基于Nrf2/HO-1信号通路探讨冠心通络方对大鼠冠脉微循环障碍的影响

Effects of Guanxin Tongluo Formula on Coronary Microcirculation Dysfunction in Rats Through Nrf2/HO⁃1 Pathway

目的:探索冠心通络方通过核因子E2相关因子(Nrf2)/血红素加氧酶1(HO-1)信号通路对冠脉微循环障碍(CMD)大鼠的保护作用及其可能机制。方法:将雄性SPF级SD大鼠按随机数字表法分为假手术组、模型组、尼可地尔(1.56 mg/kg)阳性对照组及冠心通络方低(8.663 g/kg)、中(17.325 g/kg)、高(34.650 g/kg)剂量组,每组10只,除假手术组外,其余5组采用左心室注射月桂酸钠(1 mg/kg)制备CMD大鼠模型。造模成功后冠心通络方各组分别给与相应剂量冠心通络方煎液灌胃,尼可地尔组灌胃尼可地尔混悬液,其余2组灌胃等体积生理盐水,1次/d,连续灌胃28 d。超声心动图检测心功能后留取血清及心肌组织;Carstairs染色观察心肌组织微血栓形成情况,海登汉氏(Heidenhain)染色观察心肌组织微梗死情况;相应试剂盒检测血清NO、ET-1及心肌组织SOD、GSH-Px、MDA水平;Western Blot检测心肌组织Keap1、细胞质Nrf2及细胞核Nrf2、HO-1蛋白表达;免疫组化法检测心肌组织中Nrf2、Keap1、HO-1蛋白表达。结果:与假手术组比较,模型组大鼠心功能及血清NO水平和心肌组织SOD、GSH-Px水平显著降低,心肌微梗死、心肌内微血栓阳性比例及血清ET-1水平和心肌组织MDA水平显著升高,心肌组织Keap1蛋白、细胞质Nrf2蛋白表达显著降低,细胞核HO-1、Nrf2蛋白表达显著升高(P<0.05或P<0.01)。与模型组比较,冠心通络方中、高剂量组大鼠心功能显著改善,心肌微梗死比例、微血栓阳性比例及血清ET-1和心肌组织MDA水平显著降低,血清NO水平及心肌组织SOD、GSH-Px水平显著升高,心肌组织Keap1蛋白、细胞质Nrf2蛋白表达显著降低,细胞核HO-1、Nrf2蛋白表达显著升高(P<0.05或P<0.01)。结论:冠心通络方能一定程度上改善CMD大鼠病理进程及心功能,其机制可能与激活Nrf2/HO-1信号通路表达,增加机体抗氧化能力有关。

Objective:To explore the protective effect and possible mechanism of Guanxin tongluo formula on coronary microcircu⁃lation dysfunction(CMD)in rats through nuclear factor E2⁃related factor(Nrf2/HO⁃1)signaling pathway.Methods:Male SPF SD rats were randomly divided into sham operation group,model group,nicorandil(1.56 mg/kg)posltive control group,low,medium and high dose of Guanxin tongluo formula(8.66,17.325,34.65 g/kg)groups according to the random number table,with 10 rats in each group.Except the sham operation group,the other five groups were injected with sodium silicate(1 mg/kg)into the left ventricle to prepare CMD rat models.After successful model building,rats in Guanxin tongluo formula groups were given corresponding dose of Guanxin tongluo formula by gavage,rats in nicorandil group were given gavage of nicorandil suspension,and the other two groups were given ga⁃vage of the same volume of normal saline,once a day,continuous gavage for 28 days.The serum and myocardial tissue were retained af⁃ter echocardiography.The myocardial microthrombus formation was observed by Carstairs staining,and myocardial microinfarction was observed by Heidenhain staining.The levels of NO and ET⁃1 in serum and the levels of SOD,GSH⁃Px and MDA in myocardial tissue were determined by corresponding kit.The protein expressions of Keap 1 in myocardial tissue,Nrf2 in cytoplasm,Nrf2 and HO⁃1 in nu⁃cleus were detected by Western Blot.The protein expressions of Nrf2,Keap1 and HO⁃1 in myocardial tissue were detected by immuno⁃histochemical method.Results:Compared with the sham operation group,the heart function,serum NO level,SOD and GSH⁃Px levels in myocardial tissue were significantly decreased in the model group,the positive proportion of myocardial microinfarction and intra myo⁃cardial microthrombin was significantly increased,the serum ET⁃1 level and MDA level in myocardial tissue were significantly in⁃creased,and the expressions of Keap1 protein and cytoplasmic Nrf2 protein in myocardial tissue were significantly decreased.The ex⁃pressions of HO⁃1 and Nrf2 proteins in nucleus were significantly increased(P<0.05 orP<0.01).Compared with model group,the heart function of rats in the medium and high dose of Guanxin tongluo formula groups was improved,the proportion of myocardial micro⁃infarction,the proportion of microthrombi positive,the levels of serum ET⁃1 and MDA in myocardial tissue were significantly decreased,and the levels of NO in serum and SOD and GSH⁃Px in myocardial tissue were significantly increased in each Guanxin tongluo formula group.The expressions of Keap1 protein and Nrf2 protein in myocardial tissue were significantly decreased,while the expressions of HO⁃1 and Nrf2 proteins in nucleus were significantly increased(P<0.05 orP<0.01).Conclusion:Guanxin tongluo formula can improve the pathological process and cardiac function of CMD rats,and the mechanism may be related to the activation of Nrf2/HO⁃1 signaling path⁃way expression and the increase of antioxidant capacity.