探討Lactobacillus reuteri對於心肌缺血再灌注損傷的保護作用及其機制

The effect of Lactobacillus reuteri against myocardial ischemia-reperfusion injury

洪岳慈(Yueh-Tzu Hung)

指導教授 ： 張耀仁 ； 廖娟妙

中山醫學大學/醫學院/醫學研究所/碩士(2018年)

摘要

根據2016年國際衛生組織 (WHO) 統計資料顯示缺血性心臟病名列全球十大死因第一名，發病的年齡層有逐漸下降的趨勢，且有超過半數以上的病患來不及送醫即死亡。臨床上，盡速疏通缺血心肌的血流是主要心肌梗塞的治療策略，然而會誘發心肌缺血再灌注 (myocardial ischemia-reperfusion injury) 的傷害，當心肌缺血再灌注時會誘發發炎反應，進一步引起心肌細胞的死亡，因此研究預防或治療心肌梗塞以及減少心肌缺血再灌注的傷害是非常重要的臨床議題。文獻中證實抑制發炎反應會減少心肌缺血再灌注傷害所造成的心肌梗塞體積大小及死亡率，此外，有研究顯示當腸道菌相遭受破壞時，容易引起心肌梗塞及造成較差的預後。因此，本研究擬探討具有抗發炎活性的羅伊氏乳桿菌 (Lactobacillus reuteri) 對於大鼠心肌缺血再灌注損傷是否具有保護的效果。
本實驗先給予Sprague-Dawley大鼠飲用抗生素 (Sulfamethoxazole 0.8 mg/ml, Trimethoprim 0.16 mg/ml) 10天，再飲用水2天，接著以管餵的方式給予Lactobacillus reuteri (8.2×108 CFU/kg、8.2×109 CFU/kg) 連續7天，於第8天進行心肌缺血再灌注傷害手術。實驗大鼠以urethane (1.25 g/kg) 麻醉，將左冠狀動脈下降支結紮造成缺血60分鐘，而後鬆開結紮處再灌注3小時，造成心肌缺血再灌注傷害，實驗計算心肌梗塞區域 (Infarct size) 大小、心律不整 (Arrhythmia) 及死亡率，研究評估Lactobacillus reuteri對於大鼠心肌缺血再灌注損傷是否具有心臟保護作用。研究結果發現8.2×109 CFU/kg Lactobacillus reuteri可以有效降低心肌梗塞區域大小及死亡率，Lactobacillus reuteri可能具有保護心臟對抗缺血再灌注傷害的潛力。進一步利用Western blot探討Lactobacillus reuteri保護心臟對抗缺血再灌注傷害的作用機制，結果發現Lactobacillus reuteri會抑制磷酸化NF-κB (p50)、磷酸化NF-κB (p65) 的訊息傳遞路徑，進而降低心肌缺血再灌注損傷所誘發的cyclooxygenase (COX)-2、myeloperoxidase (MPO) 之蛋白表現量，表示Lactobacillus reuteri可能透過抗發炎作用進而達到心臟保護作用，未來研究會進一步詳細探討Lactobacillus reuteri的心臟保護作用之藥理作用機轉。

關鍵字

心肌缺血再灌注損傷 ； 羅伊氏乳桿菌

並列摘要

Ischemic heart disease is the leading cause of death worldwide, according to the world health organization (WHO) statistics in 2016. The age incidence of ischemic heart disease gradually decreased and more than half of the patients died before getting medical treatment. In clinical, the strategy for treatment of myocardial infarction is to restore the blood supply of ischemic myocardium as soon as possible, however, would induce myocardial ischemia-reperfusion (I-R) injury. Myocardial I-R induce inflammation, which would further lead to the death of cardiomyocyte. Therefore, it is an important clinical issue to investigate how can prevent or treat of myocardial infarction and reduce myocardial I-R injury. Literatures have demonstrated that the inhibition of inflammatory response reduces myocardial I-R induced infarct size and mortality. In addition, studies reported that the damage of the intestinal bacterial phase is easily caused myocardial infarction and poor prognosis. This study proposes to investigate whether the administration of Lactobacillus reuteri, a probiotic with anti-inflammatory activity, is capable of cardioprotective effect against myocardial I-R injury in rats and its potential pharmacological mechanisms.
In this experiment, Sprague-Dawley rats were administrated antibiotics (Sulfamethoxazole 0.8 mg/ml, Trimethoprim 0.16 mg/ml) for 10 days and then water for 2 days. After that rats were randomly divided into groups of oral administration of water or Lactobacillus reuteri (8.2×108 CFU/kg, 8.2×109 CFU/kg) for 7 consecutive days. The myocardial I-R injury was performed on the 8th day by one hour occlusion of the left anterior descending coronary artery and followed by three hours reperfusion in anesthetized rats. We found that the group of Lactobacillus reuteri treatment significantly decreased myocardial infarct size and also significantly reduced the incidence of arrhythmia and mortality than the vehicle group. These results showed that supplementation with Lactobacillus reuteri is effective to protect the heart against myocardial I-R injury.
To address the mechanisms of the cardioprotective effect of Lactobacillus reuteri, we examined the expression of cyclooxygenase (COX)-2 myeloperoxidase (MPO), and phosphorylation NF-κB after rats subjected to myocardial I-R injury. The results showed that Lactobacillus reuteri not only significantly attenuated the increased of I-R induced the expression of COX-2, but also decreased the increased of I-R induced the expressions of MPO and p-NF-κB. Therefore, this study suggested that Lactobacillus reuteri was a potential cardioprotective agent to against myocardial I-R injury, and Lactobacillus reuteri could against myocardial I-R injury via NF-κB signaling pathway.

並列關鍵字

myocardial ischemia/reperfusion injury ； Lactobacillus reuteri

參考文獻

WHO 網址：http://www.who.int/

衛生福利部 網址：https://www.mohw.gov.tw

Burton, K.P., McCord, J.M., and Ghai, G. (1984). Myocardial alterations due to free-radical generation. American Journal of Physiology-Heart and Circulatory Physiology 246, H776-H783.

Calderon-Sanchez, E.M., Ruiz-Hurtado, G., Smani, T., Delgado, C., Benitah, J.P., Gomez, A.M., and Ordonez, A. (2011). Cardioprotective action of urocortin in postconditioning involves recovery of intracellular calcium handling. Cell Calcium 50, 84-90.

Doods, H., and Wu, D. (2013). Sabiporide reduces ischemia-induced arrhythmias and myocardial infarction and attenuates ERK phosphorylation and iNOS induction in rats. Biomed Res Int 2013, 504320.