Effects Of Vitamin D3 On Wnt Signaling Pathway In Bovine Endometrial Epithelial Cells

Endometritis is a common disease of cattle reproductive system,which restricts the development of cattle industry and causes great economic losses to cattle breeding industry.Currently,antibiotics are mainly used for treatment.Although effective,their frequent or long-term use produces drug resistance and even endangers food safety.Therefore,new methods for the prevention and treatment of endometritis need to be explored.Vitamin D(VD)is a well-known steroid hormone.In addition to regulating the balance of calcium and phosphorus,it also has anti-inflammatory and anti-tumor activities.It exists in various non-classical target tissues,including reproductive organs,and regulates many genes involved in cell growth,immunity and protein synthesis,lack of which will lead to reproductive disorders.Therefore,The aim of this study was to investigate the effect of VD3 on Wnt signaling pathway of bovine endometrial epithelial cells(BEECs)treated with lipopolysaccharide(LPS)and to elucidate the underlying molecular mechanism,so as to provide theoretical basis and data support for the use of VD in the prevention and treatment of endometritis in production practice.Experiment 1: Isolation,culture and gene screening of fetal bovine endometrial epithelial cells.In this study,fetal cattle aged 4-6 months were selected to culture BEECs by tissue fragment separation method,and the primary cells obtained were subcultured and purified.BEECs were treated with 0 ng/m L and 50 ng/m L VD3,respectively,and the differentially expressed genes were analyzed by transcription sequencing.Results:(1)bovine BEECs could be isolated and cultured successfully from fetal bovine uterus.(2)The cells grew rapidly and proliferated rapidly before P9,keeping the basic morphology unchanged,while the proliferation ability decreased after P9.(3)A total of 20 genes related to Wnt signaling pathway were screened by transcriptional sequencing.Compared with the control group,9 genes were upregulated and 11 genes were down-regulated.GO analysis showed that the most differentially expressed genes were atypical Wnt signaling pathways in biological processes and Wnt-activated receptor activities in typical Wnt signaling pathways and molecular functions.KEGG analysis showed that the most differentially expressed genes were Wnt signaling pathway and stem cell pluripotent signaling pathway,which were significantly enriched in signal transduction and endocrine system.Experiment 2: Effects of VD3 on LPS-induced Wnt pathway gene expression in endometrial epithelial cells.BEECs were treated with VD3(0,50 ng/m L)for 24-72 h,LPS(0,10,100 ng/m L),VD3 50 ng/m L + LPS 10 ng/m L,VD3 50 ng/m L + LPS 100ng/m L for 24 and 48 h,CCK-8 method was used to det ect cell proliferation at different time and concentration.The expression of Wnt pathway related genes MYC,Wnt4,GREM1,LGR5,FZD2,β-catenin,PCNA,FZD7 and VDR were detected by q RT-PCR.Results:(1)BEECs treated with VD3 for 24-72 h had no significant effect on cell proliferation(P>0.05);LPS inhibited BEECs proliferation in a time-and dosedependent manner.LPS treatment with VD3 for 24-48 h weakened the inhibitory effect of LPS on BEECs proliferation,and VD3 had protective effect on LPS-induced BEECs.(2)The expression of Wnt4 and PCNA genes showed different trends in different concentrations of LPS.MYC and GREM1 genes were only stimulated by high dose(100 ng/m L)LPS,and the expressions of FZD7,LGR5,FZD2 and β-catenin were upregulated by LPS at both concentrations.Combined with cell proliferation analysis,100 ng/m L LPS stimulated BEECs could successfully establish in vitro endometritis model.(3)LPS significantly down-regulated the expression of Wnt pathway related genes MYC,PCNA,LGR5,GREM1,β-catenin(P<0.001),Wnt4,FZD2(P<0.01)and VDR(P<0.05)under the action of VD3.VD3 has a certain regulatory effect on LPSinduced Wnt pathway related genes,which can regulate gene overexpression,thus playing a protective role on BEECs.Experiment 3: Effect of Wnt pathway inhibitor IWR-1 on expression of key genes in the pathway.IWR-1(2,4,8,16 μM)inhibitors were treated with BEECs alone;BEECs was treated with LPS(100 ng/m L)+IWR-1(2,4,8,16 μM)inhibitor for 24 and 48 h,and the action time and concentration were screened out.BEECs were treated with VD3(50 ng/m L)+LPS(100 ng/m L)+ IWR-1(8 μM),and the effect of IWR-1 on cell proliferation was detected by CCK-8 method.The expression of key genes in Wnt signaling pathway MYC,Wnt4,LGR5,β-catenin and VDR were detected by q RT-PCR.Results:(1)IWR-1 inhibited cell proliferation in a time-and concentration-dependent manner.The optimal concentration of IWR-1 was 8 μM for 24 h.(2)Compared with LPS+IWR-1,LPS+VD and IWR-1 cotreatment significantly promoted the proliferation of BEECs(P<0.01),suggesting that VD3 could alleviate the inhibitory effect of LPS+IWR-1 on BEECs proliferation.(3)Blocking Wnt signaling pathway,MYC,LGR5,β-catenin expression also showed a downregulation trend,but Wnt4 expression was up-regulated.VD mainly plays a role mediated by VDR.VD3 up-regulated the expression of VDR gene and blocked the expression of VDR in the Wnt signaling pathway.The results suggest that VD3 may play a regulatory role in other ways than the Wnt signaling pathway.Conclusion: This experiment successfully isolated and cultured BEECs and established endometritis model,and the study showed that VD3 may alleviate l PSinduced BEECs injury through a variety of ways,providing reference value and theoretical basis for clinical application of VD3 as the prevention and treatment of bovine endometritis.