以Formononetin和Salvianolic acid B靶向RhoA表达以减缓胰腺癌相关血管内皮细胞变化

根据中医理论中的气血学说，补气药与活血药的结合具有协同增强改善血瘀证的作用，尤其在肿瘤治疗中具有重要意义。经典的"黄芪根-丹参"组合正体现了这一原则，以益气和活血著称，广泛应用于肿瘤疗法中。我们先前的研究强调，来自黄芪的Formononetin与来自丹参的Salvianolic acid B在体外具有显著抑制胰腺肿瘤异种移植的作用。然而，尚不清楚该组合是否能抑制胰腺肿瘤的异常血管生成，从而发挥抗癌作用。异常血管被认为促进肿瘤生长和转移。利用血管正常化策略为抗肿瘤治疗提供了有希望的途径。本研究旨在揭示Formononetin联合Salvianolic acid B（FcS）在调控胰腺癌对血管内皮细胞影响方面的治疗潜力，阐明调控这一治疗作用的机制，从而推动肿瘤血管正常化和抗癌策略的发展。我们建立了人脐静脉血管内皮细胞（HUVECs）与PANC-1细胞的共培养系统，旨在探讨靶向异常血管作为新型抗肿瘤治疗策略的潜力。系统比较了在单一培养和共培养（H-P）条件下HUVEC的增殖、迁移、侵袭和管腔形成能力。随后对H-P体系施加FcS，评估其抗血管生成作用。采用分子对接预测Formononetin和Salvianolic acid B与RhoA的相互作用，并检测血管内皮细胞中RhoA在处理后的表达水平。此外，利用shRhoA慢病毒敲除技术阐明RhoA在FcS介导HUVEC作用中的作用。体内方面，采用斑马鱼异种移植肿瘤模型评估FcS的抗肿瘤潜力，重点观察癌细胞增殖、迁移、凋亡及血管生成。结果显示，FcS治疗显著且剂量依赖性地抑制了PANC-1诱导的HUVEC变化，包括增殖、迁移、侵袭和管腔形成能力。分子对接分析显示FcS可能与RhoA相互作用。此外，FcS还可下调RhoA的表达，并调节PI3K/AKT信号通路。值得注意的是，RhoA敲除减弱了FcS对HUVEC的表型抑制作用。体内斑马鱼模型验证了FcS的抗肿瘤活性，抑制癌细胞增殖、转移及血管新生，同时促进肿瘤细胞凋亡。总之，本研究强调FcS在对抗胰腺癌引起的内皮细胞变化中的潜在应用价值，其抗异常血管作用可能通过下调RhoA和抑制PI3K/Akt信号通路实现，为胰腺癌的治疗提供了新策略。

According to the theory of Qi and blood in Traditional Chinese Medicine (TCM), the combination of Qi-reinforcing herbs and blood-activating herbs has a synergistic effect in improving blood stasis syndrome, especially in tumor treatment. The classic "Radix Astragali - Salvia miltiorrhiza" duo exemplifies this principle, renowned for invigorating Qi and activating blood flow, employed widely in tumor therapies. Our prior research underscores the potent inhibition of pancreatic tumor xenografts by the combination of Formononetin (from Radix Astragali) and Salvianolic acid B (from Salvia miltiorrhiza) in vitro. However, it remains unclear whether this combination can inhibit the abnormal vascularization of pancreatic tumors to achieve its anti-cancer effect.Abnormal vasculature, known to facilitate tumor growth and metastasis. Strategies to normalize tumor-associated blood vessels provide a promising avenue for anti-tumor therapy. This study aimed to unravel the therapeutic potential of Formononetin combined with Salvianolic acid B (FcS) in modulating pancreatic cancer's impact on endothelial cells, illuminate the underlying mechanisms that govern this therapeutic interaction, thereby advancing strategies to normalize tumor vasculature and combat cancer progression.A co-culture system involving Human Umbilical Vein Endothelial Cells (HUVECs) and PANC-1 cells was established to investigate the potential of targeting abnormal vasculature as a novel anti-tumor therapeutic strategy. We systematically compared HUVEC proliferation, migration, invasion, and lumenogenesis in both mono- and co-culture conditions with PANC-1 (H-P). Subsequently, FcS treatment of the H-P system was evaluated for its anti-angiogenic properties. Molecular docking was utilized to predict the interactions between Formononetin and Salvianolic acid B with RhoA, and the post-treatment expression of RhoA in HUVECs was assessed. Furthermore, we utilized shRhoA lentivirus to elucidate the role of RhoA in FcS-mediated effects on HUVECs. In vivo, a zebrafish xenograft tumor model was employed to assess FcS's anti-tumor potential, focusing on cancer cell proliferation, migration, apoptosis, and vascular development.FcS treatment demonstrated a significant, dose-dependent inhibition of PANC-1-induced alterations in HUVECs, including proliferation, migration, invasion, and tube formation capabilities. Molecular docking analyses indicated potential interactions between FcS and RhoA. Further, FcS treatment was found to downregulate RhoA expression and modulated the PI3K/AKT signaling pathway in PANC-1-induced HUVECs. Notably, the phenotypic inhibitory effects of FcS on HUVECs were attenuated by RhoA knockdown. In vivo zebrafish studies validated FcS's anti-tumor activity, inhibiting cancer cell proliferation, metastasis, and vascular sprouting, while promoting tumor cell apoptosis.This study underscores the promising potential of FcS in countering pancreatic cancer-induced endothelial alterations. FcS exhibits pronounced anti-abnormal vasculature effects, potentially achieved through downregulation of RhoA and inhibition of the PI3K/Akt signaling pathway, thereby presenting a novel therapeutic avenue for pancreatic cancer management.