To Explore The Mechanism Of Angelica Sinensis Extract Promoting Angiogenesis Of Chicken Preovulatory Follicles Based On Primary Cultured FMECs And GCs

Angelica sinensis(AS)is a traditional medicine with the effect of promoting blood circulation and tonifying blood.The preovulatory follicle of laying hens needs a rich vascular network to supply nutrients for rapid growth and development,and the laying rate is closely related to the development speed of the preovulatory follicle.The molecular mechanisms by which AS promotes angiogenesis in layer preovulatory follicles are unclear.Therefore,this study explored the role of AS in promoting microvascular regeneration of preovulatory follicles,and provided a basis for AS as a dietary supplement to improve egg production rate of laying hens.Methods: The aqueous extract of AS was prepared by water extraction and alcohol precipitation method,and the effective components of the extract were determined by LC-MS analysis.The cells were identified as primary chicken preovulatory follicular microvascular endothelial cells(FMECs)and primary chicken preovulatory follicular granulosa cells(GCs)by cell morphological analysis,angiogenesis assay,Western-blot and IF detection of the expression of related factors.The CCK-8method was used to detect the effect of the AS extract on the cell viability of the isolated FMECs and GCs.Finally,the action time of AS extract was determined to be 48 h,and 100μg/m L and200μg/m L were the low and high doses of AS extract,respectively.The protein expression of pPI3 K and PI3 K in FMECs and GCs treated with LY294002 was detected by Western-blot,and the dosage of LY294002 was finally determined to be 15μM.Subsequently,Western-blot and q RT-PCR were used to detect the expression levels of angiogenesis-related regulatory factors in FMECs and GCs,and PI3K/AKT signaling pathway-related regulatory factors.Components and central targets were also validated using molecular docking.Finally,the effect of the AS extract on the angiogenesis of FMECs was further verified by a scratch test,a cell invasion test,and an angiogenesis test.The test results show that:(1)LC-MS analysis of AS extracts in this study revealed that ferulic acid and caffeic acid were the components.(2)FMECs were monolayer adherent cells with spindle-shaped and polygonal morphologies.The morphology of GCs was irregularly fusiform or polygonal,with a tiled plate bottom,and some cells were connected by filopodia.The two cell morphologies were consistent with their related characteristics.IF results showed that the expression of CD31 and v WF in the extracted FMECs was positive.The expression of FSHR in the extracted GCs was positive.The FMECs extracted in this study could be connected to form vessels after culture in Matrigel matrix.The FMECs extracted in this study could be connected to form vessels after culture in the Matrigel matrix.These results indicated that the FMECs and GCs extracted from the primary chicken could be used in the subsequent experiments.(3)The m RNA and protein expression levels of HIF1α and VEGF-A were significantly upregulated after treatment of GCs with different concentrations of AS extract.At the same time,the protein expression of p-PI3 K and p-AKT was significantly enhanced.The results of ELISA showed that AS extract could significantly promote the expression of VEGF-A.Treatment of GCs with LY294002 alone significantly downregulated the m RNA levels and protein expression of HIF1αand VEGF-A.At the same time,it significantly reduced the protein expression of p-PI3 K and AKT and inhibited the expression of VEGF-A.Furthermore,treatment of GCs with different concentrations of AS extract and LY294002 showed no significant difference in the m RNA and protein expression of HIF1-α and VEGF-A compared with LY294002 alone.The results showed that AS extract promoted VEGF-A secretion by GCs via PI3K/AKT signaling pathway.(4)After FMECs were treated with different concentrations of AS extract,the m RNA expression of VEGFR2,MMP2 and MMP9 was significantly increased,and the protein expression of p-VEGFR2,p-PI3 K,p-Akt,MMP2 and MMP9 was also significantly enhanced.In FMECs treated with LY294002 alone,the m RNA expression of VEGFR2 was significantly decreased,and the protein expression of p-VEGFR2,PI3 K and AKT was also significantly decreased.Compared with LY294002 alone,co-treatment of FMECs with different concentrations of AS extract and LY294002 did not result in significant differences in VEGFR2 m RNA levels,nor in the phosphorylated protein expression of VEGFR2,PI3 K,and AKT.These results indicated that AS extract could promote the expression of angiogenesis-related factors in FMECs.(5)The percentage of cell migration,the number of cell invasion,the number of main junctions,tubules,branches,total length and total tubule length formed in FMECs were significantly increased after treatment with different concentrations of AS extract.However,the percentage of migrating cells,the number of invasive cells,the main junctions,tubules,branches,total length and total tubule length formed in FMECs were significantly inhibited by LY294002 treatment alone.There were no significant changes in the above indexes in FMECs treated with AS extract combined with LY294002 compared with LY294002 alone.These results indicated that AS extract promoted angiogenesis in FMECs through PI3K/AKT signaling pathway.In conclusion,AS extract can upregulate the expression of angiogenesis-related factors in FMECs and promote the phosphorylation of VEGFR2 by activating PI3K/AKT signaling pathway,thereby enhancing the invasion,migration and angiogenesis ability of FMECs in vitro.In addition,AS extract activated the PI3K/AKT signaling pathway and upregulated the expression of HIF1-αand VEGF-A in GCs.AS extract can promote angiogenesis in FMECs and VEGF-A secretion by GCs.These results indicated that AS extract promoted angiogenesis of preovulatory follicles by regulating FMECs and GCs related factors.