| The uterus is an important part of the reproductive system of dairy cows and the main place for embryo attachment and development.Its physiological structure and function integrity is directly related to the reproductive rate of dairy cows and has a tremendous impact on the dairy cattle breeding industry.Postpartum factors such as cervical relaxation and endometrial epithelial damage in dairy cows can easily lead to uterine infections,leading to serious economic losses.One of the main pathogens of postpartum uterine infection are E.coli,which result in the pathogenic changes through lipopolysaccharide(LPS).Met-enkephalin(MENK)is an endogenous opioid peptide in mammals.MENK and its receptors have been confirmed to exist in endometrium.Studies have shown that MENK can inhibit the proliferation of uterine cells,but the effect of MENK on the proliferation of endometrial epithelial cells(BEEC)in dairy cows has not been studied.In this study,we aim to explore the effects of MENK on the cell proliferation activity,the cell proliferation related factors and signaling pathways in BEEC.The experiment was divided into two parts.In experiment 1,the mechanism of the effect of MENK on the proliferation of BEEC under LPS was studied.The cells were divided into the control group,the LPS group(1 μg/mL),the cotreatment groups of LPS(1 μg/mL)and various concentrations of MENK.In experiment 2,the effect of MENK on the proliferation of BEEC was further verified by the use of Naloxone(NAL),an antagonist of MENK.The cells were divided into the control group,the LPS group(1μg/mL),and the cotreatment groups of LPS(1μg/mL),MENK and NAL.The results are as follows.CCK-8 was used to detected the cell viability,the real-time PCR and Western blot was applied to detect the expressions of the cell proliferative factors and pathways,respectively.The effect of MENK on the BEEC viability were measured after 24 h treatment.Compared with the control group,the cell viability in the LPS group increased(p<0.01).Compared with the LPS group,the cell viability decreased(p<0.01)in LPS and MENK(10-6～10-4 mol/L)cotreatment group.MENK treatment alone showed no influence(p>0.05)on the cell viability of BEEC.Compared with the control group,the proportion of G1 phase cells in LPS group decreased(p<0.01),the proportion of S and G2 phase cells and PI increased(p<0.01)1).Compared with LPS group,the proportion of G1 phase cells in LPS and MENK(10-6～10-5 mol/L)co-treatment group increased(p<0.01),and PI decreased(p<0.01).The effect of MENK on gene expressions of EGFR,VEGF,FGF-2 and TGF-β3 increased(p<0.05)at 3,6 and 12 h in LPS group.The gene expressions of EGFR,VEGF and FGF-2 were lower(p<0.05)at 6 h and 12 h in LPS and MENK(10-6～10-5 mol/L)cotreatment group than those in the LPS group.No difference(p>0.05)was found in the gene expression of TGF-β3 between the LPS group and the cotreatment group.Compared with the control group,the expressions of β-catenin,c-Myc and Cyclin D1 protein,and the phosphorylation levels of PI3K and Akt in LPS treatment group increased(p<0.05).Compared with LPS treatment group,the protein expressions of β-catenin and c-Myc in LPS and MENK(10-7～10-5 mol/L)cotreatment group decreased(p<0.01).The expression of Cyclin D1 protein in LPS and MENK(10-6～10-5 mol/L)cotreatment group decreased(p<0.05).The phosphorylation levels of PI3K in LPS and MENK(10-6 mol/L-10"5 mol/L)cotreatment groups decreased(p<0.05).The phosphorylation level of Akt decreased(p<0.05)in LPS plus 10-5 mol/L MENK treatment group(p<0.05).These results showed that MENK inhibited the LPS-induced proliferation of BEEC,the cell cycle progression,the increased gene expressions of EGFR,VEGF and FGF-2,and the activations of Wnt/β-catenin and PI3K/Akt pathways.The effect of NAL on the BEEC viability were measured after 24 h treatment.Compared with the blank control,the proliferation activity of LPS treatment group increased(p<0.01).Compared with the LPS group,the cell viability showed no change(p>0.05)in the cotreatment group of LPS,MENK(10.9～10-5 mol/L)and NAL(10-9～10-5 mol/L),but decreased(p<0.05)in the cotreatment group of LPS,MENK(10-4 mol/L)and NAL(10-4 mol/L).There was no change(p>0.05)in cell viability when BEEC was treated by 10-9～10-5 mol/L NAL alone.Compared with the control group,the proportion of G1 phase cells decreased(p<0.01),and the proportion of S phase cells and PI increased(p<0.01)in LPS treatment group.Compared with LPS group,neither the proportion of cells or the PI showed any change(p>0.05)in the cotreatment group of LPS,MENK(10.6～10-5 mol/L)and NAL(10-6～10-5 mol/L.Compared with the control group,the expression levels of EGFR,VEGF,FGF-2 and TGF-β3 genes in LPS-treated cells increased(p<0.05)at 6 h.Compared with LPS-treated cells,the gene expressions of EGFR,VEGF,TGF-β3 and FGF-2 in LPS,MENK and NAL-cotreated cells showed no significant difference(p>0.05)at observed concentrations.Compared with the control group,the protein expression levels of β-catenin,c-Myc and Cyclin D1,and the phosphorylation levels of PI3K and Akt in LPS group increased(p<0.05).The expression levels of β-catenin,c-Myc and Cyclin D1 proteins,and the phosphorylation levels of PI3K and Akt in the cotreatment groups of LPS,MENK and NAL were not different(p>0.05)from those in the LPS group.The results showed that the inhibitory effect of MENK in cell proliferation could be blocked by NAL.In conclusion,MENK inhibited the LPS-induced cell proliferation,the cell cycle progression the gene expressions of EGFR,VEGF and FGF-2,and the activations of Wnt/β-catenin and PI3K/Akt pathways.This inhibitory effect of MENK can be blocked by NAL,suggesting that MENK may participate in the inhibition of LPS-induced BEEC cell proliferation through the opioid receptor mediation. |
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