银杏内酯通过抗炎和抗氧化保护缺血再灌注导致的大鼠心肌损伤

Protective effect of bilobalide on myocardial ischemia/reperfusion injury of rats via anti-inflammation and anti-oxidation

目的探究银杏内酯(BB)对心肌缺血再灌注(MI/R)模型大鼠心肌组织的影响。方法采用结扎大鼠左冠状动脉前降支30 min制备MI/R模型,按分组分别ig给予BB 2,4和8 mg·kg-1,连续4周。记录各组大鼠心率(HR),检测心肌梗死面积。用试剂盒检测血液中肌酸激酶MB型同工酶(CK-MB)及乳酸脱氢酶(LDH)浓度,HE染色观察心肌组织损伤,TUNEL染色检测心肌细胞凋亡率。ELISA法检测心肌组织超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性和丙二醛(MDA)含量;检测外周血白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)和IL-10浓度。免疫组化检测Ki67及IL-6表达水平,Western印迹法检测心肌组织中Bcl-2、Bax、活化的胱天蛋白酶3、转录因子NF-E2相关因子2(Nrf2)、血红素氧合酶(HO-1)、NAD(P)H醌脱氢酶1(NQO1)、NF-κB P65、磷酸化NF-κB抑制蛋白(p-IκBα)和IκBα激酶(p-IKK)蛋白表达水平。结果与正常对照组比较,MI/R模型组大鼠心肌梗死面积百分比及外周血中CK-MB和LDH浓度显著增加(P<0.01),HR下降(P<0.01),心肌组织出现心肌纤维断裂及炎性浸润现象。与MI/R模型组比较,MI/R模型+BB 2,4和8 mg·kg-1组中心肌梗死面积百分比及CK-MB和LDH浓度显著降低(P<0.05),心率上升(P<0.05),心肌组织纤维排列趋向规则,炎症细胞浸润明显减少损伤明显减轻。与正常对照组比较,MI/R模型组凋亡细胞百分比及Ki67阳性细胞百分比、Bax及活化的胱天蛋白酶3表达显著增加(P<0.01),Bcl-2表达显著减少(P<0.01)。与MI/R模型组比较,MI/R模型+BB 2,4和8 mg·kg-1组中Ki67阳性细胞百分比和Bcl-2表达显著增加(P<0.05),凋亡细胞百分比、Bax和活化的胱天蛋白酶3表达显著减少(P<0.05)。MI/R可降低大鼠心肌组织中SOD及GSH-Px活性(P<0.01),提高MDA浓度(P<0.01),BB可减弱MI/R对SOD,GSH-Px和MDA的影响(P<0.05,P<0.01)。与正常对照组比较,MI/R模型组大鼠外周血中IL-1β,TNF-α和IL-10浓度及心肌组织中IL-6阳性细胞百分比显著升高(P<0.01)。与MI/R模型组比较,MI/R模型+BB 2,4和8 mg·kg-1组中IL-1β,TNF-α浓度和IL-6阳性细胞百分比显著降低(P<0.05),IL-10浓度显著升高(P<0.01)。此外,MI/R模型组大鼠心肌组织中的Nrf2,HO-1和NQ1蛋白表达水平显著降低(P<0.01);NF-κB P65,p-IKK和p-IκBα1蛋白表达水平显著升高(P<0.01),BB可有效逆转MI/R对这些信号通路蛋白的作用(P<0.05,P<0.01)。结论 BB可通过抗炎及抗氧化对MI/R大鼠产生保护作用。

OBJECTIVE To investigate the effects of bilobalide(BB)on the myocardial tissue of rats with myocardial ischemia/reperfusion(MI/R).METHODS An MI/R model was prepared by ligating the anterior descending branch of the left coronary artery in rats for 30 min.BB 2,4 and 8 mg·kg-1 was adminsterted for four weeks.The heart rate of rats was recorded and myocardial infarction area was detected.Detective kits were used to evaluate the concentrations of creatine kinase MB isoenzyme(CK-MB)and lactate dehydrogenase(LDH).HE staining was performed to observe the myocardial injury,and TUNEL staining was performed to evaluate apoptosis.ELISA was performed to determine the concentrations of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),malondialdehyde(MDA),IL-1β,tumor necrosis factor-α(TNF-α)and IL-10.Immunohistochemistry was performed to evaluate the expressions of Ki67 and interleukin-6(IL-6).Western blotting was performed to evaluate the expressions of Bcl-2,Bax,cleaved-caspase 3,nuclear factor(erythroid-derived 2)-like 2 protein(Nrf2),heme oxygenase 1(HO-1),NAD(P)H quinone dehydrogenase 1(NQO1),NF-κB P65,phosphorylated inhibitor of NF-κB(pIκBα)and IκBαkinase(p-IKK).RESULTS Compared with normal control group,the percentage of the myocardial infarction area and the concentration of CK-MB and LDH were increased in MI/R model group,but heart rate was decreased(P<0.01)and myocardial tissue showed myocardial fiber rupture and inflammatory infiltration.Compared with MI/R model group,the percentage of the myocardial infarction area and the concentration of CK-MB and LDH were decreased in MI/R model+BB 2,4 and 8 mg·kg-1 groups(P<0.05),but heart rate was increased(P<0.01)and myocardial tissue fibers tended to be regular while inflammatory cell infiltration was significantly reduced.Compared with normal control group,the percentage of apoptotic cells and Ki67 expression-positive cells and the expressions of Bax and cleaved-caspase 3 were increased in MI/R model group(P<0.01),but the expression of Bcl-2 was decreased markedly(P<0.01).Compared with MI/R model group,the percentages of apoptotic cells and Ki67expression-positive cells and the expressions of Bax and cleaved-caspase 3 were decreased in MI/R model+BB 2,4 and 8 mg·kg-1 groups(P<0.01),while the expression of Bcl-2 was increased(P<0.01).MI/R could lower the activity of SOD and GSH-Px,but enhance MDA content.BB could weaken the effect of MI/R on SOD,GSH-Px and MDA.Compared with normal control group,the concentrations of IL-1β,TNF-αand IL-10 in serum and the percentage of IL-6 expression-positive cells in myocardial tissue of MI/R model group were increased(P<0.01).Compared with MI/R model group,the concentrations of IL-1βand TNF-αand the percentage of IL-6 expression-positive cells were decreased in MI/R model+BB 2,4 and 8 mg·kg-1 group(P<0.01),but the concentration of IL-10 was increased(P<0.05).Compared with mormal control group,the protein levels of Nrf2,HO-1 and NQO1 significantly decreased(P<0.01),while the levels of NF-κB P65,p-IKK and p-IκBαsignificantly increased(P<0.01).BB could effectively reverse protein changes(P<0.05,P<0.01).CONCLUSION BB can protect rats with MI/R via anti-inflammation and anti-oxidation.