植物性毒素馬兜鈴酸對於脂多醣所誘發的細胞發炎反應之抑制機轉

Inhibition of LPS-Induced Cellular Inflammation by Phytotoxin Aristolochic Acid

陳怡孜(I-Tzu Chen)

指導教授 ： 劉秉慧

中山醫學大學/醫學科技學院/生物醫學科學學系碩士班/碩士(2008年)

https://doi.org/10.6834/CSMU.2008.00105

摘要

馬兜鈴酸 (aristolochic acid，簡稱 AA) 是由馬兜鈴科植物中萃取出來的天然成份之一，主要由 aristolochic acid I (AAI) 及 aristolochic acid II (AAII) 組合而成，目前馬兜鈴酸已被證實對人類和鼠類具有嚴重的腎毒性與致癌性，但在早期研究中卻指出馬兜鈴科植物的萃取物可用於治療關節炎及風濕病，隨後也發現馬兜鈴酸能夠提高吞噬細胞的活性並具有抗發炎的特性，因此在本研究中我們專注於馬兜鈴酸在細胞株中的抗發炎作用，並且進一步探討馬兜鈴酸的作用機轉。
我們首先以脂多醣 (lipopolysaccharide，簡稱 LPS) 刺激老鼠白血球的單核球巨噬細胞 (RAW 264.7 cells) 作為發炎的模式，當 LPS 刺激 RAW 264.7 細胞時，細胞內 NO 的生成量、iNOS 的訊息 RNA (mRNA) 及蛋白質的表現量皆大量增加，接著以不同濃度的馬兜鈴酸處理已受到 LPS 刺激的 RAW 264.7 細胞，隨著馬兜鈴酸處理劑量的提高，LPS 所誘發的 NO 生成量、iNOS 蛋白質及 iNOS mRNA 量都有逐漸下降的趨勢，由於馬兜鈴酸的濃度即使提高至 100 μM 也不會造成細胞死亡率的明顯上升，所以馬兜鈴酸是在沒有細胞毒性的情況下有效抑制細胞的發炎反應。
已知 iNOS 基因的表現主要與細胞內一些轉錄因子的作用機制具有相關性，因此進一步探討馬兜鈴酸是否會影響這些轉錄因子來抑制 iNOS 基因的轉錄作用。在活體外我們藉由 Electrophoretic Mobility Shift Assays (EMSA) 發現 LPS 能夠有效誘發 NF-κB 蛋白質結合至 NF-κB 的 DNA 結合序列上，而且馬兜鈴酸的存在並不會抑制 NF-κB 的 DNA 結合能力；若是以含有 NF-κB、AP-1、ISRE 及 GAS DNA 結合序列的 luciferase 報導基因質體進行細胞內的 Luciferase reporter assay，我們發現 luciferase 的活性也不會受到馬兜鈴酸的作用而下降。此外，馬兜鈴酸不會影響 LPS 所誘發之 STAT-1 蛋白質的磷酸化表現量，而且也不能顯著地減少 NF-κB 由細胞質進入細胞核的轉移作用，但是高濃度的馬兜鈴酸卻能夠顯著地降低 NF-κB 上游抑制者 I-κB 的磷酸化表現量。
為了進一步確認馬兜鈴酸抑制 iNOS 基因轉錄作用的機轉，我們分別建構由起始點往前算含有 1588 bp、1008 bp 及 360 bp iNOS promoter 序列的 luciferase 報導者基因質體，結果發現馬兜鈴酸皆能顯著地降低不同片段長度之 iNOS promoter 對於下游基因的驅動活性，因此推論馬兜鈴酸的抑制機制可能主要作用於 360 bp iNOS promoter 的某段未知 DNA 序列上，進而負向調節下游基因的表達。

關鍵字

馬兜鈴酸 ； 脂多醣 ； 誘發性一氧化氮合成酶

並列摘要

Aristolochic acid (AA) is a group of natural compounds widely found in Artistolochia species and is mainly composed of aristolochic acid I (AAI) and aristolochic acid II (AAII). Recently AA is found to be nephrotoxic and carcinogenic to humans and rodents. However, the extract of Artistolochia serving as Chinese herbal medicine has been commonly used to treat arthritis and rheumatism for hundreds of years. In addition, AA compound has the ability to increase phagocytic activity of phagocytes and has potential anti-inflammatory properties. In the present study, we focused on the anti-inflammatory effect of AA on the rat cell line (RAW 264.7) and further studied its reaction mechanism.
Lipopolysaccharide (LPS) was used to stimulate RAW 264.7 macrophages to generate a cellular inflammation model. When RAW 264.7 macrophages were treated with LPS, the levels of nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) mRNA and protein expression were all increased. Co-treatment of RAW 264.7 macrophages with LPS and various concentrations of AA led to the down-regulation of LPS-induced NO, iNOS protein and mRNA levels in a concentration-dependent manner. Since AA above 100 μM showed no significant effect on the cellular viability of RAW 264.7, the AA concentration we used can efficiently inhibit LPS-induced cellular inflammation without showing cellular toxicity.
The induction of iNOS gene expression is known to be correlated with the regulation of various transcription factors, so both in vitro and in vivo assays were conducted to investigate if AA can affect the DNA binding activity of these transcription factors. By the in vitro Electrophoretic Mobility Shift Assays (EMSA), we found that exposure of LPS-treated cells to AA did not block the NF-κB DNA binding activity mediated by LPS. By using Luciferase reporter assay in the cellular experiment, we observed that the AA treatment did not alter the luciferase activities of reporter plasmids harboring NF-κB, AP-1, ISRE or GAS DNA binding sequences in front of luciferase gene. Besides, the presence of AA showed no influence on the status of LPS-induced STAT-1 phosphorylation and did not significantly inhibit the translocation of NF-κB from cytoplasm to nucleus. However, high concentrations of AA could obviously suppress the phosphorylation of I-κB induced by LPS; I-κB protein is known to be an inhibitor of NF-κB.
To further confirm that AA is able to decrease iNOS gene transcription, three luciferase reporter plasmids containing 1588 bp, 1008 bp or 360 bp iNOS promoter sequence from the starting site were constructed. The results demonstrated that AA could apparently decrease the LPS-induced luciferase activities of all the constructed plasmids, suggesting that the inhibitory effect of AA may be associated with an unknown DNA sequence within the 360 bp iNOS promoter.

並列關鍵字

aristolochic acid ； lipopolysaccharide ； inducible nitric oxide synthase

參考文獻

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