Protective effects of diallyl sulfide on pulmonary oxidative damage and the underlying molecular mechanisms

The lung is constantly challenged with oxidative damage due to gas exchange and exposure to exogenous air pollutants. Oxidative stress-aggravated chronic inflammatory diseases of the airway are well documented. Although the lung has a well-developed antioxidant system to protect itself against exposure to endogenous or exogenous oxidants, excessive levels of ROS can still cause inflammation in the lung. Treatment with antioxidants to ameliorate oxidative stress might be an effective strategy to reduce airway complications. Diallyl sulfide (DAS), which is enriched in garlic, is one of the natural organosulfuric compounds that are beneficial to human health. The aim of this study was to evaluate the anti-inflammatory and immunomodulation effects and mechanism of DAS in pulmonary disorders. Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a redox sensitive transcription factor, which binds to an antioxidant response element (ARE) in the promoter region of genes encoding several phase-II detoxifying/antioxidant enzymes and related stress-responsive proteins. In this study, we first investigated the promotive role of DAS in lung by evaluating the effect of DAS on expressions of antioxidant enzymes in vivo and the Nrf2 modulation in vitro in normal condition. DAS at a dosage of 500 mg/kg body weight significantly increased the pulmonic total level of Nrf2 and nuclear translocation of Nrf2. The mRNA levels of heme oxygenase-1, catalase, superoxide dismutase, glutathione peroxidase and NAD(P)H:quinone oxidoreductase 1, and GSH/GSSG ratio were also elevated. DAS level was measured in the plasma after 7 days of oral administration, and the Cmax value was 15 ± 4.2 μM. Furthermore, DAS could induce nuclear translocation of Nrf2 via extracellular signal-regulated kinase (ERK)/p38 signaling pathway in human embryonic lung fibroblasts MRC-5 cells. Our results demonstrate that DAS administration can significantly induce antioxidant enzymes in rat lung, and gives scientific support to the possible use of DAS as a dietary preventive agent against oxidative stress-induced lung injury. Due to the inflammatory events can be inhibited via a reduction in oxidative stress by antioxidant enzymes, thus, we further investigated the inhibitory effect of DAS on TNF-α- or histamine induced inflammation in rat aortic smooth muscle A7r5 cells and explored the molecular mechanism underlying this inhibition. DAS significantly blocked the accumulation of the nuclear p65 protein in TNF-α-induced rat aortic smooth muscle A7r5 cells by attenuating TNF-α receptor complex through the dissociation of the TNF receptor-associated death domain and TNF receptor-associated factor 2. Moreover, DAS inhibited histamine-induced inflammation through decreasing ROS levels by enhancing the Nrf2-related antioxidative enzyme. DAS also inhibited inflammation by suppressing interleukin-1β and TNF-α through the inhibition of ROS-induced PI3K/Akt and the downstream nuclear factor-κB (NF-κB) and activator protein 1. Our results demonstrate that DAS is a potential phytochemical to inhibit TNF-α- and histamine-induced inflammation, suggesting that DAS might be an effective dietary agent for the prevention of oxidative stress-induced inflammation of the airway. We next investigated the protective effect and mechanism of DAS against ovalbumin (OVA)-induced allergic asthma in an animal model. DAS markedly decreased the number of infiltrated inflammatory cells and the levels of type 2 T-helper cell (Th2) cytokines in bronchoalveolar lavage (BAL) fluid. The OVA-specific immunoglobin E levels in sera were also reduced by DAS. The report of lung histopathological examinations showed that the OVA-induced inflammatory cell infiltration and mucus hypersecretion were inhibited by DAS administration. Also, increased generation of ROS, 8-iso-prostaglandin F2 α and 8-hydroxy-2'-deoxyguanosine in BAL fluid as well as NF-κB nuclear translocation in lungs, by inhalation of OVA was diminished by DAS. Moreover, DAS enhanced the OVA-induced downregulation of Nrf2 activation and antioxidant enzyme mRNA levels. We further found that DAS-modulated Nrf2 activation was through regulating microRNA-144, -34a and -34b/c. These findings indicate that the pathogenesis of OVA-induced bronchial asthma is highly associated with oxidative stress, and DAS may be an effective supplementation to alleviate the disease. Excessive levels of ROS impair the cellular redox level, abnormal production of collagen and induction of extracellular matrix (ECM) accumulation, which resulting in pulmonary fibrosis. We finally investigated the effect and molecular mechanism DAS on the inhibition of transforming growth factor-beta (TGF-β)-induced fibrosis in MRC-5 cells. The results showed that DAS significantly suppressed the production of collagen in TGF-β-induced MRC-5 cells by attenuating Sma and Mad homologue (Smad) 2/3 phosphorylation through enhancing HO-1 levels. Moreover, DAS inhibited TGF-β-induced myofibroblast formation by decreasing ROS through enhancing Nrf2-related antioxidative enzyme and by promoting myofibroblast apoptosis. Our results demonstrate that DAS is a potential phytochemical to inhibit TGF-β-induced fibrosis, which suggesting that DAS might be a dietary agent to prevent oxidative stress-induced pulmonary fibrosis. This study reveals for the first time that garlic organosulfuric compound possess the promoting effect of Nrf2 activation, and the immunomodulating effects. It suggests that DAS supplementation may be useful as an adjuvant therapy for the treatment of pulmonary disorder involved bronchial asthma and pulmonary fibrosis.肺臟是氣體交換之場所，常暴露於外來汙染物中，因此須有較強的抗氧化系統來抵禦氧化傷害。氣喘 (asthma) 為國內常見之呼吸道發炎疾病，氧化壓力 (oxidative stress) 上升被認為是導致肺臟與呼吸道發炎加劇的危險因子。大蒜 (garlic) 為常見之香辛料，廣泛被應用於抗菌及抗癌相關疾病，並證實貢獻來自於其中具抗氧化與抗發炎功效之活性成分：二丙烯基硫化合物 (diallyl sulfide, DAS)。本研究將進一步評估 DAS 抑制呼吸道發炎、氣喘與肺臟纖維化等肺臟氧化傷害之多重保護功效。 轉錄因子 nuclear factor-erythroid 2 related factor 2 (Nrf2) 能經轉錄作用而產生許多抗氧化物基因，進而減少發炎反應。本研究首先探討大蒜中 DAS 對於 Nrf2 及下游抗氧化酵素之影響與其機轉。結果顯示，連續七天餵食 DAS (500 mg/kg B.W. of rats) 能顯著活化 SD 大鼠肺臟組織中 Nrf2 蛋白表現，並可增加抗氧化酵素基因如 heme oxygenase-1 (HO-1)、catalase (CAT)、superoxide dismutase (SOD)、glutathione peroxidase (GPx)、NAD(P)H:quinone oxidoreductase 1 (NQO1) 等之表現與提升GSH/GSSG 比值。由 HPLC 測定大鼠血漿發現，連續七天餵食 DAS，其濃度於血漿中可達 15 μM。此外，在 人類肺臟纖維母細胞 (MRC-5) 中亦證實，DAS 能透過 extracellular signal-regulated kinase (ERK)/p38 路徑活化 Nrf2。本研究證實大蒜有機硫化物可以透過活化肺臟 Nrf2 以提升抗氧化酵素之表現，具有減緩呼吸道發炎疾病之潛力。 已知 DAS可以透過活化肺臟 Nrf2 以提升抗氧化酵素之表現，具有減緩呼吸道發炎疾病之潛力，因此，本研究進一步探討 DAS 對於發炎介質 tumor necrosis factor-alpha (TNF-α) 與 histamine 誘導大鼠胸大動脈平滑肌 A7r5 細胞中發炎相關轉錄因子 nuclear factor-κB (NF-κB) 之抑制效應。結果顯示，DAS 能抑制 TNF-α活化之 TNF-α receptor 複合體，降低 NF-κB 之活化；此外，DAS 亦能透過活化 Nrf2 而減少以 histamine 誘導之 reactive oxygen species (ROS) 生成，降低 phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) 蛋白磷酸化表現，進而減少 NF-κB 與 activator protein 1 之活化，以減緩發炎現象。結果證實 DAS可抑制 TNF-α receptor 複合體活化並提升 Nrf2的活性，顯示大蒜於氧化壓力引起之肺部疾病中具有良好之保護功效。 DAS 具有良好提升肺臟抗氧化系統並能減少平滑肌發炎之功效，因此，本研究亦探討 DAS 對於過敏原雞卵蛋白 (ovalbumin, OVA) 誘導小鼠過敏相關免疫球蛋白E (immunoglobin E, IgE) 與發炎相關轉錄因子 NF-κB 之抑制效應。研究結果顯示，連續餵食十天 DAS (100 mg/kg B.W. of mice) 能抑制血清中 IgE 抗體含量，降低發炎細胞浸潤至肺臟組織中，並平衡輔助型 T 細胞 1/2 (hepler T cells 1/2, Th1/2) 相關之細胞激素表現而減少過敏現象。DAS 亦能透過活化 Nrf2 與提升抗氧化酵素之表現，進而減少 NF-κB 之活化並減少肺臟中氧化壓力指標 (8-iso-prostaglandin F2 α 與 8-hydroxy-2'-deoxyguanosine)，以減緩發炎現象。此外，DAS 透過調控 micro-RNA (miR-144、miR-34a 與 miR-34b/c) 之表現，而促進 Nrf2 之活性以達抗發炎之功效。綜合上述結果，DAS可抑制免疫失衡並提升 Nrf2的活性，顯示大蒜於 OVA 引起之氣喘疾病中具有良好之保護功效。 許多肺臟疾病如氣喘及慢性阻塞性肺炎等，常常最終也是因肺臟纖維化，而無法繼續救治。因此，本研究最後探討 DAS 對於發炎介質 TGF-β (transforming growth factor-β) 誘導 MRC-5 細胞纖維化現象之保護效應。實驗結果發現介入 DAS 可活化 Nrf2 並提升 HO-1蛋白表現，抑制 TGF-β 所誘發 Sma and Mad homologue (Smad) 2/3 蛋白磷酸化，進而達到減少膠原蛋白之不正常生成。TGF-β 亦會增加 ROS 的生成，而造成肺臟細胞的損傷，進而增加肌纖維細胞之活化；而 DAS 能透過活化轉錄因子 Nrf2 而轉錄出許多抗氧化及解毒酵素，因而減少 ROS 之生成。此外，DAS 亦能透過促進活化的肌纖維細胞誘導其細胞凋亡，更有助於肺臟纖維化之回復。綜合上述結果，DAS可抑制 TGF-β/Smads pathway 並提升 Nrf2 的活性，顯示大蒜於氧化壓力引起之肺臟纖維化中具有良好之保護功效。 由本論文之實驗可充分闡明大蒜之抗發炎與免疫調節能力，與其中有機硫化合物 DAS 之功能息息相關。本研究除可提供 DAS 之抗發炎與免疫調節之詳盡科學根據外，也建立肺臟纖維化之體外模式，可供未來保健食品開發與治療輔助用劑之開發參考。全文摘要 (中文) I 全文摘要 (英文) IV 目次 VIII 圖次 XIV 表次 XVII 縮寫表 XVIII 前言 1 第一章、文獻回顧 3 壹、氧化壓力及免疫反應對氣喘之影響 5 一、自由基與氧化壓力 5 二、自由基、免疫反應與呼吸道發炎 5 三、過敏反應 (Allergy) 與氣喘 8 四、過敏反應 11 五、過敏性氣喘致病機轉 14 貳、生物有機體的抗氧化防禦機制 17 一、酵素性抗氧化物 (Enzymatic antioxidants) 17 二、非酵素性抗氧化物 (Non-enzymatic antioxidants) 18 三、Nuclear factor-erythroid 2 related factor 2 (Nrf2) 18 四、抗氧化系統與氣喘 19 參、肺臟纖維化與氣喘 21 一、肺臟纖維化 21 二、MMPs (基質金屬蛋白酶) 24 三、TGF-β1 ( transforming growth factor-β1) 24 四、Sma and Mad homologue (Smad) 家族之介紹 26 五、纖維化與細胞凋亡 (apoptosis) 28 六、MRC-5 cell (human fetal lung fibroblast cell line) 人類肺臟纖維母細胞 30 肆、微型 RNA (microRNA) 與氣喘之影響 31 伍、植化素 (phytochemicals) 對氣喘之影響 33 陸、大蒜中有機硫化合物之相關研究 36 一、大蒜種類與分布 36 二、大蒜機能性成分及其生理活性 36 三、大蒜有益的生物活性 (biological actions) 38 柒、研究目的 41 捌、實驗架構 43 第二章、Diallyl sulfide 對活化肺臟 Nrf2 之機制研究 44 中文摘要 45 英文摘要 46 壹、前言 47 貳、材料與方法 49 一、材料 49 二、細胞培養 49 三、動物飼養及處理 49 四、抗氧化酵素活性測定 50 五、大鼠血漿之製備及以 HPLC 分析血漿中 DAS 之含量 51 六、細胞存活率 52 七、基因表現量分析 52 八、蛋白表現量分析 56 九、蛋白質定量 58 十、統計分析 58 參、結果 60 一、大蒜有機硫化物對 SD 大鼠相對臟器重量之影響 60 二、大蒜有機硫化物對於 SD 大鼠肺臟中抗氧化系統表現之影響 60 三、大蒜有機硫化物對於 SD 大鼠肺臟中 Nrf2 蛋白質之表現影響 61 四、大蒜有機硫化物於 SD 大鼠血漿之含量分析 61 五、大蒜有機硫化物對於 MRC-5 細胞內 Nrf2 活化之影響 61 肆、討論 63 第三章、Diallyl sulfide 抑制 TNF-α 與 histamine 誘導 A7r5 細胞發炎之機制研究 75 中文摘要 76 英文摘要 77 壹、前言 78 貳、材料與方法 80 一、材料 80 二、細胞培養 80 三、細胞存活率 80 四、基因表現量分析 81 五、免疫沉澱法 83 六、蛋白表現量分析 83 七、蛋白質定量 84 八、si-Nrf2 轉染試驗 84 九、胞內活性氧分析 85 十、統計分析 85 參、結果 86 一、TNF-α 誘導 A7r5 細胞發炎反應 86 二、TNF-α 誘導 A7r5 細胞 TNFR1 與 NF-κB 之表現 86 三、Histamine 誘導 A7r5 細胞發炎反應 86 四、Histamine誘導 A7r5 細胞 NF-κB 與 AP-1 轉錄因子活化之機轉 87 五、Histamine誘導 A7r5 細胞 Nrf2 轉錄因子及抗氧化酵素基因之表現 87 肆、討論 89 第四章、Diallyl sulfide 減緩雞卵蛋白誘導 BALB/c 小鼠氣喘之機制研究 101 中文摘要 102 英文摘要 103 壹、前言 104 貳、材料與方法 105 一、材料 105 二、動物飼養及處理 105 三、支氣管肺泡沖洗液收集與發炎細胞計數 106 四、支氣管肺泡沖洗液中 Th1/Th2 細胞激素含量與氧化傷害指標之分析 106 五、支氣管肺泡沖洗液中上皮細胞胞內活性氧分析 106 六、血清中免疫球蛋白抗體分泌量之分析 107 七、動物組織切片及觀察 107 八、基因表現量分析 107 九、蛋白表現量分析 109 十、蛋白質定量 110 十一、統計分析 111 參、結果 112 一、大蒜有機硫化物對雞卵蛋白誘導 BALB/c 小鼠相對臟器重量之影響 112 二、大蒜有機硫化物對於雞卵蛋白誘導 BALB/c 小鼠免疫調節之影響 112 三、大蒜有機硫化物抑制雞卵蛋白誘導 BALB/c 小鼠肺臟發炎細胞浸潤 112 四、大蒜有機硫化物抑制雞卵蛋白誘導 BALB/c 小鼠吸呼道杯狀細胞分泌黏液 113 五、大蒜有機硫化物對於雞卵蛋白誘導 BALB/c 小鼠肺臟中抑制發炎現象及氧化傷害之機轉 113 六、大蒜有機硫化物對於雞卵蛋白誘導 BALB/c 小鼠肺臟中抗氧化系統之機轉 114 七、大蒜有機硫化物對於雞卵蛋白誘導 BALB/c 小鼠肺臟 micro-RNA 表現之影響 115 肆、討論 116 第五章、Diallyl sulfide 經活化 Nrf2 以減低 TGF-β 誘導肺臟纖維化之機制研究 134 中文摘要 135 英文摘要 136 壹、前言 137 貳、材料與方法 138 一、材料 138 二、細胞培養 138 三、細胞存活率 138 四、基因表現量分析 139 五、蛋白表現量分析 140 六、蛋白質定量 141 七、胞內活性氧分析 141 八、明膠蛋白酶電泳活性分析 (gelatin zymography) 142 九、統計分析 143 參、結果 144 一、大蒜有機硫化合物誘導 MRC-5 細胞 heme oxygenase-1 (HO-1) 蛋白表現 144 二、大蒜有機硫化合物對 TGF-β 誘導 MRC-5 細胞 collagen 1 蛋白表現 144 三、大蒜有機硫化合物對 TGF-β 誘導 MRC-5 細胞 Smad 蛋白表現 144 四、大蒜有機硫化合物對 TGF-β 誘導 MRC-5 細胞活化纖維化相關蛋白表現 145 五、大蒜有機硫化合物對 TGF-β 誘導 MRC-5 細胞胞內 ROS 與轉錄因子 Nrf2 蛋白表現之影響 145 六、大蒜有機硫化合物對 TGF-β 誘導 MRC-5 細胞凋亡路徑蛋白表現之影響 145 肆、討論 147 總結論 158 參考文獻 16