STATIN及QUERCETIN在巨噬細胞及微神經膠細胞調節發炎相關基因表現及活化未折疊蛋白反應之訊息傳遞研究

Molecular mechanisms for statin and quercetin in the regulation of inflammation-related genes and activation of unfolded protein response in macrophages and microglial cells

陳瑞青(Jui-Ching Chen)

指導教授 ： 林琬琬

國立臺灣大學/醫學院/藥理學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.02799

摘要

HMG-CoA reductase 抑制劑 statins 可以有效抑制膽固醇的合成，且被廣泛用於治療心血管疾病。除了用於治療心血管疾病與降血脂外，近來有研究報導指出，statins 在臨床治療上仍具有多重治療效果。statins 的多重治療效果並不單純因藥物本身能降低血液中的膽固醇含量，而主要是statins 的作用機轉在於能抑制與Ras、Rho、Cdc42、Rac 等小分子G 蛋白質有關之訊息傳導路徑，進而有效抑制發炎作用。流行病學研究顯示，富含類黃酮的食物會降低罹患發炎等慢性疾病之機率。Quercetin 乃為廣泛存在於蔬菜水果的類黃酮，諸多報導指出Quercetin 可能是具有生物活性的膳食成分，可能藉由作為抗氧化劑或影響其他訊息傳遞而發揮作用。在本論文中我們分成五個研究主題，研究statins 在巨噬細胞調控與發炎反應相關的基因表現及訊息傳遞作用機轉，也研究Quercetin 在微神經膠細胞中對於iNOS及HO-1 的基因表現調控機制。希望藉由這些研究結果能更清楚闡述statins 及Quercetin 的抗發炎作用的機制與提供將來臨床應用的學理依據。
　　首先我們探討statins 在巨噬細胞內對誘導型一氧化氮合成酶﹙iNOS﹚基因表現的調節作用。本實驗使用lovastatin、pravastatin、atorvastatin 與fluvastatin，來探討它們在鼠類RAW 264.7 巨噬細胞中對NO 形成之影響。給予巨噬細胞LPS 與IFN-γ 刺激會促進iNOS 表現與大量NO 之生成，給予不同statins﹙0.1-30 μM﹚能對該刺激產生有效地抑制作用。上述的影響是作用在基因轉錄層級，藥物的抑制程度依序為︰ lovastatin ＞ atorvastatin ＞ fluvastatin ＞ ＞ pravastatin 。研究發現lovastatin 主要是抑制在經由LPS 刺激所活化的IKK/NF-κB 與IFN-γ 刺激所磷酸化的STAT1 訊息傳導路徑。再者，statins 的抑制作用可以被mevalonate、FPP、GGPP所阻斷。該實驗結果顯示statins 可能影響蛋白質isoprenylation、而抑制調控iNOS 基因表現所需訊息傳遞路徑中NF-κB 與STAT1 的活化。研究進一步發現：給予RAW 264.7 巨噬細胞statins﹙1-30 μM﹚可以誘導乙型 環氧化酶﹙COX-2﹚基因轉錄作用與PGE2 的生成，作用強度依序為︰lovastatin＞fluvastatin＞atorvastatin＞＞pravastatin。利用COX-2 基因啟動子活性分析我們發現Statins 的作用是在COX-2 基因啟動子的CREB 與C/EBPβ 結合位置，並非NF-κB結合位置。Mevalonate、FPP、GGPP 均能有效抑制statins 所活化的COX-2 基因轉錄、誘導COX-2 蛋白表現與促進PGE2 生成等作用。Manumycin A﹙Ras 抑制劑﹚與geranylgeranyltransferase 抑制劑也具有與statins 類似的作用，皆能夠誘導COX-2蛋白表現與促進PGE2 生成。其中又以toxin B﹙Rho 蛋白抑制劑﹚作用最為顯著。實驗結果亦顯示statins 的作用與tyrosine kinase、PKC、ERK、p38 MAPK 激酶之活化息息相關。總體而言，statins 可藉由促進MAPK 激酶活化，經由CREB 與C/EBPβ 啟動子結合位置來誘導COX-2 基因轉錄，同時在此過程中小分子G 蛋白扮演了負向調控角色。
　　甲型血基質氧化酶﹙HO-1﹚不只是影響血基質代謝的決定酵素，也提供細胞保護作用來對抗氧化性傷害與維持體內組織的恆定。因具有上述的防衛機制，使得「HO-1 的調控」被認為是一種極可能的切入點，將其運用到臨床上來治療因細胞凋亡、發炎反應、氧化作用或動脈粥狀硬化所造成的疾病。實驗結果顯示lovastatin、fluvastatin、atorvastatin、simvastatin、mevastatin 與pravastatin 能夠誘導HO-1 mRNA 表現。Lovastatin 的作用可以被FPP、GGPP、PKG 抑制劑﹙KT5823﹚、sGC 抑制劑﹙ODQ﹚、PKC 抑制劑﹙Ro31-8220 與GF109203X﹚、p38 MAPK 抑制劑﹙SB203580﹚與MEK 抑制劑﹙U0126 與PD98059﹚所阻斷，而不受PKA、JNK與ROCK 抑制劑所影響。實驗證實在鼠類巨噬細胞中statins 可活化ERK 與p38MAPK。實驗進ㄧ步發現statins 可經由cGMP/PKG/ERK 的訊息傳遞路徑誘導AP-1﹙HO-1 基因表現的重要轉錄因子﹚活化。此外，Ras 抑制劑﹙manumycin A﹚也可誘導相當程度的HO-1 mRNA 與蛋白表現；相反地，Rho 蛋白抑制劑﹙toxin B﹚只能促進短暫且微弱的引起HO-1 表現。Manumycin A 誘導HO-1 基因表現的訊息傳遞路徑與p38 MAPK、JNK 與ERK 活化有關。總結來說，本實驗結果是第一個證實statins 可經由調控PKG 來促進ERK 與p38 MAPK 活化，進而誘導HO-1 基因表現，該作用也為 statins 的臨床治療用途提供一種全新的抗發炎機轉。
　　GRP78 為主要的內質網伴隨蛋白之一，可提供細胞保護作用，以抵抗過度內質網壓力造成的細胞死亡。由於內質網壓力造成的巨噬細胞死亡，會使動脈粥狀硬化更趨嚴重，我們因而想了解statins 除了可以有效降低脂質含量外，是否也對未折疊蛋白反應﹙unfolded protein response，UPR﹚及其相關訊息傳遞路徑有所影響。給予RAW 264.7 巨噬細胞statins﹙3-30 μM﹚可以誘導GRP78、ATF6、XBP1與磷酸化的eIF2α 表現，而不影響ATF4、CHOP 與細胞死亡的發生。實驗結果顯xii示：1.statins 誘導GRP78 基因轉錄作用與c-Src、PI turnover、PKC、ERK與p38 MAPK有關。2.statins 可藉由調控鈣離子與抑制小分子G 蛋白作用這兩條獨立卻具有協同作用的訊息傳遞路徑，活化c-Src/PI-PLC/PKC，進而活化ERK 與p38 MAPK。3.statins 可誘導GRP78 表現來保護缺氧造成的細胞死亡，而statins 的這種細胞保護作用可被GRP78 small interfering RNA 所阻斷。4.statins 也可在內質網壓力造成腎臟病變之小鼠中提供類似的保護好處。實驗結果提供statins 一個與降低脂質含量完全無關的全新作用機轉，無論是在體外實驗模式或活體實驗模式，均能誘導具有細胞保護作用的未折疊蛋白反應。實驗結果更為臨床上運用statins 治療動脈粥狀硬化疾病提供新理論基礎。
　　另ㄧ方面，我們研究Quercetin﹙分布比例最高的類黃酮﹚與其主要代謝物Quercetin-3’-sulfate，在BV-2 微神經膠細胞中對於給予LPS 與IFN-γ 刺激，所誘導的NO 生成之影響。Quercetin 主要是抑制經由LPS 刺激所活化的IKK/NF-κB 與AP-1；與抑制經由IFN-γ 刺激所誘導的STAT1 與IRF-1 的活化以產生有效地抗發炎作用，然而Quercetin-3’-sulfate 不具抑制效果。再者Quercetin 也可以誘導HO-1表現。本實驗利用HO-1 反義質體以釐清HO-1 與iNOS 二者關係。HO-1 反義質體可以部分阻斷Quercetin 抑制NO 生成與iNOS 蛋白表現的作用；相反的，HO-1 誘導劑hemin 則具有與Quercetin 相同的抑制iNOS/NO 作用。Quercetin 誘導HO-1基因表現的訊息傳導路徑與tyrosine kinase 及MAPK 激酶活化有關。上述研究結果顯示Quercetin 能夠抑制造成神經損害之發炎作用，因此具有治療神經退化性疾病的應用價值。

關鍵字

STATIN ； Quercetin ； 發炎反應 ； 巨噬細胞 ； 未折疊蛋白反應

並列摘要

The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, statins, are potent inhibitors of cholesterol synthesis and have wide therapeutic use incardiovascular diseases. Except in coronary artery disease and hyperlipidemia management, increasing studies have demonstrated the pleiotropic benefits of statins in potential clinical uses. Some of these pleiotropic effects of statins are not attributed solely to their lipid lowering properties, but to some anti-inflammatory benefits, which are related to the inhibition of signaling pathways mediated by small G proteins (Ras, Rho, Cdc42, Rac etc.). Currently, the molecular mechanisms underlying theanti-inflammatory actions of statins are unclear and required further investigation.
Epidemiologic studies also demonstrated that foods rich in flavonoids can reduce the risk of chronic disease including inflammation. Quercetin, a flavonoid, is found ubiquitously in the vegetables and fruits, and may be a useful bioactive compound of the human diet. In the present study, we investigated the effects and action mechanisms of statins on multiple gene expressions in macrophages in order to elucidate their pleiotropic benefits relating to clinical therapy. In addition, anti-inflammatory mechanisms of quercetin such as regulation of HO-1 and iNOS gene expression in microglia were demonstrated.
In the first part, we investigated the effects of lovastatin, pravastatin, atorvastatin and fluvastatin on macrophage’s formation of NO in murine RAW 264.7 cells. Stimulation of macrophages with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) resulted in iNOS expression, which was accompanied by a large amount of NO formation. Within concentrations of 0.1-30 μM, statins can inhibit stimuli-induced NO formation and iNOS induction to ifferent extents. This inhibition occurred at the transcriptional level, and displayed potencies as ovastatin > atorvastatin > fluvastatin >> pravastatin. We found that LPS-induced IKK and NF-κB activation, as well as IFN-γ-induced STAT1 phosphorylation, were reduced by lovastatin. Moreover, inhibitions by lovastatin of NO production and NF-κB activation were reversed by mevalonate, FPP and GGPP. All these results suggest that inhibition of iNOS gene expression by statins can be attributed to the interference with protein isoprenylation, leading to uncoupling NF-κB and STAT1 activation in the upstream signaling pathways required for iNOS gene transcription.
In the second part, we investigated the effects of statins on COX-2 gene induction in murine RAW 264.7 macrophages. We found that statins within 1-30 μM stimulated COX-2 gene transcription and PGE2 formation, displaying potencies as lovastatin > fluvastatin > atorvastatin >> pravastatin. Transfection experiments with COX-2 promoter construct showed the necessity of C/EBPβ and CRE promoter sites, but not NF-κB promoter site. Effects of statins on the activation of COX-2 promoter, induction of COX-2 protein and PGE2 production were all prevented by mevalonate and prenylated metabolites, FPP and GGPP. In consistent with the effects of statins, manumycin A (a Ras inhibitor), farnesyltransferase inhibitor, and eranylgeranyltransferase inhibitor increased PGE2 production and COX-2 induction.
Likewise, toxin B, an inhibitor of Rho family members, caused a significant COX-2 induction. Results also indicated that tyrosine kinase, PKC, ERK, and p38 MAPK play essential roles in statin-mediated COX-2 induction. Taken together, these results not only demonstrate a unique action of statins in the upregulation of COX-2 expression in macrophages, but also suggest a negative role controlled by small G proteins in COX-2 gene regulation. Removing this negative regulation by impairing G protein prenylation with statins leads to MAPKs activation and promotes COX-2 gene transcription through CRE and C/EBPβ sites.
In the third part, we investigated the effects of statins on the anti-inflammatory gene heme oxygenase-1 (HO-1) expression in RAW 264.7 macrophages. HO-1 is the rate-limiting enzyme in heme catabolism, which confers cytoprotection against oxidative injury and provides a vital function in maintaining tissue homeostasis. This important defense mechanism makes it conceivable to target HO-1 induction as a promising therapeutic intervention in treating a variety of disorders related to cell apoptosis, inflammation, oxidation and atherosclerosis. We showed that lovastatin, fluvastatin, atorvastatin, simvastatin, mevastatin and pravastatin are able to up-regulate the mRNA expression of HO-1 gene. This effect of lovastatin was attenuated by FPP, GGPP, PKG inhibitor (KT5823), soluble guanylyl cyclase (sGC) inhibitor (ODQ), PKC inhibitors (Ro31-8220 and GF109203X), p38 MAPK inhibitor (SB203580), and MEK inhibitors (U0126 and PD98059), but not by inhibitors of PKA, JNK and Rho kinase.
Consistent with this notion, we found the ability of statins to activate ERK and p38 MAPK. Moreover, we demonstrated the participation of cGMP/PKG pathway for ERK activation in cells stimulated with statin, and the ability of statin to induce AP-1 activity, which is an essential transcription factor in the regulation of HO-1 gene expression. In addition, manumycin A treatment also caused a marked induction of HO-1 mRNA followed by a corresponding increase in HO-1 protein; instead, inhibition of Rho activity by toxin B only led to a transient and weak induction of HO-1. The involvement of signal pathways in manumycin A-induced HO-1 gene expression was associated with p38 MAPK, JNK and ERK activation. Taken together, these results demonstrate for the first time that PKG is an intermediate player for statins to activate ERK and p38 MAPK pathways and further induce HO-1 gene expression. HO-1 induction by statins provides a novel anti-inflammatory mechanism in the therapeutic validity.
In the fourth part, we investigated the effects of statins on 78 kDa glucose-regulated protein (GRP78) gene expression. GRP78 is a major ER chaperone whose induction by ER stress confers cytoprotection against cell death. Since ER stress-induced macrophage apoptosis contributes to advanced atherosclerotic lesions, we sought to understand the effects of statins on unfolded protein response (UPR) and the signaling cascades underlying statins’ action. Exposure of murine RAW 264.7 macrophages to statins (within 3-30 μM) increased expressions of GRP78, ATF6, XBP1, and phosphorylated eIF2α, while no effects on ATF4, CHOP and cell death. Promoter activity measurement with inhibitors indicated that phosphoinositide (PI) turnover, c-Src, PKC, ERK and p38 MAPK involve in upregulation of GRP78 gene transcription by statins. We observed that increased intracellular Ca2+ level and interruption of a small G protein pathway are two bifurcated but cooperative signaling pathways for c-Src, leading to downstream activation of phospholipase C, PKC, ERK and p38. We further demonstrated that statins protected hypoxia-mediated cell death via GRP78 induction.
Correspondingly, the downregulation of GRP78 via small interfering RNA approach decreased statins’ cytoprotection in hypoxia. Statins also conferred similar prot ective benefits on ER stress-induced kidney failure in mice model. Collectively, these results demonstrated a novel action of statins, independently of lipid lowering activity, to induce cytoprotective UPR response in vitro and in vivo. These findings provide new
insights into statins for their clinical benefits in atheroslcerosis.
In the final part, experiments were performed to explore the action of quercetin, the most widely distributed flavonoids, and its major in vivo metabolite,
quercetin-3’-sulfate, on LPS- and IFN-γ-induced NO production in BV-2 microglia. We found that quercetin could suppress LPS- and IFN-γ-induced NO production and iNOS gene transcription, while quercetin-3’-sulfate had no effect. LPS-induced IKK, NF-κB and AP-1 activation, and IFN-γ-induced NF-κB, STAT1 and IRF-1 activation were reduced by quercetin. Moreover quercetin was able to induce HO-1 expression. To address the involvement of HO-1 induction in iNOS inhibition, HO-1 antisense oligodeoxynucleotide was used. Quercetin-mediated inhibition of NO production and iNOS protein expression were partially reversed by HO-1 antisense oligodeoxynucleotide, and was mimicked by hemin, a HO-1 inducer. The involvement of signal pathways in quercetin-induced HO-1 gene expression was associated with tyrosine kinase and MAPKs activation. All these results suggest that quercetin should provide therapeutic benefits for suppression of inflammatory-related neuronal injury in neurodegenerative diseases.

並列關鍵字

STATIN ； Quercetin ； inflammation ； macrophage ； UPR

參考文獻

Alam, J., Stewart, D., Touchard, C., Boinapally, S., Choi, A. M., and Cook, J. L. (1999). Nrf2, a Cap'n'Collar transcription factor, regulates induction of the heme oxygenase-1 gene. J Biol Chem 274, 26071-26078.

Alam, J., Wicks, C., Stewart, D., Gong, P., Touchard, C., Otterbein, S., Choi, A. M., Burow, M. E., and Tou, J. (2000). Mechanism of heme oxygenase-1 gene activation by cadmium in MCF-7 mammary epithelial cells. Role of p38 kinase and Nrf2 transcription factor. J Biol Chem 275, 27694-27702.

Aliev, G., Smith, M. A., Turmaine, M., Neal, M. L., Zimina, T. V., Friedland, R. P., Perry, G., LaManna, J. C., and Burnstock, G. (2001). Atherosclerotic lesions are associated with increased immunoreactivity for inducible nitric oxide synthase and endothelin-1 in thoracic aortic intimal cells of hyperlipidemic Watanabe rabbits. Exp Mol Pathol 71, 40-54.

Álvarez de Sotomayor, M., and Andriantsitohaina, R. (2001). Simvastatin and Ca2+ signaling in endothelial cells: involvement of Rho protein. Biochem Biophys Res Commun 280, 486-490.

Amin-Hanjani, S., Stagliano, N. E., Yamada, M., Huang, P. L., Liao, J. K., and Moskowitz, M. A. (2001). Mevastatin, an HMG-CoA reductase inhibitor, reduces stroke damage and upregulates endothelial nitric oxide synthase in mice. Stroke 32, 980-986.