背景:许多研究表明,天然多糖作为宿主防御增效剂具有免疫增强作用。很少有关于天然多糖的封闭作用的报道,和潜在的机制仍然不清楚。
目的:来自Araliaelata的AELP-B6(富含阿拉伯糖和半乳糖的果胶多糖)(Miq。)以Seem为案例研究,以阐明天然多糖的角化效应的潜在机理。
方法:通过构建CTX免疫抑制小鼠模型验证AELP-B6的药效学作用。通过TMT标记的蛋白质组学探索了这种效应,能量代谢分析,流式细胞术和蛋白质印迹。通过下拉法确定AELP-B6的核心亲和力靶标,nanoLC-nanoESI+-MS,CETSA,免疫印迹和SPR测定。同时构建RAW264.7Clec4G-RFP和RAW264.7Rab1A-RFP细胞系,通过共聚焦激光扫描活细胞成像确定AELP-B6与靶标的亲和力差异。进一步使用抗体阻断测定来验证封闭效应的机制。
结果:低剂量和中等剂量的AELP-B6可能保持胸腺和脾脏的结构完整性,增加TNF-α的浓度,IFN-γ,IL-3和IL-8,并减轻CTX诱导的体内免疫细胞活力的降低。蛋白质组学和能量代谢分析显示AELP-B6调节HIF-1α介导的代谢编程,在巨噬细胞中引起Warburg效应.AELP-B6在低剂量和中等剂量下促进细胞内免疫因子的释放,并驱动巨噬细胞的M1样极化。作为对比,高剂量AELP-B6增强细胞凋亡相关蛋白的表达,表明内在凋亡级联的激活。巨噬细胞中两个高表达的跨膜蛋白,Clec4G和Rab1A,被鉴定为AELP-B6的主要结合靶标,其与细胞膜共定位并直接影响免疫细胞活化和凋亡。AELP-B6表现出与Clec4G和Rab1A的亲和力差异,这是恐怖效应的关键。
结论:我们首次观察到天然多糖(AELP-B6)的兴奋作用,和AELP-B6通过两个剂量相关的靶标介导了效应。低剂量的AELP-B6靶向Clec4G,从而通过调节NF-κB信号通路和HIF-1α介导的代谢编程来驱动M1样极化,而高剂量的AELP-B6靶向Rab1A,导致线粒体依赖性细胞凋亡。
BACKGROUND: Numerous studies indicate that natural polysaccharides have immune-enhancing effects as a host defense potentiator. Few reports are available on hormetic effects of natural polysaccharides, and the underlying mechanisms remain unclear.
OBJECTIVE: AELP-B6 (arabinose- and galactose-rich pectin polysaccharide) from Aralia elata (Miq.) Seem was taken as a case study to clarify the potential mechanism of hormetic effects of natural polysaccharides.
METHODS: The pharmacodynamic effect of AELP-B6 was verified by constructing the CTX-immunosuppressive mouse model. The hormetic effects were explored by TMT-labeled proteomics, energy metabolism analysis, flow cytometry and western blot. The core-affinity target of AELP-B6 was determined by pull down, nanoLC-nanoESI+-MS, CETSA, immunoblot and SPR assay. The RAW264.7Clec4G-RFP and RAW264.7Rab1A-RFP cell lines were simultaneously constructed to determine the affinity difference between AELP-B6 and targets by confocal laser scanning live-cell imaging. Antibody blocking assays were further used to verify the mechanism of hormetic effects.
RESULTS: AELP-B6 at low and medium doses may maintain the structural integrity of thymus and spleen, increase the concentrations of TNF-α, IFN-γ, IL-3 and IL-8, and alleviate CTX-induced reduction of immune cell viability in vivo. Proteomics and energy metabolism analysis revealed that AELP-B6 regulate HIF-1α-mediated metabolic programming, causing Warburg effects in macrophages. AELP-B6 at low and medium doses promoted the release of intracellular immune factors, and driving M1-like polarization of macrophages. As a contrast, AELP-B6 at high dose enhanced the expression levels of apoptosis related proteins, indicating activation of the intrinsic apoptotic cascade. Two highly expressed transmembrane proteins in macrophages, Clec4G and Rab1A, were identified as the primary binding targets of AELP-B6 which co-localized with the cell membrane and directly impacted with immune cell activation and apoptosis. AELP-B6 exhibits affinity differences with Clec4G and Rab1A, which is the key to the hormetic effects.
CONCLUSIONS: We observed
hormesis of natural polysaccharide (AELP-B6) for the first time, and AELP-B6 mediates the hormetic effects through two dose-related targets. Low dose of AELP-B6 targets Clec4G, thereby driving the M1-like polarization via regulating NF-κB signaling pathway and HIF-1α-mediated metabolic programming, whereas high dose of AELP-B6 targets Rab1A, leading to mitochondria-dependent apoptosis.