探討Haloperidol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作用機轉

有許多證據顯示出人類的單核球細胞和巨噬細胞皆可分泌合成出一群結構類似且帶有鋅(Zinc)金屬離子之內生性蛋白水解酵素。這群水解酵素稱之為基質金屬蛋白酵素(matrix metalloproteinases，MMPs)具有可以分解及破壞細胞外基質(extracellular matrix，ECM)之能力。因此MMPs對於組織的重組(remodeling)、修補(repairing)與破壞(destroy)上扮演著相當重要之角色。而最近研究證據顯示出在一些神經退化性疾病的病理過程中，例如：阿滋海默症(Alzheimer's disease)以及多發性硬化症(multiple sclerosis)等,皆與MMPs有關。另一方面，在癌症中，MMPs也參與著癌細胞生長及前進的許多過程且能使癌細胞在轉移(metastasis)入侵的進程中穿透周圍組織。在臨床上，用來治療精神疾病方面的藥物haloperidol和valproic acid皆發現具有抗發炎之效果。所以假設此兩種藥物haloperidol與valproic acid能抑制發炎時所誘發的不正常組織重組(remodeling)。因此本研究使用可以誘發MMP-9大量表現之腫瘤壞死因子(tumor necrosis factor-?，TNF-?)與細菌類毒素(lipopolysaccharide，LPS)作為THP-1細胞之刺激劑。由電泳酵素分析法(gelatin zymography)下，我們觀察到由TNF-?刺激MMP-9酵素之活化及表現量會隨著haloperidol與valproic acid濃度增加皆可有效地被抑制。接著利用細胞存活率測定(MTT assay)可發現haloperidol與valproic acid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)之實驗方法，發現haloperidol與valproic acid濃度增加皆可有效地抑制由TNF-?刺激MMP-9蛋白之表現量。此外在enzyme-linked immunosorbent assay (ELISA)實驗中發現，haloperidol於較高濃度20 ?M時能有意義地降低TIMP-1之產生。另外還發現，haloperidol可以隨著濃度增加而抑制LPS所誘發的MMP-9酵素活化與蛋白質之表現量。在轉錄(transcription)方面，由RT-PCR方法可得知haloperidol能抑制MMP-9之mRNA表現。綜合上述實驗結果顯示haloperidol抑制效果較valproic acid強(haloperidol IC50：5.4 ± 0.3 ?M；valproic acid IC50：255.8 ± 73.8 ?M)。藉由Western blot之實驗發現haloperidol能顯著地抑制由TNF-?或LPS刺激所導致的total inhibitor-?B??(I?B?)之降解作用。接著利用電泳移動偏向分析法(electromobility shift assay，EMSA)，分析細胞核內外NF-?B (p65)之轉位與活化情形，得知haloperidol濃度為10 ?M時，可有意義地抑制TNF-?所誘發的NF-?B之轉位與活化。在mitogen-activated protein kinases (MAPKs)方面，由目前結果觀察到haliperidol在較高濃度(20 ?M)時抑制TNF-?所誘導的c-Jun-NH2-terminal kinase (JNK)活化情形較為明顯。另外更進一步發現haloperidol並不會影響LPS在THP-1 cells中所誘發之內生性TNF-?表現量。綜合目前之實驗結果，發現haloperidol確實能選擇性地抑制人類單核球細胞(THP-1 cells)中，TNF-?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-?B之訊息傳遞路徑。期許未來能更加暸解在活體實驗中對於抗發炎反應的功能與療效。

關鍵字

基質金屬蛋白酵素；人類單核球細胞

並列摘要

Accumulating evidence indicates that human monocytes and macrophages synthesize and secrete a family of zinc-dependent neutral endopeptidases named matrix metalloproteinases (MMPs) and they are capable of degrading and disrupting most components of the extracellular matrix (ECM). Therefore MMPs play an important role in matrix remodeling, repairing and destroying. Recent evidence has indicated that MMPs are involved in the pathogenesis of neurodegenerative diseases as Alzheimer's disease and multiple sclerosis. On the other hand, in cancer, MMPs encourage tumor cells to penetrate the surrounding tissue during the invasive process of metastasis and can mediate many processes in tumor growth and progression. Haloperidol and valproic acid have been used to treat psychotic diseases clinically. These two drugs have been shown to exert anti-inflammatory effects. Therefore, we hypothesized that haloperidol and valproic acid inhibited the inflammation-induced abnormal remodeling. We used tumor necrosis factor-? (TNF-?) and lipopolysaccharide (LPS) as a stimulus, that induced expression of MMP-9 in human monocytic THP-1 cells. According to gelatin zymography method, we found that the expression and activation of MMP-9 protein induced by TNF-? were inhibited by haloperidol and valproic acid in a concentration-dependent way. We also found that the inhibitory effect of haloperidol and valproic acid were not due to impairment of cellular viability measured by MTT assays. According to Western blot analysis, we observed that the inhibition on TNF-?-induced expression of MMP-9 protein by haloperidol and valproic acid is concentration-dependent. Furthermore, we found at higher concentration of haloperidol at 20 ?M could significantly reduced TIMP-1 proteins measured by enzyme-linked immunosorbent assay (ELISA). Besides, we even more found that haloperidol suppressed MMP-9 activation and the expression of MMP-9 protein induced by LPS in a dose dependent way. In transcription level, haloperidol also suppressed the TNF-?-induced MMP-9 mRNA expression measured by RT-PCR. According to the previous experiments, we observed that haloperidol was more potent than valproic acid (haloperidol IC50：5.4 ± 0.3 ?M；valproic acid IC50：255.8 ± 73.8 ?M). We found that haloperidol significantly inhibited the degradation of total inhibitor-?B-? (I?B?? induced by TNF-??or LPS in Western blot analysis. In the nuclear aspect, we found that at 10 ?M significantly inhibited TNF-?-induced NF-?B translocation and activation measured by EMSA. In MAPKs aspect, the concentration of haloperidol at 20 ?M significantly suppressed JNK expression in TNF-? stimulation. Furthermore, we found haloperidol did not influence the LPS-induced production of TNF-? in the medium of THP-1 cells. In summary, we found that haloperidol had inhibitory effect on MMP-9 expression and activation in THP-1 cells. Its main mechanism of action might be through NF-?B signal pathway on TNF-??and LPS stimulation. It will be interesting to do further studies and investigation on haloperidol as therapeutic targets on inflammatory research in vivo.

並列關鍵字

MMP9 ； Haloperidol ； THP-1

參考文獻

Akamine, T., Nishimura, Y., Ito, K., Uji, Y., Yamamoto, T. Effects of haloperidol on K+ currents in acutely isolated rat retinal ganglion cells. Invest. Ophthalmol. Vis. Sci. 43, 1257-1261(2002).

Anthony, D.C., Miller, K.M., Fearn, S., Townsend, M.J., Opdenakker, G., Wells, G.M., Clements, J.M., Chandler, S., Gearing, A.J., Perry, V.H. Matrix metalloproteinase expression in an experimentally-induced DTH model of multiple sclerosis in the rat CNS. J. Neuroimmunol. 87, 62-72(1998).

Asahi, M., Wang, X., Mori, T., Sumii, T., Jung, J.C., Moskowitz, M.A., Fini, M.E., Lo, E.H. Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood-brain barrier and white matter components after cerebral ischemia. J. Neurosci. 21, 7724-7732(2001).

Ashkenazi, A. Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat. Rev. Cancer. 2, 420-430(2002).

Avent, K.M., Riker, R.R., Fraser, G.L., Van der Schyf, C.J., Usuki, E., Pond, S.M. Metabolism of haloperidol to pyridinium species in patients receiving high doses intravenously: is HPTP an intermediate? Life Sci. 61, 2383-2390(1997).

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探討Haloperidol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作用機轉

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