of PLGF in RASF in a time-dependent and concentration-dependent manner. PI3K-Akt and NF-κB signaling pathways were involved in the potentiation effects of 15-(S)-HETE. In addition, COX-2 was up-regulated by the treatment of 15-(S)-HETE and the increase of COX-2 expression participated in 15-(S)-HETE-induced PLGF expression, which was confirmed by COX-2 shRNA or pharmacological COX-2 inhibitor. Moreover, it was found that treatment of prostaglandin E2 (PGE2), which was the main down-stream metabolite of COX-2, increased the expression of PLGF. EP1, EP2, EP3 and EP4 agonists could up-regulate PLGF as well. In animal studies, we found that the adjuvant-induced expression of PLGF and COX-2 was inhibited in 15-LOX knockout mice. These results indicate that PLGF up-regulation by 15-LOX downstream product may be involved in inflammatory arthritis. TNF-α is one of the most important cytokines in RA pathology. To investigate the involvement of lipoxygenase pathways in TNF-α-induced inflammation in animal model, TNF-α was injected intraplantarly in wild-type or 15-LOX knockout mice. The results showed that TNF-α-induced inflammation was reduced in 15-LOX knockout mice. In addition, TNF-α-induced paw edema was also inhibited by 5-LOX inhibitors. Our results implicate novel mechanism in TNF-α-induced inflammation and suggest new therapeutic strategies targeting LOX for the treatment of rheumatoid arthritis.