UPLC-Q-TOF-MS联合网络药理学探讨当归饮子对特应性皮炎小鼠的作用机制

doi:10.16431/j.cnki.1671-7236.2025.02.037

摘要/Abstract

摘要： 【目的】探讨当归饮子(Angelica decoction,DGYZ)治疗2,4-二硝基氯苯(DNCB)诱导的小鼠特应性皮炎(atopic dermatitis，AD)皮肤病变效果，为临床宠物AD的诊断和治疗提供参考。【方法】选用90只BALB/c小鼠，分为6组：空白组(Control)、DNCB组、DGYZ低(9.8 mg/kg DGYZ)、中(19.6 mg/kg DGYZ)、高(39.3 mg/kg DGYZ)剂量组和西替利嗪组(4 mg/kg西替利嗪)，每组15只小鼠。空白组小鼠涂抹丙酮溶液作为对照，其余各组小鼠通过背部反复涂抹DNCB建立小鼠AD样皮肤病模型，并按分组进行药物治疗。治疗结束后采集小鼠背部皮肤、血液及脾脏用于后续分析。通过HE和甲苯胺蓝染色观察小鼠皮肤组织厚度和肥大细胞数；ELISA方法检测小鼠血清中免疫球蛋白E(IgE)水平；全自动血细胞分析仪测定血液中白细胞(WBC)、中性粒细胞和嗜酸性粒细胞的含量变化；流式细胞术检测小鼠脾脏CD4⁺/CD8⁺和Th1/2细胞分化；实时荧光定量PCR测定小鼠背部皮肤中细胞因子相关mRNA的表达水平；超高压液相色谱-飞行时间质谱仪(UPLC-TOF-MS)分析DGYZ的主要活性成分，联合网络药理学探讨DGYZ治疗AD的主要靶点和作用机制。最后采用实时荧光定量PCR检测JAK1-STAT3信号通路的mRNA表达水平。【结果】与空白组相比，DNCB组小鼠背部皮肤出现严重的皮肤病变和表皮增厚现象，血液中白细胞、中性粒细胞和嗜酸性粒细胞数目显著上升(P＜0.05)；与DNCB组相比，DGYZ各剂量组和西替利嗪组小鼠表皮增厚现象均得到有效缓解，血液中白细胞、中性粒细胞和嗜酸性粒细胞数目均下降，且DGYZ高剂量组及西替利嗪组差异显著(P＜0.05)；DGYZ高剂量组和西替利嗪组小鼠血清中IgE表达水平显著降低(P＜0.05)。流式细胞术结果显示，DNCB组小鼠CD4⁺/CD8⁺和Th1/2细胞的平衡受到干扰，不同剂量DGYZ均可显著改善这一情况(P＜0.05)。实时荧光定量PCR检测结果显示，与空白组相比，DNCB组小鼠皮肤组织中γ-干扰素(IFN-γ)和白细胞介素-12(IL-12)基因mRNA表达水平显著下调(P＜0.05)；IL-4、IL-13、IL-6基因mRNA表达水平显著上调(P＜0.05)。与DNCB组相比，饲喂DGYZ治疗后皮肤组织中IFN-γ和IL-12基因mRNA表达水平逐渐上调，IL-4、IL-13、IL-6基因mRNA表达水平显著下调(P＜0.05)。UPLC-TOF-MS鉴定出89种DGYZ成分，其中活性成分64种。网络药理学分析结果表明，以上64种活性成分通过作用于信号传导及转录激活蛋白3(STAT3)、蛋白激酶B(AKT1)等基因及JAK/STAT、NF-κB和Th17细胞分化信号通路，对AD样皮肤病变发挥治疗作用。实时荧光定量PCR检测结果表明，DNCB可诱导小鼠JAK1-STAT3信号通路的激活，DGYZ可抑制该途径的激活。【结论】 DGYZ可减少小鼠皮肤中肥大细胞数目和血清中IgE的表达，调节免疫平衡，降低促炎因子的mRNA表达并抑制JAK1-STAT3信号通路激活，缓解AD样皮肤病变。本研究结果可为中药治疗动物AD样皮肤病变提供依据。

关键词: 当归饮子(DGYZ); AD样皮肤病变; 2,4-二硝基氯苯(DNCB); 流式细胞术; 网络药理学

Abstract: 【Objective】 This study was aimed to investigate the effects of Angelica decoction (DGYZ) on the skin lesions of mice with atopic dermatitis (AD) induced by 2,4-dichloronitrobenzene (DNCB),provide a reference for the clinical diagnosis and treatment of AD in pets. 【Method】 A total of 90 BALB/c mice were selected and divided into six groups:Control group,DNCB group,low-dose (9.8 mg/kg DGYZ),medium-dose (19.6 mg/kg DGYZ),high-dose (39.3 mg/kg DGYZ) DGYZ groups and cetirizine group (4 mg/kg cetirizine),with 15 mice in each group.The mice in control group were treated with acetone solution as a comparison,while mice in the other groups were subjected to repeated application of DNCB on their backs to establish a mouse model of AD,followed by drug treatment according to their respective groups.After the treatment,skin samples from the back,blood and spleens were collected from the mice for subsequent analysis.Tissue thickness and the number of mast cells were observed through HE and toluidine blue staining.ELISA was used to measure serum immunoglobulin E (IgE) level.The contents of white blood cells (WBC),neutrophils and eosinophils in the blood were measured by automatic blood cell analyzer.Flow cytometry was used to detect CD4⁺/CD8⁺ cell differentiation and Th1/2 cell differentiation.Real-time quantitative PCR was used to measure the expression of cytokine-related mRNA in the back skin of mice.Ultra-high-performance liquid chromatography-time-of-flight mass spectrometer (UPLC-TOF-MS) was used to analyze the main active ingredients of DGYZ,combined with network pharmacology to explore the main targets and mechanisms of action of DGYZ in the treatment of AD.Finally,the mRNA expression of JAK1-STAT3 signaling pathway was detected by Real-time quantitative PCR. 【Result】 Compared with control group, mice in DNCB group exhibited severe skin lesions and epidermal thickening on their backs, along with a significant increase in the number of leukocytes, neutrophils, and eosinophils in the blood (P＜0.05). Compared with DNCB group,the epidermal thickening of mice in DGYZ groups at various doses and cetirizine group was effectively alleviated.And the counts of white blood cells,neutrophils and eosinophils were all decreased compared with DNCB group,DGYZ high-dose group and cetirizine groups were significantly different (P＜0.05).In addition,the serum IgE expression of mice in DGYZ high-dose group and cetirizine group were significantly reduced (P＜0.05).Flow cytometry showed that the balance of CD4⁺/CD8⁺ cells and Th1/2 cells was disrupted in DNCB group,and different doses of DGYZ significantly improved this situation (P＜0.05).Real-time quantitative PCR detection results showed that compared with blank group,the mRNA expression of interferon gamma (IFN-γ) and interleukin-12 (IL-12) genes in the skin tissue of mice in DNCB group were significantly downregulated (P＜0.05).The mRNA expression of IL-4,IL-13 and IL-6 genes were significantly increased (P＜0.05).Compared with DNCB group,the mRNA expression of IFN-γ and IL-12 genes in skin tissue were gradually increased after feeding DGYZ treatment,while the mRNA expression of IL-4,IL-13,and IL-6 genes were significantly decreased (P＜0.05).UPLC-TOF-MS identified 89 ingredients of DGYZ,including 64 active ingredients.The results of network pharmacology analysis showed that the above 64 active ingredients exerted therapeutic effects on AD-like skin lesions by acting on genes such as signal transduction and transcriptional activation protein 3 (STAT3),protein kinase B (AKT1),as well as JAK/STAT,NF-κB and Th17 cell differentiation signaling pathways.Real-time quantitative PCR detection results showed that DNCB could induce the activation of JAK1-STAT3 signaling pathway in mice,while DGYZ could inhibit the activation of this pathway. 【Conclusion】 DGYZ could alleviate AD-like skin lesions by reducing the number of mast cells in skin and the expression of IgE in serum of mice,regulating the immune balance,reducing the mRNA expression of pro-inflammatory factors and inhibiting the activation of JAK1-STAT3 signaling pathway.The results could provide a theoretical and experimental basis for the treatment of AD-like skin lesions in animals with traditional Chinese medicine.

Key words: Angelica decoction (DGYZ); AD-like skin lesions; 2,4-dichloronitrobenzene (DNCB); flow cytometry; network pharmacology

中图分类号:

S853.74

黄永熙, 闫普普, 郭伟丽, 李亚娜, 朱君, 刘满, 侯超群, 覃美林, 李蓉, 武毅, 苏应兵, 杨小林, 郭利伟, 王雄, 戴刚. UPLC-Q-TOF-MS联合网络药理学探讨当归饮子对特应性皮炎小鼠的作用机制[J]. 中国畜牧兽医, 2025, 52(2): 874-889.

HUANG Yongxi, YAN Pupu, GUO Weili, LI Yana, ZHU Jun, LIU Man, HOU Chaoqun, QIN Meilin, LI Rong, WU Yi, SU Yingbing, YANG Xiaolin, GUO Liwei, WANG Xiong, DAI Gang. Exploration of the Mechanism of Action of Angelica Decoction on Atopic Dermatitis Mice Using UPLC-Q-TOF-MS Combined with Network Pharmacology[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(2): 874-889.

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