Ligusticum chuanxiong promotes the angiogenesis of preovulatory follicles (F1-F3) in late-phase laying hens.

Ligusticum chuanxiong (CX) is a traditional Chinese medicine that is widely planted throughout the world. CX is one of the most important and commonly used drugs to enhance blood circulation. The preovulatory follicles in laying hens have a large number of blood arteries and meridians that feed the follicles' growth and maturation with nutrients, hormones, and cytokines. With the extension of laying time, preovulatory follicles angiogenesis decreased gradually. In this study, we studied the mechanism of CX on preovulatory follicles angiogenesis in late-phase laying hens. The results show that CX extract can increase the angiogenesis of preovulatory follicles (F1-F3) of late-phase laying hens. CX extract can promote vascular endothelial growth factor receptor 2 (VEGFR2) phosphorylation in preovulatory follicles theca layers, promote the proliferation, invasion and migration through PI3K/AKT and RAS/ERK signaling pathways in primary follicle microvascular endothelial-like cells (FMECs). In addition, CX extract can up-regulate the expression of hypoxia inducible factor α (HIF1α) in granulosa cells (GCs) and granulosa layers through PI3K/AKT and RAS/ERK signaling pathways, thereby promoting the secretion of vascular endothelial growth factor A (VEGFA). In conclusion, the current study confirmed the promoting effect of CX extract on the preovulatory follicles angiogenesis, which sets the stage for the design of functional animal feed for late-phase laying hens.

Effect of Angelica Sinensis extract on the angiogenesis of preovulatory follicles (F1-F3) in late-phase laying hens.

In order to form follicles and ovulate normally, there must be abundant blood vessels. Angelica sinensis (Oliv.) Diels (AS), as a traditional Chinese medicinal herb, has the effects of tonifying the blood and activating the blood circulation. However, the effect of AS on angiogenesis in hen-follicles remains to be discovered. In this study, we identified vascular richness, granulosa layer thickness, expression of platelet endothelial cell adhesion molecule-1 (CD31) and the content of vascular endothelial growth factor A (VEGFA) in granulosa layers to elucidate the effect of AS extract on angiogenesis in preovulatory follicles (F1-F3) of late-phase laying hens (75 wk). Based on network pharmacology, we predicted beta-sitosterol, ferulic acid, and caffeic acid as the main active components of AS, and hypoxia-inducible factor-1α (HIF1α), vascular endothelial growth factor receptor 2 (VEGFR2) as hub targets of AS in angiogenesis. The intersection targets were enriched by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and the hub targets were verified by immunofluorescence and western blot. Molecular docking of active components with hub targets was performed and verified in vitro. The results showed that AS extract promoted angiogenesis in preovulatory follicles and increased granulosa cell layer thickness, CD31 expression and content of VEGFA. Experiments in vitro and in vivo demonstrated that AS extract promoted the expression of HIF1α and VEGFA, up-regulated the phosphorylation levels of VEGFR2. These results further demonstrated the reliability of molecular docking and network pharmacology findings. In summary, AS extract can promote angiogenesis in the preovulatory follicles in late-phase laying hens.

Promotion effect of angelica sinensis extract on angiogenesis of chicken preovulatory follicles in vitro.

Preovulatory follicles need a network of blood vessels to growth and maturation in hens (Gallus gallus). Angelica sinensis (Oliv.) (AS), a traditional Chinese herb, displays a novel pro-angiogenic activity. The molecular mechanisms underlying AS promoting preovulatory follicles angiogenesis are poorly understand. Several recent studies investigated the expression of vascular endothelial growth factor A (VEGF-A) in angiogenesis. In order to explore the promotion effect of AS extract on angiogenesis of chicken preovulatory follicles, we studied the effect of AS extract on follicle microvascular endothelial-like cells of chicken (FMEC) and granulosa cells (GC). The current study indicated that AS extract could promote the proliferation of FMECs and GCs. The assays of wounding healing, transwell invasion and tube formation showed that AS extract could enhance the invasion and migration ability of FMECs in vitro. The results of western blot and RT-PCR showed that AS extract promoted the phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) in FMECs by activating the PI3K/AKT signaling pathway. The AS extract activated PI3K/AKT signaling pathway and up-regulated the expressions of hypoxia-inducible factor 1-α (HIF1-α) and VEGF-A in GCs. In addition, treatment of FMECs and GCs with LY294002 (a PI3K inhibitor) significantly down-regulated the phosphorylation of VEGFR2, VEGF-A, and HIF1-α. The mRNA expression levels of PI3K, AKT, VEGF-A, VEGFR2, and HIF1-α were consistent with protein expression levels. In conclusion, our research showed that AS extract can promote the follicle angiogenesis in hens in vitro, providing a basis for application of the traditional Chinese herb AS in poultry production.