Cinnamophilin 等藥物抑制人類單核球細胞 (THP-

綜合目前的實驗結果發現，天然物 cinnamophilin 與組織蛋白去乙醯化抑制劑 (Histone deacetylase inhibitor II ， HDI II) ，確實具有 抑制 MMP-9 表現之活性，而 cinnamophilin 在 LPS 所刺激方面其作由二十幾種酵素所組成的基質金屬蛋白酵素 (Matrix Metalloporteinase s ， MMPs) 能夠催化分解各種維持組織結構的細胞外基質蛋白 (extracellular matrix ECM) ，其中包括了基質 (ground tissue) 、與結締組織 (connective fibers) ，因此 MMP 在對於組織的重組 (remodeling) 、修補 (repairing) 與破壞 (destroy) 上便扮演了相當重要的角色。 MMPs 與 ECM 的結締組織分佈息息相關。 MMPs 在一般生理與病理上皆扮演著重要的角色，其中包括了基質的破壞與修補、癌細胞的入侵、

類風濕關節炎中不正常的軟骨組織破壞或粥狀動脈血管斑塊組織的剝離等。一般而言，許多病理與生理因子都會影響與調控 MMPs 的釋放 與產生。具有趨化作用的化學激素 chemokines 如 monocyte chemotactic protein-1 (MCP-1) 和發炎性細胞激素，如 tumor necorsis factor-a (TN F-a) 、 interleukin 1b (IL-1b) 、以及細菌類毒素如 lipopolysaccharide (LPS) ，均會影響與刺激發炎性細胞表現 MMPs 基因與其蛋白質的生成。

MMPs 同時也會受到相關生理性之內生性之抑制劑 (tissue inhibitor of metalloproteinases ， TIMPs) 所調節、目前所知有四種，如 TIMP-1 會 抑制 MMP-9 的表現。

在大規模中藥萃取物及化學合成藥物成分篩選實驗後，我們發現，一種由天然植物 Cinnamomum philippinense 中所粹取出來的天然物成分 cinnamophilin (MA-1) 與一種能誘發細胞生長停滯的組織蛋白去乙醯化抑制劑 Histone deacetylase inhibitor II (HDI II) ，皆具有明顯抑制 M MPs 活化之作用。 在先導試驗中我們以人類單核球細胞 (THP-1 cells) 為實驗細胞，分別以不同濃度的 LPS 或 MCP-1 處 24 小時候，

以電泳酵素分析法 (gelain zymography) 評估可發現到人類單核球細胞能誘發大量 MMPs 的活性，其中更以 MMP-9 為甚。尤其當 LPS 與 MCP-1 刺激濃度分別在 10 ng/ml 與 240 ng/ml ，細胞數為 1×106 時，此分解 gelatin 的效果最為明顯適當。隨後的實驗便以此為試驗條 件，的確在電泳酵素分析法下觀察到 cinnamophilin (1-50 mM) 與 HDI II (0.01-5 mM) 皆有意義地依濃度效應抑制 LPS 或 MCP-1 誘發人 類單核球細胞之 MMP-9 活性。另外以細胞存活率測定 (MTT assay) 發現 cinnamophilin 與 HDI II 的抑制作用並非源自細胞之損壞或死 亡。以西方點墨法 (Western blot) 的實驗發現在 LPS 的刺激下， MMP-9 的酵素蛋白表現量隨 cinnamophilin 與 HDI II 的濃度增加而降低

，故此證明天然物成分與組織蛋白去乙醯化抑制劑是作用在 MMP-9 蛋白質表現的層面。並進一步的以 RT-PCR 的實驗加以分析，發現 cinnamophilin 與 HDI II 均會抑制 MMP-9 mRNA 的表現。由此我們更深入瞭解細胞轉錄 (transcription) 之影響程度。接下來我們探討 ci nnamophilin 與 HDI II 在訊息傳遞中作用機轉的途徑。由實驗結果發現， cinnamophilin 會明顯抑制由 LPS 所刺激導致的 Inhibitor- kB-a (I kB-a) 的降解作用，使 nuclear factor-kB (NF- kB) 無法進入細胞核中與特定 MMP-9 相關 DNA 序列接合。然而， HDI II 對於 IkB-a 的 降解作用卻不明顯。在 mitogen-actived protein kinase (MAPKs) 方面，由我們目前的結果觀察到 cinnamophilin 與 HDI II 能抑制由 LPS 所刺激導致的 c-Jun-NH2-terminal kindase (JNK) 表現，但與 phosphorylated activation of extracellular signal-regulated kinases (ERKs) 並無直 接的影響。在細胞移動趨化實驗中，我們觀察到 MCP-1 能誘發 THP-1 細胞的 migration 作用。而在有 cinnamophilin 與 HDI II 存在時

， MCP-1 所誘發的細胞 migration 作用會被抑制。經由流式細胞儀的測定，我們發現藥物對 MMP-9 所產生的抑制作用與 THP-1 細胞表面 b integrin CD11b 的變化無關。

用機轉可能主要藉由影響 NF-kB 之訊息傳遞過程。未來我們將進行以 cinnamophilin 與 HDI II 為治療目標的活體實驗，期許能更加瞭解 它們在活體中對於抗發炎反應的功能與療效。

Cinnamophilin 等藥物抑制人類單核球細胞 (THP-

1) 誘發第九型基質金屬蛋白酵素活化之機轉探討

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Matrix metalloporteinases (MMPs) are a family of over 20 enzymes that cleave the various components of the extracellular matrix (ECM). The ECM serves many roles in addition to its structural and barrier functions, including ground substrates and connecting fibers, which have their function to maintain tissue structure. MMPs contr ibute to connective tissue development and proteolysis of ECM components by MMPs can alter these functions and are associated with a variety of normal and patholo gical conditions that involve matrix degradation, remodeling, tumor invasion, arthritis and atherosclerotic plaque rupture.

Many different factors have been shown to influence the transcription of MMPs, including hormones, growth factors, oncogene, and cytokines. In general, inflammator y cytokines such as tumor necrosis factor-a, (TNF- a), lipopolysaccharides (LPS), interleukin 1b (IL-1b) and chemokines such as CC chemokine monocyte chemotactic protein-1 (MCP-1) could stimulate inflammatory cells to express MMPs genes and protein, and the secretion of specific tissue inhibitors of metalloproteinases (TIMP), of which there are four to date, tightly control MMP activity. Each TIMP differs in affinity for MMP, with TIMP-1 being the major inhibitor of MMP-9.

According to previous experiments, we found that cinnamophilin (MA-1), a natural compound isolated from Cinnamomum philippinense, presented significant inhibit ory effect on MMP-9 activation. We also found that histone deacetylase inhibitor II (HDI II), as a potent inducer of growth arrest, differentiation, or apoptotic cell deat h in a variety of transformed cells in culture and in tumor bearing animals, also showed obvious inhibitory effect on MMP-9.

We used human monocyte THP-1 cells in our preliminary experiments and by using different concentration of LPS or MCP-1 treatment for 24 hours. By using gelatin e zymography analysis, we found that MCP-1- or LPS-stimulated human monocyte THP-1 cells could significantly induce MMPs activities, especially MMP-9. The ge latin effect is most appropriate and significant at the condition when LPS stimulation concentration at 10 ng/ml and MCP-1 at 240 ng/ml with 1×106 cell/ml. Then, bas ed on this experimental condition, we observed that cinnamophilin (1-50 mM) and HDI II (0.01-5 mM) both concentration-dependently inhibit MMP-9 activation indu ced by LPS and MCP-1significantly in zymography method. We also found that the inhibitory effect of cinnamophilin and HDI II were not due to impairment of cellul ar viability by MTT assays.

According to Western blot method, we found that the inhibition on expression of MMP-9 protein is concentration-dependently by cinnamophilin and HDI II in various stimulations.

By using RT-PCR method, we observed that cinnamophilin and HDI II can inhibit the expression of MMP-9 mRNA, thus they could have deeper influences on the lev el of MMP-9 transcription. At the same time, we investigated the mechanism of action of cinnamophilin and HDI II in various signaling pathways. We found that cinn amophilin could significantly inhibit the degradation of IkB-a induced by LPS, therefore nuclear factor-kB (NF- kB) may not translocate for transcription. However, th e inhibitory effect on degradation of IkB-a of HDI II is not obvious. Furthermore, in MAPKs aspect, cinnamophilin and HDI II could inhibit JNK expression, and no di rect inhibitory influence on ERKs expression was observed.

In the cell migration assay, we observed that MCP-1 produced induction of THP-1 cell migration. The presence of cinnamophilin and HDI II would reduce MCP-1-ind uced migrating effect. By flow cytometry method, we found neither cinnamophilin nor HDI II has significant effect on THP-1 cell surface CD11b expression.

In summary, we found that HDI II Chinese herbal compound, cinnamophilin, with inhibitory effect on MMP-9 expression, and cinnamophilin with its main mechanis m of action might through NF-kB signal pathway on LPS stimulation. It will be interesting to do further studies and investigation on cinnamophilin and HDI II as thera peutic targets on inflammatory research in vivo.

Investigation of the Inhibitory Mechanisms of

Cinnamophilin on Matrix Metalloprotinase-9

Activation in THP-1 Cells