紅麴發酵代謝產物 monascin 藉由調控免疫反應及減緩氧化性損傷以改善呼吸道發炎

Monascus-fermented metabolite monascin alleviates airway inflammation by modulating immune responses and attenuating oxidative injury

徐瑋萱(Wei-Hsuan Hsu)

指導教授 ： 潘子明

國立臺灣大學/生命科學院/生化科技學系/博士(2013年)

https://doi.org/10.6342/NTU.2013.10309

摘要

氧化壓力或外來抗原的刺激使呼吸道抗氧化能力失衡及發炎介質釋放，造成發炎現象，呼吸道發炎反應為氣喘等呼吸道疾病之主要症狀。紅麴發酵產物廣泛應用於亞洲，其二級代謝產物具備數種生理功效。本研究室先前研究證實紅麴黃色素 monascin 可改善數種與氧化壓力及/或發炎反應相關之疾病，推測 monascin 應具抗氧化及抗發炎等生理活性，因而具改善呼吸道發炎反應之潛力。首先，以單核球細胞評估 monascin 的抗發炎能力，並探討其詳細的分子調控機轉。接著，依據呼吸道發炎疾病相關之細胞種類，設計不同的細胞及動物模式，包含肺細胞、免疫細胞及 BALB/c 小鼠，以探討 monascin 改善呼吸道發炎疾病之可能性，分析其所扮演之角色及作用機制，並釐清 monascin 能否透過調控免疫反應及抗發炎潛力進而改善呼吸道發炎症狀。
在單核球 THP-1 細胞模式中，monascin 顯著減少因抗原刺激而造成之發炎介質大量產生，包含抑制誘導型一氧化氮合成酶 (inducible nitric oxide synthase, iNOS) 及環氧合酶 (cyclooxygenase-2, COX-2) 之表現及減少一氧化氮 (nitric oxide, NO) 及前列腺素 E2 (prostaglandin E2, PGE2) 之生成。同時，monascin 降低抗原誘導 THP-1 細胞於 mRNA 及蛋白質層次之發炎細胞激素 tumor necrosis factor-α (TNF-α) 及 interleukin 6 (IL-6) 的產生。Monascin 抑制 THP-1 細胞因抗原誘導而引起之 mitogen-activated protein kinase (MAPK) c-Jun NH2-terminal kinase (JNK) 磷酸化，但對 extracellular signal-regulated kinase (ERK) 及 p38 kinase 之活性則不具影響。進一步證實 monascin 係透過提升過氧化體增生劑活化接受器 (peroxisome proliferator activated receptor-γ, PPAR-γ) 蛋白而抑制 JNK 的活化，進而阻止發炎介質及細胞激素的釋放，因而能降低抗原引發之發炎反應，並以 PPAR-γ 拮抗劑 GW9662 印證此觀點。
為評估 monascin 保護肺部之潛力，以肺上皮細胞 A549 探討 monascin 對於氧化壓力誘導之肺細胞損傷的改善效果。Monascin 減緩過氧化氫 (hydrogen peroxide, H2O2) 引起 A549 細胞胞內活性氧 (reactive oxygen species, ROS) 之生成，並抑制氧化壓力造成之 protein kinase C (PKC) 及 JNK 磷酸化，進而抑制發炎轉錄因子 nuclear factor (NF)-κB 轉錄至細胞核中，於是降低黏附因子之表現。DNA binding assay 及 luciferase assay 證實 monascin 透過活化 PPAR-γ 而具抗發炎能力，並以 PPAR-γ siRNA 印證 monascin 之抗發炎活性係藉由調控 PPAR-γ 而達到。此外，monascin 提升核因子-紅血球之 2 相關因子-2 (nuclear factor-erythroid 2 related factor 2, Nrf-2) 活性，進而促進 Nrf-2 下游抗氧化酵素之表現及 PPAR-γ 之轉錄，因而能提升肺細胞之抗氧化能力而能抵抗氧化壓力造成之損傷。將 H2O2 以鼻腔注射之方式誘導小鼠肺部損傷，觀察到小鼠肺部有發炎細胞的浸潤，且釋放大量發炎細胞激素，氧化壓力的誘導使小鼠肺部黏附因子表現量上升，因而促使免疫細胞趨化 (recruitment) 至肺部，尤以巨噬細胞最為顯著。而 monascin 抑制黏附因子表現及免疫細胞之浸潤，進而抑制發炎細胞激素的產生，同時促進抗氧化酵素之含量，以提升小鼠之抗氧化及抗發炎能力，因而減緩氧化壓力誘發之肺組織的發炎反應。給予小鼠 siRNA 抑制 PPAR-γ 及 Nrf-2 表現，則 monascin 保護肺部的能力下降，顯示 monascin 藉由調控 PPAR-γ 及 Nrf-2 達到減緩肺部損傷之效果。
以雞卵蛋白 (ovalbumin, OVA) 誘導小鼠呼吸道發炎，探討 monascin 之改善潛力，以樹突細胞作為模式，評估 monascin 是否能影響樹突細胞之成熟與細胞激素之分泌，並以 T 細胞評估 monascin 之免疫調節能力，以釐清 monascin 於呼吸道發炎反應中扮演之角色。Monascin 顯著減緩過敏原誘導之小鼠肺部黏液細胞增生及嗜酸性球浸潤，並減少肺沖洗液中 Th2 細胞激素之釋放。在樹突細胞中，monascin 抑制 OVA 誘導之共同刺激分子 CD40、CD80 及 MHCII 表現。與 OVA 處理組相較，monascin 避免樹突細胞吞噬能力的降低及細胞激素釋放，顯示 monascin 具潛力抑制樹突細胞的成熟。於 EL-4 T 細胞模式中，monascin 顯著抑制 phorbol 12-myristate 13-acetate (PMA)/ionomycin 引起之 Th2 細胞激素產生，monascin 促進 PPAR-γ 結合至 DNA 上並引發其轉錄，且以PPAR-γ 拮抗劑處理能阻止 monascin 抑制 Th2 細胞激素生成之能力。Monascin 亦藉由避免 PKC 及訊息傳遞及轉錄活化因子 6 (signal transducer and activator of transcription 6, STAT6) 磷酸化而抑制 Th2 轉錄因子 GATA-3 及活化 T 細胞核因子 (nuclear factor of activated T cells, NF-AT) 之轉錄作用。

關鍵字

紅麴發酵代謝產物 ； monascin ； 呼吸道發炎 ； 免疫調節 ； 氧化性損傷

並列摘要

Oxidative stress and antigen stimulation cause imbalance on antioxidation and release of inflammatory mediators in the airway, resulting in inflammatory responses. Airway inflammation is the primary symptom for airway diseases such as asthma. Monascus-fermented secondary metabolites offer valuable therapeutic benefits and have been extensively used in East Asia. Our research group has reported that yellow pigment monascin derived from Monascus-fermented products has therapeutic effect on several oxidative stress- and/or inflammation-related diseases. We presumed that monascin has physiological potential on antioxidation and antiinflammation to improve airway inflammatory responses. First, we used monocytes to evaluate the effect of monascin on anti-inflammation and investigate its molecule mechanism. Further, according to the cell types involved in the airway inflammation, we designed different cell and animal models, including lung cells, immune cells, and BALB/c mice, to investigate the potential role of monascin on attenuating airway inflammatory diseases and its mechanism of action. We also clarify whether monascin could improve airway inflammation through regulating immune responses and anti-inflammatory ability.
In THP-1 monocyte model, monascin significantly attenuated several proinflammatory mediators, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression as well as nitric oxide (NO) and prostaglandin E2 (PGE2) formation caused by antigen stimulation. Further, monascin reduced the generation of tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) at both the protein and mRNA levels. Monascin decreased ovalbumin (OVA)-induced phosphorylation of mitogen-activated protein kinase (MAPK) c-Jun NH2-terminal kinase (JNK), but not that of extracellular signal-regulated kinase (ERK) or p38 kinase. We used the peroxisome proliferator activated receptor-γ (PPAR-γ) antagonist GW9662 to show that monascin inhibit JNK phosphorylation through increased expression of PPAR-γ.
We used lung epithelial A549 cell to investigate the effect of monascin on oxidative stress-induced lung injury. Monascin ameliorated hydrogen peroxide (H2O2)-induced endogenous reactive oxygen species (ROS) production in A549 cells. We found that monascin inhibited protein kinase C (PKC) and JNK phosphorylation and reduced transcription factor nuclear factor (NF)-κB translocation to the nucleus as well as decreased the expression of adhesion molecules. We used DNA binding assay, luciferase assay, and siRNA for PPAR-γ to demonstrate that monascin inhibited inflammation via activating PPAR-γ. Furthermore, monascin protect lung cell against oxidative injury through elevating Nrf-2 acitivity and promoting the expression of Nrf-2 downstream antioxidant enzymes and transcription of PPAR-γ. Mice were induced by intranasal inhalations of H2O2. Inflammatory cells infiltrated around the airways of H2O2-induced mice were noted on histopathology analysis, and inflammatory cytokines were released into bronchoalveolar lavage fluid. Adhesion molecules level were elevated in H2O2-induced lungs and caused immune cell recuitment, especially in macrophages. Monascin repressed the expression of adhesion molecules and the infiltration of immune cells to inhibit the generation of inflammatory cytokines. Monascin also elevated the level of antioxidant enzymes to accelerate antioxidative and antiinflammatory ability in the lung of mice. However, the inhibitory effect of monascin on oxidative stress-induced inflammation was abolished by using siRNA for PPAR-γ and Nrf-2, which means that monascin attenuated lung damage via regulating PPAR-γ and Nrf-2.
We evaluated the corrective potential of monascin on OVA-induced airway inflammation in mice, and used dendritic cell as the model to evaluate if monascin could influence the mature of dendritic cell inhibition, and cytokines production reduction. We also investigated the ability of monascin on immune regulation using T cells as a model to clarify the role of monascin on airway inflammatory responses. Monascin prevented eosinophilia and mucus cell hyperplasian in antigen-challenged mice markedly, and reduced Th2 cytokines generation in bronchial alveolar lavage fluid. In dendritic cells, monascin inhibited OVA-induced up-regulation of costimulatory molecules CD40, CD80, and MHCII on the cell surface. When compared with the OVA treatment group, monascin attenuated OVA-reduced the endocytotic capability and reduced levels of cytokines in supernatants of dendritic cells, revealing that monascin may has potential to inhibit dendritic cells maturation. We showed that monascin significantly inhibited the production of Th2 cytokines in phorbol 12-myristate 13-acetate (PMA)/ionomycin-activated mouse EL-4 T cells. Monascin promoted PPAR-γ-DNA interactions and cellular PPAR-γ translocation and suggest that the regulatory effects of monascin on Th2 cytokine production could be abolished with PPAR-γ antagonist treatment. Monascin also suppressed Th2 transcription factors GATA-3 and nuclear factor of activated T cells translocation by preventing the phosphorylation of PKC and signal transducer and activator of transcription 6 (STAT6).

並列關鍵字

Monascus-fermented metabolites ； monascin ； airway inflammation ； immunomodulation ； oxidative injury

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