Inflammation is an essential factor contributing to sepsis-induced endothelial cell (EC) activation. Interleukin-35 (IL-35) is an anti-inflammatory/immunosuppressive cytokine that exerts protective effects on many inflammatory diseases. In this study, we investigated the effects of IL-35 on lipopolysaccharide (LPS)-induced EC activation and the potential underlying mechanism. Human umbilical vein endothelial cells (HUVECs) were incubated with LPS (1 μg/ml) for 24 h and then cocultured with different concentrations (0, 1, 10, or 100 ng/ml) of recombinant human IL-35 (rhIL-35) for 12 h. Flow cytometry analysis revealed that IL-35 inhibited LPS-induced HUVEC apoptosis in a dose-dependent manner. RT-qPCR and Western blot analyses showed significantly higher mRNA and protein levels of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and the inflammatory factors IL-6 and IL-8 in the LPS group than in the control group. These changes were alleviated by IL-35 treatment, suggesting that IL-35 protects ECs by downregulating inflammation. Furthermore, IL-35 induced signal transducer and activator of transcription 1 (STAT1) and STAT4 activation and promoted their interaction. Blocking STAT1 or STAT4 expression by fludarabine (STAT1 inhibitor) treatment or siRNA-STAT4-interfering fragment transfection inhibited the protective effect of IL-35 on ECs. Moreover, we observed a similar protective effect of IL-35 treatment on ECs in a mouse sepsis model induced by intraperitoneal LPS injection. This study indicated that IL-35 exerts anti-inflammatory and antiapoptotic effects on LPS-induced EC activation by activating the STAT1 and STAT4 signaling pathways.

炎症是导致败血症诱导的内皮细胞 (EC) 激活的重要因素。白细胞介素-35 (IL-35) 是一种抗炎/免疫抑制细胞因子，对许多炎症性疾病具有保护作用。在这项研究中，我们研究了 IL-35 对脂多糖 (LPS) 诱导的 EC 激活的影响和潜在的潜在机制。人脐静脉内皮细胞 (HUVEC) 与 LPS (1 μg/ml) 一起孵育 24 小时，然后与不同浓度（0、1、10 或 100 ng/ml）的重组人 IL-35 (rhIL-35) 共培养) 12 小时。流式细胞术分析表明，IL-35 以剂量依赖性方式抑制 LPS 诱导的 HUVEC 凋亡。RT-qPCR 和蛋白质印迹分析显示粘附分子细胞间粘附分子 1 (ICAM-1) 和血管细胞粘附分子 1 (VCAM-1) 以及炎症因子 IL-6 和 IL- 的 mRNA 和蛋白质水平显着升高。 8 LPS组比对照组多。这些变化通过 IL-35 治疗得到缓解，表明 IL-35 通过下调炎症来保护 ECs。此外，IL-35 诱导信号转导和转录激活因子 1 (STAT1) 和 STAT4 激活并促进它们的相互作用。通过氟达拉滨（STAT1 抑制剂）处理或 siRNA-STAT4 干扰片段转染阻断 STAT1 或 STAT4 表达可抑制 IL-35 对 ECs 的保护作用。而且，我们在腹腔内 LPS 注射诱导的小鼠败血症模型中观察到 IL-35 处理对 ECs 的类似保护作用。该研究表明，IL-35 通过激活 STAT1 和 STAT4 信号通路对 LPS 诱导的 EC 激活发挥抗炎和抗细胞凋亡作用。