心肌缺血再灌注损伤（Myocardial ischemia-reperfusion injury，MIRI）是急性心肌梗死后临床预后不良的主要原因之一。血栓形成和炎症是MIRI病理生理过程的关键机制。血小板在血栓形成和炎症方面具有重要作用。在缺血再灌注过程中，活化的血小板在心脏的小血管和毛细血管内形成微血栓，导致心脏组织受损。活化的血小板还与中性粒细胞相互作用，形成 血小板-中性粒细胞复合物，促进中性粒细胞浸润到缺血再灌注组织中，并扩大炎症反应，导致心肌细胞死亡。活化的中性粒细胞可发生NETosis，释放中性粒细胞外诱捕网（Neutrophil extracellular traps, NETs），进一步增加心肌缺血再灌注损伤。研究表明，中性粒细胞的活化和NETs的形成在很大程度上依赖于血小板，干扰血小板-中性粒细胞相互作用可减轻炎症和组织损伤。CD36在血小板表面表达，与心血管疾病的进展密切相关。血小板CD36受体的激活会促进血小板活化、血栓形成和心肌梗死。抑制血小板CD36可降低血小板反应性。此外，研究表明，CD36在心脏重塑过程中刺激中性粒细胞凋亡和清除，在调节炎症方面发挥着关键作用。因此，调控CD36可能是一种通过预防血小板活化和中性粒细胞炎症治疗MIRI的潜在方法。

刺芒柄花素（Formononetin, FMN）存在于黄芪、甘草和葛根等豆科植物中，是一种异黄酮类化合物。药理研究表明，FMN具有抗氧化、抗凋亡、抑制中性粒细胞浸润的作用以及抑制血小板活化的特性。此外，我们前期通过分子对接和BLI分析发现，FMN与CD36具有良好的结合活性。以上结果表明，FMN可能通过调控血小板活化与炎症反应，对MIRI产生保护作用。然而，FMN抗MIRI的具体作用和机制尚未阐明。因此，本课题证明了FMN对MIRI的保护作用，并进一步阐明FMN通过影响血小板CD36及其下游ERK5信号通路，进而调控血小板活化和NETs形成发挥其心脏保护作用的分子机制。本研究分为以下五部分：

 

第一章 刺芒柄花素抗心肌缺血再灌注损伤的保护作用研究

实验目的：利用大鼠MI/R模型探讨FMN对MIRI的保护作用。

实验方法：SD大鼠灌胃给予FMN（10，20，40 mg/kg）7天后，心肌缺血30 min再灌注24 h，利用TTC，HE染色，超声心动，Elisa等方法评价FMN对大鼠心肌梗死面积，心脏结构与功能损伤的影响。

实验结果：

TTC染色结果表明FMN（10，20，40 mg/kg）显著缩小了大鼠心肌梗死面积。HE染色结果表明FMN（10，20，40 mg/kg）减轻了大鼠心脏的病理性损伤。

FMN（10，20，40 mg/kg）可以改善大鼠心功能，提高LVEF、LVFS水平。

Elisa结果显示，FMN（10，20，40 mg/kg）可以显著降低心肌酶水平。

结论：FMN可以减轻大鼠MIRI，FMN 40 mg/kg确定为本课题体内实验最优剂量。

 

第二章 基于4D-DIA蛋白组学解析刺芒柄花素抗心肌缺血再灌注损伤的作用机制

实验目的：利用4D-DIA定量蛋白组学，探究FMN抗MIRI的关键机制。

实验方法：收集每组大鼠心脏组织用于蛋白质组学分析。通过SDS-PAGE和胰蛋白酶酶解测定总蛋白。脱盐后采用液相色谱-串联质谱（LC-MS/MS）鉴定样品。最后通过数据库检索分析和生物信息学分析进一步解析实验结果。

实验结果：

1、火山图分析显示，相对于Sham组，MI/R组有270个蛋白表达上调，54个蛋白表达下调。与MI/R组相比，MI/R + FMN组有38个蛋白表达上调，44个蛋白表达下调。

2、维恩图分析显示，与Sham组相比，MI/R组有324个差异蛋白，与MI/R组相比，MI/R+FMN组有82个差异蛋白，值得注意的是，两组比较中共有31个相同的差异蛋白。

3、在MI/R组和Sham组之间出现明显表达差异的蛋白质主要与补体和凝血级联、NETs的形成、血小板活化等有关。MI/R+FMN组和 MI/R组的差异蛋白与血小板和炎症密切相关。

结论：FMN可能通过抑制血小板活化和NETs的形成改善MIRI。

 

第三章 刺芒柄花素抑制血小板活化抗心肌缺血再灌注损伤的作用研究

实验目的：建立SD大鼠MI/R模型，研究FMN对MIRI中血小板活化的影响。

实验方法：SD大鼠再灌注24h后，从腹主动脉采集血样，获得血浆样本，检测其Ca²⁺和CD62P水平；将新鲜血液离心得到血小板，利用血小板聚集仪和荧光显微镜检测血小板聚集率和血小板黏附率；利用流式细胞术检测血小板表面CD62P水平；利用免疫荧光技术检测SD大鼠MI/R后的心脏组织中vWF/CD62P的表达情况。

实验结果：

1、MI/R组血小板粘附率显著上升，而FMN则抑制了血小板粘附。

2、给予ADP后，MI/R组的血小板聚集明显升高，而FMN组的血小板聚集率明显下降。

3、在MI/R组中，Ca²⁺和CD62P水平都明显升高。然而，FMN处理降低了Ca²⁺含量和CD62P水平。

4、流式细胞术结果表明MI/R组CD62P阳性细胞数增加，而FMN可显著减少CD62P阳性细胞数。

5、免疫荧光结果显示，MI/R组的血小板活化和微血栓形成明显增加，而FMN给药组则明显降低血小板活化程度并抑制心脏微血栓的形成。

结论：FMN可减弱MIRI引起的血小板活化和微血栓形成。

 

第四章 刺芒柄花素抑制血小板-中性粒细胞聚集和NETs对大鼠心肌缺血再灌注损伤的保护作用研究

实验目的：基于FMN可以调控MIRI导致的血小板活化，进一步探究FMN如何通过血小板影响中性粒细胞。

实验方法：SD大鼠再灌注24h后取得新鲜血液，检测血浆IL-1β和TNF-α水平；利用流式细胞术检测外周血血小板-白细胞聚集、中性粒细胞募集和血小板-中性粒细胞聚集；利用流式细胞术和免疫荧光检测心脏组织中性粒细胞募集和血小板-中性粒细胞聚集；利用Elisa和免疫荧光检测SD大鼠MI/R后的心脏组织中MPO、NE的表达情况以及NETs的形成（MPO/CitH3）；使用TUNEL染色检测SD大鼠MI/R后的心肌细胞凋亡情况。

实验结果：

1、Elisa结果显示，MI/R组大鼠外周血中炎症因子IL-1β和TNFα明显升高，FMN给药后明显降低。

2、外周血流式细胞术结果显示，FMN改善MIRI导致的血小板-白细胞聚集、中性粒细胞募集和血小板-中性粒细胞聚集。

3、流式细胞术和免疫荧光结果显示，FMN减轻心脏组织中性粒细胞募集和血小板-中性粒细胞聚集（CD41/MPO）。

4、FMN可以减少MIRI大鼠心脏组织中MPO、NE的含量，抑制NETs的形成并改善心肌细胞凋亡情况。

结论：结果表明，FMN可抑制MIRI导致的血小板-中性粒细胞聚集和NETs的形成。

 

第五章 刺芒柄花素调控血小板CD36信号通路改善血小板活化和NETs的形成

实验目的：探究FMN通过调控血小板活化和NETs形成抗MIRI的分子机制。

实验方法：SD大鼠再灌注24h后取得血小板蛋白，WB检测CD36及其下游信号通路的蛋白表达变化；提取正常大鼠的血小板和骨髓中性粒细胞，用CD36重组蛋白和CD36抑制剂SSO处理血小板后加入至中性粒细胞中观察NETs的形成。

实验结果：

1、与MI/R组相比，MI/R + FMN 40mg/kg组的CD36表达、ERK5磷酸化水平明显降低。同时，与CD36相关的P38和JNK通路无明显变化。

2、单独的CD36重组蛋白不能激活血小板，激动剂Trap-6（25 μM）显著增加了血小板活化程度，而FMN（40 μM）有效抑制了血小板活化，SSO（200 μM）也抑制了血小板活化程度。而CD36重组蛋白则抵消了FMN对血小板活化的抑制作用。

3、活化的血小板刺激中性粒细胞形成NETs，FMN显著降低了NETs的产生，而CD36重组蛋白抵消了这一作用。

结论：FMN通过血小板CD36介导的ERK5信号通路影响NETs的形成和血小板活化。

 

总结：FMN可通过血小板CD36介导的ERK5信号通路调节NETs的形成和血小板的活化，从而减轻心肌缺血再灌注损伤，且不会增加出血风险。这些研究结果表明，FMN可能是治疗MIRI的理想候选药物。

 

Myocardial ischemia-reperfusion injury (MIRI) is one of the main causes of poor clinical prognosis after acute myocardial infarction. The pathophysiological processes that cause MIRI are not yet fully comprehended. Thrombosis and inflammation are pivotal in the pathophysiology of MIRI. Platelets are of great importance in integrating thrombosis and inflammation. During the ischemia-reperfusion process, activated platelets clump together to create microthrombi within the heart's small vessels and capillaries, resulting in damage to the cardiac tissue. Activated platelets also interact with neutrophils to form a platelet-neutrophil complex, which promotes the infiltration of neutrophils into ischemic/reperfused tissues and amplifies the inflammatory response, leading to immune-mediated cardiomyocyte death. Activated neutrophils can undergo NETosis, releasing neutrophil extracellular traps (NETs), which have been observed in patients with myocardial infarction or acute coronary syndromes. NETs link inflammation with thrombosis and have been reported to increase MIRI. Growing evidence indicates that neutrophil activation and NETs formation largely depend on platelets, and disrupting this interaction may mitigate inflammation and tissue damage. CD36 is prominently expressed on the surface of platelets. CD36 is closely linked to cardiovascular disease progression. Research indicates that platelet CD36 levels are elevated in patients with coronary artery disease compared to those without the condition. Platelet CD36 activation promotes increased platelet activity, thrombosis, and the progression of myocardial infarction. Pharmacologically inhibiting platelet CD36 showed decreased platelet reactivity and cardioprotective effects following MIRI. Moreover, a previous study indicated the key role of CD36 in regulating inflammation by stimulating neutrophil apoptosis and removal during cardiac remodeling. Therefore, focusing on CD36 may be a potential treatment approach to prevent platelet activation and neutrophil inflammation against MIRI.

Formononetin (7-hydroxy-4′-methoxyisoflavone, FMN) is commonly present in legumes like Astragalus, Glycyrrhiza glabra, and Pueraria lobata. Pharmacological studies have shown that FMN exerts anti-inflammatory, anti-oxidative, and anti-apoptotic properties in cardiovascular diseases. Studies have shown that FMN has inhibitory effects on neutrophil infiltration as well as inhibitory properties on platelet activation. In addition, our previous report suggested that FMN exhibited good binding activity with CD36 by molecular docking and BLI analysis. These results indicate that FMN may benefit from regulating platelet activation and inflammatory responses generated by MIRI. However, the specific roles and mechanisms of FMN concerning platelet activation and neutrophil inflammation against MIRI have not yet been elucidated. Therefore, the present work demonstrated the protective effect of FMN against MIRI both in vivo and in vitro and further elucidated the molecular mechanisms by which FMN regulates platelet activation and NETs exert cardioprotection. This study is divided into the following five parts:

 

Chapter 1. Study on the protective effect of formononetin on myocardial ischemia-reperfusion injury

Objective: To investigate the protective effect of FMN against MIRI using a rat MIRI model.

Method: SD rats were given FMN (10, 20, 40 mg/kg, i.g.) for 7 days, myocardial ischemia for 30 min, and reperfusion for 24 h. The effects of FMN on the area of myocardial infarction, the damage to cardiac structure and function, and the level of apoptosis of cardiomyocytes in rats were investigated by using TTC, HE staining, echocardiography, western blot, and ELISA.

Results:

TTC and HE staining results showed that FMN (10, 20, 40 mg/kg) significantly attenuated the area of myocardial infarction and cardiac pathological injury induced by MIRI in rats.

FMN (10, 20, 40 mg/kg) can significantly improve the cardiac function, LVEF, and LVFS levels in rats.

Elisa results showed that FMN (10, 20, 40 mg/kg) could significantly reduce the level of cardiac enzymes.

Conclusion: The results showed that FMN could reduce MIRI in rats, and FMN 40 mg/kg was the optimal dose for the in vivo experiments.

 

Chapter 2. Analysis of the mechanism of anti myocardial ischemia-reperfusion injury effect of formononetin based on 4D-DIA proteomics

Objective: To investigate the key pathway of FMN anti-MIRI using 4D-DIA quantitative proteomics.

Method: Heart tissues of each group of rats were collected for proteomics analysis. Total protein was determined by SDS-PAGE and trypsin digestion. After desalting, the samples were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Finally, the results were further analysed by database search analysis and bioinformatics analysis.

Results:

Volcano plot analysis showed that 270 proteins were up-regulated and 54 proteins were down-regulated in the MI/R group relative to the Sham group. Compared with MI/R group, MI/R + FMN group had 38 proteins expression up-regulated and 44 proteins expression down-regulated.

Venn diagram analysis showed that there were 324 differential proteins in the MI/R group compared with the Sham group, and 82 differential proteins in the MI/R + FMN group compared with the MI/R group, of which 31 proteins were common in the comparison of the two groups.

The proteins that showed significant expression differences between MI/R and Sham groups were mainly related to complement and coagulation cascades, formation of NETs, platelet activation, etc. Differential proteins in MI/R+FMN and MI/R groups were closely related to platelets and inflammation.

Conclusion: The results suggest that FMN may improve MIRI by inhibiting platelet activation and NETs.

 

Chapter 3. Study on the inhibitory effect of formononetin on platelet activation against myocardial ischemia-reperfusion injury

Objective: To establish SD rat MIRI model and study the effect of FMN on platelet activation due to MIRI.

Method: Fresh blood was obtained after MIRI modeling in SD rats, and Ca²⁺ and CD62P levels were detected; platelets were obtained by centrifugation of fresh blood, and platelet aggregation rate and platelet adhesion rate were detected using platelet aggregometer and fluorescence microscopy; CD62P levels on the surface of platelets were detected using flow cytometry; and vWF/CD62P levels were detected using immunofluorescence in cardiac tissues of SD rats after MIRI vWF/CD62P expression.

Results:

MI/R surgery significantly enhanced platelet adhesion to fibrinogen, while FMN significantly reduced platelet adhesion to fibrinogen.

After administration of ADP, platelet aggregation was significantly increased in the MI/R group, while it was significantly decreased in the FMN group.

Ca²⁺ and CD62P are key indicators of platelet activation. In the MI/R group, the levels of both markers were significantly elevated. However, FMN treatment decreased Ca²⁺ levels and CD62P expression.

Flow cytometry results showed that the number of CD62P+ cells increased in the MI/R group, whereas FMN reduced the number of CD62P+ cells.

Immunofluorescence results showed a significant increase in platelet activation and microthrombosis in the MI/R group, whereas it was significantly reduced in the FMN-administered group.

Conclusion: The results showed that FMN attenuated platelet activation and microthrombosis induced by cardiac injury.

 

Chapter 4. Study on the protective effects of formononetin inhibition of platelet-neutrophil aggregation and NETs formation on myocardial ischemia-reperfusion injury in rats

Objective: Based on the fact that FMN can modulate platelet activation caused by MIRI, this chapter further explores how FMN affects neutrophils via platelets.

Methods: Blood was obtained from SD rats after MIRI modeling, and IL-1β and TNF-α levels were detected. Peripheral blood platelet-leukocyte aggregation, neutrophil recruitment, and platelet-neutrophil aggregation were detected using flow cytometry. Neutrophil recruitment and platelet-neutrophil aggregation were detected in cardiac tissues using flow cytometry and immunofluorescence. The expression of MPO and NE and the formation of NETs (MPO/CitH3) were detected in the cardiac tissues of SD rats after MIRI. Cardiomyocyte apoptosis was detected in SD rats after MIRI.

Results:

Elisa's results showed that inflammatory factors IL-1β and TNF-α were significantly elevated in the peripheral blood of rats in the MI/R group, and significantly reduced after FMN administration.

Peripheral blood flow cytometry results showed that FMN ameliorated platelet-leukocyte aggregation, neutrophil recruitment, and platelet-neutrophil aggregation caused by MIRI.

Flow cytometry and immunofluorescence results showed that FMN attenuated neutrophil recruitment and platelet-neutrophil aggregation (CD41/MPO) in cardiac tissue.

FMN reduced the expression of MPO, NE, and CitH3, inhibited the formation of NETs, and improved cardiomyocyte apoptosis in cardiac tissues of MIRI rats.

Conclusion: The results suggest that FMN inhibits platelet-neutrophil aggregation and NETs formation caused by MIRI.

 

Chapter 5. Formononetin modulates platelet CD36 signaling pathway and ERK5 phosphorylation activation to improve platelet activation and NETs

Objective: It has been demonstrated that FMN regulates platelet activation and NETs to exert cardioprotective effects, and this section further determines the molecular mechanism of FMN anti-MIRI.

Methods: Platelet proteins were obtained after MIRI modeling in SD rats, and protein expression changes of CD36 and its downstream signaling pathway were detected by WB; platelets and bone marrow neutrophils from normal rats were extracted, and platelets were treated with CD36 recombinant protein and CD36 inhibitor SSO and then added to neutrophils to observe the formation of NETs.

Results:

Compared with the MI/R group, the expression of CD36 and the phosphorylation level of ERK5 were significantly reduced in the MI/R + FMN group. Meanwhile, there were no significant changes in P38 and JNK pathways associated with CD36.

CD36 recombinant protein alone did not activate platelets, and the addition of the agonist Trap-6 (25 μM) significantly increased platelet activation, whereas the addition of FMN (40 μM) effectively inhibited platelet activation, and SSO (200 μM) also suppressed the degree of platelet activation. In contrast, CD36 recombinant protein counteracted the inhibitory effect of FMN on platelet activation.

Activated platelets stimulated neutrophils to form NETs, and FMN significantly reduced the production of NETs, which was counteracted by CD36 recombinant protein.

Conclusion: FMN affects NETs formation and platelet activation through the platelet CD36-mediated ERK5 signaling pathway.

 

Summary: FMN mitigates myocardial ischemia/reperfusion injury by modulating NETs formation and platelet activation through platelet CD36-mediated ERK5 signaling, without elevating bleeding risk. These findings indicate that FMN could be a promising candidate for developing MIRI therapeutics.