血府逐瘀胶囊通过调控Sirt3/EPAC1信号通路对动脉粥样硬化小鼠的影响

Xuefu Zhuyu Capsules Ameliorate Atherosclerosis in Mice by Regulating Sirt3/EPAC1 Signaling Pathway

摘要:

目的 观察血府逐瘀胶囊通过调控沉默信息调节因子3（Sirt3）/鸟嘌呤核苷酸转化因子交换蛋白1（EPAC1）信号通路对高脂饲料诱导的动脉粥样硬化小鼠血脂、主动脉斑块的影响，探讨血府逐瘀胶囊改善动脉粥样硬化的作用机制。方法 将小鼠分为正常组，模型组，空白组，瑞舒伐他汀组，血府逐瘀胶囊低、中、高剂量组，正常组用C57BL/6J正常小鼠，其余各组为同品系载脂蛋白E敲除（ApoE^-/-）小鼠。正常组和空白组常规饲养，其余各组采用高脂饲料喂养，连续24周构建小鼠动脉粥样硬化模型，血府逐瘀胶囊以0.3、0.6、1.2 g·kg^-1·d^-1剂量灌胃，瑞舒伐他汀组以0.05 g·kg^-1·d^-1剂量灌胃，正常组、模型组和空白组给予等体积去离子水灌胃，连续灌胃6周。采用小动物B超仪评估小鼠心功能和主动脉斑块情况；全自动生化分析仪检测小鼠总胆固醇（CHOL）、甘油三酯（TG）、高密度脂蛋白-胆固醇（HDL-C）、低密度脂蛋白-胆固醇（LDL-C）、极低密度脂蛋白（VLDL）等血脂指标；油红O染色观察主动脉脂质沉积情况；苏木素-伊红（HE）染色评估小鼠组织病理学改变；马松（Masson）染色观察小鼠血管内胶原沉积程度；透射电镜观察小鼠主动脉线粒体损伤情况；酶联免疫吸附测定法（ELISA）检测促肾上腺皮质激素（ACTH）、腺嘌呤核苷三磷酸（ATP）、烟碱型胆碱受体α1（CHRNα1）、总超氧化物歧化酶（T-SOD）等指标；实时荧光定量聚合酶链式反应（Real-time PCR）检测小鼠主动脉及心脏组织中Sirt3、EPAC1、胱天蛋白酶-3（Caspase-3）、B细胞淋巴瘤-2（Bcl-2）、Bcl-2关联X蛋白（Bax） mRNA相对表达；蛋白免疫印迹法（Western blot）检测Sirt3、EPAC1、Caspase-3、Bcl-2、Bax蛋白相对表达。结果 模型组主动脉弓多处形成动脉粥样硬化斑块，提示造模成功；经血府逐瘀胶囊治疗后，与模型组比较，斑块明显缩小，斑块数量也明显减少。生化结果显示，与正常组比较，模型组CHOL含量显著升高（P<0.01）；与模型组比较，瑞舒伐他汀组、血府逐瘀胶囊低剂量组中CHOL和TG含量均显著降低（P<0.01）。油红O染色结果显示，与正常组比较，模型组主动脉血管壁上可见大量凸起的红色脂质斑块，其动脉粥样硬化斑块面积占组织总面积的百分比显著高于正常组（P<0.01）；与模型组比较，血府逐瘀胶囊低剂量组斑块负荷明显减少（P<0.05）。与模型组比较，血府逐瘀胶囊各剂量组脂质斑块和胶原沉积显著减少。与正常组比较，模型组内皮细胞可见线粒体嵴减少或消失，膜结构损伤严重；各给药组血管内皮细胞线粒体形态趋近于正常结构，隐约可见线粒体嵴。与正常组比较，模型组小鼠心肌线粒体ATP活性显著下降（P<0.01），各给药组与模型组比较均有显著提高（P<0.01）。与正常组比较，模型组Sirt3含量显著降低（P<0.01），EPAC1的表达量显著升高（P<0.01）；与模型组比较，血府逐瘀胶囊低剂量组Sirt3含量显著升高（P<0.01），血府逐瘀胶囊中剂量组EPAC1含量显著降低（P<0.01）。提示经血府逐瘀胶囊治疗后小鼠心脏组织的Sirt3蛋白及基因表达量明显升高，EPAC1的蛋白及基因表达量明显降低。结论 血府逐瘀胶囊可明显改善动脉粥样硬化小鼠血脂异常、主动脉脂质沉积、动脉粥样硬化斑块面积、内皮细胞线粒体形态和功能，增加ATP活性，提高Sirt3表达，同时抑制EPAC1的表达，其作用机制可能与通过调控Sirt3/ EPAC1信号通路调节线粒体能量代谢有关。

Abstract:

Objective To observe the effects of Xuefu Zhuyu capsules （XFZY） on blood lipid levels and aortic plaques in the mouse model of atherosclerosis （AS） induced by a high-fat diet by regulating the silencing regulatory factor 2-like protein 3 （Sirt3）/exchange protein directly activated by cAMP 1 （EPAC1） signaling pathway and explore the mechanism of XFZY in ameliorating AS.Method Mice were assigned into normal， model， blank， rosuvastatin （0.05 g·kg^-1·d^-1）， and low-， medium-， and high-dose （0.3， 0.6， 1.2 g·kg^-1·d^-1， respectively） XFZY groups. The normal group consisted of normal C57BL/6J mice， while the other groups consisted of ApoE^-/- C57BL/6J mice. The normal group and blank group were fed routinely， and the rest groups were fed with a high-fat diet for 24 consecutive weeks for the modeling of AS. The drug intervention groups were administrated with corresponding drugs by gavage， and model group and blank group with an equal volume of deionized water for 6 consecutive weeks. The small animal B-ultrasound was used to evaluate the mouse heart function and aortic plaque condition. A fully automated biochemical analyzer was used to measure the levels of blood lipids such as total cholesterol （CHOL）， triglycerides （TG）， high-density lipoprotein cholesterol （HDL-C）， low-density lipoprotein cholesterol （LDL-C）， and extremely low-density lipoprotein （VLDL） in mice. Oil red O staining was employed to observe lipid deposition in the aorta. Hematoxylin-eosin staining and Masson staining were employed to observe the pathological changes and collagen deposition in mouse blood vessels. Transmission electron microscopy was employed to observe the mitochondrial damage in mouse aorta. The levels of adrenocorticotropic hormone （ACTH）， adenosine triphosphate （ATP）， and nicotinic choline receptor α1 （CHRNα1）， and total superoxide dismutase （T-SOD） were measured by enzyme-linked immunosorbent assay （ELISA）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot were performed to determine the mRNA and protein levels， respectively， of Sirt3， EPAC1， Caspase-3， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax） in the mouse aorta and heart.Result Multiple AS plaques were observed in the aortic arch， indicating that the model was successfully established. Compared with the model group， the XFZY groups showed reduced and narrowed plaques. Compared with the normal group， the model group showed elevated CHOL level （P<0.01）. Compared with the model group， rosuvastatin and low-dose XFZY lowered the CHOL and TG levels （P<0.01）. Compared with the normal group， the model group presented a large number of protruding red lipid plaques on the aortic wall and increased percentage of AS plaque area to total tissue area （P<0.01）. Compared with the model group， low-dose XFZY reduced the plaque load （P<0.05）. Compared with the model group， XFZY at different doses reduced the lipid plaques and collagen deposition. Compared with the normal group， the model group showed decreased or disappeared mitochondrial cristae and presented severe damage of the membrane structure in endothelial cells. The mitochondria of endothelial cells in each treatment group approached the normal structure， with mitochondrial cristae faintly visible. Compared with the normal group， the model group showcased reduced myocardial mitochondrial ATP activity （P<0.01）， which were rescored in the drug intervention groups （P<0.01）. Compared with the normal group， the modeling inhibited the expression of Sirt3 （P<0.01） and promoted the expression of EPAC1 （P<0.01）. Compared with the model group， low-dose XFZY increased the Sirt3 content （P<0.01） and medium-dose XFZY increased the EPAC1 content （P<0.01）， which indicated that XFZY treatment upregulated the mRNA and protein levels of Sirt3 and downregulated the mRNA and protein levels of EPAC1.Conclusion XFZY can alleviate the aortic lipid deposition， reduce the AS plaque area， improve the mitochondrial morphology and functions in endothelial cells， increase the ATP activity， upregulate the expression of Sirt3， and downregulate the expression of EPAC1 in AS mice by regulating mitochondrial energy metabolism via the Sirt3/EPAC1 signaling pathway.