白藜芦醇调控细胞自噬对大鼠心肌缺血再灌注无回流的改善作用研究

Improving effect of resveratrol on regulating cell autophagy for no-reflow after myocardial ischemia-reperfusion in rats

目的 研究白藜芦醇对大鼠心肌缺血再灌注的保护作用及其机制。方法按照随机数表法将大鼠分为3组:假手术组、模型组和实验组,每组12只。造模前,实验组灌胃给予白藜芦醇50 mg·kg-1·d-1,模型组和假手术组灌胃给予等体积的0. 9%Na Cl,1次/天,连续7 d。建立心肌缺血再灌注无回流大鼠模型。记录心电图,测量左心室内压最大上升速率(+dp/dtmax)和最大下降速率(-dp/dtmin);TTC染色法观察无回流区(ANR)和缺血区(AI)情况。用试剂盒法检测肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)、C反应蛋白(CRP)、心肌钙蛋白I(c Tn I)水平及丙二醛(MDA)、超氧化物歧化酶(SOD)和髓过氧化物(MPO)活性。以免疫印迹法检测LC3-II和Becl-1自噬相关蛋白水平;电镜下观察大鼠心肌细胞超微结构改变和自噬体。结果 干预后,实验组的T波和ST波显著降低(P <0. 05),+dp/dtmax和-dp/dtmin显著下降(P <0. 05);实验组和模型组的ANR/AI分别为(11. 78±2. 10)%,(21. 52±4. 07)%,组间比较差异有统计学意义(P <0. 05)。实验组和模型组的CK-MB分别为(503. 35±200. 23),(725. 39±159. 79) U·L-1;这2组的LDH分别为(1290. 41±270. 71),(2115. 68±879. 51) U·L-1;这2组的CRP分别为(38. 85±3. 12),(17. 86±3. 57)μmol·g-1;这2组的c Tn I蛋白水平分别为(6. 68±0. 78),(8. 05±0. 76) U·L-1;这2组的MDA活性分别为(2. 35±0. 32),(5. 17±0. 37) U·m L-1;这2组的SOD活性分别为(1. 68±0. 25),(2. 25±0. 43) U·L-1;这2组的MPO活性分别为(1. 57±0. 27),(2. 85±0. 54)μmol·g-1,2组间比较,上述指标的差异均有统计学意义(均P <0. 05)。实验组LC3-II和Beclin 1蛋白表达水平显著升高;电镜观察发现,实验组心肌细胞自噬体数量显著少于模型组。结论 白藜芦醇通过抑制心肌缺血再灌注期间自噬,减少心肌损伤。

Objective To study the protective effect of resveratrol onregulating cell autophagy for no-reflow after myocardial ischemia-reperfusion in rats,and to explore the mechanism.Methods Rats were divided into three groups:sham operation group,model group and experimental group,each group had 12 rats.Before modeling,rats in experimental group were given resveratrol 50 mg·kg-1·d-1,while rats in sham operation group and model group were given an equal volume of normal saline for 7 d,one time for a day.A rat model of myocardial ischemia-reperfusion was established.Record the electrocardiogrammeasure the maximum rate of increase of left ventricular pressure(+dp/dtmax)and the maximum rate of decline(-dp/dtmin).The area of no-reflow(ANR),area of ischemic(AI)were measured with TTC staining.The levels of creatine kinase isoenzyme(CK-MB),lactate dehydrogenase(LDH),C-reactive protein(CRP),cardiac troponin I(cTnI)and the activity of malondialdehyde(MDA),superoxide dismutase(SOD),and myeloperoxidase(MPO)were measured with kits.The levels of autophagy-related proteins in LC3-II and Becl-1 were detected by Western blot.Ultrastructural changes and autophagosomes of rat cardiomyocytes observed under electron microscope.Results After intervention,the T wave and ST wave of the experimental group were significantly decreased(P<0.05),and+dp/dtmax and-dp/dtmin were significantly decreased(P<0.05).the value of ANR/AI in experimental group and model group were(11.78±2.10)%,(21.52±4.07)%,there was significant difference between the two groups(P<0.05).The levels of CK-MB in experimental group and model group were(503.35±200.23),(725.39±159.79)U·L-1;the levels of LDH in the two groups were(1290.41±270.71),(2115.68±879.51)U·L-1;the levels of CRP in the two groups were(38.85±3.12),(17.86±3.57)μmol·g-1;the levels of cTnI protein in the two groups were(6.68±0.78),(8.05±0.76)U·L-1;the activity of MDA in the two groups were(2.35±0.32),(5.17±0.37)U·mL-1;the activity of SOD in the two groups were(1.68±0.25),(2.25±0.43)U·L-1;the activity of MPO in the two groups were(1.57±0.27),(2.85±0.54)μmol·g-1,there was significant difference of the factors between the two group(all P<0.05).The expression levels of LC3-II and Beclin 1 in the experimental group were significantly increased.Electron microscopy revealed that the number of autophagosomes in the experimental group was significantly less than that in the model group.Conclusion Resveratrol reduces myocardial damage by inhibiting autophagy during myocardial ischemia-reperfusion.