探讨麻黄连翘赤小豆汤通过NLRP3细胞焦亡对特应性皮炎样小鼠模型的作用

Exploring the effect of Mahuang Lianqiao Chixiaodou decoction on NLRP3 cell pyroptosis in an atopic dermatitis-like mouse model

目的:探讨麻黄连翘赤小豆汤(Mahuang Lianqiao Chixiaodou decoction,MLCD)通过NOD样受体蛋白3(NOD-like receptor protein 3,NLRP3)角质形成细胞焦亡途径干预特应性皮炎样(atopic dermatitis-like,AD样)小鼠皮肤屏障功能障碍与免疫炎症“内外串扰”的疗效及机制。方法:用2,4-二硝基氟苯诱导AD样模型小鼠,分别用MLCD或糠酸莫米松凝胶(mometasone furoate,MF,阳性对照组)处理7天。采用马松染色法、甲苯胺蓝染色法检测皮肤组织的病理变化与肥大细胞浸润情况。采用智能皮肤分析仪、流式细胞术、荧光定量聚合酶链反应和免疫印迹法观察和评估AD样小鼠的皮肤屏障功能障碍、免疫炎症反应和皮肤细胞焦亡。结果:MLCD和MF均可不同程度地改善AD样小鼠的皮损状态,减少病理组织损伤和肥大细胞浸润。MLCD显著降低皮肤色素沉着和炎症状态(P=0.005,P=0.038),增加脾脏中CD4^(+)CD3^(+)T细胞的百分比(P=0.022),降低CD8^(+)CD3^(+)T细胞百分比(P=0.044),降低CD8^(+)CD3^(+)/CD3^(+)的比值(P=0.031),并增加CD4^(+)CD3^(+)/CD8^(+)CD3^(+)的比值(P=0.027)。MLCD还可显著降低角质形成细胞焦亡相关因子NLRP3、casp-1、白细胞介素(IL)-1β和IL-18得mRNA相对表达水平(P=0.027、P<0.001、P=0.012和P=0.039)以及NLRP3、casp-1、凋亡相关斑点蛋白和IL-1β的蛋白表达水平(P=0.002、P=0.006、P=0.004和P=0.035)。结论:MLCD通过干预NLRP3角质形成细胞焦亡介导的皮肤屏障功能障碍和免疫炎症的“内外串扰”机制,对AD样小鼠的治疗具有疗效。

Objective:To investigate the efficacy and mechanism of Mahuang Lianqiao Chixiaodou decoction(MLCD)in intervening in the“internal and external crosstalk”between skin barrier dysfunction and immune inflammation in an atopic dermatitis-like(AD-like)mouse model via the NOD-like receptor protein 3(NLRP3)pyroptosis pathway.Methods:AD-like model mice were induced with 2,4-dinitrofluorobenzene and treated with MLCD or mometasone furoate gel(MF,positive control)for 7 days.Pathological changes in skin tissue were examined using Masson or methamphetamine blue staining.A smart skin analyzer,flow cytometry,fluorescence quantitative polymerase chain reaction,and western blotting were used to observe and evaluate skin barrier dysfunction,immune inflammatory responses,and skin cell pyroptosis in AD-like mice.Results:MLCD and MF improved skin damage and reduced pathological tissue damage and mast cell infiltration in AD-like mice to varying degrees.MLCD significantly reduced skin pigmentation and inflammatory status(P=.005 and P=.038,respectively),increased the percentage of splenic CD4^(+)CD3^(+)T cells(P=.022),decreased the CD8^(+)CD3^(+)T cell percentage(P=.044),decreased the CD8^(+)CD3^(+)/CD3^(+)ratio(P=.031)and increased the CD4^(+)CD3^(+)/CD8^(+)CD3^(+)ratio(P=.027).MLCD also significantly decreased the mRNA expression levels of cell scorch-related factors NLRP3,casp-1,interleukin(IL)-1β,and IL-18(P=.027,P<.001,P=.012,and P=.039,respectively),as well as the protein expression of NLRP3,casp-1,apoptosis-associated speck-like protein containing a CARD,and IL-1β(P=.002,P=.006,P=.004,and P=.035,respectively).Conclusion:MLCD achieved efficacy in the treatment of AD-like mice by interfering with the“internal and external crosstalk”mechanisms of skin barrier dysfunction and immune inflammation mediated by NLRP3 pyroptosis.