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標題: 綠茶多酚保護H9c2心肌細胞免於氧化壓力的機制探討
Mechanistic studies of cardio-protection of EGCg by attenuating H2O2-induced oxidative stress in H9c2 cardiomyoblasts
作者: 許建生
Hsu, Chien-Sheng
關鍵字: H9c2 cardiomyoblasts
oxidative stress
intracellular calcium
gap junction proteins
adherens junction proteins
出版社: 生命科學系所
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摘要: EGCg是綠茶中主要多酚物質,能有效的清除自由基。當心臟面臨因缺血再灌流所導致的心肌傷害,常伴隨著產生細胞過氧化物的自由基,例如:hydrogen peroxide、superoxy anions、以及 hydroxyl radicals。這些具有氧化活性的氧化物質(ROS),會對心臟細胞造成氧化壓力,讓心臟細胞造成損傷。本研究論文旨在:藉由建立外加雙氧水造成氧化刺激, 使心臟細胞株 (H9c2,大鼠心肌纖維母細胞) 產生氧化壓力的細胞模式,來探討綠茶多酚對心臟細胞面臨氧化壓力的保護機制。首先,檢視及量化綠茶多酚─EGCg和雙氧水影響H9c2心臟細胞的存活率。結果發現:心臟細胞受到氧化壓力會降低細胞存活率;然而,心臟細胞在面臨氧化壓力前事先添加綠茶多酚,卻能增加心臟細胞存活。另外,實驗結果發現:心臟細胞受到氧化壓力使細胞內產生過多的ROS及胞內鈣離子;然而,EGCg能降低細胞內ROS產生,以及降低氧化壓力所造成胞內鈣離子過多的現象。為了瞭解EGCg進入心臟細胞以及可能的作用機制,將H9c2心臟細胞轉染帶有綠螢光蛋白(EGFP)基因的轉殖載體,再利用螢光顯微鏡來觀察表現綠螢光蛋白基因的心臟細胞,並且利用螢光光譜儀來分析EGCg作用在H9c2心臟細胞所產生的影響。結果發現:逐漸增加綠茶多酚劑量會造成螢光強度呈現遞減的現象;而逐漸增加雙氧水劑量並沒有造成螢光強度產生變化,但這些氧化刺激後的心臟細胞對EGCg產生反應的敏感性卻有增加的情形。此外,加入Triton X-100破壞細胞膜時,EGCg對心臟細胞反應的敏感性也有類似增加的結果。綜合這些實驗結果,顯示:心臟細胞細胞膜受到傷害壓力時會增加對EGCg反應的敏感性,另外EGCg可以經由Triton X-100可溶及非可溶路徑影響心臟細胞。因為這些EGCg作用在氧化傷害的心臟細胞的保護機轉,可能會經由影響細胞膜上的貼附蛋白(beta-catenin、N-cadherin)及間隙蛋白( Cx43),進而改變心臟細胞間的訊息聯繫以及相關活性的表現。因此,利用西方轉滯法以及相關的專一性抗體來分析EGCg可能的作用路徑。實驗結果發現,受雙氧水處理的心臟細胞事先處理EGCg,會影響細胞膜上貼附蛋白(beta-catenin、N-cadherin)及間隙蛋白( connexin 43)蛋白表現量;也影響GSK-3beta蛋白的磷酸化,因而保護氧化傷害心臟細胞間的貼附。另外,EGCg也會影響心臟細胞間的Cx43磷酸化進而增加心臟細胞間的訊息聯繫。這些實驗研究結果,顯示: EGCg可以免於心臟細胞內氧化壓力產生的自由基及胞內鈣離子,進而經由抑制GSK-3beta/beta-catenin以及Cx43磷酸化來調控心臟細胞間的貼附以及訊息聯繫,終而達到保護氧化傷害所造成心臟細胞的死亡。
(-)-Epigalocathine-3-gallate (EGCg) is the major polyphenols in green tea, containing many OH- groups for a strong free radical scavenging capacity. When the heart is damaged by ischemia-reperfusion injury, the cells are accompanied by the generation of intracellular hydrogen peroxide, superoxi anions, and hydroxyl radicals and other reactive oxygen species (ROS). ROS would cause oxidative stress and the consequent damage on the heart. The main goal of the present study was to determine the potential mechanism for cardioprotection of EGCg on hydrogen peroxide (H2O2)-induced oxidative stress in H9c2 rat cardiomyoblasts. The results using the MTT assay showed that H9c2 cells exposing to H2O2 suppressed cell viability, while EGCg pretreatment for 30 min prior to oxidative stress effectively improved cell survival of cardiac cells. EGCg prevented ROS formation and attenuated cytosolic Ca2+ overload in H2O2-treated cells. To identify the putative mechanisms underlying the EGCg signaling pathways, EGFP (enhanced green fluorescence protein) was ectopically expressed in H9c2 cells. EGCg exerted a dose-dependent decrease in the EGFP fluorescence. In contrast, H2O2 did not cause significant spectral changes in EGFP fluorescence, but an increase in the sensitivity for the EGCg induced decrease in EGFP fluorescence. Using Triton X-100 to remove plasma membrane of the cells the similar result was obtained for the increased sensitivity of the EGCg induced response. Apparently, both Triton X-100-soluble and -insoluble fractions involved in transmission of the EGCg signals to regulate EGFP fluorescence in H9c2 cells, and the cell membrane damage caused by oxidative stress could enhance the sensitivity of EGCg. Intercellular junctional complexes in the intercalated discs play an important role in modulation of functional recovery of the cardiac injury. To examine whether EGCg could act to maintain the integrity of the intercalated disc and transmitting cell-cell adhesion signals during oxidative stress, immunoblotting with specific antibody were used to analyze the phosphorylated and non-phosphorylated protein levels of the major ventricular gap junction protein connexin 43 (Cx43) and adhesion molecule beta-catenin、N-cadherin and GSK-3beta in cells. Results obtained showed that EGCg could increase cell-cell adhesion by decreasing GSK-3beta dependent pathway to maintain adherens junction, and increase direct communication between adjacent cells by increasing phosphorylated level of Cx43. In summary, EGCg could protect cardiac cells from oxidative stress by preventing ROS formation and cytosolic Ca2+ overload, and by modulating adherens and gap junction proteins on cell membrane.
其他識別: U0005-1008201114162600
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