| Ketogenesis of lamb rumen epithelium plays a central role in the genomic activity regulating rumen development,but the specific mechanism is still unclear.This paper systematically analyzed the regulatory effect of rumen ketogenesis on the proliferation and differentiation of epithelial cells and the molecular mechanism,providing new evidence for the in-depth and systematic understanding of the physiological significance of rumen ketogenesis,and providing new ideas for optimizing lamb breeding strategies.In this paper,according to the purpose and significance of the experiment,the following two-part experiment was designed to study the molecular mechanism of dietary ketogenesis regulating the proliferation and differentiation of rumen epithelial cells of lambs through in vivo feeding experiment and in vitro cell experiment.The details are as follows:Experiment 1: Relationship between dietary ketogenesis and ruminal epithelial cell development in lambsTwelve pairs(24)of 14-day-old male weaned Hu sheep with similar body weight were selected and divided into 2 groups with 4 replicates per group and 3 lambs per replicate using a paired experiment design.All lambs were fed Forage based Starter and forage based Starter,which had the same nutritional composition and were fed forage based starter type(CS,Corn based starter,NDF/NFC=15/55)or forage based starter type(FS,Forage based starter,NDF/NFC=30/30).Eight pairs of twin lambs at 56 days of age were slaughtered to determine the relevant indexes.The results showed as follows: roughad-type feeding had a tendency to increase feed intake and daily gain of lambs(0.05<P<0.1),and increased rumen papilla length,papilla width,rumen p H,ammonia nitrogen,acetic acid and ethylene to propyl ratio of lambs(P<0.05).Concentrate type feeding increased rumen muscle layer thickness,cortical thickness and rumen propionic acid,butyric acid and total acid concentrations(P<0.05),and the contents of HMGCS2 and β-hydroxybutyric acid in rumen epithelial cells of lambs in this group were all coarse type lambs(P<0.05).Single-cell transcriptomic sequencing,concentrate feeding increased the proportion of rumen epithelial keratocytes(20.63% vs 14.65%)and macrophages(9.24% vs 4.32%),while decreased the proportion of proliferating cells(2.43% vs 11.12%).Compared with concentrate type treatment,m RNA expressions of proliferating genes Cyclin A,Cyclin B1 and P21 were significantly up-regulated,while protein expressions of proliferating genes Cyclin D1 and CDK2 were significantly down-regulated in rumen epithelial cells of lambs in coarse type treatment group(P <0.05).The m RNA expression of apoptotic gene Bcl-2 and the protein expressions of Caspase3,Caspase9 and Bax were significantly down-regulated(P < 0.05).m RNA expression levels of differentiated genes MUC2,CK-18,E-Cadherin and HDAC1 were significantly down-regulated,while m RNA expression levels of CDX2 gene were significantly up-regulated(P < 0.05).The protein expressions of IAP and E-Cadherin were significantly decreased,while the protein expressions of CK-18,CDX2 and H3K9 ac were significantly increased(P < 0.05).The results showed that the difference model of rumen ketogenesis was established successfully.The concentrate group(CS)promoted the formation of ketobodies(β-hydroxybutyric acid)in rumen epithelial cells,the thickness of rumen epithelium increased,but the shape of rumen papillae was smaller.Dietary type can change rumen epithelial cell typing of lambs.Ketogenesis induced by concentrate type(CS)diet slowed down the proliferation of rumen epithelial cells,but accelerated the apoptosis and differentiation of rumen epithelial cells.Experiment 2: Molecular mechanism of ketone body metabolism regulating rumen epithelial cell proliferation and differentiationThe aim of this study was to investigate the molecular mechanism of ketone body metabolism regulating the proliferation and differentiation of rumen epithelial cells.The results show that:Increasing the concentration of β-hydroxybutyric acid increased the protein expression of key ketogenic enzymes HMGCS2,HMGCL and BDH1.The protein expression of PPAR-β was decreased in the inhibitor group,and the protein expression of key ketogenic enzymes(HMGCS2,HMGCL and BDH1)was also decreased.The protein expression of HDAC2 was up-regulated in the activator group.The protein expression of key ketogenic enzymes(HMGCS2,HMGCL and BDH1)was decreased.PPARs and HDACs mediated the regulation of β-hydroxybutyric acid on ketogenic enzymes,which further affected the ketogenic process of rumen epithelium.β-hydroxybutyric acid at 10 m M/L decreased the m RNA and protein expressions of proliferating genes Cyclin D1,Cyclin E1,CDK2 and CDK6,and the m RNA and protein expressions of apoptotic genes Caspase-3 and Caspase-9 were significantly up-regulated with the increase of β-hydroxybutyric acid concentration.The addition ofβ-hydroxybutyrate in vitro inhibited the proliferation of rumen epithelial cells and promoted the apoptosis of rumen epithelial cells.With the increase of β-hydroxybutyric acid concentration,the protein expressions of IAP,CK-18,E-Cadherin,CDX2 and P21,the key genes of rumen epithelial cell differentiation,were increased.In terms of protein expression,HMGCS2 inhibitor decreased the protein expressions of E-Cadherin,IAP and CDX2.β-hydroxybutyric acid(2.5,5,10 m M/L)increased the expression levels of NOTCH1,JAG1,HES1 and NICD proteins in the Notch signaling pathway,and inhibited Notch signaling decreased the expression levels of rumen epithelial differentiation genes HMGCS2 and H3K9 ac proteins.The expression levels of CK-18,CDX2 and E-Cadherin were increased,and the expression levels of HDAC1,HDAC2 and HDAC3 genes in rumen epithelial cells were decreased by inhibiting the expression of HDAC(indicating obvious inhibitory effect),while the expression levels of NOTCH protein were decreased.β-hydroxybutyric acid activates the NOTCH signaling pathway by inhibiting HDAC,which promotes the differentiation of rumen epithelial cells in lambs by regulating target genes(HMGCS2,IAP,E-Cadherin,etc.).. |
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