探討運動訓練介入對放射照射誘發之心肌功能異常之成效

Abstract

研究背景與目的：放射治療是常見的胸腔癌症治療方式之一。放射照射會誘發生成大量的游離自由基，造成細胞凋亡和壞死。由於照射過程中，心臟會受到非標的劑量之照射造成傷害。受傷後的心肌會進行重塑，過程中所形成的纖維化會破壞正常組織結構，造成心臟功能異常，最終可能導致心臟衰竭。前置性運動訓練已知可以增加心肌對放射照射傷害的抵抗力，但放射照射後再介入運動訓練，是否可以減緩放射相關的心肌毒性則尚不清楚。因此本研究的目的是以動物模式探討放射照射後介入有氧運動訓練對心肌功能異常的影響並探討可能之相關機制。研究方法：本研究將18隻7-8週大之成年雄性Wistar大鼠，以每組6隻隨機分配到控制組（Ctrl）、放射照射組（IR）及放射照射後介入運動訓練組（IREx）。IR及IREx組的大鼠先接受心臟分次放射照射，劑量為5 Gy/次、1次/天、5天總量共25 Gy。IREx組大鼠在完成放射照射一週後，進行為期6週、每週5天、每次60分鐘之有氧運動訓練。所有組別的大鼠在IR和IREx組放射照射4週後（T1）和7週後（T2）兩個時間點進行心臟超音波測量。所有組別的大鼠在IREx組運動訓練後，以導電心導管進行活體心臟功能測試（壓力-體積分析）。實驗結束後，犧牲大鼠取出其左心室組織進行後續生化分析。心肌的氧化壓力傷害以化學冷光分析儀檢測自由基含量；抗氧化能力以反轉錄聚合酶連鎖反應分析CuZnSOD及MnSOD mRNA表現量；心肌重塑和纖維化則利用Masson's trichrome染色分析膠原蛋白容積分數（collagen volume fraction，CVF），及西方墨點法分析MMP-2、MMP-9、type I collagen和type III collagen蛋白表現量。研究結果：放射照射後，心臟超音波結果顯示，和Ctrl組相比，IR組在T1和T2時的FS（p=0.01和p=0.02）、EF（皆為p=0.02）、SV（皆為p=0.002）、CO（p=0.01和p=0.001）和EDV（p=0.05和p=0.03）均顯著較低，IVRT則在T2時顯著較長（p<0.001）；導電心導管結果顯示，和Ctrl組相比，IR組+dP/dtmax（p=0.001）和-dP/dtmax（p=0.01）顯著較低且EDP（p<0.001）顯著較高。氧化壓力結果顯示，IR組的總氧自由基（p=0.02）和超氧陰離子（p<0.001）含量均顯著較Ctrl組高。IR組的CVF（p<0.001）和type III collagen蛋白表現量（p=0.03）皆顯著較Ctrl組高，IR組的MMP-2蛋白表現量顯著較Ctrl組低（p=0.04）。放射照射後介入6週有氧運動訓練，心臟超音波顯示，和IR組相比，IREx組在T2的FS（p=0.02）和EF（p=0.01）顯著較高，ESV顯著較低（p=0.04），IVRT有較短的趨勢（p=0.07）。導電心導管結果顯示，IREx組的+dP/dtmax（p<0.001）和-dP/dtmax（p=0.002）顯著較IR組高，EDP顯著較IR組低（p<0.001）。IREx組的CuZnSOD（p=0.03）和MnSOD（p=0.02）mRNA表現量均顯著較IR組高。纖維化分析結果顯示，IREx組的CVF顯著較IR組低（p=0.003）。結論：放射照射後介入6週有氧運動訓練可透過減少心肌之氧化壓力和正調控心肌之抗氧化能力來降放射照射的心臟毒性，改善心臟功能。

Background: Radiotherapy (RT) is one of common treatments for patients with thoracic cancers. During mediastinal radiation, heart is often included in the irradiation field and all components of the heart are susceptible to radiation-related oxidative stress damage. Exercise preconditioning has been shown to enhance the resistance of heart to radiation-induced myocardial injury. However, whether exercise intervention after irradiation would attenuate radiation-induced cardiotoxicity remains to be determined. The purpose of this study was to investigate the effects of exercise intervention after irradiation on radiation-induced myocardial dysfunction and related mechanisms were also explored in the animal model. Methods: A total of 18 adult male Wistar rats aged 7 to 8 week-old were randomly assigned to control (Ctrl), irradiation (IR), or exercise training after irradiation (IREx) groups (n=6 per group). The rats in IR and IREx received irradiation to the heart region once daily for 5 days with a dose of 5 Gy/day (total dose of 25 Gy). One week after irradiation, the rats in IREx underwent 60 min/day treadmill exercise training 5 days per week for 6 weeks. Cardiac functions were determined by non-invasive transthoracic echocardiogram performed at 4 weeks (time point 1, T1) and 7 weeks (time point 2, T2) post irradiation. Invasive hemodynamic measurements using in vivo pressure-volume analysis were performed after all interventions were completed. The rats were sacrificed immediately after invasive hemodynamic measurements and the hearts were removed en bloc. Oxidative stress was determined by reactive oxygen species (ROS) level. Antioxidant capacity was determined by analyzing mRNA expression levels of CuZnSOD and MnSOD. Myocardial remodeling and fibrosis were examined by analyzing collagen volume fraction (CVF) from Masson’s trichrome staining, and protein expression levels of MMP-2, MMP-9, type I and type III collagen using Western blotting. Results: Echocardiographic data showed that FS (p=0.01 and p=0.02 for T1 and T2, respectively), EF (both p=0.02 for T1 and T2), SV (both p=0.002 for T1 and T2), CO (p=0.01 and p=0.001 for T1 and T2, respectively), and EDV (p=0.05 and p=0.03 for T1 and T2, respectively) were significantly lower at T1 and T2, and IVRT was significantly longer (p<0.001) at T2 in IR group than those of Crtrl group. In IREx group, FS (p=0.02) and EF (p=0.01) were significantly higher, and ESV (p=0.04) was significantly lower at T2 compared to those of IR group. Pressure-volume analysis showed that +dP/dtmax (p=0.001) and -dP/dtmax (p=0.01) were significantly lower, and EDP (p<0.001) was significantly higher in IR group than those of Ctrl group. In IREx group, +dP/dtmax (p<0.001) and -dP/dtmax (p=0.002) were significantly higher, and EDP (p<0.001) was significantly lower than those of IR group. In IR group, the ROS level (p<0.05), CVF (p<0.001), type III collagen protein expression level (p=0.03) were significantly higher than those of Ctrl group, and MMP-2 protein expression level (p=0.04) was significantly lower than those of Ctrl group. IREx group demonstrated significantly lower ROS level (p<0.05) and CVF (p=0.003) compared to those of IR group. Expression levels of CuZnSOD (p=0.03) and MnSOD (p=0.02) were significantly higher in IREx than those of IR group. Conclusions: Exercise training after irradiation could attenuate radiation-induced cardiotoxicity and preserve cardiac function by reducing oxidative stress and improving antioxidant defense capacity of myocardium.