富马酸二甲酯减轻糖尿病大鼠心肌缺血再灌注损伤及其相关机制研究

doi:10.3877/cma.j.issn.1674-6880.2021.02.002

目的

探讨富马酸二甲酯（DMF）通过调节核因子E2相关因子2（Nrf2）对糖尿病大鼠心肌缺血再灌注（MI/R）损伤的保护作用。

方法

60只Sprague-Dawley大鼠均予高脂饮食喂养4周，禁食12 h后，一次性腹腔注射1%链脲佐菌素柠檬酸缓冲液25 mg/kg制备糖尿病大鼠模型，继续给予高脂饲养4周后制备MI/R损伤模型。将60只糖尿病大鼠分成假手术组（S组）、糖尿病大鼠MI/R组（MI/R组）、DMF + MI/R组（R组）和DMF + ML385 + MI/R组（RE组），每组15只大鼠。R组、RE组大鼠给予DMF 25 mg/kg灌胃，连续7 d，1次/d；S组与MI/R组大鼠给予等容量等渗NaCl溶液灌胃。其中，RE组大鼠于缺血前30 min腹腔注射Nrf2抑制剂（ML385）30 mg/kg。MI/R组、R组和RE组大鼠予结扎左冠状动脉前降支30 min，恢复灌注120 min以制备MI/R模型；S组大鼠只开胸，不结扎冠状动脉。比较4组大鼠心肌梗死面积、心率、左心室收缩压（LVSP）、左心室射血分数（LVEF）、左心室短轴缩短率（LVFS）、乳酸脱氢酶（LDH）、肌酸激酶同工酶MB（CK-MB）、心肌肌钙蛋白I（cTnI）、丙二醛、活性氧自由基（ROS）、超氧化物歧化酶（SOD）水平以及Nrf2、血红素氧合酶1（HO-1）蛋白表达水平。

结果

S组大鼠无心肌梗死，MI/R组、R组和RE组大鼠心肌梗死面积比较，差异有统计学意义[（32.5 ± 2.2）%、（23.2 ± 1.6）%、（29.5 ± 1.7）%，F = 98.364，P < 0.001]，且MI/R组和RE组大鼠心肌梗死面积均较R组大鼠显著增加（P均< 0.05）。4组大鼠心率、LVSP、LVEF、LVFS、LDH、CK-MB、cTnI、丙二醛、ROS、SOD水平及Nrf2、HO-1蛋白表达水平比较，差异均有统计学意义（F = 198.124、103.884、86.052、39.256、109.126、164.241、98.673、132.102、108.146、76.535、61.132、120.401，P均< 0.001）。与S组相比，MI/R组、R组及RE组大鼠心率[（404 ± 30）、（264 ± 27）、（331 ± 22）、（270 ± 23）次/min]、LVSP [（127 ± 19）、（73 ± 17）、（98 ± 10）、（80 ± 10）mmHg]、LVEF [（68.1 ± 3.2）%、（39.5 ± 4.5）%、（48.4 ± 1.9）%、（38.6 ± 2.3）%]、LVFS [（32.4 ± 1.2）%、（18.5 ± 0.8）%、（25.6 ± 0.7）%、（20.6 ± 1.1）%]、SOD水平均显著下降，LDH [（0.21 ± 0.04）、（0.54 ± 0.07）、（0.37 ± 0.06）、（0.52 ± 0.08）U/mg]、CK-MB [（1 783 ± 41）、（4 357 ± 523）、（3 068 ± 276）、（4 198 ± 490）ng/L]、cTnI [（406 ± 45）、（1 437 ± 251）、（748 ± 167）、（1 520 ± 190）ng/L]、丙二醛、ROS水平及Nrf2、HO-1蛋白表达水平均显著升高（P均< 0.05）；与R组相比，MI/R组和RE组大鼠心率、LVSP、LVEF、LVFS、SOD水平及Nrf2、HO-1蛋白表达水平均明显降低，LDH、CK-MB、cTnI、丙二醛、ROS水平均显著升高（P均< 0.05）。

结论

DMF可通过调节Nrf2减轻MI/R损伤，发挥其心肌保护作用。

关键词: 糖尿病, 富马酸二甲酯, 核因子E2相关因子2, 心肌缺血再灌注损伤

Objective

To investigate the protective effect of dimethyl fumarate (DMF) on myocardial ischemia/reperfusion (MI/R) injury in diabetic rats by regulating the nuclear factor E2-related factor 2 (Nrf2).

Methods

Sixty Sprague-Dawley rats were fed with a high-fat diet for 4 weeks. After fasting for 12 h, 1% streptozotocin citrate buffer solution was injected intraperitoneally for 25 mg/kg to prepare a diabetic rat model, and then a MI/R injury model was prepared after high-fat feeding for 4 weeks. These 60 diabetic rats were divided into a sham operation group (S group), a diabetes with MI/R group (MI/R group), a DMF + MI/R group (R group) and a DMF + ML385 + MI/R group (RE group), 15 rats in each group. Rats in the R and RE groups were given DMF (25 mg/kg) by intragastrical administration once a day for 7 d, and rats in the S and MI/R groups were given isosmotic NaCl solution in equal volume. Then rats in the RE group were injected intraperitoneally with the Nrf2 inhibitor ML385 (30 mg/kg) 30 min before ischemia. Rats in the MI/R, R and RE groups were ligated with the left anterior descending coronary artery for 30 min, and then restored perfusion for 120 min to prepare the MI/R model. Rats in the S group only opened the chest without ligating the coronary artery. The levels of myocardial infarction area, heart rate, left ventricular systolic pressure (LVSP), left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), lactate dehydrogenase (LDH), creatine kinase isoenzyme-MB (CK-MB), cardiac troponin I (cTnI), malondialdehyde, reactive oxygen species (ROS), superoxide dismutase (SOD) and the protein levels of Nrf2, heme oxygenase-1 (HO-1) were compared among the four groups.

Results

No myocardial infarction occurred in the S group. The myocardial infarction area in the MI/R, R and RE groups showed statistically significant differences [(32.5 ± 2.2)%, (23.2 ± 1.6)%, (29.5 ± 1.7)%; F = 98.364, P < 0.001], and it was significantly higher in the MI/R and RE groups than in the R group (both P < 0.05). There were significant differences in the levels of heart rate, LVSP, LVEF, LVFS, LDH, CK-MB, cTnI, malondialdehyde, ROS, SOD and the protein levels of Nrf2, HO-1 in the four groups (F = 198.124, 103.884, 86.052, 39.256, 109.126, 164.241, 98.673, 132.102, 108.146, 76.535, 61.132, 120.401; all P < 0.001). Further pairwise comparison revealed that compared with the S group, the levels of heart rate [(404 ± 30), (264 ± 27), (331 ± 22), (270 ± 23) beats/min], LVSP [(127 ± 19), (73 ± 17), (98 ± 10), (80 ± 10) mmHg], LVEF [(68.1 ± 3.2)%, (39.5 ± 4.5)%, (48.4 ± 1.9)%, (38.6 ± 2.3)%], LVFS [(32.4 ± 1.2)%, (18.5 ± 0.8)%, (25.6 ± 0.7)%, (20.6 ± 1.1)%] and SOD were decreased significantly, while the levels of LDH [(0.21 ± 0.04), (0.54 ± 0.07), (0.37 ± 0.06), (0.52 ± 0.08) U/mg], CK-MB [(1 783 ± 41), (4 357 ± 523), (3 068 ± 276), (4 198 ± 490) ng/L], cTnI [(406 ± 45), (1 437 ± 251), (748 ± 167), (1 520 ± 190) ng/L], malondialdehyde, ROS and the protein levels of Nrf2, HO-1 were increased significantly in the MI/R, R and RE groups (all P < 0.05). As compared with the R group, the levels of heart rate, LVSP, LVEF, LVFS, SOD and the protein levels of Nrf2, HO-1 were decreased significantly, while the levels of LDH, CK-MB, cTnI, malondialdehyde and ROS were increased significantly in the MI/R and RE groups (all P < 0.05).

Conclusion

DMF can reduce MI/R injury in diabetic rats by regulating Nrf2 and thus exert its myocardial protective effect.

Key words: Diabetes mellitus, Dimethyl fumarate, Nuclear factor E2-related factor 2, Myocardial ischemia/reperfusion injury

表1 4组大鼠心脏功能及超声心动图结果比较（ ± s）

组别	只数	心率（次/min）	LVSP（mmHg）	LVEF（%）	LVFS（%）
S组	10	404 ± 30	127 ± 19	68.1 ± 3.2	32.4 ± 1.2
MI/R组	10	264 ± 27^a	73 ± 17^a	39.5 ± 4.5^a	18.5 ± 0.8^a
R组	10	331 ± 22^ab	98 ± 10^ab	48.4 ± 1.9^ab	25.6 ± 0.7^ab
RE组	10	270 ± 23^ac	80 ± 10^ac	38.6 ± 2.3^ac	20.6 ± 1.1^ac
F值		198.124	103.884	86.052	39.256
P值		<0.001	<0.001	<0.001	<0.001

表1 4组大鼠心脏功能及超声心动图结果比较（ ± s）

组别	只数	心率（次/min）	LVSP（mmHg）	LVEF（%）	LVFS（%）
S组	10	404 ± 30	127 ± 19	68.1 ± 3.2	32.4 ± 1.2
MI/R组	10	264 ± 27^a	73 ± 17^a	39.5 ± 4.5^a	18.5 ± 0.8^a
R组	10	331 ± 22^ab	98 ± 10^ab	48.4 ± 1.9^ab	25.6 ± 0.7^ab
RE组	10	270 ± 23^ac	80 ± 10^ac	38.6 ± 2.3^ac	20.6 ± 1.1^ac
F值		198.124	103.884	86.052	39.256
P值		<0.001	<0.001	<0.001	<0.001

表2 4组大鼠LDH、CK-MB、cTnI表达水平比较（ ± s）

组别	只数	LDH（U/mg）	CK-MB（ng/L）	cTnI（ng/L）
S组	10	0.21 ± 0.04	1 783 ± 41	406 ± 45
MI/R组	10	0.54 ± 0.07^a	4 357 ± 523^a	1 437 ± 251^a
R组	10	0.37 ± 0.06^ab	3 068 ± 276^ab	748 ± 167^ab
RE组	10	0.52 ± 0.08^ac	4 198 ± 490^ac	1 520 ± 190^ac
F值		109.126	164.241	98.673
P值		<0.001	<0.001	<0.001

表2 4组大鼠LDH、CK-MB、cTnI表达水平比较（ ± s）

组别	只数	LDH（U/mg）	CK-MB（ng/L）	cTnI（ng/L）
S组	10	0.21 ± 0.04	1 783 ± 41	406 ± 45
MI/R组	10	0.54 ± 0.07^a	4 357 ± 523^a	1 437 ± 251^a
R组	10	0.37 ± 0.06^ab	3 068 ± 276^ab	748 ± 167^ab
RE组	10	0.52 ± 0.08^ac	4 198 ± 490^ac	1 520 ± 190^ac
F值		109.126	164.241	98.673
P值		<0.001	<0.001	<0.001

图1 4组大鼠心肌组织丙二醛、ROS含量及SOD活性的比较（n = 10）

图2 4组大鼠心肌组织Nrf2、HO-1蛋白表达水平比较（n = 10）

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Protective effect of dimethyl fumarate on myocardial ischemia/reperfusion injury in diabetic rats and its mechanism

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Protective effect of dimethyl fumarate on myocardial ischemia/reperfusion injury in diabetic rats and its mechanism