UNASSIGNED: We developed an in vitro cell culture disease model by using LAD-2 MC, HaCaT keratinocytes and RAW 264.7 macrophage cell lines. We challenged keratinocytes and macrophage cells with CST-14 treated MC supernatant in the presence and absence of GE1111 and measured the expression of tight junction protein claudin 1, inflammatory cytokines and macrophage phagocytosis activity through immunohistochemistry, western blotting, RT-qPCR and fluorescence imaging techniques. In addition to this, we developed a DFNB-induced AD model in mice and evaluated the protective effect and underlying mechanism of GE1111.
UNASSIGNED: Our in vitro findings demonstrated a potential therapeutic effect of GE1111, which inhibits the expression of TSLP, IL-13, MCP-1, TNF-a, and IL-1ß in MC and keratinocytes. In addition to this, GE1111 was able to preserve the expression of claudin 1 in keratinocytes and the phagocytotic activity of macrophage cells. The in vivo results demonstrated that GE1111 treatment significantly reduced phenotypic changes associated with AD (skin thickening, scaling, erythema and epidermal thickness). Furthermore, immunohistochemical analysis demonstrated that GE1111 treatment preserved the expression of the tight junction protein Involucrin and reduced the expression of the inflammatory mediator periostin in the mouse model of AD. These findings were supported by gene and protein expression analysis, where GE1111 treatment reduced the expression of TSLP, IL-13, and IL-1ß, as well as downstream signalling pathways of MRGPRX2 in AD skin lesions. In conclusion, our findings provide compelling in vitro and in vivo evidence supporting the contribution of MRGPRX2-MC interaction with keratinocytes and macrophages in the pathogenesis of AD.
■我们使用LAD-2MC建立了体外细胞培养疾病模型,HaCaT角质形成细胞和RAW264.7巨噬细胞系。在存在和不存在GE1111的情况下,我们用CST-14处理的MC上清液攻击角质形成细胞和巨噬细胞,并通过免疫组织化学测量紧密连接蛋白claudin1,炎性细胞因子和巨噬细胞吞噬活性的表达,西方印迹,RT-qPCR和荧光成像技巧。除此之外,我们建立了DFNB诱导的小鼠AD模型,并评估了GE1111的保护作用和潜在机制。
■我们的体外研究结果证明了GE1111的潜在治疗作用,它抑制了TSLP的表达,IL-13,MCP-1,TNF-a,MC和角质形成细胞中的IL-1β。除此之外,GE1111能够保留角质形成细胞中claudin1的表达和巨噬细胞的吞噬活性。体内结果表明,GE1111治疗可显着减少与AD相关的表型变化(皮肤增厚,缩放,红斑和表皮厚度)。此外,免疫组织化学分析表明,在AD小鼠模型中,GE1111治疗保留了紧密连接蛋白Involucrin的表达,并降低了炎症介质骨膜素的表达。这些发现得到了基因和蛋白质表达分析的支持,其中GE1111处理降低了TSLP的表达,IL-13和IL-1β,以及AD皮肤病变中MRGPRX2的下游信号通路。总之,我们的研究结果提供了令人信服的体外和体内证据,支持MRGPRX2-MC与角质形成细胞和巨噬细胞相互作用在AD发病机制中的作用.