Amaral Ana, Fernandes Carina, Lukasik Karolina, Szóstek-Mioduchowska Anna, Baclawska Agnieszka, Rebordão Maria Rosa, Aguiar-Silva Joana, Pinto-Bravo Pedro, Skarzynski Dariusz J, Ferreira-Dias Graça
CIISA - Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine, University of Lisbon, Lisbon, Portugal.
Institute of Animal Reproduction and Food Research, Polish Academy of Science, Olsztyn, Poland.
Reprod Domest Anim. 2018 Sep;53 Suppl 2:66-69. doi: 10.1111/rda.13258.
We have shown that bacteria induce neutrophil extracellular traps (NETs) in mare endometrium. Besides killing pathogens, NETs may contribute for endometrosis (chronic endometrium fibrosis). Since elastase (ELA) is a NETs component that regulates fibrosis and prostaglandin (PG) output, the aim was to evaluate if inhibition of ELA would affect collagen 1 (COL1) transcription and PGs secretion by endometrium explants, in different estrous cycle phases. Follicular-FP (n = 8) and mid luteal-MLP (n = 7) phases explants were cultured for 24-48 hr with medium alone (Control), ELA (0.5 μg/ml,1 μg/ml), sivelestat - ELA inhibitor (INH,10 μg/ml), or ELA (0.5 μg/ml,1 μg/ml) + INH (10 μg/ml). COL1 gene transcription was done by qRT-PCR and PGE and PGF α determination in culture medium by EIA. In FP, at 24 hr, ELA0.5 increased COL1 transcription (p < 0.001) but its inhibition (ELA0.5 + INH10) decreased COL1 transcription (p < 0.01) and PGF α production (p < 0.05). Also, ELA0.5 + INH10 or ELA1 + INH10 raised PGE production (p < 0.01). At 48 hr, ELA1 increased COL1 transcription (p < 0.01) and PGF α production (p < 0.001), but its inhibition (ELA1 + INH10) decreased these actions (p < 0.01; p < 0.05, respectively). Besides, ELA1 + INH10 incubation increased PGE (p < 0.05). PGF α also augmented with ELA0.5 (p < 0.001), but lowered with ELA0.5 + INH10 (p < 0.01). In MLP, ELA0.5 up-regulated COL1 transcription (24 hr, p < 0.01; 48 hr, p < 0.001), but ELA0.5 + INH10 decreased it (24 hr, p < 0.05; 48 hr, p < 0.001). At 48 hr, incubation with ELA1 also increased COL1 transcription and PGF α production (p < 0.05), but PGF α production decreased with ELA1 + INH10 incubation (p < 0.05). PGE production was higher in ELA1 + INH10 incubation (p < 0.05). Therefore, ELA inhibition may reduce the establishment of mare endometrial fibrosis by stimulating the production of anti-fibrotic PGE and inhibiting pro-fibrotic PGF α.
我们已经表明，细菌可诱导母马子宫内膜中的中性粒细胞胞外陷阱（NETs）形成。除了杀死病原体外，NETs可能与子宫内膜纤维化（慢性子宫内膜纤维化）有关。由于弹性蛋白酶（ELA）是一种调节纤维化和前列腺素（PG）分泌的NETs成分，本研究旨在评估在不同发情周期阶段，抑制ELA是否会影响子宫内膜外植体中I型胶原蛋白（COL1）的转录和PGs的分泌。将卵泡期-FP（n = 8）和黄体中期-MLP（n = 7）阶段的外植体分别与单独的培养基（对照）、ELA（0.5μg/ml、1μg/ml）、西维来司他-ELA抑制剂（INH，10μg/ml）或ELA（0.5μg/ml、1μg/ml）+INH（10μg/ml）一起培养24 - 48小时。通过qRT-PCR检测COL1基因转录，并通过酶免疫分析（EIA）测定培养基中的前列腺素E（PGE）和前列腺素Fα（PGFα）。在卵泡期，培养24小时时，ELA0.5增加了COL1转录（p < 0.001），但其抑制作用（ELA0.5 + INH10）降低了COL1转录（p < 0.01）和PGFα的产生（p < 0.05）。此外，ELA0.5 + INH10或ELA1 + INH10增加了PGE的产生（p < 0.01）。在48小时时，ELA1增加了COL1转录（p < 0.01）和PGFα的产生（p < 0.001），但其抑制作用（ELA1 + INH10）降低了这些作用（分别为p < 0.01；p < 0.05）。此外，ELA1 + INH10孵育增加了PGE（p < 0.05）。PGFα也随着ELA0.5增加（p < 0.001），但随着ELA0.5 + INH10降低（p < 0.01）。在黄体中期，ELA0.5上调了COL1转录（24小时，p < 0.01；48小时，p < 0.001），但ELA0.5 + INH10降低了它（24小时，p < 0.05；48小时，p < 0.001）。在48小时时，与ELA1孵育也增加了COL1转录和PGFα的产生（p < 0.05），但ELA1 + INH10孵育使PGFα的产生降低（p < 0.05）。ELA1 + INH10孵育时PGE的产生更高（p < 0.05）。因此，抑制ELA可能通过刺激抗纤维化PGE产生和抑制促纤维化PGFα来减少母马子宫内膜纤维化的发生。
