探討Resveratrol抑制黴漿菌MALP-2成分刺激人類單核球細胞(THP-1)誘發第九型基質金屬蛋白酵素活化之作用機轉

Investigation of the Inhibitory Mechanisms of Resveratrol on Mycoplasma fermentans-Derived Lipoprotein MALP-2-Induced Matrix Metalloproteinase-9 Activation in THP-1 Cells

江妤璇(Yu-Hsuan Chiang)

指導教授 ： 蕭哲志

臺北醫學大學/醫學院/藥理學研究所/碩士(2006年)

摘要

細菌性感染已知會引起人類疾病例如嚴重的發炎反應，典型的發炎反應會讓單核球細胞及巨噬細胞釋放多種的發炎性cytokines及chemokines。這些發炎反應多是由細菌之細胞壁成分引起，例如lipopolysaccharide、bacterial lipoprotein或lipoteichoic acids。黴漿菌(mycoplasmas)為一種缺乏細胞壁的菌類，而現在也證實mycoplasma lipoproteins也會刺激單核球細胞及誘發其分泌proinflammatory cytokine。已有證據顯示人類的單核球細胞和巨噬細胞皆可分泌合成基質金屬蛋白酵素(matrix metalloproteinases, MMPs)，MMPs為一群結構相似且含有鋅(Zinc)金屬離子之內生性蛋白水解酵素。MMPs在基質的修補、重組及破壞中扮演非常重要的角色。 Resveratrol為一種天然的的polyphenolic phytoalexin，可在許多植物發現，且具有抗發炎及抗癌作用。實驗中證明，在電泳酵素分析法(gelatin zymography)下，可以觀察到由Mycoplasma fermentans分離出的macrophage-activating lipopeptide-2 (MALP-2)成分刺激人類單核球細胞(THP-1)後，MMP-9酵素之活化及表現量會隨著resveratrol濃度之增加而有效的被抑制。接著利用細胞存活率測定(MTT)可發現resveratrol對MMP-9的抑制作用並非源自於細胞的死亡或損壞。接著用西方墨點法(Western blot)之實驗發現resveratrol的濃度增加，能顯著地降低由MALP-2刺激所誘發的MMP-9蛋白之表現。在初步的實驗中可以證實resveratrol對MALP-2有濃度梯度抑制(concentration-dependent inhibition)的效應。此外在enzyme-linked immunosorbent assay (ELISA)實驗中發現resveratrol於較高濃度20 micro Molar時能有意義地降低tissre inhibitor of metalloproteinases-1 (TIMP-1)之產生。在轉錄(transcription)方面，由reverse transcription-polymdrase chain reaction (RT-PCR)方法可得知resveratrol能抑制MMP-9之mRNA表現。 藉由Western blot之實驗發現resveratrol能顯著地抑制由MALP-2刺激所導致的total inhibitor-kappa B alpha 之降解作用及其在較高濃度10 micro Molar時抑制MALP-2所誘導的Akt活化情形較為明顯。在mitogen-activated protein kinases (MAPKs)方面，由目前結果觀察到resveratrol在較高濃度時抑制MALP-2所誘導的Extracellular signal-regulated kinase 1 and 2 (ERK1/2)活化情形較為明顯。 綜合目前之實驗結果，發現resveratrol確實能抑制人類單核球細胞(THP-1 cells)中，MALP-2所誘發的MMP-9活性與表現，而此抑制之機轉可能主要是影響NF-kappa?B與MAPKs之訊息傳遞路徑，期許未來能更加暸解resveratrol在活體實驗中對於抗發炎與抗癌反應的功用與療效。

關鍵字

第九型基質金屬蛋白酵素,黴漿菌,人類單核球細胞

並列摘要

Infection with bacteria have been reported to be associated with human inflammation reaction. Typicallys macrophages and monocytes are activated by components of the microbial cell wall such as peptidoglycan fragments, LPS, lipoteichoic acid, and bacterial lipoprotein. Surprisingly, cell wall-less mycoplasmas can also activated mcarophages very efficiently. Mycoplasma lipoproteins have been demonstrated to activated monocytic cells and induce proinflammatory chtokine secretion. Accumulating evidence indicates that human monocytes and macrophages synthesize and secrete a family of zinc-dependent neutral endopeptidases named matrix metalloproteinases (MMPs), which play an important role in matrix remodeling, repairing and destroying. Resveratrol, a naturally occurring polyphenolic phytoalexin, is found in a wide variety of plants, which exhibits a number of biological activites, including anti-inflammatory and anticarcinogenic properties. We used Mycoplasma fermentans-derived macrophage-activating lipopeptide (MALP-2) as a stimulus that induced expression of MMP-9 in human monocytic THP-1 cells. According to gelatin zymography, we found that the expression and activation of MMP-9 protein induced by MALP-2 were inhibited by resveratrol in a concentration-dependent way. We also found that the inhibitory effect of resveratrol was not due to an impairment of cellular viability as measured by MTT assays. According to Western blot analysis, we observed that the inhibition on MALP-2-induced expression of MMP-9 protein by resveratrol was concentration-dependent. Furthermore, we found that at higher concentration (20?micro Molar), resveratrol significantly reduced TIMP-1 proteins measured by enzyme-linked immunosorbent assay (ELISA). In the transcription level, resveratrol also suppressed the MALP-2-induced MMP-9 mRNA expression measured by RT-PCR. We found that resveratrol significantly inhibited the degradation of total inhibitor-kappa?B-alpha? (IkappaBalpha) and suppressed Akt expression induced by MALP-2?in Western blot analysis. Regarding MAPKs, resveratrol suppressed extracellular signal-regulated kinase 1 and 2 (ERK1/2) expression in MALP-2 stimulation. In summary, we found that resveratrol had inhibitory effect on MMP-9 expression and on activation in THP-1 cells. Its main mechanism of action might be through NF-kappa?B and MAPK signal pathway on MALP-2?stimulation. It is interesting to do further studies and investigations on resveratrol as therapeutic targets in inflammatory and cancer research in vivo.

並列關鍵字

resveratrol,MALP-2,THP-1

參考文獻

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