Kunxian Capsule Extract Inhibits Angiogenesis in Zebrafish Embryos via PI3K/AKT-MAPK-VEGF Pathway

Objective: To investigate the anti-angiogenic activity of Kunxian Capsule(KX) extract and explore the underlying molecular mechanism using zebrafish. Methods: The KX extract was prepared with 5.0 g in 100 mL of 40% methanol followed by ultrasonication and freeze drying. Freeze dried KX extract of 10.00 mg was used as test stock solution. Triptolide and icariin, the key bioactive compounds of KX were analyzed using ultra-high performance liquid chromatography. The transgenic zebrafish Tg(flk1:GFP) embryos were dechorionated at 20-h post fertilization(hpf) and treated with PTK 787, and 3.5, 7, 14 and 21 μg/m L of KX extract, respectively.After 24-h post exposure(hpe), mortality and malformation(%), intersegmental vessels(ISV) formation, and m RNA expression level of angiogenic pathway genes including phosphoinositide 3-kinase(PI3K), protein kinase B(AKT),extracellular signal-regulated kinases(ERKs), mitogen-activated protein kinase(MAPK), vascular endothelial growth factor(VEGF) and fibroblast growth factor(FGF-2) were determined. Further, the embryos at 72 hpf were treated with KX extract to observe the development of sub-intestinal vein(SIV) after 24 hpe. Results: The chromatographic analysis of test stock solution of KX extract showed that triptolide and icariin was found as 0.089 mg/g and 48.74 mg/g, respectively, which met the requirements of the national drug standards. In zebrafish larvae experiment, KX extract significantly inhibited the ISV(P<0.01) and SIV formation(P<0.05). Besides, the m RNA expression analysis showed that KX extract could significantly suppress the expressions of PI3K and AKT,thereby inhibiting the m RNA levels of ERKs and MAPK. Moreover, the downstream signaling cascade affected the expression of VEGF and its receptors(VEGFR and VEGFR-2). FGF-2, a strong angiogenic factor, was also downregulated by KX treatment in zebrafish larvae. Conclusion: KX extract exhibited anti-angiogenic effects in zebrafish embryos by regulating PI3K/AKT-MAPK-VEGF pathway and showed promising potential for RA treatment.