Mechanism Of LPS Induced Downregulation Of ER And PGR Expression In Rabbit Endometrial Stromal Cells

Genital tract of female animal is susceptible to various pathogenic microorganisms such as,bacteria,fungi,and viruses due to it directly accesses to the outside.Lipopolysaccsride(LPS)is the main component of the cell wall of Gram negative bacteria.Previous studies have shown that LPS-induced immunoresponse downregulated the expression of ER-α and PGR in rabbit endometrial stromal cells(ESCs),but its mechanism is still unclear.This study aims to explore the mechanism of LPS inhibiting the expression of ER-α and PGR in ESCs,providing theoretical support for studying the molecular mechanism of uterine reproductive dysfunction caused by Gram negative bacterial infection,and thus providing a basis for treating reproductive dysfunction caused by Gram negative bacterial infection in the reproductive tract of female rabbits.In this study,these proteins which bind to the 5-upstream expression regulatory regions(5-UERRs)of ER-α and PGR gene were pull down from nuclear proteins of LPS-treated endometrial stromal cells,and then the pulldown protein was analyzed by mass spectrometry to screen out the possible specific binding proteins with the 5-UERRs of ER-α and PGR,and the possible binding proteins were detected by the dual luciferase reporter assay,and verified by CHIP-q PCR.The results are as follows:(1)LPS treat down-regulated the expression levels of ER-α and PGR in endometrial stromal cells.193 specific binding proteins with 5-UERRs of ER-α were identified,and 304 specific binding protein with 5-UERRs of PGR were identified.(2)GO and KEGG analysis of theses binding proteins with 5-UERRs of ER-αwere annotated to a total of 37 GO entries and 191 KEGG pathways.Among them,13proteins(HSP90A,HSP90 B,HMGB1,CSE1 L,CYFIP1,EIF5 A,FLNC,TUBB6,TUBA4 A,ARHGEF2,ANXA1,IPO7 and TUBA1C)were enriched in inflammation and immune related functions and pathways,and LPS-induction significant increase their expression(P<0.01)in ESCs.(3)These binding proteins with 5-UERRs of PGR were performed GO and KEGG analysis,and a total of 37 GO entries and 243 KEGG pathways were annotated.Among them,16 proteins(HSP90B,HMGB1,NCKAP1,ACTR2,ARPC2,RHOA,HMGB2,IPO7,CSE1 L,CYFIP1,TUBB6,TUBA1,RAC1,ACTR1 A,ARPC4 and FHOD1)were enriched in inflammation and immunity related functions and pathways,and their expression were significant up-regulated by LPS in ESCs(P<0.01).(4)Four protein,HSP90 B,HMGB1,HSP90 A and EIF5 A,specially bind to 5-UERRs of ER-α,and two proteins,HSP90 B and HMGB1 PGR,specially bind to 5-UERRs of PGR.The four proteins(HSP90B,HMGB1,HSP90 A and EIF5A)were further verified by dual Luciferase reporting experiment and CHIP-q PCR.The results showed that the binding of HMGB1 to 5-UERRs of ER-α significantly decreased the expression of the downstream region and the binding of HSP90 B and HMGB1 to 5-UERRs of PGR significantly decreased the expression of the downstream region.HMGB1 overexpression significantly decreased m RNA expression of ER-α and PGR(P<0.05)and HSP90 B overexpression significantly decreased m RNA expression of PGR(P<0.01).Result suggests that the binding of HMGB1 to 5-UERRs of ER-α inhibit the expression of ER-α,and HMGB1 and HSP90 B may also down regulate the expression of PGR by the binding to 5-UERRs of PGR.