To Investigate The Mechanism Of Angelica Sinensis Extract Promoting Follicular Microvascular Regeneration In Laying Hens Based On Network Pharmacology

Primordial and primary follicles obtain hormones,oxygen,and a large amount of yolk material required for follicular development by passive diffusion,and secondary follicles transport these nutrients into follicles through a separate microvascular network generated.Therefore,angiogenesis is necessary for normal follicular development.In the late laying period of laying hens,the egg production rate gradually decreased due to the aging of ovaries.Thinning of granular layer and decreased angiogenesis are important reasons for ovulation reduction in Late-phase laying hens.Angelica sinensis is a traditional Chinese medicinal herb,which has the effect of tonifying blood and activating blood circulation.Angelica sinensis is also widely used as a functional food or dietary supplement.This study investigated the effects of Angelica sinensis extract on egg production performance and angiogenesis of preovulatory follicles(F1-F3)in late-phase laying hens,providing a basis for Angelica sinensis to be used as a feed supplement for late-phase laying hens.Methods:(1)Effect of aqueous extract of Angelica sinensis on the performance of late-phase laying hens.In this experiment,hens were divided into positive control group(30 W),control group(70W),low-dose intervention group(70W + L),medium-dose intervention group(70W + M)and high-dose intervention group(70W + H),with 10 hens in each group.Three replicates were set in each group.The low,medium and high dose groups were fed with Angelica sinensis extract at doses of 1 g/chicken,2 g/chicken and 4 g/chicken for 28 days,respectively.The egg production rate was recorded and egg quality was measured to calculate the differences among the groups.(2)Effect of Angelica sinensis extract on serum sex hormone parameters.Serum E2 and FSH were detected by ELISA kit.(3)Effect of Angelica sinensis extract on angiogenesis.The apparent number of blood vessels in follicles was observed,and the thickness of granular layer and cell structure of preovulatory follicles(F1-F3)in each group were detected by HE staining.VEGFA content in granular layer tissue was detected using an ELISA kit.Immunofluorescence and Western blot were used to detect the expression of vascular endothelial marker protein CD31.Thus,the effect of Angelica sinensis extract on angiogenesis of preovulatory follicles(F1-F3)in late-phase laying hens was elucidated.(4)Network pharmacology predicts the mechanism of angelica sinensis affecting angiogenesis.The active components of Angelica sinensis extract and the possible targets of Angelica sinensis in angiogenesis were predicted by network pharmacology.GO functional analysis and KEGG pathway enrichment analysis were performed on target genes.(5)Key targets and pathways were validated by immunofluorescence and Western Blot.The experimental results were as follows:(1)Angelica sinensis extract significantly increased egg production rate in laying hens.After 28 days of intervention,the egg production rate was 60% in the 70 W group and 73%,83%,and 83% in the low dose group,medium dose group and high dose group of Angelica sinensis,respectively;Angelica sinensis extract significantly increased the number of LYF,SYF and SWF in the ovary,and at the same time,increased eggshell thickness eggshell strength and Haugh Unit.(2)Angelica sinensis extract significantly increased E2 and FSH levels in the serum of 70 W laying hens.(3)Angelica sinensis extract increased the apparent vascular richness of preovulatory follicles(F1-F3)and significantly increased the thickness of granular layer and the content of VEGFA in granular layer of preovulatory follicles(F1-F3)in 70 W laying hens.Angelica sinensis extract significantly increased total CD31 expression in F1-F3 theca layer.(4)Network pharmacology analysis showed that the active components of Angelica sinensis were Beta-sitosterol,Ferulic acid and Caffeic acid.216 targets were predicted for the three components,including 54 targets associated with angiogenesis.GO enrichment analysis and KEGG enrichment analysis showed that 54 target genes were involved in angiogenesis related biological functions such as response to hypoxia and transmembrane receptor protein tyrosine kinase activity in a variety of structures of cells,and regulated through VEGF signaling pathway,HIF-1 signaling pathway and other signaling pathways.Topological analysis screened key targets of angiogenesis as HIF1α,VEGFR2,MMP2,and MMP9.(5)Angelica sinensis extract significantly up-regulated the expression of HIF1α and VEGFA in the granular layer of F1-F3 follicles,and significantly up-regulated the expression of MMP2 and MMP9 and the phosphorylation level of VEGFR2 in the theca layer of F1-F3 follicles.Conclusion: Angelica sinensis extract can improve the egg production rate of late-phase laying hens,delay the decline of egg production after the peak laying period,and improve egg quality.Angelica sinensis extract can increase the content of E2 and FSH in serum,increase the thickness of granular layer of preovulatory follicles F1-F3,and promote the generation of follicular vessels in late-phase laying hens by up-regulating the expression of HIF1α,VEGFA in granular layer and MMP2,MMP9,p-VEGFR2 in theca layer.