Supporting data for “Network pharmacology prediction and biological validation of the molecular mechanisms underlying the cardioprotection of botanicals against myocardial injury”

Acute myocardial infarction (AMI) is a leading cause of human death worldwide. Provided that timely medical interventions are in place, contemporary therapies could effectively reduce the rates of morbidity and mortality. However, new therapies are pressingly needed to prevent heart failure and improve the life quality of AMI patients. Botanicals are proven to be effective for treating myocardial injury and other heart diseases. Thus, the objective of this study was to characterize the cardioprotective properties of the well-documented botanicals and elucidate the underlying molecular mechanisms.Firstly, we investigated the herbal medicine formula Baoyuan Decoction (BYD) for cardioprotective compounds and potential molecular targets. We validated that BYD not only protected cardiomyocyte H9C2 cells against oxygen glucose deprivation but also effectively reduced infarct size and preserved cardiac functions in a mouse model of myocardial ischemia reperfusion injury (MIRI). UPLC-MS/MS analysis identified 122 compounds from ethanolic BYD extract while network pharmacology approach predicted 37 MI-related target proteins in the regulation of inflammation and apoptosis. Pathway enrichment analysis revealed that BYD most likely targeted the tumor necrosis factor-alpha (TNF-α)/ nuclear factor-κB (NF-κB) pathway. Adenosine, ginsenoside Rh2, isoliquiritigenin, and licochalcone A were selected to form the four-compound mixture AGILe for targeting TNF-α/NF-κB pathway. Indeed, the formula AGILe effectively inhibited the TNF-α/NF-κB pathway and manifested the anti-inflammatory functions of BYD in the context of myocardial infarction.Secondly, we previously discovered that C-glycosylated isoflavone puerarin preserved cardiac function against ischemia-reperfusion injury by upregulating SUMO2, a small ubiquitin-like modifier. We hypothesized that SUMO2 might activate endogenous cardioprotective mechanisms during myocardial infarction. We generated cardiac-specific SUMO2 knockout mice and evaluated their performance in myocardial infarction. As results, SUMO2 KO mice experienced severe myocardial injury and cardiac dysfunction, while SUMO2 overexpression markedly improved the performance of SUMO2 KO mice during ischemia reperfusion challenge. In vitro SUMO2 silencing increased ROS generation, disrupted the Bax/Bcl-2 signaling pathway, and exacerbated intrinsic cell apoptosis during oxygen glucose deprivation. These results confirmed the importance of SUMO2 in cardiac homeostasis and resistance. Ultimately, botanic drug puerarin may be a potential therapyvagainst myocardial injury for targeting SUMO2.Thirdly, we prepared recombinant CRPPR-tagged human serum albumin (HSA) and puerarin-loaded HSA-CRPPR nanoparticles (HSA-CRPPR-PUE) for heart-targeting delivery of puerarin. Based on pharmacological evaluation in mouse model of MIRI, HSA-CRPPR-PUE showed better heart-targeting efficiency compared to free puerarin and HSA-PUE nanoparticles. Consistently, HSA-CRPPR-PUE more effectively protected the hearts against MIRI.In conclusion, this thesis employed multiple state-of-art technologies to elucidate the molecular mechanisms underlying the cardioprotective effects of botanicals drugs. The key findings include: 1) A four-compound remedy AGILe was developed to manifest the potential of BYD for treating myocardial injury via regulating the TNFα/NF-Κb pathway; 2) Expression level of SUMO2 positively correlated with the health and functions of the hearts during myocardial infarction; 4) HSA-CRPPR-PUE nanoparticles supported heart-targeting delivery of puerarin for treating myocardial injury. Overall, these findings paved the way to develop botanical drugs for the therapy of myocardial infarction.

急性心肌梗死（AMI）是全球人类死亡的主要原因。鉴于及时的医疗干预措施得以实施，当代疗法能够有效降低患病率和死亡率。然而，迫切需要新的疗法来预防心力衰竭并提高急性心肌梗死患者的生命质量。植物药已被证实对治疗心肌损伤和其他心脏病有效。因此，本研究的目标是对已广泛记录的植物药的心脏保护特性进行表征，并阐明其潜在的分子机制。首先，我们研究了草药配方保元汤（BYD）中的心脏保护化合物和潜在的分子靶点。我们验证了BYD不仅能保护心肌细胞H9C2细胞免受缺氧糖剥夺的影响，还能在心肌缺血再灌注损伤（MIRI）的动物模型中有效减少梗死面积并保护心脏功能。UPLC-MS/MS分析从乙醇BYD提取物中鉴定出122种化合物，而网络药理学方法预测了37种与MI相关的靶蛋白，这些靶蛋白参与炎症和细胞凋亡的调节。通路富集分析显示，BYD很可能靶向肿瘤坏死因子-α（TNF-α）/核因子-κB（NF-κB）通路。选择腺苷、人参皂苷Rh2、异甘草素和甘草查尔酮A组成四元混合物AGILe，以靶向TNF-α/NF-κB通路。确实，配方AGILe有效抑制了TNF-α/NF-κB通路，并在心肌梗死背景下表现出BYD的抗炎功能。其次，我们先前发现C-糖基化异黄酮大豆苷元通过上调SUMO2（一种小泛素化修饰蛋白）来保护心脏功能，以抵抗缺血再灌注损伤。我们假设SUMO2可能在心肌梗死期间激活内源性心脏保护机制。我们生成了心脏特异性SUMO2敲除小鼠，并评估了它们在心肌梗死中的表现。结果发现，SUMO2 KO小鼠经历了严重的心肌损伤和心脏功能障碍，而SUMO2过表达显著改善了SUMO2 KO小鼠在缺血再灌注挑战中的表现。体外SUMO2沉默增加了ROS的产生，破坏了Bax/Bcl-2信号通路，并在缺氧糖剥夺期间加剧了细胞的内在凋亡。这些结果证实了SUMO2在心脏稳态和抵抗力中的重要性。最终，植物药物大豆苷元可能是一种针对SUMO2的潜在治疗心肌损伤的药物。第三，我们制备了重组CRPPR标记的人血清白蛋白（HSA）和负载大豆苷元的HSA-CRPPR纳米粒子（HSA-CRPPR-PUE），用于大豆苷元的心脏靶向递送。基于MIRI小鼠模型中的药理学评估，HSA-CRPPR-PUE与游离大豆苷元和HSA-PUE纳米粒子相比，表现出更好的心脏靶向效率。一致地，HSA-CRPPR-PUE更有效地保护心脏免受MIRI的影响。总之，本论文采用了多种最先进的技术来阐明植物药物心脏保护作用背后的分子机制。主要发现包括：1）开发了一种四元疗法AGILe，通过调节TNFα/NF-Κb通路来展现BYD治疗心肌损伤的潜力；2）SUMO2的表达水平与心肌梗死期间心脏的健康和功能呈正相关；4）HSA-CRPPR-PUE纳米粒子支持大豆苷元的心脏靶向递送，以治疗心肌损伤。总的来说，这些发现为开发治疗心肌梗死的植物药物铺平了道路。