探討黃芩素抑制第九型基質金屬蛋白酵素活化與脾臟細胞活化的作用機轉

Investigation of the Inhibitory Mechanisms of Baicalein on Matrix Metalloproteinase-9 and Splenocyte Activation

王鈞賜(Chun-Szu Wang)

指導教授 ： 蕭哲志副教授

臺北醫學大學/醫學院/藥理學研究所/碩士(2004年)

摘要

基質金屬蛋白酵素(Matrix metalloproteinases，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。同時MMP的含量與活性表現受到許多方式嚴密地調節控制。許多文獻指出，類風濕性關節炎的軟骨組織不正常破壞或粥狀動脈血管斑塊組織的剝離以及癌細胞的生長與惡性轉移皆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、癌細胞、血管內皮細胞或巨噬細胞)產生及釋放大量MMPs所致。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。 許多文獻指出，漢方草藥黃芩根部類黃酮素萃取物之黃芩素(baicalein; 5, 6, 7-trihydroflavone)，具有抗發炎、抗氧化及抗癌的作用。因此，本實驗將探討黃芩素(baicalein)是否具有影響MMPs活性之作用機轉及調節非專一性免疫功能。在此實驗中我們利用電泳酵素分析法中觀察到黃芩素(baicalein 1-50 ?M)確實有意義地依濃度效應抑制對於腫瘤壞死因子(tumor necrosis factor-?，TNF-?)誘發人類單核球細胞之MMP-9酵素活性。此外以細胞存活率測定(MTT assay)發現黃芩素(baicalein)的抑制作用並非源自細胞之損害。且黃芩素也僅在高濃度(50 ?M)時對於單純MMP-9酵素活性有部份抑制作用。 由西方點墨法(Western blotting)實驗中，發現在TNF-?刺激下細胞內MMP-9 protein的表現量會隨著黃芩素(baicalein)濃度的增加而降低，故可證實作用在MMP-9蛋白質表現層面。並進一步以反轉錄鏈鎖反應(reverse transcription-polymerase chain reaction，PT-PCR)的實驗加以分析，發現黃芩素(baicalein)會抑制MMP-9 mRNA的表現，由此瞭解此成分作用在細胞轉錄(transcription)之上游。因此，我們將更進一步探討黃芩素(baicalein)在訊息傳遞中作用機轉的方式。由實驗結果發現，baicalein 會部份抑制由TNF-? 所刺激導致的Inhibitor-?B-? (I?B-?)的降解作用。然而，在baicalein 高濃度(20 ?M)對於I?B-??降解的抑制作用卻不明顯，同樣的我們也在細胞核內蛋白發現低濃度的baicalein會抑制NF-?B的表現與轉移(translocation)。在Mitogen-activated protein kinase (MAPKs)方面，由目前的實驗結果觀察到baicalein能部份抑制由TNF-?所刺激導致的c-Jun-NH2-terminal kindase (JNK)與phosphorylated activation of extracellular signal-regulated kinases (ERKs) 表現。?另外在細胞移動趨化功能性的實驗?migration assay?中，我們觀察到Monocyte Chemotactic Protein-1 ?MCP-1)能誘發THP-1細胞進行migration作用，而在baicalein存在時，MCP-1所誘發的細胞migration作用會被抑制。 針對baicalein對於脾臟細胞活化和細胞激素釋放的實驗中，取自Balb/c小鼠脾臟細胞(Splenocytes)的初代細胞為實驗細胞，以concanavalin A (Con A)和lipopolysaccharide (LPS)為刺激劑，在46小時細胞培養中，發現各具有促進脾臟細胞(Splenocytes)的增殖(proliferation)。隨後以Con A (17 ?g/ml)為實驗條件，利用MTT assay觀察到黃芩素(baicalein)具有意義的依濃度效應抑制脾臟細胞增殖的反應，而針對以LPS (100 ?g/ml)為實驗條件下，baicalein則具較弱的抑制作用。另在Con A (17 ?g/ml)的刺激下，以酵素免疫分析法(Enzyme Linked Immunosorbent Assay，ELISA)也發現黃芩素具有濃度效應抑制IFN-?釋出的濃度，然而在IL-2釋出的濃度則呈現相反的現象。 綜合目前實驗的結果，發現黃芩素(baicalein)的確具有抑制MMP-9表現之活性，而其作用機轉可能是藉由影響NF-?B的訊號傳遞過程。另外在經由Con A 所刺激脾臟細胞增殖的條件下也發現具有抑制的作用。因此在未來則將進行相關之訊息與基因性實驗及活體collagen誘發關節炎實驗，以瞭解其具有治療發炎性疾病之功能。

關鍵字

黃芩素 ； 第9型基質金屬蛋白酵素 ； 脾臟細胞

並列摘要

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. The levels and activities of MMPs are strictly regulated and controlled in various ways. Many evidences indicate that monocyte cell, cancer cells, human endothelial cells and macrophages synthesize and secrete several MMPs which participate in the degradation of ECM components in rheumatoid arthritis tissues or atherosclerosis or during cancer growth and metastasis. In general, inflammatory cytokines and several growth factors can stimulate MMPs gene expression and biosynthesis Baicalein (5, 6, 7-trihydroflavone) as a flavonoid originated from the root of Chinese medicinal herd Scutellaria baicalensis, has been shown to exert anti-inflammatory and anti-oxidant effects. Therefore, we investigated the hypothesis that baicalein could modulate the activity of MMP-9 and immune responses. We found that baicalein 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 baicalein were not mediated by reduction of cellular viability. According to Western blotting method, we found that baicalein had the inhibitory effect in TNF-?-induced-MMP-9 expressions. By using RT-PCR method, we also found that baicalein 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 baicalein in various signaling pathways. We also found that baicalein could partially inhibit the degradation of I?B-? induced by TNF-???Simultaneously, from extracted nuclear protein, evidence showed low concentration of baicalein inhibit the expression of NF-?B. Furthermore, in Mitogen-activated protein kinase (MAPKs) aspect, baicalein could partially inhibit c-Jun-NH2-terminal kindase (JNK) and phosphorylated activation of extracellular signal-regulated kinases (ERKs) expression was observed. In the cell migration assay, we observed that Monocyte Chemotactic Protein-1 (MCP-1) produced induction of THP-1 cell migration. The presence of baicalein would reduced MCP-1-induced migrating effect. According to the experiment of splenocytes avtivation, we also show that baicalein (1-50 ?M) could significant concentration-dependently inhibition on concanavalin A (Con A) (17 ?g/ml)-induced proliferation of spleen cells, but on lipopolysaccharide (LPS) (100 ?g/ml)-induced spleen cells proliferation show no significant inhibition . The production of Interferron-? (IFN-?) induced by Con A were also concentration- dependently inhibited by baicalein, whereas the expression of Interleukin- 2 (IL-2) induced by Con A, was not inhibited by baicalein. In summary, we found that baicalein have inhibitory effect on MMP-9 expression and activation, and baicalein with its mechanism of action might through NF-?B signal pathway on TNF-? stimulation, Further we found that baicalein could inhibit Con A-induced spleen cells proliferation . Therefore, it will be interesting to study further on baicalein by in vivo and in vitro experiments to see its potential as a therapeutic agent for inflammatory diseases.

並列關鍵字

Baicalein ； Matrix Metalloproteinase-9 ； Splenocyte

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