The Effect Of Ufmylation Modification On TLR4/NF-κB Pathway And Pyroptosis Pathway In Goat Endometritis

Ruminant endometritis usually occurs after childbirth,it is an inflammatory reaction caused by pathogenic microorganism infection.Endometritis can damage the function of the endometrium and ovaries,thereby affecting the embryonic development and attaching ability,leading to a decline in animal reproduction.Ruminants have a high incidence of endometritis and the causes are complex.Pathogenic microorganisms and their virulence factors are the main reasons for uterine tissue damage and endometrial inflammation.Lipopolysaccharide(LPS)is the main pathogenic factor of the endometritis pathogenic bacteria E coli.LPS can activate the TLR4/NF-κB pathway and the pyroptosis pathway after entering the body.Although there have been many reports on the inflammatory response caused by LPS,the mechanism of LPS-induced endometritis in ruminants is still not fully understood.Ufmylation is a conservative post-translational ubiquitination-like modification,and ubiquitin folding modifier factor 1(UFM1)is a substrate binding protein for Ufmylation.Studies have confirmed that UFM1-mediated modification of Ufmylation plays an important regulatory role in the inflammatory response caused by LPS,but the specific molecular mechanism remains to be elucidated.In this study,immortalized goat endometrial epithelial cells(gEECs)were used as experimental materials to analyze the regulatory effects of UFM1 on the TLR4/NF-κB pathway and pyroptosis pathway in the inflammatory response of gEECs induced by LPS.To lay the foundation for further revealing the molecular mechanism of Ufmylation regulates goat endometritis.The main research results are as follows:(1)Using 5 μg/mL LPS to treat gEECs,the results of qRT-PCR and Western blotting showed that LPS activates the TLR4/NF-κB pathway in gEECs and promotes the mRNA expression of TLR4,My D88,TRIF,p65 and NF-κB in this pathway;LPS treatment significantly increased the protein expression of phosphorylated p65.LPS can significantly up-regulate the mRNA expression of NLRP3,caspase-1,and GSDMD in the pyroptosis pathway;up-regulate the proteins expression of pro caspase-1,cleavage caspase-1,FL GSDMD and cleavage GSDMD.The above results prove that LPS activates the TLR4/NF-κB and pyroptosis pathways in gEECs.In order to explore the relationship between the TLR4/NF-κB pathway and the pyroptosis pathway,TAK-242,a specific inhibitor of TLR4 pathway,was used to inhibit the activation of TLR4 pathway,and qRT-PCR technology was used to screen the optimal TAK-242 concentration to be 1 μM.The 1 μM TAK-242 can significantly inhibit the mRNA expression of TLR4,p65,NF-κB and the mRNA expression of NLRP3,caspase-1,and GSDMD caused by LPS,indicating that there is a close relationship between the TLR4/NF-κB pathway and the pyroptosis pathway.(2)Using 5 μg/mL LPS to treat gEECs,the results of qRT-PCR and Western blotting showed that UFM1 was significantly up-regulated in the LPS group.The si RNA and overexpression plasmid were used to interfere or overexpress UFM1 in gEECs,then LPS was added to detect the effect of UFM1 on the activation state of TLR4/NF-κB pathway and pyroptosis pathway induced by LPS.The results of qRT-PCR showed that the mRNA expression of TLR4 and p65 significantly increased after interference with UFM1,and the mRNA expression levels of NLRP3 and caspase-1 also increased significantly;the mRNA expression of TLR4,TRIF,p65 and NF-κB after overexpression of UFM1 was decreased significantly,and the mRNA expression of NLRP3 and GSDMD also decreased significantly.Double immunofluorescence was used to detect the nuclear translocation of the p65 subunit of NF-κB.The results showed that LPS caused the nuclear translocation of p65;compared with the LPS group,interference with UFM1 promoted the nuclear translocation of p65,and overexpression of UFM1 inhibited the nuclear translocation of p65.After LPS treatment,Western blotting results showed that interference with UFM1 increased the protein expression of pro caspase-1,cleavage caspase-1 and FL GSDMD;and after overexpression of UFM1,the protein expression of cleavage caspase-1,FL GSDMD and cleavage GSDMD protein was down-regulated.The above results indicate that UFM1 has an inhibitory effect on the TLR4/NF-κB and pyroptosis pathway induced by LPS.In summary,this study shows that Ufmylation-modified substrate binding protein UFM1 has an inhibitory effect on the TLR4/NF-κB pathway and pyroptosis pathway in goat endometrial epithelial cells caused by LPS.This study laid a theoretical foundation for exploring the pathological mechanism of ruminant endometritis caused by E.coli.