Journal Pre-proof Decreasing of S100A4 in bovine endometritis in vivo and in vitro

Endometritis is a prevalent reproductive disease in dairy cows, and is a 21 superficial inflammation of the endometrium. S100 calcium-binding protein A4 22 (S100A4) is suggested to be implicated in the progression of inflammation. However, 23 to our knowledge, no study has reported the changes of S100A4 during bovine 24 endometritis. The objective of this study was to investigate S100A4 gene expression 25 and protein levels in the uterus with endometritis in dairy cows. Vaginal mucus 26 samples were collected for diagnosis of the severity degree of endometritis and the 27 detection of S100A4 protein content. Blood samples and endometrial biopsies were 28 collected and divided into the control (CN), mild endometrtis (M), and severe 29 endometritis (S) groups according to the characteristics of the vaginal mucus type. 30 The isolated bovine endometrial epithelial cells (BEECs) were challenged with E. coli 31 (2×10 6 CFU/mL, 2×10 7 CFU/mL) or lipopolysaccharide (LPS, 3 and 10 μ g/mL) as an 32 inflammatory model. RT-qPCR was used to detect the gene expression levels of 33 S100A4 and cytokines, including interleukin-1 β (IL-1 β ), interleukin-6 (IL-6), 34 interleukin-10 (IL-10), and tumour necrosis factor-alpha (TNF- α ), in tissues or cells. 35 Enzyme-linked immunosorbent assay (ELISA) was used for S100A4 protein level 36 detection in tissues, cells, cell supernatant, vaginal mucus, and serum samples. The 37 results showed that S100A4 gene and protein levels decreased in bovine endometrium 38 with endometritis and in E. coli - or LPS-stimulated BEECs. We failed to detect 39 S100A4 in the cell supernatant, vaginal mucus, and serum samples. This study 40 suggested that S100A4 is a pathogenesis-related protein of endometritis, and decreased expression of S100A4 may pave the way for the development of 42 endometritis in dairy cows. 43


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Enzyme-linked immunosorbent assay (ELISA) was used for S100A4 protein level 36 detection in tissues, cells, cell supernatant, vaginal mucus, and serum samples. The 37 results showed that S100A4 gene and protein levels decreased in bovine endometrium 38 with endometritis and in E. coli-or LPS-stimulated BEECs. We failed to detect 39 S100A4 in the cell supernatant, vaginal mucus, and serum samples. This study 40 suggested that S100A4 is a pathogenesis-related protein of endometritis, and including pyometra, anovulation and pregnancy loss, as well as reduce productive 51 performance [2,3]. 52 S100 calcium-binding proteins are widely reported to be crucial to many diseases 53 and are considered potential biomarkers in the diagnosis and treatment of several 54 diseases [4]. S100A4 is an S100 protein family member that functions in both 55 intracellular and extracellular pathways [5]. Existing studies have shown that S100A4 56 protein is expressed in immune cells, such as monocytes, macrophages, and 57 polymorphonuclear granulocytes, and is also expressed at low levels in normal tissues 58 [6,7]. S100A4 plays important roles in a range of biological functions, including cell 59 adhesion, movement, invasion, and metastasis [8]. Numerous studies on S100A4 have 60 focused on tumours and cancer [5]. S100A4 has also been reported tobe involved in 61 inflammation progression. For example, S100A4 can aggravate the clinical symptoms 62 of colitis in mice [9], and it may be a potent trigger of inflammatory processes and 63 4 induce the release of cytokines [10]. It has also been reported that S100A4 promoted 64 the progression of endometritis, while deficiency of S100A4 reduced the mRNA 65 expression levels of IL-1β and TNF-α in the endometrium of mice [11]. However, the 66 roles of S100A4 in bovine endometritis have not been reported. To investigate the 67 roles of S100A4 in endometritis of dairy cows, we determined the gene and protein 68 expression of S100A4 in endometrial tissues, vaginal mucus, serum, E.     Kit (Takara, Japan) was used to measure the concentration of total protein in mucus.

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Many S100 calcium-binding protein family members have potent antimicrobial 163 properties and are essential components of the immune response to invading 164 pathogens during infection and inflammation [10]. For example, S100A12 can 165 promote vascular and valve calcification to regulate vascular inflammation 166 in non-oxidized low-density lipoprotein-induced vascular smooth muscle cells [14].

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Exogenous S100A12 significantly upregulated MMP-13 and VEGF via the p38 168 MAPK and NF-κB pathways [15], and high expression of S100A8/9 was observed in 169 osteoarthritis [16]. While some evidences strongly support S100A4 as an inflammation-related 171 protein, the regulatory mechanism of S100A4 is unclear [10]. One study reported that 172 S100A4 was upregulated in LPS-treated endometritis, and a lack of S100A4 resulted 173 in an impaired inflammatory response in mice models of acute endometritis [11].
174 S100A4 has also been reported to induce a network of inflammatory cytokines or 175 promote the inflammatory response in mononuclear cells [17]. However, another 176 study found that S100A4 decreased the release of cytokines in a high fat-diet 177 inflammatory model [18]. Therefore, the inflammatory regulation of S100A4 may be 178 different under various conditions, suggesting that the relative mechanisms still need 179 further study.

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Bovine endometritis often occurs postpartum within 60 days. However, the 181 alteration of S100A4 in bovine endometritis is unknown. In this study, we chose cows 182 40to 60 days postpartum that experienced the prolonged uterine inflammation [12]. 183 We first found that S100A4 mRNA expression levels and protein contents decreased 184 in bovine endometrium with endometritis ( Fig. 2), but increased in some reports [9, 185 11]. Furthermore, combined with pathological grade, S100A4 decrease was closely 186 related to the intensity of inflammation in bovine endometrium ( Fig. 1 and Fig.2).

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Thus, decreased S100A4 may contribute to deterioration of endometritis in dairy 188 cows.

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Because endometritis is an inflammatory response of endometrial epithelial cells, 190 an inflammatory cell model should be used to research S100A4. We successfully 191 developed a cell model using E. coli or LPS stimulation and evaluated it by cytokine 192 10 detection (Fig. 3). In vitro results showed that cytokines increased with the dosage of 193 bacteria or LPS, however, S100A4 was negatively correlated with the dosage of E. 194 coli or LPS in BEECs (Fig. 4). Thus, the decrease of S100A4 was also closely related 195 to the intensity of cellular inflammation in vitro. In conclusion, the decrease of intracellular S100A4 was closely related to 208 endometritis in dairy cows, and S100A4 is a pathogenesis-related protein of 209 endometritis. We tentatively proposed that decreased expression of S100A4 may play 210 an important role in the development of bovine endometritis. However, the function 211 and mechanism of S100A4 in bovine endometritis still need further investigation.    Images were magnified 400×.