Effects of the combination of tanshinone IIA and puerarin on cardiac function and inflammatory response in myocardial ischemia mice
Effects of the combination of tanshinone IIA and puerarin on cardiac function and inflammatory response in myocardial ischemia mice
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丹参酮IIA联合葛根素对心肌缺血小鼠心功能及炎症反应的影响
DOI：
10.1016/j.yjmcc.2019.09.012
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发表时间：
2019-12-01
影响因子：
5
通讯作者：
Fan, Guanwei
中科院分区：
文献类型：
--
作者：
Gao, Shan;Li, Lingyan;Fan, Guanwei
背景：心室重构是正常心力衰竭的主要病理过程。随着我国社会老龄化、饮食控制不良、社会、心理等危险因素的影响，心肌梗死和高血压的发病率逐年上升。许多治疗方法有效地延缓了心室重构的发生。然而，为了预防和延缓心室重构的发生和发展，新的治疗策略不容忽视。方法：本研究选用雄性C57 BL/6小鼠（8周龄），体重23 g-27 g，SPF级。按照本课题组建立的方法，采用冠状动脉左前降支分支（LAD）制作心肌缺血模型，以EF值的变化来评价心肌缺血的程度。实验分为七组：假手术组、模型组、美托洛尔组、葛根素组、丹参酮Ⅱ A组、丹参酮Ⅱ A：葛根素= 1：1组、丹参酮Ⅱ A：葛根素= 1：2组。分别于给药周期3d、7 d、14 d、28 d，通过超声心动图和血流动力学观察各组心功能的变化。全自动生化分析仪检测3d血清酶指标LDH、CK、CK-MB。流式细胞术检测3 d和7 d心肌组织中CD 11b、F4/80、Ly 6C的表达。ELISA法检测血清中IL-1 β、TNF-α的表达。采用RT-PCR方法检测IL-1 β、IL-6、IL-10、iNOS等相关基因的表达。采用HE、Masson染色及免疫组化染色观察小鼠心肌组织形态学变化。我们还研究了不同药物处理对Raw 264.7细胞、H9 C2细胞和HUVECs的增殖和功能的影响。Western blot检测不同药物处理对炎症通路相关蛋白TLR 4和C/EBP-β表达的影响。超声心动图结果显示，随着缺血时间的延长，模型组的射血分数、左室短轴缩短率、流出道流速均明显降低，心室结构明显改变。血流动力学检测显示，缺血后模型组最大和最大下降速率明显降低，收缩期和舒张期容积增加，压差减小。经药物治疗后，各组均有所改善，但丹参酮IIA：葛根素= 1：1组在给药28 d后可明显改善上述指标，可有效缓解急性心肌梗死引起的心功能恶化。2.给药3 d和7 d后，流式细胞仪检测心脏组织中炎性细胞CD 11b单核细胞和F4/80表型巨噬细胞，发现丹参酮IIA：葛根素= 1：1可抑制炎性细胞的释放。RT-PCR结果显示，丹参酮IIA：葛根素= 1：1组能显著提高IL-1 β、IL-6、IL-10和iNOS等炎性细胞因子的表达。在iNOS和Arg-1的免疫组化分析中，丹参酮IIA和葛根素1：1治疗组能够在炎症早期抑制M1巨噬细胞的表达，促进M2巨噬细胞的表达。3. 28 d后心脏指数显著升高，血清TGF-β水平升高。丹参酮IIA与葛根素合用可显著降低上述指标。HE、Masson、天狼猩红及免疫组化染色均发现丹参酮IIA和葛根素联合应用可明显减轻急性缺血心肌细胞结构损伤和间质水肿，减少胶原合成，及成纤维细胞释放，从而抑制心肌纤维化和心脏重构。4. MTT法显示，丹参酮IIA：葛根素= 1：1组细胞增殖明显高于其他组，且丹参酮IIA：葛根素= 1：1组细胞内可见较多的结节和网状结构。5.结论：丹参酮IIA与葛根素联合应用可通过抑制TLR 4蛋白的表达，上调C/EBP-β蛋白的表达来调节炎症反应。通过改善心功能改善血流动力学，减少心肌细胞的破坏，减少胶原合成，抑制心肌纤维化和心室重构。通过整个实验，丹参酮IIA：葛根素= 1：1为最佳。
Background: Ventricular remodeling is a major pathological process of normal heart failure. With the aging of society, poor diet control, social, psychological and other risk factors in our country, the incidence of myocardial infarction and hypertension is reported to increase yearly. Many treatment methods have effectively delayed the occurrence of ventricular remodeling. However, in order to prevent and delay the occurrence and development of ventricular remodeling, the new treatment strategy cannot be ignored.Methods: In this study, we used male C57BL/6 mice (8 weeks old), weight 23 g-27 g, SPF grade. According to the established methods of the research group, the left anterior descending branch of the coronary artery (LAD) was used to make the model of myocardial ischemia, and which was evaluated by the change of EF value in mice. The experiment included seven groups: sham operation group, model group, metoprolol group, puerarin group, tanshinone IIA group, tanshinone IIA: puerarin = 1:1 group, tanshinone IIA: puerarin = 1:2 group. The changes of cardiac function in each group were observed by echocardiography and hemodynamics after the drug delivery cycle was 3d, 7d, 14d and 28d. Detection of 3d serum enzyme indexes LDH, CK and CK-MB by automatic biochemical analyzer. The expression of CD11b, F4/80, Ly6C in cardiac tissues were detected by flow cytometry at 3d and 7d. The expression of IL-1 beta and TNF-alpha in serum were detected by ELISA. IL-1 beta, IL-6, IL-10, iNOS and other related genes were detected by RT-PCR method. HE, Masson staining and immunohistochemical staining were used to observe the changes of myocardial histomorphology in mice. We also examined the effects of different drug treatments on the proliferation and function of Raw264.7 cells, H9C2 cells and HUVECs. Western blot examined the effects of different drug treatments on the expression of inflammatory pathway related proteins TLR4 and C/EBP-beta.Results: 1. Echocardiographic results showed that with the prolongation of ischemic time, the ejection fraction of the model group, the shortening rate of the short axis of the left ventricle, the flow rate of the outflow tract were significantly decreased, and the structure of the ventricle was significantly changed. Hemodynamic tests showed that the maximum and maximum rate of decline in the post-ischemia model group were significantly reduced, with increased systolic and diastolic volume, and a decrease in pressure difference. After treatment with drugs, all groups improved, but tanshinone IIA: puerarin = 1:1 group can significantly improve the above indicators after 28d of administration, which can effectively relieve the deterioration of cardiac function caused by acute myocardial infarction. 2. After administration for 3 and 7 days, the inflammatory cell CD11b monocytes and the F4/80 phenotype macrophages in heart tissue were detected by flow cytometry, and it was found that tanshinone IIA: puerarin = 1:1 can inhibit the release of inflammatory cells. The results of RT-PCR showed that the tanshinone IIA: puerarin = 1:1 group significantly improved the expression of inflammatory cytokines such as IL-1 beta, IL-6, IL-10, and iNOS. In the immunohistochemical analysis of iNOS and Arg-1, the tanshinone IIA and puerarin 1:1 treatment group was able to inhibit the expression of M1 macrophages in the early stage of inflammation and promote the expression of M2 macrophages. 3. The cardiac index increased significantly and the serum TGF-beta increased after 28d. The combination of tanshinone IIA and puerarin could significantly reduce these indexes. HE, Masson, Sirius red and immunohistochemical staining were found in the combination of tanshinone IIA and puerarin can significantly reduce the structure of acute ischemic myocardial cell damage and interstitial edema, reduce collagen synthesis, and fibroblasts release, thereby inhibiting myocardial fibrosis and heart remodeling. 4. MTT assay showed a significantly greater proliferation of above two cells types treated with tanshinone IIA: puerarin = 1:1 and more nodes and meshes were found in tanshinone IIA: puerarin = 1:1 group compared with other groups. 5. The combination of tanshinone IIA and puerarin could regulate inflammation through inhibiting the expression of TLR4 protein, but up-regulating the expression of C/EBP-beta protein.Conclusion: The combination of tanshinone IIA and puerarin inhibits the immersion of inflammatory cells. Improving hemodynamics by improving cardiac function, reducing the destruction of cardiac myocytes, reducing collagen synthesis, inhibiting myocardial fibrosis and ventricular remodeling. Through the whole experiment, tanshinone IIA: puerarin = 1:1 is the best.