Screening And Verification Of Hypothalamic Signaling Pathways Of Glucosamine Promoting Feed Intake In Laying Hens

Feed intake is one of the key factors affecting the production performance in laying hens.Previous studies confirmed that glucosamine(GlcN)has anti-inflammatory and through upregulates hypothalamus m RNA expression of NPY gene and downregulates hypothalamus m RNA expression of POMC gene to promote feeding intake in laying hens,but its upstream signaling pathway is unclear.The paper analyzed the correlation relationship between feeding intake and inflammatory factor;hypothalamus were collected and 4D-Fast DIA phosphorylation modified proteomics were conducted to screen the potential signaling pathway of GlcN of regulate feeding intake in laying hens;Chicken hypothalamic neuronal cells were cultured and verified the pathway by q RT-PCR and Western Blotting,in order to analyze the molecular mechanism of GlcN promoting feed intake in laying hens and provide technical support for the application of GlcN in laying hens.Trial 1 Based phosphorylated proteomics analyzed hypothalamic signaling pathways of GlcN promoting feed intake in laying hens To investigate the molecular mechanism of GlcN promoting feed intake in laying hens,promoting feed intake model of GlcN will be established in laying hens and the correlation relationship between feeding intake and inflammatory factor will be analyzed,and 4D-Fast DIA phosphorylation proteomics analysis will be conducted to screen signaling pathways of GlcN involved in feeding intake.A paired T-test design was conducted and forty healthy,with similar body weight,Guizhou yellow×Lohman hybrid laying hens aged 420 day,with similar egg production rate were divided into control and GlcN groups,each group has 20 hens respectively.Birds consumed basal diet and 0.45% GlcN diet,respectively.The pre-feeding period was 7 days and the trial period was 30 days.The feed intake of the hens was recorded daily.Blood and Hypothalamic samples were sampled at the end of the trial period.The results showed that 0.45%GlcN promoted the feed intake of hens(P<0.01),the concentration of IL-1β and TNF-α of serum were attenuated(P<0.01),and there is a negative correlation between feed intake and inflammatory factor(P<0.05).Phosphorylated proteins analyzed that a total of 95 differentially phosphorylated proteins were identified,including 39 were upregulated and 56 were downregulated.103 differentially phosphorylated sites were identified,of which 45 were upregulated sites and 58 were downregulated sites.KEGG enrichment and enrichment clustering analysis found that the signaling pathway associated with promoted the feed intake of hens were Fox O signaling pathway,mTOR signaling pathway,phosphatidylinositol signaling pathway and Toll-like receptor signaling pathway.Trial 2 Verification of mTOR signaling pathway of GlcN promoting feed intake in laying hens In this study,we examined whether the mTOR signaling in the hypothalamus neuronal cells is involved in the regulation feed intake of GlcN.Chicken hypothalamic neuronal cells were isolated,then hypothalamic neuronal cells were stimulated by adding different concentrations of GlcN,mTOR inhibitor and mTOR activator,respectively.q RT-PCR and Western blotting were used to detect the expression of genes and proteins related with feed intake.The results showed that: GlcN significantly increased the m RNA expression of NPY(P<0.05)and decreased the m RNA expression of POMC,CART and Ag RP(P<0.05).Similar with the function of the GlcN,the mTOR inhibitor significantly increased the m RNA expression of NPY,POMC,CART and Ag RP genes(P<0.05),and the mTOR activator MHY1485 significantly curb the m RNA expression of NPY and CART genes(P<0.05),significantly promote the m RNA expression of POMC gene(P<0.05),but had no significant effect on the m RNA expression of Ag RP gene(P>0.05).GlcN inhibited p-mTOR protein level in the hypothalamic neuronal cells and a significant interaction was noted in GlcN and MHY1485 on gene expression of POMC and Ag RP.These data suggest that GlcN can promote the feed intake via regulated expression of genes by inhibiting p-mTOR protein level.In conclusion:(1)The effect of promoting feed intake of GlcN was related to the decrease of inflammatory response in laying hens;(2)The mTOR signaling pathway,phosphatidylinositol signaling pathway,Fox O signaling pathway,and Toll-like receptor signaling pathway were involved in the regulation of feeding intake by GlcN in laying hens;(3)GlcN can promote feed intake via downregulating p-mTOR protein level.