DataSheet_1_LPS Mediates Bovine Endometrial Epithelial Cell Pyroptosis Directly Through Both NLRP3 Classical and Non-Classical Inflammasome Pathways.docx

As a highly inflammatory form of programmed cell death, pyroptosis is triggered by pro-inflammatory signals and associated with inflammation. It is characterized by cell swelling and large bubbles emerging from the plasma membrane, which release cytokines during inflammation. Compared with other types of cell death, pyroptosis has a distinct morphology and mechanism and involves special inflammasome cascade pathways. However, the inflammasome mechanism through which endometrial epithelial cell pyroptosis occurs in LPS-mediated inflammation remains unclear. We confirmed that there was an increased mRNA and protein expression of the IL-6, TNF-α, IL-1β, IL-18 cytokines, the inflammasome molecules NLRP3, CASPASE-1, CASPASE-4, and GSDMD in LPS-induced primary bovine endometrial epithelial cells (BEECs) in an in vitro established inflammatory model using ELISA, real-time PCR (RT-PCR), vector construction and transfection, and Western blotting. Scanning electron microscopy and lactate dehydrogenase (LDH) activity assays revealed induced cell membrane rupture, which is the main characteristic of pyroptosis. In conclusion, the cytolytic substrate GSDMD’s cleavage by caspase-1 or caspase-4 through the NLRP3 classical and non-classical inflammasome pathways, GSDMD N-terminus bind to the plasma membrane to form pores and release IL -18, IL-1β cause cell death during LPS induced BEECs inflammation.作为一类高度炎症性的程序性细胞死亡形式，细胞焦亡（pyroptosis）由促炎信号所触发，并与炎症反应紧密关联。其典型特征为细胞肿胀，细胞膜表面向外形成巨大囊泡，并在炎症过程中释放细胞因子。相较于其他类型的细胞死亡，细胞焦亡具有独特的形态学特征与分子机制，并涉及特殊的炎症小体级联通路。然而，在脂多糖（LPS）介导的炎症反应中，子宫内膜上皮细胞发生细胞焦亡的具体炎症小体机制仍未明确。本研究通过酶联免疫吸附试验（ELISA）、实时荧光定量聚合酶链式反应（RT-PCR）、载体构建与转染以及蛋白质印迹法（Western blotting），在体外构建的炎症模型中开展实验，证实脂多糖诱导的原代牛子宫内膜上皮细胞（BEECs）中，IL-6、TNF-α、IL-1β、IL-18等细胞因子，以及炎症小体分子NLRP3、半胱天冬酶-1（CASPASE-1）、半胱天冬酶-4（CASPASE-4）与GSDMD的mRNA及蛋白表达水平均显著上调。扫描电子显微镜与乳酸脱氢酶（LDH）活性检测结果显示，细胞发生了细胞膜破裂——这正是细胞焦亡的核心特征之一。综上，在脂多糖诱导的BEECs炎症反应中，溶细胞底物GSDMD可通过NLRP3经典与非经典炎症小体通路，被半胱天冬酶-1或半胱天冬酶-4切割；其N端结构域结合细胞膜并形成孔道，进而释放IL-18与IL-1β，最终诱导细胞死亡。