探討Benzydamine抑制單核球第九型基質金屬蛋白酵素活化及血管舒張作用之機轉

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1 探討Benzydamine抑制單核球第九型基質金屬蛋白酵素活化及血管舒張作用之機轉
許多文獻指出，粥狀動脈血管斑塊組織的剝離與異常基質崩解作用有關，其中主要病理因素源自發炎性相關細胞(單核球或巨噬細胞)產生及釋放大量基質金屬蛋白酵素(Matrix metalloproteinases，MMPs)所致。基質金屬蛋白酵素(MMPs)，是重要且含鋅(zinc)金屬離子之蛋白水解酵素，他們能夠分解細胞外基質(Extracellular Matrix，ECM)，包括基質與結締纖維組織，故其對於組織之結構重組、修補與破壞都扮演相當重要的角色。一般而言，發炎性細胞激素以及生長因子等，均會刺激這些細胞表現MMPs基因及其酵素蛋白之生合成。 Benzydamine是一個屬於NSAID的抗發炎藥物，具有抗發炎、止痛、鎮熱和局部麻醉的作用。本實驗將探討benzydamine是否影響MMP-9的作用機轉以及對血管收縮作用有無影響。我們利用電泳酵素分析法中觀察到benzydamine (10-50 M)的確有意義地依濃度效應抑制由於腫瘤壞死因子(tumor necrosis factor-，TNF-)誘發人類單核球細胞之MMP-9酵素活性，並且以細胞存活率測定(MTT assay)發現benzydamine的抑制作用應非源自對細胞之損害。在西方點墨法(Western blotting)實驗中，發現由TNF-刺激之細胞內MMP-9 protein的表現量會隨著benzydamine濃度的增加而降低，所以可證實影響於MMP-9蛋白質表現層面。另外在若以反轉錄鏈鎖反應(Reverse transcription-polymerase chain reaction，RT-PCR)的實驗加以分析，發現benzydamine會抑制MMP-9 mRNA的表現，由此瞭解此成分影響於細胞轉錄(transcription)之上游。因此，我們將進一步探討benzydamine在訊息傳遞中作用機轉的方式。由實驗結果發現，benzydamine並未能抑制由TNF-刺激導致的Inhibitor-B- (IB-)的降解作用。而在MAPK的訊息傳遞上，發現其對於磷酸化P38的路徑有抑制作用。另外，在THP-1細胞給予LPS刺激後測量所釋放之TNF-含量，發現並未有明顯的差異，因此認為benzydamine可能具有選擇性細胞發炎介質之表現。 在血管的舒張作用方面，我們藉由從大鼠身上分離出來的主動脈環測量血管收縮及舒張的變化，發現benzydamine具有濃度效應抑制phenylephrine所造成之血管收縮作用。不論有無血管內皮細胞的存在下，皆能造成血管之舒張作用。在平滑肌的部分，當給予sGC抑制劑時，雖對benzydamine所造成的血管舒張作用有部分抑制，但是benzydamine所造成的血管舒張作用比結構相似的sGC活化劑YC-1還要強，因此我們認為benzydamine在血管所造成的舒張作用應該還有其他的機轉。實驗結果亦顯示，benzydamine能抑制高鉀所引起的收縮作用，並且藉由在無鈣之Krebs溶液中，經benzydamine前處理的血管給予高鉀刺激之後，再給予鈣離子刺激血管收縮，發現其能抑制外鈣內流所造成的血管收縮作用。而藉由共軛焦雷射顯微鏡系統觀察鈣離子進入平滑肌細胞的情形，我們發現前處理benzydamine (30 的平滑肌細胞再給予KCl (60 mM)刺激，其鈣離子進入細胞的狀況不變，與鈣離子通道阻斷劑nifedipine (10 所造成的抑制效果相較有明顯的差異。 在活體動物血壓測量實驗時，發現benzydamine依濃度效應造成血壓下降且心跳變慢。而在給予lipopolysaccharide (LPS)所引起的敗血性休克動物模式上，給予benzydamine未能發現有減緩或加強LPS所造成的血壓?降的情形。 根據目前實驗的結果，發現benzydamine的確具有抑制MMP-9表現之活性，而其作用機轉可能是抑制經由MAPK之p38路徑的訊號傳遞過程。另外具有調節血管舒張之作用，且其主要作用機轉為刺激血管平滑肌sGC活化，並且抑制血管平滑肌細胞之Ca2+離子釋出後之下游機制，但仍需要進一步研究探討。

2 Investigation of the Inhibitory Mechanisms of Benzydamine on Monocytic Matrix Metalloproteinase-9 Activation and Vasorelaxation Many evidences indicate that monocyte cell, human endothelial cells and macrophages synthesize and secrete several MMPs which participate in the degradation of ECM components in atherosclerosis. Matrix metalloproteinases (MMPs) are important group of zinc-containing proteinases responsible for degradation of the extracellular matrix (ECM), including ground substances and connecting fiber. Thus, it plays an important role in tissue structure remodeling, repairing and destroys. In general, inflammatory cytokines and several growth factors can stimulate MMPs gene expression and biosynthesis. Benzydamine is a topical nonsteroidal anti-inflammatory drug (NSAID) with anti-inflammatory, analgesic, antipyretic and local anesthetic activity. Therefore, we investigated the hypothesis that benzydamine could modulate the activity of MMP-9 and vascular activity. We found that benzydamine could inhibit TNF--induced MMP-9 activation on human monocytic THP-1 cells by gelatin zymography in the concentration-dependent manner. The inhibitory activities of benzydamine were not mediated by reduction of cellular viability. According to Western blotting method, we found that benzydamine had the inhibitory effect in TNF--induced-MMP-9 expressions. By using RT-PCR method, we also found that benzydamine could significantly inhibit the expression of MMP-9 mRNA, thus have deeper influence on the level of MMP-9 transcription. Therefore, we will investigate the mechanism of action of benzydamine in some signaling pathways. We found that benzydamine could not inhibit the degradation of IB- induced by TNF-Besides, we found that benzydamine inhibits p38 MAPK activation induced by TNF-. By TNF- ELISA assay, benzydamine could not reduce LPS-induced TNF-So we considered that improved inflammation indipendent with TNF- releasing. On the aspect of vascular relaxation, we used the isolated rat aortic rings to investigate the change of vascular activity, found that benzydamine concentration-dependently inhibite phenylephrine-induced contraction, and induced vasodilation no mater endothelium cells exist or not. In the part of smooth muscle cells, when we pretreat aorta rings with sGC inhibitor (ODQ), found that ODQ inhibits the relaxation induced by benzydamine. However, benzydamine-induced relaxation was stronger than soluble guanylyl cyclase (sGC) activator--YC-1. Thus, we considered that should be other mechanism involved in vasodilation by benzydamine. Our experiment showed that addition of benzydamine induced vasorelaxation in the denuded aorta precontracted with KCl in the normal krebs. Benzydamine affected the Ca2+ response strongly , suggesting that the inhibition of benzydamine on agonist response was due to the alteration of Ca2+ sensitivity of the contractile proteins. By confocal, pretreatment of benzydamine (30 M) did not affect the Ca2+ influx compared with calcium channel blocker (nifedipine 10 M). In vivo, we found that injection of benzydamine could interfere the blood pressure and heart beats. On the lipopolysaccharide (LPS)-induced sepsis animal models, injection of benzydamine could not inhibit downregulation of blood pressure in duced by LPS. In summary, we found that benzydamine have inhibitory effect on MMP-9 expression and activation, and benzydamine with its mechanism of action might through p38 MAPK signal pathway on TNF- stimulation. Furthermore, we found that major mechanism of benzydamine-induced vasorelaxation could stimulate activity of sGC and inhibite the downstream of Ca2+ release on vascular smooth muscle cell .