Houshiheisan Modulates Angiogenic Responses After Ischemic Stroke in Association with miR-126/PIK3R2 and miR-21/Hmga2/WWP1 Signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Houshiheisan (HSHS), a classical formula from the Synopsis of the Golden Chamber, is traditionally used for stroke and is known for its wind-dispelling and deficiency-tonifying properties. While HSHS has been confirmed to significantly reduce tissue necrosis following cerebral ischemia, its precise effects on post-ischemic angiogenic responses remain incompletely understood. AIM OF THE STUDY: This study aimed to investigate the effects of HSHS on post-ischemic angiogenic responses and to explore the underlying mechanisms. MATERIALS AND METHODS: For in vivo experiments, male Sprague-Dawley rats underwent permanent occlusion of the middle cerebral artery (MCAO) to establish an ischemic stroke model. HSHS was administered intragastrically at doses of 5.25 g/kg and 10.5 g/kg once daily for 7 days. Pathological changes were assessed using HE staining. Cortical vascular density after cerebral ischemia was evaluated by CD31 immunofluorescence staining. Western blot and qRT-PCR techniques were employed to examine the expression of miRNAs and proteins within the relevant signaling pathways. For in vitro studies, primary BMECs were cultured, and an oxygen-glucose deprivation (OGD) model was established. CCK-8, wound healing, and tube formation assays were utilized to detect the functions of BMECs. Lentiviral transfection was employed to inhibit miR-126 or overexpress miR-21, thereby investigating the mechanism by which HSHS enhances BMEC angiogenic activity. RESULTS: In vivo, HSHS attenuated vascular injury, increased cortical vascular density, and modulated the expression of angiogenesis-related proteins in the ischemic region. HSHS significantly elevated miR-126 expression in brain tissue while inhibiting the expression of its target gene PIK3R2. This was accompanied by increased PI3K expression and AKT phosphorylation. Additionally, HSHS decreased miR-21 expression in brain tissue and increased the expression of its target genes, Hmga2 and WWP1. These changes were associated with the downregulation of p16 and suppression of the TGF-β/SMAD7 signaling axis. In vitro, HSHS-serum enhanced the proliferation, migration, and tube formation of BMECs. Both miR-126 inhibition and miR-21 overexpression attenuated the HSHS-serum-induced angiogenic activity of BMECs. CONCLUSION: HSHS modulated post-ischemic angiogenic responses, a process that potentially involves miR-126/PIK3R2- and miR-21/Hmga2/WWP1-related regulation of BMEC function.