Preventive effects of phytochemicals on ketoprofen-induced gastrointestinal mucosa oxidative damage and its underlying mechanisms

Non-steroidal anti-inflammatory drugs (NSAIDs), such as ketoprofen, are generally used to alleviate swelling and pain of rheumatoid arthritis and other inflammatory diseases in clinical medicine. However, these drugs may damage the gastrointestinal (GI) mucosa. The previously study indicated that digestive diseases, such as ulcers, are associated with oxidative damage in the mucosa. Phytochemicals are considered as dietary antioxidants that possess many beneficial characteristics, such as antioxidant and anti-inflammatory capabilities, and they may play an important role in GI protection. According to our preliminary work, catechin, caffeic acid and protocatechuic acid (PCA) were the most effective antioxidants, and they significantly increased the levels of intracellular glutathione peroxidase (GPx), glutathione reductase (GR), and total sulfhydryl groups (TSH) in ketoprofen-damaged human Int-407 cells. Thus, the objective of this study was to investigate the preventive effects of phytochemicals on ketoprofen-induced GI mucosa oxidative injury in vitro and in vivo. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical transcription factor involved in the regulation of antioxidant enzymes. In vitro treatment of the cells with catechin, caffeic acid and PCA for 1-4 h significantly increased the nuclear/cytosol Nrf2 ratio and totoal HO-1 protein expression. Moreover, our results also indicate that treatment of Sprague-Dawley (SD) rats with catechin (35 mg/kg b.w.), caffeic acid (120 mg/kg b.w.), and PCA (100 mg/kg b.w.) prior to the administration of ketoprofen (50 mg/kg b.w.) successfully inhibited oxidative damage and also reversed the impairment of the antioxidant enzymes expression in the GI mucosa. The results suggest that increasing Nrf2 activation and HO-1 expression may reduce oxidative damage caused by ROS in the GI tract. DJ-1/PARK7 is a multi-functional protein in association with promotion of cell-growth, anti-oxidant, anti-apoptotic and preservation of mitochondrial integrity in healthy subjects. Recent reports have suggested that Nrf2 is unstable in lack of DJ-1, which would decrease the expression of Nrf2-associated antioxidant enzymes and lead to oxidative stress. However, data concerning the effects of DJ-1 on ketoprofen-induced GI mucosa oxidative lesion remain unknown. In this study, we proposed that increasing DJ-1/Nrf2 expression by phytochemicals might represent a potential therapeutic approach to prevent patients against oxidative stress-associated development of GI ulcers. The results indicated that caffeic acid increased the nuclear/cytosolic Nrf2 ratio and the mRNA expression of the downstream antioxidant enzymes by JNK/p38 pathway. Moreover, PCA could prevent human Int-407 cells against ketoprofen-induced oxidative stress by regulating DJ-1/phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway. Pre-treatment with PCA could both inhibit caspase-8, caspase-9 and caspase-3 activity, and reverse the impaired DJ-1 protein expression in human Int-407 cells induced by ketoprofen. These results indicate that increasing Nrf2 translocation by DJ-1 may represent a novel means of GI protection. In conclusion, our observations suggest that increasing DJ-1/Nrf2 expression by exogenous antioxidant compounds may provide a potential therapeutic approach to protect patients against oxidative stress-associated injury followed by disorders from developing GI ulcers. Moreover, our results support the possible use of catechin, caffeic acid and PCA to be a dietary preventive agent against GI injuries caused by oxidative stress.非類固醇抗發炎藥物 (non-steroidal anti-inflammatory drugs, NSAIDs)，如 ketoprofen 為醫療常用於止痛、抗發炎及治療類風濕性關節炎等臨床用藥，但易引起腸胃潰瘍之潛在副作用，故往往影響了其藥物使用之潛能。消化性潰瘍目前已證實與黏膜氧化壓力有關，而蔬果中富含多種抗氧化及抗發炎之生理活性物質，可能具預防腸胃黏膜氧化傷害之能力。在本研究預試驗結果中發現，catechin、caffeic acid 及 protocatechuic acid (PCA)，具較佳抗氧化能力，與 ketoprofen 誘導組相比，可顯著提升人類腸道 Int-407 細胞麩胱甘肽過氧化酶 (glutathione peroxidase, GPx)、麩胱甘肽還原酶 (glutathione reductase, GR) 及總硫醇基 (total sulfhydryl groups, TSH) 表現量。故本論文將更進一步以體外及體內試驗，評估這些植化素對 ketoprofen 造成腸道黏膜氧化傷害之預防能力。 轉錄因子 nuclear factor erythroid 2-related factor 2 (Nrf2) 具調控多種抗氧化酵素能力，為生物體抵禦氧化傷害重要之機轉。在本研究結果顯示，細胞與 catechin、caffeic acid 及 PCA 共培養 1-4 小時，能顯著提升人類腸道 Int-407 細胞 Nrf2 蛋白及血紅素氧化酶-1 (heme oxygenase-1, HO-1) 表現。動物實驗結果顯示，餵食 catechin (35 mg/kg b.w.)、caffeic acid (120 mg/kg b.w.) 及 PCA (100 mg/kg b.w.) 能有效抵抗 ketoprofen 對腸胃黏膜造成之氧化傷害，並回復組織抗氧化酵素活性 (p < 0.05)。由上述數據推測，catechin、caffeic acid 及 PCA 可能藉由提升腸胃道黏膜 Nrf2 及 HO-1 蛋白表現，進而達到預防腸胃潰瘍之能力。 DJ-1 (又稱為 PARK7) 蛋白，目前已知具有多種生理活性，包括促進細胞增生、增加組織修復、提升體內抗氧化及預防細胞凋亡之潛能。近期報導更指出，Nrf2 轉錄作用及誘導下游抗氧化酵素表現亦與 DJ-1 蛋白有關；當 DJ-1 基因突變或蛋白表現功能異常時，將使細胞胞內 ROS 大量累積；反之，提升 DJ-1 則能幫助細胞抵禦氧化傷害。然而，提升 DJ-1 是否有助於改善腸胃黏膜細胞抵禦氧化傷害，目前仍不清楚。因此，本研究更進一步探討 DJ-1 蛋白對於 ketoprofen 誘導腸胃氧化傷害之保護效應。試驗結果發現，caffeic acid 調控 Nrf2 轉錄及下游相關抗氧化酵素表現，主要透過 DJ-1/JNK 及 p38 訊息傳導路徑。而 PCA 則可透過 DJ-1/phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) 訊息傳導路徑，以減輕 ketoprofen 誘導腸胃細胞氧化傷害及減輕組織間凋亡反應 (抑制 caspase-8、caspase-9 及 caspase-3 凋亡蛋白活性)。因此，提升腸胃細胞 DJ-1 蛋白表現，將有助於提升腸胃黏膜抵禦氧化物質對組織造成之傷害，而 DJ-1 具抗氧化及抗凋亡特性，更可作為未來腸胃保健研究之重要生物指標。 總結以上結果，提升腸胃細胞 DJ-1/Nrf2 訊息傳導，並調控抗氧化酵素表現，可能有助於預防 ketoprofen 對黏膜造成之氧化傷害。此外，本論文研究結果更提供天然植化素，如 catechin、caffeic acid 及 PCA 在預防或減緩 ketoprofen 誘導人類腸胃黏膜氧化傷害之應用依據。目次 全文摘要 I Abstract III 目次 VI 圖次 X 表次 XIII 附圖次 XIV 縮寫表 (Abbreviation) XV 前言 1 第一章 文獻回顧 6 壹、消化性潰瘍 (Peptic ulcer) 7 一、消化系統常見的疾病 7 二、潰瘍致病原因 7 三、潰瘍之治療 14 四、瘍藥治潰之臨床用藥 15 貳、NSAIDs 誘發腸胃潰瘍之致病機轉 18 一、非類固醇抗發炎藥物 18 二、抗氧化劑與潰瘍 24 三、氧化傷害與疾病 24 四、生物體內抗氧化防禦機制 26 五、胃腸道之抗氧化防禦機制 29 六、轉錄因子Nrf2之化學預防角色 29 七、血基質氧化酶-1 30 八、micro RNA (mi-RNA) 調控 Nrf2/HO-1 之影響 30 九、生長因子與潰瘍修復 36 十、細胞之生長與死亡 39 十一、PARK7 (DJ-1) 蛋白之生理活性 41 參、人類腸道 Int-407 細胞 47 肆、植化素於腸胃潰瘍相關之運用研究 48 伍、研究目的 49 陸、研究架構 50 第二章 Catechin抵抗 ketoprofen 誘導腸胃黏膜氧化傷害藉由提升Nrf2 轉錄作用之研究 51 摘要 52 Abstract 53 前言 54 材料方法 56 結果 63 一、Catechin 調控人類腸道 Int-407 細胞 Nrf2 轉錄作用及 HO-1 蛋白之表現 63 二、餵食 catechin 對於 ketoprofen 誘導大鼠腸胃黏膜氧化傷害之保護效應 63 三、餵食 catechin 對於 ketoprofen 誘導大鼠腸胃黏膜 COX-1 及 COX-2 酵素之影響 64 四、組織病理判讀 catechin 對於 ketoprofen 誘導大鼠腸腸胃潰瘍之保護作用 64 討論 65 第三章 Caffeic acid 可透過提升 DJ-1 蛋白並達到預防 ketoprofen 誘導腸胃黏膜氧化傷害 77 中文摘要 78 Abstract 79 前言 80 材料方法 83 結果 93 一、Caffeic acid 經由 DJ-1/Nrf2 訊息傳導路徑調控人類腸道 Int-407 細胞抗氧化酵素表現 93 二、Caffeic acid 經由 DJ-1/mitogen-activated protein kinases (MAPKs) 訊息傳導路徑調控人類腸道 Int-407 細胞 HO-1 蛋白表現 94 三、預先處理 Caffeic acid 對 ketoprofen 誘導人類腸道 Int-407 細胞存活率表現影響 95 四、餵食 caffeic acid 對 ketoprofen 誘導腸黏膜氧化傷害之保護效應 95 五、餵食 caffeic acid 對於 ketoprofen 誘導大鼠腸胃黏膜 COX-2 及 NO 表現之影響 96 六、組織病理判讀 caffeic acid 對於 ketoprofen 誘導大鼠腸腸胃潰瘍之保護作用 96 七、免疫組織染色 (Immunohistochemical, IHC) 判讀 caffeic acid 對於 ketoprofen 誘導大鼠腸腸胃潰瘍之保護作用 97 討論 98 第四章 原兒茶酸調控 DJ-1/PI3K/mTOR 訊息傳導路徑以抵禦黏膜氧化傷害 117 摘要 118 Abstract 119 前言 121 材料方法 125 結果 130 一、PCA 調控人類腸道 Int-407 細胞抗氧化酵素透過 DJ-1/Nrf2 訊息傳導路徑 130 二、PCA 減輕 ketoprofen 誘導人類腸道 Int-407 細胞毒性，經由 PI3K/mTOR 訊息路徑 130 三、PCA 透過提升 DJ-1 以抑制 ketoprofen 誘導人類腸道 Int-407 細胞凋亡之保護效應 131 四、透過組織病理 (H&E) 及免疫組織染色 (IHC) 評估 PCA 預防 ketoprofen 誘導腸胃黏膜損傷之保護作用 132 五、探討 lansoprazole 及 EGCG 等天然植化素對 ketoprofen 誘導腸胃黏膜 DJ-1 蛋白之表現 132 討論 134 總結論 150 參考文獻 15