Mechanism And Effect Of Progesterone On The Proliferation Of Bovine Endometrial Epithelial Cells

Bovine endometritis is a reproductive disease that often occurs in dairy cows.It is related to factors such as occurrence and abnormal production,retention of placenta,postpartum uterine infection,imbalance of hormone levels,environmental factors,and nutritional imbalance.Endometritis is often accompanied by uterine infection and damage.After the uterus is damaged or even infected,it undergoes self-repair under the control of various hormones and factors to restore the normal structure and function of the uterus.Progesterone is the main hormone produced by the corpus luteum,which together with estrogen is an important hormone for maintaining the female reproductive system.Previous study have clear evidence for the pro-proliferative effects of estrogen or estrogen together with progesterone on endometrium,but the pro-proliferative effect of progesterone on endometrial epithelial cells alone is not yet clear.Therefore,in this experiment,the effect of progesterone on cell cycle,proliferation factors and related pathways in bovine endometrial epithelial cells(BEECs)were studied in vitro,which provided a theoretical basis for the proliferative effect of progesterone.In this experiment,the BEECs were divided into blank group,LPS-treated(1μg/mL)group,progesterone-treated(1,3,5ng/mL)groups and LPS-progesterone co-treatment(1,3,5ng/mL progesterone and 1μg/mL LPS)groups.The activity of the cells was detected by CCK-8 assay at 24h.The cell cycle was detected by flow cytometry.As a result,progesterone of various concentration or LPS have no effect on cell viability(p>0.05).The number of cells in G1 phase decreased(p<0.01),G2 phase cells were increased(p<0.01),and PI was significantly elevated(p<0.01).The whole cells developed to G2 phase,and the overall proliferation level increased.After LPS treatment,the cells in G1 phases increased significantly(p<0.01).The cell growth in S phase was not significant(p>0.05).The cells in G2 phases decreased significantly(p<0.01).PI was significantly degraded(p<0.01),the overall proliferation level decreased.When 1ng/mL progesterone and LPS were co-treated,G1 cells decreased significantly(p<0.01),S phase did not change significantly(p>0.05),G2 phase cells were increased(p<0.01).The overall PI elevated significantly(p<0.01),the proliferation level increased.When treated with 3ng/mL progesterone and LPS,G1 phase cells decreased significantly(p<0.01),S phase and G2 phase cells significantly increased(p<0.01).The overall PI significantly increased(p<0.01),the overall proliferation level increased.When treated with 5ng/mL progesterone and LPS,no significant change in G1 phase cells were measured(p>0.05),whereas the numbers of S phase cells were significantly decreased(p<0.01),and G2 phase cells were increased(p<0.01).The overall PI did not change significantly.In order to study the effect of progesterone on the proliferative factors of BEECs,the mRNA expressions of vascular endothelial growth factor(VEGF),connective tissue growth factor(CTGF),transforming growth factor-β(TGF-1β),epidermal growth factor receptor(EGFR)and epidermal growth factor receptor(PR)were measured.The cells were treated the same as previous experiment.The result showed that treatment with progesterone alone showed no significant change at 3h and 18h(p>0.05),but a significant decrease of CTGF and PR at 18h(p<0.01).The mRNA expressions of VEGF,CTGF,TGF-β,EGFR and PR of cells treated with progesterone showed a significant increase at 12h(p<0.01)in a concentration-dependent manner.In the LPS group,The expression of VEGF,TGF-β,EGFR and PR was generally elevated at 3h and 12h,generally decreased at 18h,and CTGF was consistently decreased.This shows that LPS played a promoting role at 3h and 12h as a whole,and as time progressed,the promotion effect weakened.In co-treatment groups,the mRNA expressions of VEGF and PR increased,whereas the VTGF,TGF-β,EGFR declined at 3h.The mRNA expressions of VEGF,TGF-β,EGFR and PR decreased at 12h and 18h,whereas the CTGF gene expression increased in 3ng/mL progesterone-and-LPS-cotreated group.This shows that after LPS triggers cell damage,progesterone can affect the self-repair and recovery of cells by inhibiting the expression of partial repair factors and receptors.Signal transduction of the Wnt/β-catenin and PI3K/Akt pathways is one of the important pathways for cell repair and inflammatory injury.The BEECs were treated with LPS and/or progesterone for 15min.the protein expressions of β-catenin,cyclin D1,c-Myc,PI3K,p-PI3K,Akt and p-Akt were detected by Western blot.As a result,the expression level of β-catenin significantly increased in 1ng/mL and 3ng/mL progesterone treatment group(p<0.01),but decreased(p<0.01)in 5ng/mL progesterone-treated groups.The expression levels of cyclin D1 and c-Myc were increased(p<0.01),where the 3ng/mL progesterone treatment group was the most significant.The relative expression of PI3K decreased significantly(p<0.01).The relative expression of Akt increased(p<0.01)in 1ng/mL progesterone treatment group,but decreased(p<0.01)in 3ng/mL and 5ng/mL progesterone treatment groups.It shows that progesterone can promote cell proliferation by activating Wnt/β-catenin pathway.But progesterone also inhibits PI3K/Akt signal transduction,indicating that it can negatively regulate the proliferation and differentiation of cells through the signal transduction pathway..The expression levels ofβ-catenin,cyclin D1 and c-Myc increased(p<0.01)after LPS stimulation,whereas the relative expressions of PI3K and Akt decreased(p<0.01).In LPS and progesterone cotreatment groups,there were decreases(p<0.01)in the expression levels of β-catenin and c-Myc.The cyclin D1 level increased(p<0.01)in 1ng/mL progesterone and LPS treated group,but decreased(p<0.01)in 3ng/mL and 5ng/mL progesterone and LPS cotreatment groups.The PI3K levels decreased(p<0.01),whereas the Akt levels increased with various degree in these groups.The activation ofβ-catenin was visualized by immunofluorescence,which is consistent with the Western blot results.β-catenin was mainly concentrated on the cell membrane in normal BEECs.β-catenin was widely distributed in cytoplasm when progesterone and LPS respectively acted on BEECs,and Wnt/β-catenin pathway was activated.When progesterone and LPS were co-treated,there was no significant β-catenin distribution in cytoplasm.Progesterone could inhibit LPS-activated Wnt/β-catenin pathway.In summary,1ng/mL and 3ng/mL progesterone can promote cell proliferation by activating the Wnt/β-catenin pathway.However,when inflammation is present,progesterone inhibits the activation of β-catenin and PI3K,thereby reducing cell self-propagation and repair through this pathway and aggravating BEECs damage.This study provides new ideas for exploring the self-repair of dairy cows after endometrial injury,and provides guidance for the prevention and treatment of bovine endometritis.