优化新生脉散方调控β1-AR/cAMP/PKA/CREB信号通路对心力衰竭大鼠心肌纤维化的影响

Effects of Optimized New Shengmai Powder in Modulatingβ1-AR/cAMP/PKA/CREB Signaling Pathway on Myocardial Fibrosis in Rats with Heart Failure

目的 基于β1-AR/cAMP/PKA/CREB信号通路探讨优化新生脉散方对心力衰竭大鼠心肌纤维化的影响。方法 50只SD大鼠随机分为假手术组10只和手术组40只,采用左冠状动脉前降支结扎法建立心力衰竭大鼠模型。将成模大鼠随机分为模型组、卡托普利组和中药低、高剂量组,每组8只,给药组分别予相应药物灌胃4周。超声心动图测定大鼠左室射血分数(LVEF)、左室短轴缩短率(LVFS),测定心、肺质量并计算心、肺脏器系数,组织病理学观察心肌纤维化程度,ELISA测定血清N端脑利钠肽前体(NT-ProBNP)及心肌组织环磷酸腺苷(cAMP)、Ⅰ型胶原(ColⅠ)、Ⅲ型胶原(ColⅢ)含量,免疫组化检测心肌组织β1肾上腺素受体(β1-AR)、cAMP阳性表达,Western blot检测心肌组织β1-AR/cAMP/PKA/CREB信号通路相关蛋白表达。结果 与假手术组比较,模型组大鼠LVEF、LVFS明显降低(P<0.01),心、肺脏器系数明显升高(P<0.01);心肌细胞数目减少、胶原容积分数升高、Ⅰ/Ⅲ型胶原纤维占比增加(P<0.01),血清NT-ProBNP和心肌组织ColⅠ、ColⅢ含量明显升高,心肌组织cAMP含量明显降低(P<0.01),心肌组织β1-AR和cAMP阳性表达明显降低(P<0.01),β1-AR、腺苷酸环化酶(AC)1、cAMP、p-蛋白激酶A(PKA)、p-环磷腺苷反应元件结合蛋白(CREB)蛋白表达明显降低,p-Smad2、ColⅠ、ColⅢ、α平滑肌肌动蛋白(α-SMA)蛋白表达明显升高(P<0.05,P<0.01)。与模型组比较,给药组不同程度增加大鼠LVEF、LVFS,降低心、肺脏器系数;增加心肌细胞数目、减少胶原容积分数和Ⅰ/Ⅲ型胶原纤维占比,下调血清NT-ProBNP和心肌组织ColⅠ、ColⅢ含量,上调cAMP含量,增加心肌组织β1-AR和cAMP阳性表达,上调β1-AR、AC1、cAMP、p-PKA、p-CREB蛋白表达,抑制p-Smad2、ColⅠ、ColⅢ、α-SMA蛋白表达,其中中药高剂量组和卡托普利组作用更明显(P<0.01,P<0.05)。结论 优化新生脉散方能减轻心力衰竭大鼠心肌纤维化程度,改善心肌肥厚和重构,增加左心室收缩力,其机制可能与激活β1-AR/cAMP/PKA/CREB信号通路有关。

Objective To investigate the effects of Optimized New Shengmai Powder on myocardial fibrosis in rats with heart failure based on theβ1-AR/cAMP/PKA/CREB signaling pathway.Methods Totally 50 SD rats were randomly divided into sham-operation group(10 rats)and operation group(40 rats).The left anterior descending coronary artery was ligated to establish a rat model of heart failure. The modeling rats were randomly divided into themodel group, the captopril group, and TCM low- and high-dosage groups, with 8 rats in each group. Theadministration groups received relevant medicine for gavage for 4 weeks. LVEF and LVFS in rats were detected byechocardiography, and measurement of heart and lung mass and calculation of heart and lung organ coefficients wereperformed, myocardial fibrosis degree was observed by histopathology, serum NT-ProBNP and cAMP, Col Ⅰ, and Col Ⅲcontent in myocardial tissue were detected by ELISA, immunohistochemical was used to detect β1-AR, cAMPpositive expression, and Western blot was used to detect the expression of β1-AR/cAMP/PKA/CREB signalingpathway related proteins. Results Compared with the sham-operation group, the LVEF and LVFS of the model grouprats were significantly decreased (P<0.01), and the heart and lung organ coefficient significantly increased (P<0.01);the number of myocardial cells decreased, collagen volume fraction increased, and the proportion of type Ⅰ/Ⅲcollagen fibers increased (P<0.01), the contents of serum NT-ProBNP and myocardial tissue Col Ⅰ and Col Ⅲincreased significantly, while the cAMP content in myocardial tissue decreased significantly (P<0.01), the positiveexpressions of β1-AR and cAMP were significantly decreased (P<0.01), the expressions of β1-AR, AC1, cAMP,p-PKA, and p-CREB proteins were significantly decreased, while protein expressions of p-Smad2, Col Ⅰ, Col Ⅲ, andα-SMA significantly increased (P<0.05, P<0.01). Compared with the model group, the administration groups couldincrease LVEF and LVFS and decrease heart and lung organ coefficient to different degrees in rats;increase thenumber of myocardial cells, decrease collagen volume fraction and the proportion of type Ⅰ/Ⅲ collagen fibers, downregulateserum NT-ProBNP and the content of Col Ⅰ and Col Ⅲ in myocardial tissue, up-regulate the content ofcAMP, increase the positive expressions of β1-AR and cAMP in myocardial tissue, up-regulate β1-AR, AC1,cAMP, p-PKA, p-CREB protein expression, and inhibit p-Smad2, Col Ⅰ, Col Ⅲ, and α-SMA protein expression, inwhich the effects of the TCM high-dosage group and captopril group were more pronounced (P<0.01, P<0.05).Conclusion Optimized New Shengmai Powder can effectively reduce myocardial fibrosis in heart failure rats,improve myocardial hypertrophy and remodeling, and increase left ventricular contractility, and the mechanism maybe related to the activation of the β1-AR/cAMP/PKA/CREB signaling pathway.