Da chuanxiong formula improves vascular remodeling via promoting angiogenesis and vasodilation mediated by PI3K/Akt/eNOS signaling.

Abstract

Vascular cognitive impairment (VCI) is a vast disease with chronic cerebral hypoperfusion (CCH) being the major driver. In our previous study, we have verified that Da ChuanXiong (DCX) formula could alleviate VCI, and obviously upregulate the decreased cerebral blood flow, indicating its potential benefits on vascular remodeling. This study aims to explore the pro-angiogenic and vasodilatory effects of DCX formula after CCH injuries, and clarify its pharmacodynamic substances and potential mechanisms. The rat model of CCH was established by bilateral common carotid artery occlusion. CD31⁺ immunofluorescent staining and quail chorioallantoic membrane (qCAM) experiments were conducted to assess the angiogenic effect of DCX. Isolated vascular ring experiments and NO assay in qCAM were performed to evaluate the vasodilatory effect of DCX. Then we analyzed the chemical components absorbed in the blood of DCX under CCH conditions by UPLC-Q-TOF/MS, and further combined network pharmacology, molecular docking to predict the potential mechanisms and effective components. Western blot was performed in CCH rats to verify the possible mechanisms and in vitro pharmacodynamic verification experiment was conducted for innovative active ingredients in DCX. Our results revealed that DCX obviously increased the number of CD31⁺ cells in the hippocampus and brain cortex of CCH rats, and significantly increased the vascular areas in qCAM. DCX significantly dilated the pre-constricted vascular ring and increased NO contents in qCAM. Besides, Senkyunolide A, Butylphthalide, Ferulic acid, Senkyunolide I, Levistilide A, Ligustilide, Senkyunolide H and Gastrodin were identified as the 8 major blood-absorbed compounds of DCX under CCH condition. Next, network pharmacology and molecular docking revealed NOS3 and PI3K/Akt signaling as the critical targets and pathway of DCX improving vascular remodeling against CCH injuries, which was further validated by western blot. Moreover, we firstly discovered that Senkyunolide A and Senkyunolide H could obviously promote cell migration and enhance the tube formation ability of HUVECs in vitro. Taken together, DCX formula effectively enhanced the angiogenesis and vasodilation via activating PI3K/Akt/eNOS signaling, which might be the important pathways for DCX promoting vascular remodeling against CCH injuries. Notably, Senkyunolide A and Senkyunolide H were firstly identified as innovative components responsible for the pro-angiogenic effects of DCX.