The Effect Of Meloxicam On The Proliferation Of Bovine Endometrial Epithelial Cells In Lipopolysaccharide Treated

Uterine infection is common in postpartum cows,causing diseases such as metritis or endometritis,resulting in decreased reproductivity of cows and increased elimination rate,bringing huge economic losses to the dairy industry.The common clinical factors of endometritis in dairy cows include pathogenic microorganism infection,secondary disease and improper feeding management.Escherichia coli(E.coli)is a common pathogenic microorganism,which plays a pathogenic role by releasing lipopolysaccharides(LPS).Under normal physiological conditions,postpartum uterine involution is affected by a variety of factors,including individual differences,hormone levels and feeding management level.The degree and speed of uterine involution are of great significance to the whole breeding cycle of dairy cows.Improving the level of uterine involution has a positive effect on the development of dairy industry.Non-steroidal anti-inflammatory drugs(NSAIDs)are often used to relieve dystocia,treat mastitis and endometritis in cows,among which meloxicam(MEL)has been used to treat metritis and endometritis in cows.Studies have found that MEL can inhibit the growth of tissues and cells in the treatment of cow mastitis and human cancer,but the effects of MEL on endometrial regeneration and postpartum uterine involulation in cows have not been reported.Therefore,it is of great significance to reveal the mechanism of MEL on the proliferation and repair of bovine endometrium.In the present study,we aims to explore the mechanism of MEL on the proliferation of primary bovine endometrial epithelial cells(BEECs).The inflammatory response of cells was induced by LPS.This study could provide more theoretical basis for the scientific and reasonable use of MEL in clinical treatment of endometritis.To investigate the effect of MEL on the proliferation of BEECs,CCK-8 method,flow cytometry,and cell scratch test were used to detect cell viability,cell cycle,and proliferation.The results showed that MEL treatment(5×10-2 μM,5×10-1 μM,5 μM,or 5×101 μM)for 24 h had no effect(P>0.05)on the cell viability.LPS treatment 10 μg/mL alone or cotreatment of LPS and MEL(0.5 μM or 5 μM)for 24,48 and 72 h had no effect(P>0.05)on cell viability.The effects of 0.5 μM and 5 μM MEL on cell migration rate were investigated at 0,3,6,12 and 24 h.The results showed that MEL alone treatment had no effect(P>0.05)on cell migration at 24 h;Cotreatment of LPS and MEL for 24 h reduced(P<0.01)the cell migration rate.The results of cell cycle showed that MEL(0.5μM and 5 μM)had no effect(P>0.05)on the cell cycle of BEECs for 24 h.When treated with 10 μg/mL LPS or LPS combined with MEL(0.5 μM and 5 μM)for 24 h,the cell numbers decreased(P<0.05)in G1 phase,and increased(P<0.01)in S phase.The cell cycle was arrested in G0/G1 phase.The effects of MEL on the mRNA transcription levels of cyclooxygenase-1(COX-1),cyclooxygenase-2(COX-2),toll like receptor 4(TLR4)and proliferation related factors in BEECs were detected by quantitative PCR.As a result,compared with LPS treatment group,cotreatment of LPS and MEL for 3,12 and 24 h have no influence(P>0.05)on the expressions of COX-1 and TLR4,whereas the expression of COX-2 gene decreased(P<0.01).The cells were treated with LPS alone or cotreated with LPS and MEL for 3,12 and 24 h to detect the expression of proliferation-related genes,including vascular endothelial growth factor(VEGF),connective tissue growth factor(CTGF),transforming growth factor-β1(TGF-β1),TGF-β3,and insulin-like growth factor receptor(IGFR).The results showed that compared with the blank control group,the expression of genes in LPS treated group unchanged or decreased(P<0.05).Compared with the LPS group,the expression levels of these growth factors decreased(P<0.05)in the LPS and MEL cotreatment groups.The effects of MEL on the protein levels of Wnt/β-catenin and PI3K/AKT signaling pathway were investigated in BEECs.The results showed that MEL(0.5 μM and 5 μM)alone had no effect(P>0.05)on the protein expression levels of β-catenin,c-Myc,cycIinD1,or glycogen synthase kinase-3β(GSK-3β)the protein distribution of β-catenin,or the phosphorylation levels of PI3K and AKT.Compared with the blank control group,the expression levels of proteins in LPS treatment group increased(P<0.01);Compared with LPS group,these protein expressions and phosphorylation levels decreased(P<0.01)in BEECs cotreated with LPS and MEL(0.5 μM and 5 μM).MEL prevented LPS-inducedβ-catenin from entering the nucleus,and β-catenin was distributed on the cell membrane.In conclusion,MEL alone has no effect on cell viability,cell cycle,or cell proliferation of BEECs.In the event of inflammatory response,MEL inhibited the proliferation of BEECs,blocked the cell cycle process,reduced the cell migration rate,inhibited the inflammatory response and the gene expressions of VEGF,CTGF,IGFR,TGF-β1 and TGF-β3,as well as the activation of Wnt/β-catenin and PI3K/AKT pathways.These results suggest that MEL has an inhibitory effect on the proliferation of inflammatory response state BEECs,possibly through the inhibitory effect of Wnt/β-catenin and PI3K/AKT pathways.