基于Notch1-Jagged1通路探讨鹿鹅鼻炎方对变应性鼻炎小鼠Th17/Treg的影响

Effect of Lu'e Rhinitis Formula on Th17/Treg Immune Imbalance in Mice With Allergic Rhinitis Based on Notch1-Jagged1 Signaling Pathway

目的 探讨鹿鹅鼻炎方对变应性鼻炎(AR)小鼠Notch1-Jagged1通路及辅助性T细胞17(Th17)/调节性T细胞(Treg)免疫失衡的影响。方法 将30只BALB/c小鼠随机分为空白组、模型组、鹿鹅鼻炎方低、中、高剂量组,每组6只。以卵清蛋白(OVA)为致敏原诱导建立AR模型小鼠,鹿鹅鼻炎方低、中、高剂量组每天分别给与鹿鹅鼻炎方0.5、1、2 g/kg灌胃。通过行为学观察评估各组小鼠鼻部症状(打喷嚏、流鼻涕)改变;进行鼻灌洗液(NLF)细胞计数;观察各组小鼠鼻黏膜组织形态学变化并计算嗜酸性粒细胞、杯状细胞和肥大细胞数;采用酶联免疫吸附法(ELISA)检测各组小鼠血清中OVA特异性IgE(OVA-sIgE)、OVA特异性IgG(OVA-sIgG)水平以及NLF中白细胞介素(IL)-10、IL-17水平;Western Blot检测各组小鼠鼻黏膜中Notch1、Jagged1、视黄酸相关孤儿受体γt(RORγt)及叉头状螺旋转录因子3(Foxp3)蛋白表达。结果 与空白组比较,模型组小鼠可观察到鼻黏膜上皮细胞显著增生和化生,纤毛脱落不完整,黏膜下层血管扩张明显,出现以嗜酸性细胞为主的炎性细胞浸润;经鹿鹅鼻炎方干预后,黏膜上皮损伤好转。与空白组比较,模型组小鼠末次滴鼻激发后10 min内喷嚏和抓鼻次数、鼻黏膜上皮厚度、嗜酸性粒细胞数、杯状细胞数、肥大细胞数、血清OVA-sIgE及OVA-sIgG水平、NLF IL-17水平、鼻黏膜RORγt、Notch1、Jagged1蛋白表达增加(P<0.05),NLF IL-10水平、鼻黏膜Foxp3蛋白表达降低(P<0.05)。与模型组比较,鹿鹅鼻炎方低、中、高剂量组小鼠末次滴鼻激发后10 min内喷嚏和抓鼻次数、鼻黏膜上皮厚度、嗜酸性粒细胞数、血清OVA-sIgE及OVA-sIgG水平、NLF、IL-17水平、鼻黏膜RORγt、Notch1、Jagged1表达降低,鼻黏膜Foxp3表达水平升高(P<0.05);鹿鹅鼻炎方中、高剂量组鼻黏膜杯状细胞数、鼻黏膜RORγt蛋白表达降低,NLF IL-10水平升高(P<0.05);鹿鹅鼻炎方高剂量组鼻黏膜肥大细胞数降低(P<0.05)。结论 鹿鹅鼻炎方干预AR小鼠的机制可能是通过抑制Notch1-Jagged1表达,恢复Th17/Treg免疫平衡实现的。

Objective To investigate the effects of Lu'e Rhinitis Formula(LEBY) on Notch1-Jagged1 signaling pathway and helper T cell 17(Th17)/regulatory T cell(Treg) immune imbalance in allergic rhinitis(AR)mice. Methods Thirty BALB/c mice were randomly divided into the sham group,model group,LEBY low,medium,high dose(LEBYL,LEBYM,LEBYH) group,6 in each group. AR mice model was established by inducing ovalbumin(OVA) as the allergen. LEBYL,LEBYM,LEBYH groups were given intragastric administration of LEBY as 0.5,1,2 g/kg per day. The nasal symptoms(sneezing and runny nose) were assessed by behavioral observation in each group. Nasal lavage fluid(NLF) cell count was performed.The morphological changes of nasal mucosa and the numbers of eosinophils,goblet cells and mast cells in each group were assessed. The enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of OVAspecific IgE(OVA-sIgE),OVA-specific IgG(OVA-sIgG) in serum,interleukin-10(IL-10) and interleukin-17(IL-17) in NLF. The relative protein expressions of Notch1,Jagged1,and retinoid related orphan receptor γt(RORγt) and fork head box transcription factor 3(Foxp3) in nasal mucosa were measured by Western Blot.Results Compared with the sham group,significant proliferation and metaplasia of nasal mucosal epithelial cells as well as eosinophils dominating the inflammatory infiltrating cells were observed in the model group,with cilium shedding and submucosal vessels dilatation. After intervention with LEBY,the damage of the mucosal epithelial has been partly restored. Compared with the sham group,the quantities of nasal itching and sneezing 10 min after the last stimulation,the thickness of nasal mucosal epithelium,the quantities of eosinophils,globet cells and mast cells,the levels of serum OVA-s Ig E and OVA-sIgG,IL-17 in NLF,and the expression of RORγt,Notch1,and Jagged1 proteins in the nasal mucosa increased(P<0.05),the level of IL-10 in NLF and the expression of Foxp3 protein in the nasal mucosa decreased in model group(P<0.05). Compared with the model group,the quantities of nasal itching and sneezing 10 min after the last stimulation,the thickness of nasal mucosal epithelium and the quantities of eosinophils,the levels of serum OVAs Ig E and OVA-sIgG,IL-17 in NLF,and the expression of RORγt,Notch1 and Jagged1 proteins in the nasal mucosa decreased,the expression of Foxp3 proteins in the nasal mucosa increased in LEBYL,LEBYM,LEBYH groups(P<0.05).The quantities of globet cells and the expression of RORγt protein in the nasal mucosa decreased,the level of IL-10in NLF increased in LEBYM and LEBYH group(P<0.05). The mast cells in nasal mucosa decreased in LEBYH group(P<0.05). Conclusion The mechanism of LEBY intervention in AR mice may be achieved by inhibiting Notch1-Jagged1 expression and regulating Th17/Treg immune homeostasis.