Molecular Mechanisms Of Estrogen Regulating Inflammatory Endometrial Cells In Pigs

Sow endometritis refers to the myometrial mucosa mucinous or suppurative inflammation,can cause sow estrus abnormalities,is caused by large-scale pig production in the sow high elimination rate of one of the most important diseases,to pig Industry caused great losses.A survey shows that in the pig industry,with endometriosis in the delivery of sows as high as 20%.Endometritis is mainly caused by viruses,bacteria and parasites.At present,the treatment of endometritis mainly antibiotics,Chinese medicine,biological agents for treatment,although with good results,but the above treatment can not completely treat the disease,and there are some side effects,seriously affected the disease After the reproductive capacity.Therefore,for livestock endometritis,should be anti-rule,in the understanding of its pathogenesis on the basis of a more effective means of prevention and treatment,and ultimately the effective control of the disease.The main component of estrogen is 17β-estradiol(17β-E2),the uterus is an important target organ of estrogen.Studies have shown that a variety of uterine diseases are closely linked with estrogen,estrogen secretion abnormalities usually lead to certain uterine diseases.Domestic and foreign studies have shown that estrogen can regulate endometritis.In this study,we used in vitro culture of pig endometrial epithelial cells to explore 17β-E2 on sow endometrial epithelial cell inflammatory factors in the regulatory mechanism.(2)to establish an inflammatory model of porcine endometrial epithelial cells under in vitro conditions;(3)to explore the effect of 17β-E2 on endometrial epithelial cells of endometrial epithelial cells NO,TNF-α and IL-1 expression;(4)To explore the role of estrogen receptor antagonist in 17β-E2 regulation of the expression of inflammatory factors in endometrial epithelial cells;(5)to explore the JNK signal Pathway in 17β-E2 regulation of endometrial epithelial cells in the process of expression of inflammatory factors.The study is as follows:(1)The normal uterus of healthy sow was selected and its endometrial peeling was carried out.The primary culture,purification and subculture were carried out.The endometrial epithelial cells were obtained by immunofluorescence staining.The endometrial epithelial cells Of the purification rate of 95% or more,for the follow-up study to provide a high purity,meet the requirements of cells;(2)In order to explore the required LPS dose for the preparation of porcine endometrial epithelial cell inflammation model,1μg/mL,10μg/mL and 100μg/m L lipopolysaccharide were used to stimulate endometrial epithelial cells cultured in vitro for 12 h.The activity of NO,TNF-α,IL-1 and NOS,TNF-α and IL-1 mRNA in cell culture medium were detected by MTT and RT-PCR.The res μ Lts showed that 10μg/mL LPS could significantly increase the activity of endometrial epithelial cells and increase the expression of NO,TNF-α,IL-1 and NOS,TNF-α and IL-1 mRNA in the control group(P <0.05).The concentration of LPS and the secretion of inflammatory factor were dose-dependent,but the cell activity was significantly decreased by 100 μg /ml LPS.Therefore,10 μg/mL LPS was used in the uterus Membrane epithelial cells were stimulated to produce inflammatory models;(3)In order to explore the effect of 17β-E2 on the inflammatory cytokines of endometrial epithelial cells,10 mg / ml LPS stimulated endometrial epithelial cells for12 h,10-6M,10-7M,10-8M 17β-E2 The levels of NO,TNF-α,IL-1 and NOS,TNF-α and TNF-α were measured by NO kit,fluorescence quantitative PCR and ELISA at 2h,6h,12 h and 24 h respectively.Α,IL-1 mRNA expression levels.The results showed that all concentrations of 17β-E2 decreased the expression of inflammatory factors in varying degrees,and this degree of reduction was proportional to the concentration of 17β-E2.(4)In order to explore the role of estrogen receptor α in 17β-E2 regulation of endometrial epithelial cell inflammatory factors,the experiment was carried out with 10μg/mL LPS for 12 h,then the concentration was 10-6M 17β-E2 The expression of NO,NOS,TNF-α and IL-1 were detected by ELISA and fluorescence quantitative PCR.The expression of NO,NOS,TNF-α and IL-1 was detected by NO kit and ELISA.The results showed that the secretion of inflammatory factors in LPS + 17β-E2 group was significantly lower than that in LPS + 17β-E2 group and LPS + 17β-E2 group,and there was no significant difference between LPS + 17β-E2 group and LPS + 17β-E2 group Hormone receptor α plays an important role in 17β-E2 regulation of inflammatory factor expression.(5)In order to understand the role of JNK signal transduction pathway in the regulation of 17β-estradiol on inflammatory factors of endometrial cells,LPS + 17β-E2 and LPS + TAM + 17β-E2 P-JNK and JNK protein expression in LPS group were significantly increased,while the addition of 17β-E2 significantly reduced the expression of P-JNK,and the protein expression ratio of P-JNK was increased by adding TAM LPS + 17β-E2 group was significantly improved,and LPS group was no significant difference.Suggesting that estrogen reacts with estrogen receptor alpha and inhibits JNK pathway,thereby inhibiting the expression of cytokines under LPS stimulation.To sum up,the experiment can get the following conclusions:1.This experiment successfully cultivated the porcine endometrial epithelial cells and established the cell inflammation model.2.Estrogen can inhibit the secretion of NO,TNF-α and IL-1 and the expression of NOS,TNF-α and IL-1 mRNA in the epithelial cells of LPS-induced endometrial epithelial cells.3.Estrogen inhibits the secretion of NO,TNF-α and IL-1 and the expression of NOS,TNF-α and IL-1 mRNA by binding to receptor α on endometrial epithelial cells.4.Estrogen inhibits JNK pathway by inhibiting the expression of cytokines stimulated by LPS by interacting with estrogen receptor alpha.