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随着临床肿瘤放射治疗的广泛应用,胸部肿瘤患者生存时间明显延长,而在治疗过程中位于纵隔的心脏不可避免地要受到射线照射。大量临床资料显示,由放射治疗所导致的放射性心脏损伤(radiation-induced heart disease,RIHD)已成为患者非肿瘤死亡的重要原因,这部分患者心血管疾病的发病率和病死率都明显升高,特别是心功能不全的患者[1]。有研究者认为,能量代谢的改变在心功能不全的发病机制中起着重要作用,调节心肌能量代谢有望成为一种治疗心功能不全的新策略,而线粒体作为心肌能量供应的主要细胞器,其结构和功能的正常对于维持心肌细胞正常的能量代谢具有重要作用[2]。因此,本研究旨在观察X射线照射对心肌细胞线粒体功能的影响以及自噬在线粒体功能改变中的作用。
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照射组大鼠经20 Gy X射线照射后7 d、14 d左心室各功能参数与假照射组比较无明显变化,而照射后21 d大鼠的LVDP、±dP/dtmax均低于假照射组,且差异显著(表 1)。
组别 数量
(只)0 d 7 d LVDP
(mmHg)+dP/dt max
(mmHg/s)-dP/dt max
(mmHg/s)LVDP
(mmHg)+dP/dt max
(mmHg/s)-dP/dt max
(mmHg/s)假照射组 10 107.5±12.6 3060.0±280.1 -1999.0±241.4 108.5±10.8 3043.3±258.3 -2016.0±211.7 照射组 80 108.5±10.3 3060.0±304.9 -1999.0±222.2 102.5±8.7 2813.5±366.4 -2031.0±252.4 组别 14 d 21 d LVDP
(mmHg)+dP/dt max
(mmHg/s)-dP/dt max
(mmHg/s)LVDP
(mmHg)+dP/dt max
(mmHg/s)-dP/dt max
(mmHg/s)假照射组 106.3±13.0 3093.3±225.7 -2033.0±236.8 105.5±11.7 3093.3±306.9 -2016.0±261.5 照射组 104.2±9.3 2893.3±252.5 -2066.0±297.1 87.5±11.6 2060.0±266.6 -1666.0±243.5 注:表中,1 mmHg=0.133 kPa;LVDP:左心室舒张压;+dP/dtmax:左心室压力上升的最大速率;-dP/dtmax:左心室压力下降的最大速率。20 Gy X射线照射后21 d,大鼠的LVDP、±dP/dtmax均低于假照射组。 表 1 经20 Gy X射线照射后大鼠离体心功能参数的变化(x±s)
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线粒体膜电位常作为评估线粒体功能的重要参数之一。本实验采用JC-1荧光染色来反映心肌细胞线粒体膜电位的变化。本实验中假照射组大鼠心肌组织呈高红荧光,而照射组大鼠经20 Gy X射线照射后1 d的心肌组织绝大部分呈绿色荧光,提示照射后大鼠线粒体功能损伤。
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本实验选用LC3Ⅱ和Beclin 1作为自噬形成的标志物。与假照射组比较,照射组大鼠经X射线照射后3 h、6 h、12 h、1 d、2 d、4 d和7 d,LC3Ⅱ及Beclin 1的蛋白表达水平和mRNA水平均明显升高,在照射后6 h达到最高值(图 1)。
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3-MA是一种特异性的自噬抑制剂。与照射组相比,照射+3-MA组经X射线照射6 h后,大鼠心肌细胞LC3Ⅱ及Beclin 1蛋白表达水平明显下降,大鼠离体心功能参数出现异常的时间提前至照射后14 d:LVDP=(84.2±9.3)mmHg(1 mmHg=0.133 kPa);+dP/dtmax=(1993.3±167.8)mmHg/s;-dP/dtmax=(-1599.0±185.4)mmHg/s。
自噬在X射线照射所致心肌线粒体损伤中的作用
Effects of autophagy in myocardial mitochondrial injury caused by X-ray irradiation
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摘要:
目的 观察X射线照射对大鼠心肌线粒体功能的影响以及自噬在线粒体功能损伤中的作用。 方法 将135只实验大鼠分成假照射组(10只)、照射组(80只)和照射+3-甲基腺嘌呤(3-MA)组(45只),假照射组接受0 Gy X射线照射,照射组给予20 Gy X射线照射,照射+3-MA组接受20 Gy X射线照射前30 min腹腔注射2 μl 3-MA。采用蛋白免疫印迹法和实时定量PCR法检测心肌细胞的LC3Ⅱ及Beclin 1蛋白表达及其mRNA水平变化,检测左心室舒张压及±dP/dtmax(左心室压力上升及下降的最大速率)等离体心功能指标。 结果 与假照射组相比,照射组大鼠经20 Gy X射线照射后离体心功能在21 d时出现降低;而在1 d时心肌组织线粒体功能便出现了损伤;同时,心肌细胞自噬水平在3 h、6 h、12 h、1 d、2 d、4 d及7 d时出现明显上升,与假照射组之间的差异显著,且在6 h达到最高值。与照射组相比,照射+3-MA组大鼠心肌LC3Ⅱ及Beclin 1表达水平明显下降,且心功能出现异常的时间提前至14 d。 结论 X射线照射损伤大鼠心肌线粒体功能并诱导自噬发生,可能是心肌保护的重要因素。 Abstract:Objective To observed the effects of X-ray irradiation on myocardial mitochondrial function and role of autophagy in mitochondrial injury. Methods The rats were divided into sham-irradiation control group, irradiation group and irradiation+3-methyladenine(3-MA) group. Rat in sham-irradiation control group accepted no irradiation, while rat in irradiation group accepted local 20 Gy X-ray irradiation in the chest, the last group accepted 20 Gy X-ray irradiation prior to intraperitoneal injection 2 μl 3-MA. Western blot and real time PCR were used to detect the expression of LC3Ⅱ and Beclin 1 in protein and their mRNA levels. In vitro cardiac function was used to detect the changes of the left ventricular diastolic pressure(LVDP), the maximal rate of rise of pressure in the isovolumetric systolic left ventricle(+dP/dtmax)and the maximal rate of rise of pressure in the isovolumetric diastolic left ventricle(-dP/dtmax). Results Compared with the sham-irradiation control group, there were the decreased of the LVDP, +dP/dtmax and -dP/dtmax at 21 d, and myocardial mitochondrial injury at 1 d after 20 Gy irradiation. The expression of LC3 and Beclin 1 in protein and mRNA levels increased significantly at 3 h, 6 h, 12 h, 1 d, 2 d, 4 d and 7 d after 20 Gy irradiation, up to peak at 6 h. Compared with irradiation group, intraperitoneal injection 2 μl 3-MA 30 min before irradiation, the expression of LC3Ⅱ and Beclin 1 in protein decreased significantly at 6 h after irradiation, there were the decreased of the LVDP, +dP/dtmax and -dP/dtmax at 14 d, then the irradiation group for 21 days. Conclusion X-ray irradiation could injury myocardial mitochondrial function and induce autophagy in rat myocardial cell, which was an important factor of myocardial protection. -
Key words:
- Mitochondria, heart /
- Radiation injury /
- Autophagy /
- X-rays
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表 1 经20 Gy X射线照射后大鼠离体心功能参数的变化(x±s)
组别 数量
(只)0 d 7 d LVDP
(mmHg)+dP/dt max
(mmHg/s)-dP/dt max
(mmHg/s)LVDP
(mmHg)+dP/dt max
(mmHg/s)-dP/dt max
(mmHg/s)假照射组 10 107.5±12.6 3060.0±280.1 -1999.0±241.4 108.5±10.8 3043.3±258.3 -2016.0±211.7 照射组 80 108.5±10.3 3060.0±304.9 -1999.0±222.2 102.5±8.7 2813.5±366.4 -2031.0±252.4 组别 14 d 21 d LVDP
(mmHg)+dP/dt max
(mmHg/s)-dP/dt max
(mmHg/s)LVDP
(mmHg)+dP/dt max
(mmHg/s)-dP/dt max
(mmHg/s)假照射组 106.3±13.0 3093.3±225.7 -2033.0±236.8 105.5±11.7 3093.3±306.9 -2016.0±261.5 照射组 104.2±9.3 2893.3±252.5 -2066.0±297.1 87.5±11.6 2060.0±266.6 -1666.0±243.5 注:表中,1 mmHg=0.133 kPa;LVDP:左心室舒张压;+dP/dtmax:左心室压力上升的最大速率;-dP/dtmax:左心室压力下降的最大速率。20 Gy X射线照射后21 d,大鼠的LVDP、±dP/dtmax均低于假照射组。 -
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