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克弗爾Kefir(芯蕊康)於缺血-再灌流損傷個體之應用

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【摘要】:
欧洲医学和药理学评论?2015;19:887-896克弗爾Kefir(芯蕊康)於缺血-再灌流損傷個體之應用A.U.YENER1,M.H.SEHITOGLU2,M.T.A.OZKAN1,A.BEKLER3,A.EKIN1,O.COKKALENDER1,M.DENIZ4,M.SACAR1,T.KARACA5,S.OZCAN1,T.KURT11DepartmentofCardiovascularSurg

欧洲医学和药理学评论 2015; 19: 887-896

克弗爾Kefir(芯蕊康)於缺血-再灌流損傷個體之應用

A.U. YENER1, M.H. SEHITOGLU2, M.T.A. OZKAN1, A. BEKLER3, A. EKIN1,O. COKKALENDER1, M. DENIZ4, M. SACAR1, T. KARACA5, S. OZCAN1, T. KURT1

1Department of Cardiovascular Surgery, Canakkale Onsekiz Mart University, Medical Faculty,

Canakkale, Turkey

2Department of Biochemistry, Canakkale Onsekiz Mart University, Medical Faculty, Canakkale, Turkey

3Department of Cardiology, Canakkale Onsekiz Mart University, Medical Faculty, Canakkale, Turkey

4Department of Physiology, Canakkale Onsekiz Mart University, Medical Faculty, Canakkale, Turkey

5Department of Histology and Embryology, Trakya University, Medical Faculty, Edirne, Turkey

摘要團隊利用大鼠模式分析食用克弗爾發酵乳缺血/再灌流引起肺臟、腎臟損傷過程之預防效果實驗用大鼠分為三組,包含對照組、缺血/再灌流實驗組(I/R)、與克弗爾發酵乳餵食+缺血/再灌流實驗組(Kefir+I/R)。在缺血/再灌流實驗進行前,三組大鼠需先經過60前處理,包含前30天以相同飼料飼養,而在往後30天中Kefir+I/R組大鼠另以克弗爾發酵乳灌食。結束60天前處理後進行缺血/再灌流之實驗施作,並取其肺臟與腎臟進行分析。結果顯示事先以克弗爾發酵乳餵食可有效降低缺血/再灌流對大鼠肺臟、腎臟之損傷。主要係透過提升大鼠體內抗氧化物質如超氧歧化物(SOD)、過氧化氫酶(Catalase)、與鼓胱甘肽(GSH, Glutathione)之含量與活性,藉以抑制體內升高之氧化壓力,避免過多的過氧化脂質產生,維持細胞壁完整性以避免細胞損傷發生。最終,用克弗爾發酵乳可保護缺血/再灌流發生時對於腎臟之損傷,並避免大鼠體內炎症之發生。上述結果顯示,克弗爾發酵乳相當適合有缺血/再灌流患者(如腦梗、心梗高風險族群)平日之保養,除可降低血管硬化、血栓形成、血管內徑縮小等造成缺血之風險外,若發生缺血性栓塞可避免血液再灌流對於器官的二度傷害。

关键词:克弗尔;缺血再灌注损伤;肺;肾

 

目的:

腦梗、心梗患者在疾病發生時,除了器官缺血時造成的第一波傷害死亡外,在器官恢復血流後,更會面臨缺血/再灌流後形成大量氧化壓力,釋放促進細胞炎症發生的炎症因子,對細胞與組織造成二度傷害。因此本實驗即在探討,擁有良好抗氧化、抗炎症發生功能之克弗爾發酵乳是否可提供給腦梗、心梗之高風險族群日常食用,除了可以減少上述疾病發生外,亦可避免發生阻塞缺血後因血液再灌流造成之二度傷害

 

Introduction

Kefir grains had been found for the first time by the tribes living in the mountainous regions of the North Caucasus. Kefir is a creamy looking and acidic drink which is fermented naturally from milk. Kefir consists of two microorganisms; lactic acid bacteria and yeast1,2. As a result of metabolic activity of yeast and lactic acid bacteria in kefir, lactic acid, ethyl alcohol and carbon dioxide is formed3. There are a variety of useful biological activities of kefir (commercially available in Danem Ltd. Sti., Isparta, Turkey). Acetic acid bacteria and yeasts in kefir microflora has antibiotic activity. Together with these, as shown in studies in animals, kefir has antidiabetic, antibacterial and antifungal effects. Kefir is shown to have strong antioxidant effect4. Ischemia is reversible or irreversible cell/tissue damage caused by an insufficient blood flow perfusing organ or tissue5. During ischemia, adenine nucleotide degradation in hypoxic tissues is increased. As a result, precursor of reactive oxygen species (ROS) increases intracellular accumulation of hypoxanthine. After ischemia, by restoration of blood flow in that area (reperfusion) and re-introduction of molecular oxygen, intracellular ROS is formed quickly6. Performing reperfusion may lead to more damage than ischemia in tissues. Basically, in post-ischemic reperfused tissue, inflammatory and metabolic damage characterized by edema and dysfunction begins as a result of disruption of cellular integrity. Events often are not confined to those tissues. Various organs such as lungs, kidneys, liver and heart, and cells those may be in every organ such as endothelium and epithelium are affected by many activated systems and toxic mediators. After reperfusion injury, clinical complications due to the failure of distant organs may even lead to death7,8. By the help of several drugs and agents, protection of tissues against reperfusion injury which is formed by providing blood after ischemia, is targeted. In this study, we tried to demonstrate the benefits of kefir that is available easily and also the beneficial effects of it on ischemia reperfusion model.

Study Groups

Twenty-four rats were divided into three groups, randomly and in equal numbers (n = 8). In the control group, laparotomy and dissection of infrarenal abdominal aorta (IAA) was performed, but occlusion was not performed (Sham group). In the group of aortic ischemia reperfusion (A/R), dissection of abdominal aorta (AA) was performed, 180 minutes of ischemia was performed by placing cross-clamp to AA, and 60 minutes of reperfusion was performed after removing crossclamp (Control group). In the group of A/R + kefir, 180 minutes of ischemia was performed by placing cross-clamp to AA, and 60 minutes of reperfusion was performed after removing the cross-clamp (Operations Group).

Operative Procedures and Techniques

 Ketamine (50 mg/kg) and xylazine (5 mg/kg) were used intraperitoneally for premedication of rats. Anesthesia was maintained with intermittent injections of ketamine, without intubation or mechanical ventilation. Temperature probes were inserted into the rectum. The procedures were performed to the rats in the supine position. After preparing sterile operative field, laparotomy was made with a standard midline incision. After pulling the intestines aside, the retroperitoneum was reached, and by opening the retroperitoneum abdominal aorta was reached. During the surgical procedure, cross-clamp was placed just below the renal arteries. Ischemia time was kept 180 minutes, and then cross-clamp was taken and the organs were allowed to be reperfused for 60 minutes. During this process, the body temperature was kept between 36.5 and 37.5°C. After the procedure, the abdominal wall was closed by suturing with 5/0 prolene. 12 hours after the procedure, the animals were given phenobarbital (20 mmg/kg) and sacrificed. The kidneys and lungs were carefully dissected and were fixed in formalin for 7 days.

 

實驗方法:

本團隊將24SD雄性大鼠分為三組,分別為對照組、缺血-再灌流實驗組(I/R)、與缺血-再灌流但有事先餵食克弗爾發酵乳之實驗組(Kefir+I/R)。在缺血-再灌流實驗進行前,三組大鼠需先經過60天前處理,在前30天中三組大鼠以相同飼料飼養之,在後30天的前處理期間內,Kefir+I/R組大鼠除了正常飼料外另以克弗爾發酵乳處理。結束60天前處理後進行缺血-再灌流之實驗施作,將大鼠昏迷後針對腹主動脈進行180分鐘的缺血處理,並接續60分鐘血液灌流處理,並於缺血/再灌流手術處理後12小時犧牲大鼠,取其肺臟與腎臟進行分析,觀察克弗爾發酵乳對於缺血-再灌流造成損傷是否具有保護效果。

 

實驗結果:

1. 克弗爾發酵乳保護肺臟,避免缺血/再灌流再成之損傷:

由組織病理分析發現,缺血/再灌流會使I/R組大鼠肺臟出現明顯的浮腫與白血球浸潤,除了使肺泡空間萎縮外,更會造成肺泡細胞壞死(Necrosis)。然而,前處理期間餵食克弗爾發酵乳可維持Kefir+I/R組大鼠肺部正常肺泡結構,同時大幅避免浮腫、白血球浸潤、與肺泡細胞壞死情形,顯示先以克弗爾發酵乳餵食可減緩缺血/再灌流對肺臟組織造成的傷害。

2. 克弗爾發酵乳減輕缺血/再灌流造成的氧化壓力上升與炎症發生

缺血/再灌流造成的自由基增加,會與細胞膜上磷脂質作用形成過氧化脂質,進而破壞細胞膜完整性而誘發細胞壞死。本團隊亦利用MDA分析腎臟與肺臟組織萃取物過氧化脂質含量發現,缺血/再灌流後會造成肺臟與腎臟中MDA含量上升,顯示兩臟器中過氧化的脂質含量大幅增加。進一步分析兩臟器中具抗氧化物質如SODCatalase、與GSH等,均可發現在經過缺血/再灌流的I/R組大鼠中,上述抗氧化物質含量均顯著下降。令人意外的是,若在前處理階段以克弗爾發酵乳餵食Kefir+I/R組大鼠,可以避免兩臟器中過氧化脂質含量的上升,同時可以維持上述抗氧化物質的表現量,顯示食用克弗爾發酵乳有助於體內維持良好的抗氧化能力。

3. 克弗爾發酵乳避免缺血/再灌流造成體內發炎與保護腎臟功能

利用犧牲取得之血清進行尿素、肌酸(Creatine)、與TNF-α等可反映腎臟功能與體內發炎情況的因子。結果顯示缺血/再灌流處理會使I/R組大鼠血清中TNF-α含量上升,顯示該組大鼠因缺血/再灌流造成體內發炎情況加劇;此外,該組大鼠血清中的尿素與肌酸含量亦同步上升,顯示經缺血/再灌流處理破壞了腎臟正常生理功能之運作。相較之下,前處理階段接受克弗爾發酵乳餵食之Kefir+I/R組大鼠,可顯著降低原本因缺血/再灌流而升高之TNF-α、尿素、與肌酸,顯示事前食用克弗爾發酵乳可避免缺血/再灌流發生時造成的體內炎症發生,並保護腎臟之正常生理功能。

 

結論:

缺血/再灌流於肺臟、腎臟造成之損傷均相當嚴重,尤其在腎臟造成之傷害更可能嚴重衝擊腎臟功能與患者生命。此中,相關器官損傷主要由氧化壓力造成,因此各種抗氧化物質的使用至今仍是醫學領域嘗試用於減輕缺血/再灌流患者症狀之標的。

缺血/再灌流影響腎臟正常組織型態與其功能,缺血造成的急性腎功能衰竭包含了,腎絲球過濾率(Glomerular filtration rate)下降、腎小管細胞壞死、腎血管抗性等。目前觀察腎臟功能維持主要係以血清中肌酸之水平還評估。在缺血/再灌流造成腎損傷後,遠端腎小管的損傷可由血清中肌酸含量上升來觀察,過去同樣是科學家嘗試用抗氧化劑之服用來避免之疾病。在缺血/再灌流過程中大量產生之自由基會與細胞膜上脂質作用形成過氧化脂質,使得細胞膜的完整性出現缺陷,進一步造成細胞傷害與死亡。抗氧化劑理論上可避免自由基對於細胞中抗氧化物質如:SODCaralaseSGH之活性抑制作用,減輕缺血/再灌流過程中自由基產生之傷害,但目前仍無顯著有效之案例

克弗爾發酵乳擁有長久的飲用歷史,經過相當多研究顯示該發酵乳蘊含豐富抗氧化物質。在前人研究中發現,克弗爾發酵乳具有相當良好活性以提升GSHGSH還原酶之活性。由於還原型GSH具有對抗氧化作用之保護效果,因此GSHGSH還原酶之含量增加,顯示克弗爾發酵乳有助於強化體內抗氧化能力。在Paller 博士團隊研究已發現克弗爾發酵乳可以透過增加SOD含量來達到保護肝臟之功能,本團隊進一步證實克弗爾發酵乳同樣可以提供優良抗氧化活性以保護肺臟、腎臟等器官之組織與功能。綜合前人研究與本文結果,均顯示食用克弗爾發酵乳可提供良好抗氧化與清除自由基之能力,有助於保護各種器官免於缺血/再灌流發生時之傷害。對於腦梗、心梗之高風險患者而言,若可平時使用克弗爾發酵相關營養補充,優良的抗氧化能力除了可以減輕動脈粥狀硬化,降低未來腦梗、心梗發生機率外,亦可在腦梗、心梗不幸發生時,可以減輕對於腦部與心臟之傷害。