Jiawei guomin decoction regulates the degranulation of mast cells in atopic dermatitis mice via the HIS/PAR-2 pathway
Jiawei guomin decoction regulates the degranulation of mast cells in atopic dermatitis mice via the HIS/PAR-2 pathway
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DOI：
10.1016/j.jep.2023.117485
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发表时间：
2023-11-28
影响因子：
5.4
通讯作者：
Wu，Xian-bo
中科院分区：
文献类型：
--
作者：
Huang，Jian-li;Xu，Yi-hua;Wu，Xian-bo
民族药理学相关性国民汤是临床常用的传统中药。它传统上用于治疗所有过敏性疾病。目前，加味国民汤（JWGMD）用于治疗初始治疗后的敏感皮肤。虽然它具有显著的临床治疗效果，但肥大细胞脱颗粒在治疗特应性皮炎（AD）中的确切作用尚不清楚。本研究的目的GMD和JWGMD均能治疗过敏性疾病，而JWGMD侧重于皮肤过敏。本研究旨在探讨加味颗粒微丸对2，4-二硝基氟苯（DNFB）诱导的AD小鼠模型肥大细胞脱颗粒的影响，探讨加味颗粒微丸对AD的缓解作用，为进一步治疗AD提供特异性治疗靶点。并收集皮肤组织用于随后的测量。苏木精-伊红（H&E）染色观察小鼠背部皮肤组织学变化，甲苯胺蓝染色检测小鼠皮肤肥大细胞脱颗粒情况，酶联免疫吸附试验（ELISA）分析组胺（HIS）、肥大细胞类胰蛋白酶（MCT）表达与肥大细胞脱颗粒的关系。Western blot检测AD小鼠脑组织中蛋白酶激活受体2（PAR-2）、组胺1受体（H1 R）、H2 R、H4 R和MCT蛋白的表达。免疫荧光分析（IFA）进一步证实PAR-2、H1 R、H2 R、H4 R和MCT蛋白在皮肤中的定位。采用实时定量PCR（qPCR）测定皮损中PAR-2、H1 R、H2 R和H4 R mRNA水平，以进一步阐明JWGMD在AD中放大肥大细胞脱颗粒的机制。此外，还建立了可靠的超高效液相色谱-四极杆静电场轨道阱质谱联用技术进行（UPLC-QE-MS）非靶向代谢组学分析以分析GMD和JWGMD之间代谢物丰度的差异，并且这些结果用于鉴定JWGMD中可能具有抗炎和抗炎性质并抑制肥大细胞脱颗粒的活性成分。皮损表现为广泛脱屑、干燥、结痂、皮肤增厚和轻微出血。两种治疗方法都缓解了这种现象并减少了划痕的数量，其中JWGMD最有效。加味颗粒微丸能明显减轻小鼠炎症细胞浸润、水肿和部分毛细血管新生，并能减少肥大细胞的脱颗粒。ELISA结果表明，加味甘草汤能提高小鼠血清中MCT和HIS蛋白水平。WB和IFA结果表明，JWGMD减少PAR-2，H1 R，H4 R和MCT蛋白的表达水平在皮损中，蛋白定位主要在表皮层，而H2 R蛋白水平增加，主要定位在真皮层。此外，JWGMD下调PAR-2，H1 R，H2 R和H4 R的mRNA表达。结论JWGMD可能通过甲基丁香酚、白藓碱、芥子碱等物质发挥止痒作用，其机制可能与抑制AD模型小鼠HIS/PAR-2通路，进一步调节肥大细胞脱颗粒的自身放大作用有关。JWGMD是一种潜在的药物，用于...
Ethnopharmacological relevanceGuomin decoction (GMD) is a traditional Chinese medicine commonly used in clinical practice. It has traditionally been used to treat all allergic diseases. Currently, Jiawei Guomin Decoction (JWGMD) is used to treat sensitive skin after initial therapy. Although it has a significant clinical therapeutic effect, the exact role of mast cell degranulation in treating atopic dermatitis (AD) is still unclear.Aim of the studyGMD and JWGMD can both treat allergic diseases, while JWGMD focuses on skin allergies. This study aims to explore the potential effect of JWGMD on the degranulation of mast cells in an AD mouse model induced by 2,4-dinitrofluorobenzene (DNFB) and investigate the effectiveness of JWGMD in alleviating disease progression to further provide specific therapeutic targets for treating AD.Materials and methodsThe scratching times and skin lesions of model mice induced by DNFB were observed, and skin tissues were collected for subsequent measurement. Histopathological changes in the back skin of mice were observed by haematoxylin eosin (H&E) staining, Toluidine blue staining was used to detect the degranulation of mouse skin mast cells, and the relationship between the expression of histamine (HIS), mast cell tryptase (MCT) and mast cell degranulation was analysed by enzyme-linked immunosorbent assay (ELISA). The expression of protease-activated receptor-2 (PAR-2), histamine 1 receptor (H1R), H2R, H4R and MCT proteins in AD mice was detected by Western blot (WB). Immunofluorescence assay (IFA) further confirmed the localization of PAR-2, H1R, H2R, H4R, and MCT proteins in the skin. Quantitative real-time PCR (qPCR) was used to determine PAR-2, H1R, H2R and H4R mRNA levels in skin lesions to further clarify the mechanism by which JWGMD amplifies mast cell degranulation in AD. In addition, a reliable ultrahigh-performance liquid chromatography-quadrupole electrostatic field orbitrap mass spectrometry (UPLC-QE-MS) nontargeted metabolomics analysis was performed to analyse the differences in metabolite abundance between GMD and JWGMD, and these results were used to identify the active components in JWGMD that may have antipruritic and anti-inflammatory properties and inhibit mast cell degranulation.ResultsAfter intermittent stimulation with DNFB, the skin lesions showed extensive desquamation, dryness, scabbing, skin thickening, and slight bleeding. Both treatments alleviated this phenomenon and reduced the number of scratches, with JWGMD being the most effective. JWGMD can significantly reduce inflammatory cell infiltration, oedema, and some capillary neogenesis in mice and reduce the degranulation of mast cells. The ELISA results showed that JWGMD can increase the levels of MCT and HIS proteins. The WB and IFA results demonstrated that JWGMD reduced the expression levels of PAR-2, H1R, H4R, and MCT proteins in skin lesions, with protein localization mainly in the epidermal layer, while H2R protein levels were increased and mainly localized in the dermis. In addition, JWGMD downregulates the mRNA expression of PAR-2, H1R, H2R, and H4R. Interestingly, through UPLC-QE-MS nontargeted metabolomic analysis, we detected the anti-inflammatory and antiallergy active substances in JWGMD, such as methyl eugenol, dictamnine and sinapine.ConclusionsJWGMD may alleviate itching through methyl syringol, dictamnine, sinapine and other substances, and its mechanism may be related to inhibiting the HIS/PAR-2 pathway in AD model mice and further regulating the self-amplification of mast cell degranulation. JWGMD is a potential drug for …