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Abstract：
UNASSIGNED: Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterised by itching, erythema, and epidermal barrier dysfunction. The pathogenesis of AD is complex and multifactorial; however,mast cell (MC) activation has been reported to be one of the crucial mechanisms in the pathogenesis of AD. The MC receptor Mas related G protein-coupled receptor-X2 (MRGPRX2) has been identified as a prominent alternative receptor to the IgE receptor in causing MC activation and the subsequent release of inflammatory mediators. The current study aimed to evaluate the therapeutic effect of a novel small molecule MRGPRX2 antagonist GE1111 in AD using in vitro and in vivo approaches.
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.
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.
摘要：
■特应性皮炎（AD）是一种以瘙痒为特征的慢性炎症性皮肤病，红斑,和表皮屏障功能障碍。AD的发病机制是复杂和多因素的;然而,肥大细胞(MC)活化是AD发病的重要机制之一。MC受体Mas相关的G蛋白偶联受体X2（MRGPRX2）已被确定为IgE受体的主要替代受体，可引起MC活化和随后的炎症介质释放。本研究旨在使用体外和体内方法评估新型小分子MRGPRX2拮抗剂GE1111在AD中的治疗效果。
■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.
■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.
