Green tea extracts provide antioxidative protection on myocardial ischemia associated with ligation of anterior descending coronary artery in rats

Containing large amounts of polyphenol compounds, green tea could help scavenge free radicals and reduce oxidative damage such as to protect from cardiovascular diseases, cancer and chronic diseases. Myocadial infarction (MI) resulting from myocardial ischemia often undergoes cardiac remodeling process, by which secondary damage to the heart might occur by generating excessive reactive oxygen species (ROS) and free radicals. Previously, we have established MI animal models associated with ligation of the left anterior descending coronary artery (LAD) in rats, and used that model to demonstrate that tube feeding green tea extract (GTE) could reduce heart tissue remodeling, ventricular hypertrophy, infarct size, as well as significantly improve the left ventricular functions. In this thesis, my study aimed to further understand the mechanism of GTE acting to ameoliate myocardial ischemia induced oxidative stress by using rats with sham control group (only thoracotomy), post MI group with GTE treatment or with water (H2O group). After three days without GTE feeding, cTnI was found significantly reduced in myocardial cells, suggesting cTnI degradation occurring in acute myocardial ischemia injured cells. In contrast, gelsolin (GSN), an actin severing protein, was found increased in ischemic myocardial cells from post MI rats. This is consistent with the role of GSN in cardiac apoptosis occurring in post MI hearts. Using myocardial cells freshly isolated from rat hearts showed that EGCG, a major green tea polyphenol, efficienly reduced formation of free radicals in cells disturbed by 100 µM H2O2. In addition, the rate of intracellular free radicals in the GTE groups (for three days, two weeks, and three weeks) all became slower in comparison with the H2O groups. The increase of GTE feeding time also increased efficiency in reducing the rate of free radical production in post MI cells. To assess antioxidative capacity of GTE in myocardial ischemia injured cells, the level of superoxide dismutase (SOD) activity in myocardial tissues was measured. Results showed that GTE treatment could help maintain the activity of SOD in cells at the remote region of the heart in post MI rats for the time periods from three days to three weeks, while in H2O group the SOD activity was found significantly decreased in post MI rats. However, the measured SOD mRNA level in myocardial tissues was not significantly different in control, post MI rats with or without GTE treatments. For measuring another antioxidatant enzyme, heme oxygenase-1 (HO-1), the mRNA level was found significantly reduced for the H2O group, while GTE treatment protecting the decrease of mRNA expression in the post MI myocardium. To further examine oxidative stress in cardiac cells, 4-hydroxynonenal (4HNE) post-translational modification on myocardial proteins were determined. Results showed that 4-HNE modified proteins were increased for the H2O group but decreased for the GTE group, as compared to the sham controls. Proteomic analysis also revealed that post MI animals for three days or three weeks significantly altered the myocardial proteins characterized with cytoskeletal function and energy metabolism as compred with control and post MI rats with GTE treatment. Finally, the potential signaling pathways by caveolin, Akt and GSK3β dependent events were determined for the involenent in the GTE-mediated antioxidation against the myocardial ischemia stress in post MI rats. Taken together, the data found in this study suggested that the caveolin, Akt and GSK3β signaling pathways might act to modulate the antioxidaticve protection of GTE against the myocardia ischemia induced oxidative stress in the heart.利用綠茶研究心血管疾病、癌症和慢性疾病的許多相關實驗證實綠茶具有抗氧化功效，因為含有大量多酚化合物幫助細胞清除自由基，減少氧化傷害，進而保護健康細胞。心臟缺血傷害導致心肌梗塞，後期心臟重塑過程對心臟組織造成二次傷害，伴隨著過量活性氧化物與自由基產生。本實驗室先前研究建立結紮左前降支冠狀動脈心肌梗塞大鼠，利用心肌梗塞大鼠動物模式探討灌食綠茶萃取物的功效，發現綠茶萃取物有效降低心臟組織重塑的傷害、避免心室擴大、減少梗塞區域，以及顯著改善左心室收縮功能 欲瞭解綠茶萃取物如何對心肌梗塞大鼠心臟給予保護效果。因此，本研究利用相同模式結紮左前降支冠狀動脈的心肌梗塞大鼠，比較控制組(只有進行開胸手術未結紮， Sham) 、心肌梗塞大鼠灌食綠茶萃取物組(GTE) 或灌食水組 (H2O) 探討綠茶對於心臟缺血傷害的保護機制。手術後灌食有效劑量綠茶萃取物 (400 mg/kg) 或單純餵水的灌食時間為 3 天、2 週和 3 週。實驗結果顯示：大鼠於手術後灌食 3 天時間點， H2O 組減少心肌細胞內 cTnI 蛋白，可能是缺血傷害之心肌細胞中 cTnI 產生裂解。另外心臟缺血傷害的細胞內增加肌絲調控蛋白 GSN 表現量，可能會導致心臟缺氧傷害誘發的細胞凋亡。利用萃取控制組心肌細胞的實驗，證實兒茶素 EGCG 降低 100 µM H2O2 所誘發的胞內自由基產生速率。犧牲各時間點的實驗動物後，再萃取心肌細胞來檢測 100 µM H2O2 溶液刺激產生氧化壓力下胞內自由基生成的變化速率。實驗結果顯示： GTE 組在氧化壓力下產生的胞內自由基速率較慢；且隨著增加灌食綠茶萃取物的時間顯著減少胞內自由基產生的速率。另外評估心臟細胞抗氧化能力，利用偵測超氧化物歧化酶 (SOD) 活性，結果發現：隨著增加灌食時間 GTE 組相較 H2O 組更能維持超氧化物歧化酶活性；但是 SOD mRNA 基因並沒有顯著差異。然而，第一型血紅素氧化酶 (HO-1) mRNA 基因，在 H2O 組心臟非梗塞區域相對的基因表現量顯著減少。分析心臟細胞內 4-羥基壬烯酸 (4HNE) 後修飾氧化蛋白，發現 H2O 組心臟細胞蛋白明顯被 4-HNE所氧化修飾，而 GTE 組減少蛋白氧化修飾。繼由蛋白質體學分析，灌食 3天和 3 週的實驗動物組與控制組具明顯差異的蛋白，大多與細胞骨架和能量代謝等功能相關。最後探討綠茶萃取物對心臟細胞抗氧化相關訊息路徑；檢測實驗動物心臟細胞內 Caveolin 、 Akt 和 GSK3β 蛋白表現量與蛋白磷酸化與去磷酸化活性 結果發現 GTE 組非梗塞區 CAV3 明顯增多；。pAkt/Akt 比值具有趨勢變化，在 H2O 組梗塞區域顯著減少； pGSK3β(Ser9) /GSK3β 比值，於灌食 3 週時間點發現， GTE 組相較 H2O 組顯著增加。綜觀以上結果證實綠茶萃取物可經由影響 Caveolin 、 Akt 和GSK3β 訊息路徑對心臟缺血傷害的心肌組織提供抗氧化保護作用。第一章 前言 ................1 第一節 研究概要................1 第二節 綠茶萃取物成份與機能................1 第一項 綠茶及兒茶素................1 第二項 綠茶的健康效益................3 第三節 心血管疾病................4 第一項 心臟構造及功能................4 第二項 心肌梗塞................4 第三項 心臟受損常見診斷及治療................6 第四項 心臟受損後期心臟重塑現象................8 第四節 氧化壓力 (Oxidative stress) ................9 第五節 心肌梗塞相關的訊息傳遞................11 第六節 心肌梗塞模式的氧化壓力及炎症反應 ................12 第七節 天然物的抗氧化及抗發炎作用 ................13 第八節 研究方向................14 第二章 材料與方法................16 第一節 實驗流程................16 第二節 實驗動物................17 第三節 綠茶萃取物................17 第四節 心肌梗塞之動物模式................17 第五節 心臟組織之細胞分離................18 第六節 胞內自由基測定................18 第七節 超氧化物歧化酶活性測試(SOD activity kit)................20 第八節 蛋白質體學分析................20 第九節 西方轉漬法 (Western blot) ................22 第十節 基因表現檢測................24 第三章 實驗結果................27 第一節 心肌梗塞模式確立及綠茶萃取物保護作用 ................27 第一項 結紮 LAD 之心肌缺血模式 ................27 第二項 手術後血液生化檢查及生理值影響 ................28 第三項 綠茶萃取物對於心臟保護的效果 ................28 第四項 心肌梗塞病程時間點的變化................28 第五項 心臟肌鈣蛋白 (Cardiac troponin I, cTnI) 蛋白表現 ....... 29 第六項 Gelsolin (GSN) 蛋白和基因表現 ................30 第二節 心肌梗塞後綠茶萃取物產生抗氧化作用................32 第一項 心肌細胞於氧化壓力下 EGCG 的作用................32 第二項 胞內自由基測定................33 第三項 自由基產生速率的比較................35 第四項 超氧化物歧化酶活性測試................36 第五項 超氧化物歧化酶基因差異變化 ................37 第六項 第一型血紅素氧化酶 (HO-1) 基因差異變化 ................. 39 第七項 4-羥基壬烯酸 (4HNE) 修飾蛋白的表現 ................40 第三節 蛋白質體學分析................41 第四節 綠茶萃取物影響心肌梗塞後訊息傳遞 ................45 第一項 Caveolin 蛋白和基因表現 ................45 第二項 Akt 蛋白表現................48 第三項 GSK3β 蛋白和基因表現 ................49 第四章 討論................51 第一節 缺血傷害與心臟組織重塑................51 第二節 綠茶萃取物提供的抗氧化保護作用................53 第三節 氧化還原蛋白質體學應用................55 第四節 心臟保護相關訊息傳遞的影響 ................57 第五章 結論 ................60 第六章 參考文獻 ................6