硫化氢后处理对2型糖尿病大鼠心肌缺血再灌注损伤的保护作用及其与自噬潮的相关性研究

doi:10.3877/cma.j.issn.1674-6880.2016.02.002

中华危重症医学杂志(电子版) ›› 2016, Vol. 09 ›› Issue (02) : 81 -86. doi: 10.3877/cma.j.issn.1674-6880.2016.02.002

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论著

硫化氢后处理对2型糖尿病大鼠心肌缺血再灌注损伤的保护作用及其与自噬潮的相关性研究

赵雯洁¹, 王琛², 孙波¹, 单海华¹, 王安¹, 乔世刚²^,^†(

)

^1. 215004　江苏苏州，苏州大学附属第二医院麻醉科
^2. 215163　江苏苏州，苏州科技城医院的麻醉科

收稿日期:2015-10-11 出版日期:2016-04-01
通信作者: 乔世刚
基金资助:
江苏省自然科学基金面上研究项目(BK20141187); 苏州市科技计划项目(SYS201473); 苏州大学附属第二医院博士、留学归国人员预研项目(SDFEYBS1401)

Correlative study of autophagy flux and hydrogen sulfide postconditioning protecting on type 2 diabetic rats with myocardial ischemia reperfusion injury

Wenjie Zhao¹, Chen Wang², Bo Sun¹, Haihua Shan¹, An Wang¹, Shigang Qiao²^,^†()

^1. Department of Anesthesiology, Second Affiliated Hospital, Soochow University, Suzhou 215004, China
^2. Department of Anesthesiology, Suzhou Science and Technology City Hospital, Suzhou 215000, China

Received:2015-10-11 Published:2016-04-01
Corresponding author: Shigang Qiao
About author:
Corresponding author: Qiao Shigang, Email: qiaoshigang@163.com

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引用本文：

赵雯洁, 王琛, 孙波, 单海华, 王安, 乔世刚. 硫化氢后处理对2型糖尿病大鼠心肌缺血再灌注损伤的保护作用及其与自噬潮的相关性研究[J/OL]. 中华危重症医学杂志(电子版), 2016, 09(02): 81-86.

Wenjie Zhao, Chen Wang, Bo Sun, Haihua Shan, An Wang, Shigang Qiao. Correlative study of autophagy flux and hydrogen sulfide postconditioning protecting on type 2 diabetic rats with myocardial ischemia reperfusion injury[J/OL]. Chinese Journal of Critical Care Medicine(Electronic Edition), 2016, 09(02): 81-86.

目的

观察硫化氢后处理对2型糖尿病大鼠心肌缺血再灌注损伤（I/R）的保护作用及其与自噬潮的关系。

方法

将60只成年雄性SD大鼠分为五组：假手术组：仅开胸并分离冠状动脉左前降支，但不结扎阻断血流；I/R组：于冠状动脉左前降支阻断30 min，再灌注4 h；氯喹（CQ）组：于术前1 h腹腔注射10 mg/kg，其余操作同I/R组；硫氢化钠（NaHS）组：于开放左冠状动脉再灌注即刻1 min内静脉注射硫氢化钠0.05 mg/kg，再灌注4 h；CQ + NaHS组：于术前1 h腹腔注射CQ，其余操作同NaHS组；每组12只。连续监测并记录大鼠平衡期15 min、缺血期15 min、再灌注1、2、4 h时大鼠心率、平均动脉压和心率-收缩压乘积（RPP）。再灌注4 h结束后分批处死大鼠并取心脏测定心肌梗死范围，并采用Western blotting法测定微管相关蛋白1轻链3（LC3）、Cathepsin B、Beclin-1、P62表达水平。

结果

各组大鼠在各时间点心率的比较，差异无统计学意义（F=0.854，P=0.512），而平均动脉压及RPP的比较差异均有统计学意义（F=5.182，P=0.007；F=5.082，P=0.008）。且再灌注期2、4 h时，NaHS组平均动脉压为（87 ± 8）mmHg、（91 ± 10）mmHg，RPP为（35.4 ± 4.6）次·mmHg·10³/min、（36.2 ± 5.8）次·mmHg·10³/min，NaHS组明显高于I/R组[平均动脉压分别为（72 ± 10）mmHg、（63 ± 6）mmHg，RPP分别为（28.7±5.8）次·mmHg·10³/min、（26.8 ± 3.8）次·mmHg·10³/min，P均< 0.05]。各组灌注期4 h时，心肌梗死范围区域所占比例及LC3、Cathepsin B、Beclin-1、P62比较差异均有统计学意义（F=96.907、71.164、43.594、57.180、35.967，P均< 0.05），且NaHS组上述各指标均明显低于I/R组、CQ组、CQ + NaHS组（P均< 0.05）。

结论

硫化氢可能通过修复自噬潮对2型糖尿病大鼠心肌I/R损伤发挥保护作用。

关键词: 硫化氢, 缺血后处理, 心肌再灌注损伤, 糖尿病，2型, 自噬, 大鼠

Objective

To investigate the correlative of autophagy flux and hydrogen sulfide postconditioning protecting against myocardial ischemia reperfusion (I/R) injury in type 2 diabetic rats in vivo.

Methods

Sixty adult male Sprague-Dawley rats were randomly divided into five groups (n=12): sham group, I/R group, chloroquine (CQ) group, sodium hydrosulfide (NaHS) group and CQ + NaHS group. Rats in the sham group only gave thoracotomy and separation of the left anterior descending coronary artery; In the I/R group rats were occluded the left anterior descending coronary artery for 30 min, followed by 4 h of reperfusion; In the CQ group rats received CQ 10 mg/kg by intraperitoneal injection at 1 h before the I/R operation; In the NaHS group rats were injected NaHS 0.05 mg/kg intravenously within 1 min after releasing the left anterior descending coronary artery undergoing 30 min occlusion, then followed by 4 h of reperfusion; and in the CQ + NaHS group rats received CQ 10 mg/kg by intraperitoneal injection at 1 h before the I/R operation additionally on the basis of NaHS group. The heart rate, mean arterial pressure and rate-pressure product (RPP) were detected and recorded at 15 min of balance period, ischemic period, and 1, 2, 4 h after reperfusion. After reperfusion for 4 h, the rats were sacrificed and the hearts were used to calculate the range of myocardial infarction and the express of microtubule-associated protein 1 light chain 3 (LC3), Cathepsin B, Beclin-1 and P62 were determined by Western blotting.

Results

The heart rates in the five groups had no siginficant differences at each time point (F=0.854, P=0.512), but the mean arterial pressure and RPP showed siginficant differences among the five groups at each time point (F=5.182, P=0.007; F=5.082, P=0.008). Furthermore, the mean arterial pressure[(87 ± 8) mmHg vs. (72 ± 10) mmHg, (91±10) mmHg vs. (63 ± 6) mmHg] and RPP [(35.4 ± 4.6)·10³ mmHg·beat/min vs. (28.7 ± 5.8)·10³ mmHg·beat/min, (36.2 ± 5.8)·10³ mmHg·beat/min vs. (26.8 ± 3.8)·10³ mmHg·beat/min] in the NaHS group at 2, 4 h after reperfusion were much higher than those in the I/R group (all P<0.05). The range of myocardial infarction and the express of LC3, Cathepsin B, Beclin-1 and P62 at 4 h after reperfusion all had statistical significance obviously among five groups (F=96.907, 71.164, 43.594, 57.180, 35.967, all P<0.05) , and above indicators in the NaHS group were much lower than those in the I/R group, CQ group and CQ + NaHS group (all P<0.05).

Conclusion

Hydrogen sulfide postconditioning play a protective role through repairinng autophagy flux in type 2 diabetic rats with I/R injury.

Key words: Hydrogen sulfide, Ischemic postconditioning, Myocardial reperfusion injury, Diabetes mellitus, type 2, Autophagy, Rats

表1 各组大鼠血流动力学变化的比较（±s）

组别		例数	平衡期	缺血期	再灌注期1 h	再灌注期2 h	再灌注期4 h
心率（min^-1）		?	?	?	?	?	?
?	假手术组	8	392 ± 32	382 ± 49	393 ± 51	382 ± 37	381 ± 38
?	I/R组	8	388 ± 45	385 ± 39	379 ± 51	381 ± 41	375 ± 37
?	CQ组	8	382 ± 51	375 ± 47	382 ± 44	379 ± 58	388 ± 35
?	NaHS组	8	388 ± 47	385 ± 41	379 ± 53	381 ± 43	375 ± 39
?	NaHS + CQ组	8	380 ± 32	386 ± 57	382 ± 52	385 ± 28	377 ± 46
平均动脉压（mmHg）		?	?	?	?	?	?
?	假手术组	8	118 ± 15	115 ± 16	110 ± 10	113 ± 12	110 ± 17
?	I/R组	8	122 ± 17	102 ± 11^a	88 ± 12^a	72 ± 10^a	63 ± 6^a
?	CQ组	8	123 ± 16	108 ± 13^a	90 ± 9^a	75 ± 14^ab	65 ± 9^ab
?	NaHS组	8	125 ± 18	101 ± 9^a	99 ± 10^a	87 ± 8^ac	91 ± 10^ac
?	NaHS + CQ组	8	124 ± 12	103 ± 12^a	92 ± 11^a	76 ± 10^ab	66 ± 12^ab
RPP（次·mmHg·10³/min）		?	?	?	?	?	?
?	假手术组	8	48.8 ± 5.8	47.8 ± 7.2	45.0 ± 7.1	45.5 ± 8.0	44.6 ± 8.3
?	I/R组	8	51.2 ± 8.2	42.3 ± 6.5^a	38.8 ± 6.3^a	28.7 ± 5.8^a	26.8 ± 3.8^a
?	CQ组	8	51.3 ± 7.8	41.9 ± 6.8^a	38.6 ± 7.1^a	28.8 ± 6.2^ab	27.4 ± 6.3^ab
?	NaHS组	8	48.8 ± 8.5	42.9 ± 7.1^a	39.9 ± 7.5^a	35.4 ± 4.6^ac	36.2 ± 5.8^ac
?	NaHS + CQ组	8	49.5 ± 8.0	42.8 ± 8.6^a	39.4 ± 4.5^a	30.2 ± 4.6^ab	26.5 ± 4.8^ab

表1 各组大鼠血流动力学变化的比较（±s）

组别		例数	平衡期	缺血期	再灌注期1 h	再灌注期2 h	再灌注期4 h
心率（min^-1）		?	?	?	?	?	?
?	假手术组	8	392 ± 32	382 ± 49	393 ± 51	382 ± 37	381 ± 38
?	I/R组	8	388 ± 45	385 ± 39	379 ± 51	381 ± 41	375 ± 37
?	CQ组	8	382 ± 51	375 ± 47	382 ± 44	379 ± 58	388 ± 35
?	NaHS组	8	388 ± 47	385 ± 41	379 ± 53	381 ± 43	375 ± 39
?	NaHS + CQ组	8	380 ± 32	386 ± 57	382 ± 52	385 ± 28	377 ± 46
平均动脉压（mmHg）		?	?	?	?	?	?
?	假手术组	8	118 ± 15	115 ± 16	110 ± 10	113 ± 12	110 ± 17
?	I/R组	8	122 ± 17	102 ± 11^a	88 ± 12^a	72 ± 10^a	63 ± 6^a
?	CQ组	8	123 ± 16	108 ± 13^a	90 ± 9^a	75 ± 14^ab	65 ± 9^ab
?	NaHS组	8	125 ± 18	101 ± 9^a	99 ± 10^a	87 ± 8^ac	91 ± 10^ac
?	NaHS + CQ组	8	124 ± 12	103 ± 12^a	92 ± 11^a	76 ± 10^ab	66 ± 12^ab
RPP（次·mmHg·10³/min）		?	?	?	?	?	?
?	假手术组	8	48.8 ± 5.8	47.8 ± 7.2	45.0 ± 7.1	45.5 ± 8.0	44.6 ± 8.3
?	I/R组	8	51.2 ± 8.2	42.3 ± 6.5^a	38.8 ± 6.3^a	28.7 ± 5.8^a	26.8 ± 3.8^a
?	CQ组	8	51.3 ± 7.8	41.9 ± 6.8^a	38.6 ± 7.1^a	28.8 ± 6.2^ab	27.4 ± 6.3^ab
?	NaHS组	8	48.8 ± 8.5	42.9 ± 7.1^a	39.9 ± 7.5^a	35.4 ± 4.6^ac	36.2 ± 5.8^ac
?	NaHS + CQ组	8	49.5 ± 8.0	42.8 ± 8.6^a	39.4 ± 4.5^a	30.2 ± 4.6^ab	26.5 ± 4.8^ab

图1 2型糖尿病大鼠心肌缺血区范围及梗死范围比较。注：I/R：心肌缺血再灌注损伤（ischemia reperfusion）；CQ：氯喹（chloroquine）；NaHS：硫氢化钠（sodium hydrosulfide）；与假手术组比较，^*P<0.05；与NaHS组比较，^#P<0.05；n= 8

图2 2型糖尿病大鼠缺血再灌注4 h后自噬标志蛋白LC3表达变化比较。注：I/R：心肌缺血再灌注损伤（ischemia reperfusion）；CQ：氯喹（chloroquine）；NaHS：硫氢化钠（sodium hydrosulfide）；与假手术组比较，^*P<0.05；与NaHS组比较，^#P<0.05；n=4

图3 2型糖尿病大鼠缺血再灌注4 h后自噬标志蛋白Cathepsin B表达变化比较。注：I/R：心肌缺血再灌注损伤（ischemia reperfusion）；CQ：氯喹（chloroquine）；NaHS：硫氢化钠（sodium hydrosulfide）；与假手术组比较，^*P<0.05；与NaHS组比较，^#P<0.05；n=4

图4 2型糖尿病大鼠缺血再灌注4 h后自噬标志蛋白Beclin-1表达变化比较。注：I/R：心肌缺血再灌注损伤（ischemia reperfusion）；CQ：氯喹（chloroquine）；NaHS：硫氢化钠（sodium hydrosulfide）；与假手术组比较，^*P<0.05；与NaHS组比较，^#P<0.05；n=4

图5 2型糖尿病大鼠缺血再灌注4 h后自噬标志蛋白P62表达变化比较。注：I/R：心肌缺血再灌注损伤（ischemia reperfusion）；CQ：氯喹（chloroquine）；NaHS：硫氢化钠（sodium hydrosulfide）；与假手术组比较，^*P<0.05；与NaHS组比较，^#P<0.05；n=4

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Correlative study of autophagy flux and hydrogen sulfide postconditioning protecting on type 2 diabetic rats with myocardial ischemia reperfusion injury

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Correlative study of autophagy flux and hydrogen sulfide postconditioning protecting on type 2 diabetic rats with myocardial ischemia reperfusion injury