Chrysin promotes angiogenesis in rat hindlimb ischemia: Impact on PI3K/Akt/mTOR signaling pathway and autophagy

Abstract Limb ischemia occurs due to obstruction of blood perfusion to lower limbs, a manifestation that is associated with peripheral artery disease (PAD). Angiogenesis is important for adequate oxygen delivery. The present study investigated a potential role for chrysin, a naturally occurring flavonoid, in promoting angiogenesis in hindlimb ischemia (HLI) rat model. Rats were allocated into four groups: (1) sham‐operated control, (2) HLI: subjected to unilateral femoral artery ligation, (3) HLI + chrysin: received 100 mg/kg, i.p. chrysin immediately after HLI, and (4) HLI + chrysin + rapamycin: received 6 mg/kg/day rapamycin i.p. for 5 days then subjected to HLI and dosed with 100 mg/kg chrysin, i.p. Rats were killed 18 h later and gastrocnemius muscles were collected and divided into parts for (1) immunohistochemistry detection of CD31 and CD105, (2) qRT‐PCR analysis of eNOS and VEGFR2, (3) colorimetric analysis of NO, (4) ELISA estimation of TGF‐β, VEGF, ATG5 and Beclin‐1, and (5) Western blot analysis of p‐PI3K, PI3K, p‐Akt, Akt, p‐mTOR, mTOR, and HIF‐1α. Chrysin significantly enhanced microvessels growth in HLI muscles as indicated by increased CD31 and CD105 levels and decreased TGF‐β. Chrysin's proangiogenic effect is potentially mediated by increased VEGF, VEGFR2 and activation of PI3K/AKT/mTOR pathway, which promoted eNOS and NO levels as it was reversed by the mTOR inhibitor, rapamycin. Chrysin also inhibited autophagy as it decreased ATG5 and Beclin‐1. The current study shows that chrysin possesses a proangiogenic effect in HLI rats and might be useful in patients with PAD.