维生素C注射液调节氧化应激抑制心肌缺血再灌注损伤大鼠的作用及机制研究

The effect and mechanism of vitamin C injection regulating oxidative stress response to inhibit myocardial ischemia reperfusion injury in rats

目的探究维生素C(VC)注射液通过调节氧化应激反应抑制心肌缺血再灌注损伤大鼠的作用及机制。方法将24只wistar大鼠进行麻醉,建立心肌缺血再灌注损伤模型,并分为空白对照组(在左冠状动脉中上1/3处穿线但不结扎)、模型组(在左冠状动脉中上1/3处穿线并结扎)、VC治疗组(在再灌注时静脉注射VC 100 mg/kg)。通过TTC染色计算各组大鼠心肌梗死区在心室总面积的占比;采用免疫组织化学检测各组大鼠心肌肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)的表达;采用酶联免疫吸附试验(ELISA)检测分析氧化应激指标[丙二醛(MDA)和超氧化物歧化酶(SOD)]含量变化;采用Western blotting检测各组大鼠组织中炎症关键指标[核因子κB(NF-κB)p65及自噬过度激活蛋白(Beclin-1)]的表达。结果心肌缺血再灌注大鼠的心肌梗死面积增加。空白对照组大鼠心肌结构正常,心肌细胞无肿胀,间质未见炎性细胞及纤维化改变。模型组可见大鼠心肌细胞肿胀,胞质淡染,心肌间质弥漫性出血,伴炎性细胞浸润,局部纤维化明显;TNF-α、IL-6表达水平上升,心室组织中MDA含量显著上升,SOD含量显著下降,NF-κB p65和Beclin-1的蛋白表达水平均显著上调。VC治疗组大鼠心肌梗死面积减少,心肌细胞肿胀改善明显,伴有轻度间质水肿和少量炎性细胞浸润,心肌出血及纤维化改善明显;TNF-α、IL-6表达水平下降,抑制了氧化应激,并下调了心肌组织中NF-κB p65和Beclin-1的蛋白表达。VC治疗组上述指标与空白对照组及模型组比较,差异均有统计学意义(P<0.05)。结论VC能够显著抑制心肌细胞缺血再灌注后氧化应激损伤,此过程与下调大鼠炎症关键指标(NF-κB p65和Beclin-1)表达密切相关。

Objective To explore the effect and mechanism of vitamin C(VC)injection regulating oxidative stress response to inhibit myocardial ischemia reperfusion injury in rats.Methods A total of 24 wistar rats were anesthetized to establish a model of acute myocardial ischemia-reperfusion injury model,and divided into the normal control group(threading but not ligated in the upper 1/3 of the left coronary artery),the model group(threading and ligated in the upper 1/3 of the left coronary artery),and the VC treatment group(100 mg/kg VC intravenously during reperfusion).Myocardial infarction area was measured by TTC staining,and the percentage of myocardial infarction area in ventricular area was calculated.Immunohistochemistry was used to detect the expression of myocardial tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in each group of rats.The enzyme-linked immunosorbent assay(ELISA)was used to analyze the content changes of oxidative stress indexes[malondialdehyde(MDA)and superoxide dismutase(SOD)].Western blotting was used to detect the expression of key indicators of inflammation[nuclear factor-κB(NF-κB)p65 and autophagy-hyperactivated proteins(Beclin-1)]in the tissues of each group of rats.Results The area of myocardial infarction increased in myocardial ischemia-reperfusion rats.The myocardial structure of the normal control group was normal,myocardial cells showed no swelling,inflammatory cells and fibrosis changes were not observed in the interstitium.In the model group,myocardial cells were swollen,cytoplasmic staining,myocardial interstitial bleeding with inflammatory cell infiltration and obvious local fibrosis were observed.The expression of TNF-αand IL-6 increased,MDA content increased significantly,SOD content decreased significantly,NF-κB p65 and Beclin-1 protein expression were significantly up-regulated.In the VC treatment group,the area of myocardial infarction was reduced,and myocardial cell swelling was significantly improved with interstitial edema and a small amount of inflammatory cell infiltration,and myocardial bleeding and fibrosis were significantly improved.The expression of TNF-αand IL-6 decreased,which inhibited oxidative stress and down-regulated the protein expression of NF-κB p65 and Beclin-1 in myocardial tissue.The above indexes in the VC treatment group were statistically significant compared with the normal control group and the model group(P<0.05).Conclusion VC can significantly inhibit the oxidative stress injury after myocardial ischemia reperfusion which was closely related to down-regulating the expression of key indicators of inflammation(NF-κB p65 and Beclin-1)in rats.