■随着全球生育率的下降,异位妊娠的药物治疗具有重要意义。紫花苜蓿作为草药用于杀死胚胎。紫草素是紫草的关键核;然而,机制尚不清楚。本研究旨在探讨紫草素抗异位妊娠的作用机制。
■在这项研究中,我们通过实验检查了HTR-8/SVneo细胞的活力和LDH释放,通过显微镜成像和PI染色观察细胞膜中的孔形成,和IL-1β释放通过WB和ELISA检测试剂盒。然后,我们使用网络药理学来分析紫草素之间的潜在相互作用,异位妊娠和焦亡,并使用分子对接技术验证紫草素与核心共同靶标之间的相互作用。最后,采用免疫印迹法和免疫荧光法探讨紫草素诱导HTR-8/SVneo细胞凋亡的机制。
■紫草素可能以浓度和时间依赖性方式引起HTR-8/SVneo细胞活力的显着抑制。在HTR-8/SVneo细胞中,紫草素诱导的细胞肿胀,气泡形成,乳酸脱氢酶(LDH)释放的增加和几种焦亡相关因子的上调。而网络药理学显示紫草素-异位妊娠-焦亡的主要作用靶点是IL-1β和caspase-1,分子对接结果显示紫草素可以与IL-1β紧密结合,caspase-1和GSDMD。此外,坏死性凋亡抑制剂GSK\'872不能抑制成熟IL-1β的表达并阻止焦亡表型的发展。然而,核苷酸寡聚化结构域样受体家族含pyrin结构域3(NLRP3)抑制剂MCC-950可下调焦亡相关因子的表达并阻止焦亡表型的发展.紫草素导致组织蛋白酶B(CTSB)的表达升高,CTSB抑制剂CA-074消除了紫草素诱导的焦亡;然而,NLRP3抑制剂MCC-950不能抑制CTSB的表达。
■我们的结果表明,紫草素激活CTSB以诱导HTR-8/SVneo细胞中NLRP3依赖性的焦亡。本研究对异位妊娠的治疗具有重要的临床意义。
UNASSIGNED: With the decline of global fertility, drug therapeutic of ectopic pregnancy is of great significance. Lithospermum erythrorhizon is using for embryo killing as herbal medicine. Shikonin is the critical nucleus of Lithospermum erythrorhizon; however, the mechanism is still unclear. The study aimed to explore the mechanism of shikonin against ectopic pregnancy.
UNASSIGNED: In this study, we examined the viability and LDH release of HTR-8/SVneo cells by assays, observed pore formation in cell membranes by microscopy imaging and PI staining, and IL-1β release by WB and ELISA assay kit. Then, we used network pharmacology to analyse the potential interaction between shikonin, ectopic pregnancy and pyroptosis and used molecular docking techniques to verify interactions between shikonin and core common targets. Finally, western blotting and immunofluorescence assay were used to explore the mechanism of shikonin-inducing pyroptosis of HTR-8/SVneo cells.
UNASSIGNED: Shikonin could cause a significant inhibition of HTR-8/SVneo cell viability in a concentration- and time-dependent manner. In HTR-8/SVneo cells, shikonin-induced cell swelling, bubble formation, an increase in the release of lactate dehydrogenase (LDH) and up-regulation of several pyroptosis-associated factors. And network pharmacology showed that The main targets of shikonin-ectopic pregnancy-pyroptosis were IL-1β and caspase-1, and molecular docking results showed that shikonin can closely bind to IL-1β, caspase-1 and GSDMD. Additionally, the necroptosis inhibitor GSK\'872 could not suppress the expression of mature-IL-1β and prevent the pyroptosis phenotype from developing. However, the nucleotide oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inhibitor MCC-950 could downregulate the expression of pyroptosis-associated factors and prevent the pyroptosis phenotype from developing. Shikonin led to an elevation in the expression of cathepsin B (CTSB), and the CTSB inhibitor CA-074 abolished pyroptosis induced by shikonin; however, the NLRP3 inhibitor MCC-950 could not inhibit the expression of CTSB.
UNASSIGNED: Our results suggest that shikonin activates CTSB to induce NLRP3-dependent pyroptosis in HTR-8/SVneo cells. This study has important clinical implications for the treatment of ectopic pregnancy.