Many species of pathogenic bacteria secrete toxins that form pores in mammalian cell membranes. These membrane pores enable the delivery of virulence factors into cells, result in the leakage of molecules that bacteria can use as nutrients, and facilitate pathogen invasion. Inflammatory responses to bacteria are regulated by the side-chain-hydroxycholesterols 27-hydroxycholesterol and 25-hydroxycholesterol, but their effect on the intrinsic protection of cells against pore-forming toxins is unclear. Here, we tested the hypothesis that 27-hydroxycholesterol and 25-hydroxycholesterol help protect cells against pore-forming toxins. We treated bovine endometrial epithelial and stromal cells with 27-hydroxycholesterol or 25-hydroxycholesterol, and then challenged the cells with pyolysin, which is a cholesterol-dependent cytolysin from Trueperella pyogenes that targets these endometrial cells. We found that treatment with 27-hydroxycholesterol or 25-hydroxycholesterol protected both epithelial and stomal cells against pore formation and the damage caused by pyolysin. The oxysterols limited pyolysin-induced leakage of potassium and lactate dehydrogenase from cells, and reduced cytoskeletal changes and cytolysis. This oxysterol cytoprotection against pyolysin was partially dependent on reducing cytolysin-accessible cholesterol in the cell membrane and on activating liver X receptors. Treatment with 27-hydroxycholesterol also protected the endometrial cells against Staphylococcus aureus α-hemolysin. Using mass spectrometry, we found 27-hydroxycholesterol and 25-hydroxycholesterol in uterine and follicular fluid. Furthermore, epithelial cells released additional 25-hydroxycholesterol in response to pyolysin. In conclusion, both 27-hydroxycholesterol and 25-hydroxycholesterol increased the intrinsic protection of bovine endometrial cells against pore-forming toxins. Our findings imply that side-chain-hydroxycholesterols may help defend the endometrium against pathogenic bacteria.

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氧甾醇保护牛子宫内膜细胞免受来自病原菌的成孔毒素

许多种类的病原菌分泌的毒素会在哺乳动物细胞膜上形成孔。这些膜孔能够将毒力因子输送到细胞中，导致细菌可以用作营养物质的分子泄漏，并促进病原体入侵。对细菌的炎症反应受侧链羟基胆固醇 27 -羟基胆固醇和 25 -羟基胆固醇的调节，但它们对细胞对成孔毒素的内在保护作用尚不清楚。在这里，我们检验了 27-羟基胆固醇和 25-羟基胆固醇有助于保护细胞免受成孔毒素侵害的假设。我们用 27 -处理牛子宫内膜上皮细胞和基质细胞羟基胆固醇或 25-羟基胆固醇，然后用溶菌素攻击细胞，溶菌素是一种来自化脓性细菌的胆固醇依赖性溶细胞素，可靶向这些子宫内膜细胞。我们发现用 27-羟基胆固醇或 25-羟基胆固醇处理可以保护上皮细胞和造口细胞免受孔形成和溶菌素引起的损伤。氧甾醇限制了溶菌素诱导的钾和乳酸脱氢酶从细胞中的泄漏，并减少了细胞骨架的变化和细胞溶解。这种氧甾醇对溶菌素的细胞保护作用部分依赖于减少细胞膜中溶细胞素可接近的胆固醇和激活肝 X 受体。治疗 27 -羟基胆固醇还保护子宫内膜细胞免受金黄色葡萄球菌α-溶血素的侵害。使用质谱法，我们在子宫和卵泡液中发现了 27-羟基胆固醇和 25-羟基胆固醇。此外，上皮细胞响应溶菌素释放额外的 25-羟基胆固醇。总之，27-羟基胆固醇和25-羟基胆固醇都增加了牛子宫内膜细胞对成孔毒素的内在保护作用。我们的研究结果表明，侧链羟基胆固醇可能有助于保护子宫内膜免受病原菌的侵害。