关键词: 阴道毛滴虫, 巨噬细胞迁移抑制因子, THP?1巨噬细胞, NLRP3炎症小体, 细胞凋亡, 细胞焦亡

Abstract: Objective　To investigate the effect of Trichomonas vaginalis macrophage migration inhibitory factor (TvMIF) on THP⁃1 macrophages. Methods　Recombinant TvMIF protein was prokaryotic expressed and purified, and endotoxin was removed after identification. Following exposure to TvMIF at concentrations of 0, 1, 5, 10, 50 and 100 ng/mL, the cytotoxicity of the recombinant TvMIF protein to THP⁃1 macrophages was tested using cell counting kit (CCK)⁃8 assay, and the apoptosis of THP⁃1 macrophages and reactive oxygen species (ROS) were detected using flow cytometry. The relative expression of nucleotide⁃binding oligomerization domain⁃like receptor protein 3 (NLRP3), caspase⁃1, interleukin⁃1β (IL⁃1β) and IL⁃18 genes was quantified using real⁃time fluorescent quantitative  PCR (qPCR) assay, and the expression of caspase⁃1, NLRP3, gasdermin D (GSDMD), gasdermin D N⁃terminal (GSDMD⁃NT) and pro⁃IL⁃1β proteins were determined using Western blotting assay. Results　Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS⁃PAGE) displayed successful expression and purification of the recombinant TvMIF protein with a molecular weight of 15.5 kDa, and the endotoxin activity assay showed the successful removal of endotoxin in the recombinant TvMIF protein (endotoxin concentration < 0.1 EU/mL), which was feasible for the subsequent studies on protein functions. Flow cytometry revealed that the recombinant TvMIF protein at a concentration of 10 ng/mL and less promoted the apoptosis of THP⁃1 macrophages, and the highest apoptotic rate of THP⁃1 macrophages was seen following exposure to the recombinant TvMIF protein at a concentration of 5 ng/mL, while the recombinant TvMIF protein at concentrations of 50 and 100 ng/mL inhibited the apoptosis of THP⁃1 macrophages. Exposure to the recombinant TvMIF protein at a concentration 1 ng/mL resulted in increased ROS levels in THP⁃1 macrophages. qPCR assay quantified significantly elevated caspase⁃1, NLRP3, IL⁃18 and IL⁃1β expression in THP⁃1 macrophages 8 hours post⁃treatment with the recombinant TvMIF protein at a concentration 1 ng/mL, and Western blotting determined increased caspase⁃1, NLRP3, pro⁃IL⁃1β, GSDMD and GSDMD⁃NT protein expression in THP⁃1 macrophages following exposure to the recombinant TvMIF protein at a concentration 1 ng/mL. Pretreatment with MCC950 significantly reduced GSDMD and GSDMD⁃NT protein expression. Conclusions　High⁃concentration recombinant TvMIF protein inhibits macrophage apoptosis, while low⁃concentration recombinant TvMIF protein activates NLRP3 inflammasome and promotes macrophage pyroptosis.

Key words: Trichomonas vaginalis, Macrophage migration inhibitory factor, THP?1 macrophage, NLRP3 inflammasome, Apoptosis, Pyroptosis