Roles and mechanism of mitophagy in myocardial injury in mice following delayed resuscitation after severe burns
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摘要:
目的 探讨线粒体自噬在严重烧伤后延迟复苏小鼠心肌损伤中的作用及其机制。 方法 该研究为实验研究。取45只6~8周龄雄性C57BL/6J小鼠,按照随机数字表法（分组方法下同）分为假伤组、伤后0 h补液组、伤后3 h补液组、伤后6 h补液组、伤后不补液组,每组9只。将假伤组小鼠致假伤,其余4组小鼠均致30%体表总面积Ⅲ度烫伤（以下称烧伤）。伤后0 h补液组、伤后3 h补液组、伤后6 h补液组小鼠分别于伤后0（即刻）、3、6 h开始补液,假伤组、伤后不补液组小鼠伤后不予补液。伤后12 h,采用全自动生化分析仪测定血清中乳酸脱氢酶（LDH）和肌酸激酶同工酶（CK-MB）水平；行苏木精-伊红染色后采用Kishimoto评分行心肌组织病理学评分；采用蛋白质印迹法检测心肌组织中线粒体自噬相关蛋白线粒体外膜转位酶20（TOM20）、微管相关蛋白1轻链3B（LC3B）、P62和苄氯素1的蛋白表达,并计算LC3B-Ⅱ/LC3B-Ⅰ比值。取27只6~8周龄雄性C57BL/6J小鼠,分为单纯伤后6 h补液组、伤后6 h补液+3-甲基腺嘌呤（3-MA）组、伤后6 h补液+雷帕霉素组,每组9只。在烧伤造模前1周,对伤后6 h补液+3-MA组和伤后6 h补液+雷帕霉素组小鼠分别经腹腔注射线粒体自噬抑制剂3-MA、线粒体自噬激动剂雷帕霉素。对3组小鼠均造成严重烧伤,且均于伤后6 h开始补液。伤后12 h,同前测定血清中LDH和CK-MB水平,行心肌组织病理学评分,检测心肌组织中线粒体自噬相关蛋白的蛋白表达；另经铀铅双染色后于透射电子显微镜下观察心肌组织中线粒体超微结构变化并行Flameng评分。 结果 伤后12 h,与伤后0 h补液组比较,伤后6 h补液组和伤后不补液组小鼠血清中LDH、CK-MB水平均明显升高（P<0.05）。伤后12 h,与伤后0 h补液组比较,假伤组小鼠心肌组织病理学评分明显降低（P<0.05）,伤后3 h补液组、伤后6 h补液组和伤后不补液组小鼠心肌组织病理学评分均明显升高（P<0.05）。伤后12 h,与伤后0 h补液组比较,假伤组小鼠心肌组织中苄氯素1的蛋白表达明显降低（P<0.05）,TOM20、P62的蛋白表达均明显升高（P<0.05）；伤后3 h补液组小鼠心肌组织中苄氯素1的蛋白表达明显升高（P<0.05）,TOM20、P62的蛋白表达均明显降低（P<0.05）；伤后6 h补液组和伤后不补液组小鼠心肌组织中苄氯素1的蛋白表达、LC3B-Ⅱ/LC3B-Ⅰ比值均明显升高（P<0.05）,TOM20、P62的蛋白表达均明显降低（P<0.05）。伤后12 h,与单纯伤后6 h补液组比较,伤后6 h补液+3-MA组小鼠血清中LDH、CK-MB水平均明显降低（P<0.05）,伤后6 h补液+雷帕霉素组小鼠血清中LDH、CK-MB水平均明显升高（P<0.05）。伤后12 h,与单纯伤后6 h补液组比较,伤后6 h补液+3-MA组小鼠心肌组织病理学评分明显降低（P<0.05）,伤后6 h补液+雷帕霉素组小鼠心肌组织病理学评分明显升高（P<0.05）。伤后12 h,与单纯伤后6 h补液组比较,伤后6 h补液+3-MA组小鼠心肌组织中苄氯素1的蛋白表达和LC3B-Ⅱ/LC3B-Ⅰ比值均明显降低（P<0.05）,TOM20、P62的蛋白表达均明显升高（P<0.05）；伤后6 h补液+雷帕霉素组小鼠心肌组织中苄氯素1的蛋白表达和LC3B-Ⅱ/LC3B-Ⅰ比值均明显升高（P<0.05）,TOM20、P62的蛋白表达均明显降低（P<0.05）。伤后12 h,与单纯伤后6 h补液组[（2.67±0.11）分]比较,伤后6 h补液+3-MA组小鼠心肌组织中线粒体Flameng评分[（2.07±0.11）分]明显降低（P<0.05）,伤后6 h补液+雷帕霉素组小鼠心肌组织中线粒体Flameng评分[（3.60±0.20）分]明显升高（P<0.05）。 结论 延迟复苏可导致严重烧伤后小鼠心肌损伤和线粒体自噬过度激活,抑制线粒体自噬能够减轻烧伤后延迟复苏造成的心肌损伤。 Abstract:Objective To explore the roles and mechanism of mitophagy in myocardial injury in mice following delayed resuscitation after severe burns. Methods This study was an experimental study. Forty-five 6-8-week-old male C57BL/6J mice were divided into sham injury group, 0 h post-injury rehydration group, 3 h post-injury rehydration group, 6 h post-injury rehydration group, and no post-injury rehydration group, with 9 mice in each group, according to the random number table (the same grouping method below). The mice in sham injury group were subjected to a sham injury, while the mice in the remaining 4 groups were subjected to full-thickness scald (hereinafter referred to as burn) of 30% total body surface area. The mice in 0 h post-injury rehydration group, 3 h post-injury rehydration group, and 6 h post-injury rehydration group received rehydration starting from 0 (immediately), 3, and 6 h post-injury, respectively. The mice in sham injury group and no post-injury rehydration group did not receive rehydration after injury. At 12 h post-injury, the serum levels of lactate dehydrogenase (LDH) and creatine kinase isoenzyme (CK-MB) were detected using an automated biochemical analyzer. Myocardial histopathological scoring was performed using Kishimoto scores after hematoxylin-eosin staining. The protein expressions of mitophagy-related proteins, including translocase of outer mitochondrial membrane 20 (TOM20), microtubule-associated protein 1 light chain 3B (LC3B), P62, and Beclin-1 in myocardial tissue were detected by Western blotting, and the ratio of LC3B-Ⅱ/LC3B-Ⅰ was calculated. Twenty-seven 6-8-week-old male C57BL/6J mice were divided into 6 h post-injury rehydration alone group, 6 h post-injury rehydration+3-methyladenine (3-MA) group, and 6 h post-injury rehydration+rapamycin group, with 9 mice in each group. One week before burn modeling, mice in 6 h post-injury rehydration+3-MA group and 6 h post-injury rehydration+rapamycin group were injected intraperitoneally with the mitophagy inhibitor 3-MA or mitophagy activator rapamycin. All the mice in 3 groups received rehydration starting from 6 h post-injury following severe burns. At 12 h post-injury, the serum levels of LDH and CK-MB were detected, myocardial histopathological scoring was performed, and the protein expressions of mitochondrial autophagy-related proteins were detected as before. The mitochondrial ultrastructure changes in myocardial tissue were observed under a transmission electron microscope after uranium-lead double staining, and Flameng scoring was performed. Results At 12 h post-injury, compared with those in 0 h post-injury rehydration group the serum levels of LDH and CK-MB in mice in 6 h post-injury rehydration group and no post-injury rehydration group were significantly increased (P<0.05). At 12 h post-injury, compared with that in 0 h post-injury rehydration group, the myocardial histopathological score in mice in sham injury group was significantly decreased (P<0.05), and the myocardial histopathological scores in mice in 3 h post-injury rehydration group, 6 h post-injury rehydration group, and no post-injury rehydration group were significantly increased (P<0.05). At 12 h post-injury, compared with those in 0 h post-injury rehydration group, the protein expression of Beclin-1 was significantly decreased (P<0.05), while the protein expressions of TOM20 and P62 were significantly increased (P<0.05) in myocardial tissue of mice in sham injury group; the protein expression of Beclin-1 was significantly increased (P<0.05), while the protein expressions of TOM20 and P62 were significantly decreased (P<0.05) in myocardial tissue of mice in 3 h post-injury rehydration group; the protein expressions of Beclin-1 and ratios of LC3B-Ⅱ/LC3B-Ⅰwere significantly increased (P<0.05), while the protein expressions of TOM20 and P62 were significantly decreased (P<0.05) in myocardial tissue of mice in 6 h post-injury rehydration group and no post-injury rehydration group. At 12 h post-injury, compared with those in 6 h post-injury rehydration alone group, the serum levels of LDH and CK-MB in mice in 6 h post-injury rehydration+3-MA group were significantly decreased (P<0.05), while the serum levels of LDH and CK-MB in mice in 6 h post-injury rehydration+rapamycin group were significantly increased (P<0.05). At 12 h post-injury, compared with that in 6 h post-injury rehydration alone group, the myocardial histopathological score in mice in 6 h post-injury rehydration+3-MA group was significantly decreased (P<0.05), while the myocardial histopathological score in mice in 6 h post-injury rehydration+rapamycin group was significantly increased (P<0.05). At 12 h post-injury, compared with those in 6 h post-injury rehydration alone group, the protein expression of Beclin-1 and ratio of LC3B-Ⅱ/LC3B-Ⅰ were significantly decreased (P<0.05), while the protein expressions of TOM20 and P62 were significantly increased (P<0.05) in myocardial tissue of mice in 6 h post-injury rehydration+3-MA group; the protein expression of Beclin-1 and ratio of LC3B-Ⅱ/LC3B-Ⅰwere significantly increased (P<0.05), while the protein expressions of TOM20 and P62 were significantly decreased (P<0.05) in myocardial tissue of mice in 6 h post-injury rehydration+rapamycin group. At 12 h post-injury, compared with 2.67±0.11 in 6 h post-injury rehydration alone group, the mitochondrial Flameng score (2.07±0.11) in myocardial tissue of mice in 6 h post-injury rehydration+3-MA group was significantly decreased (P<0.05), while the mitochondrial Flameng score (3.60±0.20) in myocardial tissue of mice in 6 h post-injury rehydration+rapamycin group was significantly increased (P<0.05). Conclusions Delayed rehydration leads to myocardial injury and excessive activation of mitophagy in mice with severe burns. The inhibition of mitophagy can mitigate myocardial injury caused by delayed rehydration after severe burns. -
Key words:
- Burns /
- Myocardium /
- Mitophagy /
- Shock /
- Delayed resuscitation
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参考文献
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图 1 蛋白质印迹法检测的5组小鼠伤后12 h心肌组织中线粒体自噬相关蛋白的蛋白表达
注：TOM20为线粒体外膜转位酶20,LC3B为微管相关蛋白1轻链3B；条带上方1、2、3、4、5分别指示假伤组、伤后0 h补液组、伤后3 h补液组、伤后6 h补液组、伤后不补液组；假伤组小鼠致假伤,其余4组小鼠均造成严重烧伤,伤后0 h补液组、伤后3 h补液组、伤后6 h补液组小鼠分别于伤后0（即刻）、3、6 h开始补液,伤后不补液组小鼠不予补液
图 2 蛋白质印迹法检测的3组严重烧伤后延迟复苏小鼠伤后12 h心肌组织中线粒体自噬相关蛋白的蛋白表达
注：TOM20为线粒体外膜转位酶20,LC3B为微管相关蛋白1轻链3B,3-MA为3-甲基腺嘌呤；条带上方1、2、3分别指示伤后6 h补液+3-MA组、单纯伤后6 h补液组、伤后6 h补液+雷帕霉素组；于烧伤造模前1周,先对伤后6 h补液+3-MA组和伤后6 h补液+雷帕霉素组小鼠分别经腹腔注射线粒体自噬抑制剂3-MA、线粒体自噬激动剂雷帕霉素,然后对单纯伤后6 h补液组、伤后6 h补液+3-MA组、伤后6 h补液+雷帕霉素组小鼠均造成严重烧伤且均于伤后6 h开始补液
图 3 3组严重烧伤后延迟复苏小鼠伤后12 h心肌组织中线粒体自噬情况 醋酸铀-柠檬酸铅×5 000。3A.伤后6 h补液+3-MA组小鼠心肌纤维排列疏松、局部断裂,部分线粒体肿胀、嵴模糊或减少,少部分线粒体膜破裂；3B.单纯伤后6 h补液组小鼠心肌纤维排列轻度紊乱,大量线粒体肿胀、嵴稀疏,部分线粒体膜破裂；3C.伤后6 h补液+雷帕霉素组小鼠心肌纤维排列明显紊乱,大部分线粒体膜破裂,嵴严重紊乱、广泛断裂,可见线粒体自噬小体
注：于烧伤造模前1周,先对伤后6 h补液+3-甲基腺嘌呤（3-MA）组和伤后6 h补液+雷帕霉素组小鼠分别经腹腔注射线粒体自噬抑制剂3-MA、线粒体自噬激动剂雷帕霉素,然后对单纯伤后6 h补液组、伤后6 h补液+3-MA组、伤后6 h补液+雷帕霉素组小鼠均造成严重烧伤且均于伤后6 h开始补液；黄色箭头指示心肌纤维,蓝色箭头指示线粒体膜,红色箭头指示线粒体嵴,绿色箭头指示线粒体自噬小体
图 4 3组严重烧伤后延迟复苏小鼠伤后12 h心肌细胞凋亡情况 异硫氰酸荧光素-4',6-二脒基-2-苯基吲哚×40。4A、4B、4C.分别为伤后6 h补液+3-MA组、单纯伤后6 h补液组、伤后6 h补液+雷帕霉素组TUNEL荧光强度,图4B中TUNEL荧光强度明显高于图4A,但明显低于图4C
注：于烧伤造模前1周,先对伤后6 h补液+3-甲基腺嘌呤（3-MA）组和伤后6 h补液+雷帕霉素组小鼠分别经腹腔注射线粒体自噬抑制剂3-MA、线粒体自噬激动剂雷帕霉素,然后对单纯伤后6 h补液组、伤后6 h补液+3-MA组、伤后6 h补液+雷帕霉素组小鼠均造成严重烧伤且均于伤后6 h开始补液；细胞核阳性染色为蓝色,细胞原位末端标记（TUNEL）阳性染色为绿色；以阳性染色区域的平均荧光强度表示细胞凋亡情况
Table 1. 5组小鼠伤后12 h心肌组织中线粒体自噬相关蛋白的蛋白表达比较（
组别 样本数 苄氯素1 P62 TOM20 LC3B-Ⅱ/LC3B-Ⅰ比值 假伤组 3 0.118±0.031 1.13±0.13 1.30±0.11 0.93±0.06 伤后0 h补液组 3 0.349±0.046 0.99±0.03 1.06±0.16 1.08±0.14 伤后3 h补液组 3 0.556±0.051 0.63±0.05 0.71±0.07 1.22±0.03 伤后6 h补液组 3 1.020±0.073 0.43±0.07 0.57±0.10 1.35±0.07 伤后不补液组 3 1.248±0.192 0.27±0.05 0.43±0.04 1.39±0.04 F值 68.64 70.53 35.18 17.70 P值 <0.001 <0.001 <0.001 <0.001 P1值 0.048 0.041 0.043 0.145 P2值 0.048 <0.001 0.015 0.145 P3值 <0.001 <0.001 0.001 0.011 P4值 <0.001 <0.001 <0.001 0.006 注：假伤组小鼠致假伤,其余4组小鼠均造成严重烧伤,伤后0 h补液组、伤后3 h补液组、伤后6 h补液组分别于伤后0（即刻）、3、6 h开始补液,伤后不补液组、假伤组小鼠不予补液；TOM20为线粒体外膜转位酶20,LC3B为微管相关蛋白1轻链3B；F值、P值为5组间各指标总体比较所得；P1值、P2值、P3值、P4值分别为假伤组、伤后3 h补液组、伤后6 h补液组、伤后不补液组与伤后0 h补液组各指标比较所得 
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Table 2. 3组严重烧伤后延迟复苏小鼠伤后12 h心肌组织中线粒体自噬相关蛋白的蛋白表达比较（
组别 样本数 苄氯素1 P62 TOM20 LC3B-Ⅱ/LC3B-Ⅰ比值 伤后6 h补液+3-MA组 3 0.112±0.015 0.94±0.12 1.16±0.08 0.95±0.07 单纯伤后6 h补液组 3 0.459±0.051 0.71±0.04 0.64±0.10 1.37±0.05 伤后6 h补液+雷帕霉素组 3 0.835±0.101 0.47±0.05 0.36±0.06 1.83±0.27 F值 89.02 27.18 75.54 22.24 P值 <0.001 0.001 <0.001 0.002 P1值 <0.001 0.018 <0.001 0.026 P2值 <0.001 0.018 0.006 0.026 注：于烧伤造模前1周,对伤后6 h补液+3-甲基腺嘌呤（3-MA）组和伤后6 h补液+雷帕霉素组小鼠分别经腹腔注射线粒体自噬抑制剂3-MA、线粒体自噬激动剂雷帕霉素,然后对单纯伤后6 h补液组、伤后6 h补液+3-MA组、伤后6 h补液+雷帕霉素组小鼠均造成严重烧伤且均于伤后6 h开始补液；TOM20为线粒体外膜转位酶20,LC3B为微管相关蛋白1轻链3B；F值、P值为3组间各指标总体比较所得；P1值、P2值分别为伤后6 h补液+3-MA组、伤后6 h补液+雷帕霉素组与单纯伤后6 h补液组比较所得
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