The Role Of Ufmylation In The Inflammatory Response Of Goat Endometrial Epithelial Cells

Endometrial cell injury and chronic inflammation caused by postpartum infection are the main causes of repeated infertility in ruminants,seriously endanger the productivity of dairy cows or milk goats.Endometritis is often caused by bacterial infections like Escherichia coli,Pseudomonas pyogenes,Bacillus,Streptococcus and Staphylococcus,but the inflammatory mechanism of that remains not well understood.Ufmylation is a kind of newly discovered post-translational modification which takes the UFM1 as a signal molecule and functions through a three-step enzymatic reaction to covalently bind to the target protein like DDRGK1.Various cellular functions such as immune recognition and DNA repair,as well as endoplasmic reticulum stress are regulated by the enzymatic reaction,and the Ubiquitin-like modifier-activating enzyme 5 act as E1,the UFM1-conjugating enzyme 1 act as E2 and the UFM1-specific ligase 1 act as E3 are neccessary to be involved in this process.Recently,evidences have shown that inflammatory response is closely related to endoplasmic reticulum stress and the role of Ufmylation in the regulation of endoplasmic reticulum stress and inflammantory response were also revealed in studies.UFM1,the key protein of Ufmylation and the substrate protein DDRGK1 play an important role in the body's inflammatory response by regulating the nuclear import process of NF-?B and the stability of I?B.Here,we established a mouse endometritis model to analyze the correlation between the Ufmylation and endometritis,and immortalized goat endometrial epithelial cells(gEECs)were also used to further investigate the role of Ufmylation in LPS-induced inflammation.The main findings obtained are as follows:(1)The UFM1 and the substrate protein DDRGK1 of Ufmylation are mainly expressed in mice's and goats' endometrial epithelial cells.(2)Genes and proteins of Ufmylation-related is up-regulated in the endometritis.Prerequisites for the induction of endometritis related to LPS were screened and the results showed that intrauterine perfusion with 20 ?L of 1.25-5.00 mg/mL LPS can successfully construct a mouse endometritis model;The protein expression levels of UFM1 and DDRGK1 were significantly increased and a dose-dependent with LPS was found.Meanwhile,the mRNA expression levels of Ufmylation and protein expression of free-UFM1 were significantly up-regulated in the inflammatory response of gEECs induced by 5 ?g/mL LPS.(3)LPS can significantly up-regulate the expression of UFM1.Overexpressing UFM1 can markedly increase the mRNA expression levels of IL-1? and IL-6 but down-regulate TNF-? mRNA;Protein detection showed no obvious effect on the expression of TNF-? and IL-1?.In summary,Immunohistochemistry,Real-time quantitative PCR,Western blotting,RNA Interference and Overexpression were uesd to investigate the relationship and mechanism between Ufmylation and endometritis that inducted by LPS in mice and goats,the results showed that the Ufmylation is up-regulated in endometritis,and over-expressed UFM1 can regulate goat endometritis by up-regulating the mRNA expression levels of IL-1?,IL-6 and down-regulate the one of TNF-?.