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研究生:周蓓莉
研究生(外文):Chou, Pei-Li
論文名稱:缺氧前處理或抗氧化水給予以降低氧化損傷之策略應用
論文名稱(外文):The Strategy and Application of Hypoxic Preconditioning or Antioxidant Water Administration on Ameliorating Oxidative Injury
指導教授:鄭劍廷
指導教授(外文):Chien, Chiang-Ting
學位類別:博士
校院名稱:國立臺灣師範大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文出版年:2020
畢業學年度:109
語文別:英文
論文頁數:85
中文關鍵詞:缺氧前處理1α缺氧誘發因子活性氧凋亡醋酸十二指腸潰瘍含氫水錳超氧化物歧化酶大鼠慢性腎衰竭抗氧化水電解還原水
外文關鍵詞:hypoxic preconditioningHIF-1αBcl-xLduodenal ulcerhydrogen-rich waterMnSODchronic renal failureantioxidant waterelectrolyzed reduced waterH2
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嚴重缺氧/缺血會引起組織病理變化和細胞死亡,重複缺氧前處理(HP)可防止隨後嚴重的缺氧/缺血壓力。我假設缺氧前處理可以經由與1α缺氧誘發因子(HIF-1α)/活性氧(reactive oxygen species, ROS)相關的防禦機制來保護大鼠肝臟免於受到缺血/再灌注損傷。過多的活性氧(ROS)透過增加凋亡相關的Bax/Bcl-2比例可能會引發十二指腸和腎臟損傷。含有溶解氫分子的抗氧化水(antioxidant water, AW)具有安全的抗氧化活性,可保護十二指腸組織和腎臟免受氧化損傷。
缺氧前處理是讓雌性Wistar大鼠進入低壓缺氧室(5500米,每天15小時),而其他大鼠則保持在海平面高度(SL)。夾住大鼠的肝門靜脈導致肝缺血45分鐘,然後重新輸注6小時。我以70μL, 100%的醋酸,刺激十二指腸粘膜表面10秒建立了雌性Wistar大鼠十二指腸潰瘍模型。大鼠接受5/6腎臟移除術來引發慢性腎衰竭。我使用通過鎂合金濾心的含氫抗氧化水體外實驗來評估清除過氧化氫和次氯酸活性能力以及體內實驗探討飲用抗氧化水對醋酸誘發大鼠十二指腸潰瘍的影響。我使用西方墨點法和硝基藍四氮唑(NBT)/3-硝基酪氨酸(3-NT)染色來評估細胞核萃取物中的1α缺氧誘發因子、錳超氧化物歧化酶(MnSOD)、銅鋅超氧化物歧化酶(CuZnSOD)、過氧化氫酶、Bad、Bcl-xL、Caspase 3、聚腺苷酸二磷酸核糖基聚合酶(PARP)、Bax/Bcl-2、粒線體Bcl-xL和細胞質細胞色素C的表現量。透過免疫細胞化學法觀察庫佛氏細胞浸潤和TUNEL檢測細胞凋亡。通過超靈敏的化學發光放大法檢測肝腎表面、膽汁和血液的活性氧數值。檢測血漿中的ALT和AST值來評估肝功能。我測量了醋酸誘導大鼠十二指腸潰瘍的十二指腸近端黏液中的錳超氧化物歧化酶量。
隨著缺氧前處理的時間,細胞核轉譯1α缺氧誘發因子、Bcl-xL、錳超氧化物歧化酶、銅鋅超氧化物歧化酶、過氧化氫酶蛋白表現顯現增加。缺氧前處理誘發增加的肝臟1α缺氧誘發因子和Bcl-xL表現可以經由1α缺氧誘發因子抑制劑YC-1而抑制。肝缺血/再灌注增強了細胞Bad轉錄到粒線體,細胞色素C釋放到細胞質, 並活化Caspase 3/聚腺苷酸二磷酸核糖基聚合酶相關的細胞凋亡, 造成活性氧、骨髓過氧化酵素活性、 庫佛氏細胞浸潤、ALT和AST值增加。缺氧前處理顯著改善肝缺血/再灌注引發的氧化壓力、細胞凋亡、粒線體和肝臟功能障礙。與蒸餾水或自來水相比, 抗氧化水含有約50 ppb的溶解氫分子, 顯示出較低的氧化還原電位(-150 mV)和有效清除過氧化氫而不是次氯酸的活性。醋酸損傷1天和3天引起體重減輕並導致嚴重的十二指腸潰瘍和膠原蛋白沉積。與自來水飲用相比,每天飲用抗氧化水可以顯著緩解體重減輕和十二指腸纖維化,有效保留十二指腸完整性和錳超氧化物歧化酶量。我的電解還原水溶解了35ppb氫, 酸鹼pH值大於9.0。體外和體內結果顯現,慢性腎功能衰竭增加了活性氧, Bax/Bcl-2比例、Caspase 3表現和細胞凋亡,進而導致腎損傷。在腎臟表面檢測到的活性氧量與腎組織和血液中硝基藍四氮唑沉積相關,如果每天攝入電解還原水就會減少。
總之, 缺氧前處理增強了缺氧誘發因子1α /活性氧相關反應, 增強粒線體 Bcl-xL 蛋白表現,保護肝臟免受缺血/再灌注損傷。飲用抗氧化水可以透過溶解的氫分子直接清除自由基的能力來提供十二指腸保護以及減少慢性腎衰竭產生的大量活性氧和細胞凋亡。
Severe hypoxia/ischemia can initiate tissue pathology change and cell death, but repeated hypoxic preconditioning (HP) protected against subsequent severe hypoxia/ischemic stress. I hypothesized that HP can protect the liver of rats from ischemic/reperfusion damage through the defense mechanism associated with hypoxia-induced factor 1α (HIF-1α)/reactive oxygen species (ROS). Exaggerated reactive oxygen (ROS) production may initiate duodenal damage caused by acetic acid and kidney damage by increasing the Bax/Bcl-2 ratio of pro-apoptosis factor. Antioxidant water (AW) containing dissolved molecular hydrogen had a safe antioxidant activity and protected duodenal tissue and kidney from oxidative damage.
HP induction was to keep female Wistar rats in a hypobaric hypoxia chamber (5, 500 m, 15 hours/day), while other rats remain at sea level (SL). The rats' portal veins were blocked, causing liver ischemia for 45 minutes and then re-infusion for 6 hours. I established a model of duodenal ulcer in female Wistar rats, with a content of 70 μL, 100% acetic acid, and 10 s on the surface of the duodenum mucosa. The rats received 5/6 nephrectomy to induce chronic renal failure. I used antioxidant water made from a modified magnesium alloy to assess the in vitro H2O2 and HOCl scavenging activity and the effect of AW drinking on the in vivo rat acetic acid-induced duodenal ulcer. I used Western blot and nitroblue tetrazolium (NBT)/3-nitrotyrosine (3-NT) stain to evaluate HIF-1α in nuclear extract, MnSOD, CuZnSOD, catalase, Bad/Bcl-xL/Caspase 3/poly-(ADP-ribose) polymerase (PARP), Bax/Bcl-2, mitochondrial Bcl-xL and cytosolic cytochrome c expression. Kupffer cell infiltration and TUNEL-apoptosis were observed by immunocytochemistry. The ROS values of liver surface, renal surface, bile and whole blood were detected by ultrasensitive chemiluminescence-amplification method. Liver function was assessed with plasma ALT and AST. The expression of manganese superoxide dismutase (MnSOD) was measured in the scraping of the proximal duodenal mucus of rats duodenum ulcers in acetic acid induction.
The nuclear translocation of HIF-1α and Bcl-xL, MnSOD, CuZnSOD, and Catalase protein expression were increased after HP in a time-dependent manner. The response of HP enhanced hepatic HIF-1α and Bcl-xL expression was inhibited by HIF-1α inhibitor YC-1. ROS levels, myeloperoxidase activity, Kupffer cell infiltration, ALT and AST levels were increased by hepatic ischemia/ reperfusion with the enhancement of cytosolic Bad translocation to mitochondria, release of cytochrome c to cytosol, and activation of Caspase 3/PARP mediated apoptosis. HP significantly improved liver ischemic/ reperfusion -enhanced oxidative stress, apoptosis, mitochondrial, and liver dysfunction. Compared to distilled water or tap water, the established AW contained dissolved molecular hydrogen of about 50 ppb and exhibited a lower redox potential (-150 mV) and an efficient H2O2 instead of HOCl scavenging activity. After 1 day and 3 days of acetic acid injury caused weight loss, it induced severe duodenal ulcers and collagen deposition. Compared with tap water drinking, daily AW drinking significantly reduced weight loss and duodenal fibrosis, effectively preserving the duodenal integrity and MnSOD expression. The electrolyzed reduced water had 35 ppb dissolved hydrogen and pH>9.0. In vitro and in vivo results showed that chronic renal failure increased the production of ROS and increased the mechanism of apoptosis, including increased Bax/Bcl-2 ratio, Caspase 3 expression and apoptosis formation, which in turn leaded to kidney damage. The ROS levels detected on the kidney surface was associated with increased ROS levels in NBT accumulation in kidney tissue and blood, which were reduced by daily intake of electrolyzed reduced water.
In summary, HP enhanced HIF-1α/ROS dependent cascades to enhance mitochondrial Bcl-xL protein expression and protect liver from ischemia/reperfusion damage. AW drinking may give the duodenal protection and reduce the apoptosis in chronic renal failure through the direct removal capacity of the molecular hydrogen it dissolved.
Chapter 1 Introduction 1
Chapter 2 Methods and Materials 7
Chapter 3 Results 26
Chapter 4 Discussion 34
References 47
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