Postpartum endometritis in dairy cows has been defined as inflammation of endometrium occurring 21 days or more after parturition without systemic signs of illness (Mandhwani et al., 2017). Endometritis is a kind of mucinous or purulent inflammation, which typically results from the entry of air, urine, semen, bacteria, fungi or yeast into the uterus (Woodward and Troedsson, 2015). According to histopathology, endometritis can be divided into acute and chronic (Kitaya et al., 2018). Whether it is acute endometritis with fever, pelvic pain and vaginal secretion or chronic endometritis with subtle and unexplainable symptoms, it will cause great harm to the health of human beings and animals, and may even be fatal (Mackeen et al., 2015). The bacteria most frequently grown from animals with uterine illness are Escherichia coli, T. pyogenes, Fusobacterium necrophorum, and Prevotella and Bacteroides species (Huszeniczal, 1999). Moreover, Escherichia coli has been generally recognized as the main reason for clinical endometritis in dairy cows (Sheldon et al., 2009a, Sheldon et al., 2009b, Sheldon et al., 2009a, Sheldon et al., 2009b, Wathes et al., 2020). Although endometritis can be asymptomatic, it can be found in many infertile patients and lead to repeated implantation failure and habitual abortion (Moreno et al., 2018). In addition, endometritis history lengthens the time between inseminations and delays conception. Additionally, cows with clinically relevant endometritis had a lower pregnancy rate, necessitating more inseminations per conception than healthy cows (Borsberry S, 1989, Gautam et al., 2009). Cows with postpartum clinical endometritis are more likely to be eliminated due to reproductive failure than cows without endometritis (S.J. LeBlanc et al., 2002). The above negative effects of endometritis lead to serious economic losses in the dairy industry

Lipopolysaccharide (LPS) is typically composed of hydrophobic domain called lipid A (or endotoxin), non-repeating "core" oligosaccharide and a distal polysaccharide (or O- antigen) (Raetz and Whitfield, 2002). As the crucial component of the outer membrane of pathogenic Gram-negative bacteria, LPS constitutes a unique asymmetric structure (Guest et al., 2021), and is recognized as the major virulence factor of Gram-negative bacteria (Klein et al., 2021). LPS is an important tool for the induction of inflammation, it could induce the activation of nuclear factors, which then triggers downstream pathways that culminate in a plethora of pro-inflammatory genes (Batista et al., 2019, Chen et al., 2019, Chen et al., 2020). Hence, LPS is commonly used to stimulate cells and establish animal inflammatory models, endometritis model is also included (Liang et al., 2018, Liu et al., 2021).

Mitogen-Activated Protein Kinase Kinase Kinase 8 (MAP3K8, also known as COT or TPL2) is a serine/threonine kinase that belongs to the MAP3K family (Yan et al., 2019). In the steady state, both isoforms of MAP3K8 forms a complex with p105 (NFκB1) and ABIN2 (A20-binding inhibitor of NF-κB), which stabilizes MAP3K8 and keeps it in an inactive form (Lang et al., 2004, Yang et al., 2012, Webb et al., 2019). Upon an array of proinflammatory factors stimulation, releasing an active MAP3K8 that exerts effector functions (Vougioukalaki et al., 2011). The released MAP3K8 is involved in both innate and adaptive immune responses by activating ERK, JNK, NF-κB and p38 pathway in a stimulus- and cell-specific manner (Xu et al. 2018). Since MAP3K8 is the sole serine/threonine kinase involved in ERK1/2 activation, MAP3K8 is essential during the production of TNF through ERK1/2 in macrophages stimulated by LPS (Rousseau et al., 2008). Futhermore, MAP3K8 regulates ERK and p38 pathways to control production of inflammatory cytokines and chemokines such as IL8, MIP-1α, MIP-1β (Xu et al., 2018). These results suggest that the MAP3K8 axis may play a crucial role in regulating pro-inflammatory responses.

Small endogenous RNAs called microRNAs (miRNAs), which are 19 to 25 nucleotides in size, control posttranscriptional gene expression (Lu and Rothenberg, 2018). Although miRNAs account for only 3% of the human genome, they regulate about 90% of genes (Singh et al., 2013). A single miRNA can bind to hundreds of target mRNAs, this process requires the seed region of miRNA to match the target site on the 3'-UTR of mRNA (Bartel, 2009). Indeed, studies have linked miRNAs to the development and operation of the innate and adaptive immune systems (Carissimi et al., 2009). In the analysis of miRNA expression profile in monocyte cell lines treated with LPS, miR-146 and miR-155 have been shown to be induced by proinflammatory stimuli such as IL-1β, TNF-α, and TLRs (Roy and Sen, 2011). The activity of several profiles of miRNA, including Let-7c, miR-148a and miR-488, can affect the activation of NF-κB and the development of bovine endometritis (Umar et al., 2021).

MiR-26a is a multifunctional miRNA belonging to the highly conserved miR-26 family, which has been proved to be related to tumorigenesis and immunoreaction. Recently, it has been reported that there is an inverse relationship between the levels of miR-26 and interleukin-6 (IL-6) in tumor cells (Zhang et al., 2013). In particular, miR-26a is up-regulated in the process of TnP (a candidate peptide for anti-inflammatory therapy) treating inflammation of LPS-Challenged zebrafish (Disner et al., 2021). And it can reduce the production of inflammatory factors by silencing NF-κB signaling related factors (Chen et al., 2016). However, the regulatory function of miR-26a in LPS-induced endometritis has not been investigated. In this study, we screened the possible targets of miR-26a using the online miRNA target prediction program and identified MAP3K8 as a potential target of miR-26a. Notably, we found that the expression of miR-26a was prominently down-regulated in uterine tissues of endometritis and in LPS-stimulated bEECs. Thus, miR-26a may be a new therapeutic target for the therapeutic accesses to inflammatory diseases including endometritis.