基于Toll样受体4/髓样分化因子88/核转录因子κB信号通路探讨针刺“蝶腭穴”改善变应性鼻炎大鼠免疫炎性反应的机制

Acupuncture at “Die E acupoint” alleviates inflammatory reaction via inhibiting TLR4/MyD88/NF-κB signaling in rats with allergic rhinitis

目的:探讨针刺“蝶腭穴”对变应性鼻炎(AR)大鼠Toll样受体4(TLR4)、髓样分化因子88(MyD88)、核转录因子κB(NF-κB)、转录因子T-bet(T-bet)、GATA结合蛋白-3(GATA-3)的蛋白表达水平及相关炎性细胞因子的影响。方法:SD大鼠随机分为空白组、模型组、针刺组、假针刺组,每组6只。采用卵清蛋白诱导建立AR大鼠模型。针刺组大鼠行双侧“蝶腭穴”针刺,假针刺组仅行假针刺,均每日1次,共6次。记录各组大鼠行为学评分;HE染色法观察大鼠鼻黏膜形态变化;ELISA法检测大鼠血清免疫球蛋白E(IgE)、卵清蛋白特异性IgE(OVA-sIgE)、干扰素(IFN)-γ、白细胞介素(IL)-4、IL-10、IL-17的含量;Western blot法检测大鼠鼻黏膜中TLR4、MyD88、NF-κB p65、T-bet、GATA-3蛋白表达水平。结果:与正常组比较,模型组行为学评分升高(P<0.05),血清IgE、OVA-sIgE、IL-4、IL-17的含量及鼻黏膜中GATA-3、TLR4、MyD88、NF-κB p65蛋白表达水平升高(P<0.05),血清IFN-γ、IL-10含量及鼻黏膜T-bet蛋白表达水平降低(P<0.05)。与模型组比较,针刺组行为学评分降低(P<0.05),血清IgE、OVA-sIgE、IL-4、IL-17的含量及鼻黏膜中GATA-3、TLR4、MyD88、NF-κB p65蛋白表达水平降低(P<0.05),血清IFN-γ、IL-10的含量及鼻黏膜T-bet蛋白表达水平升高(P<0.05);假针刺组以上指标差异均无统计学意义。模型组和假针刺组可见鼻黏膜炎性浸润,针刺组鼻黏膜炎性反应明显减轻。结论:针刺“蝶腭穴”可减轻OVA诱导的AR大鼠的症状,改善鼻黏膜炎性反应,其机制可能与抑制TLR4/MyD88/NF-κB信号通路,有效调控Th1/Th2、Treg/Th17细胞因子及T-bet/GATA-3的平衡有关。

Objective To observe effects of acupuncture at “Die E acupoint” on the protein expression levels of Toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),nuclear transcription factor κB(NF-κB),transcription factor T-bet(T-bet),and GATA-binding protein-3(GATA-3) in the nasal mucosa and the serum contents of related inflammatory cytokines in rats with allergic rhinitis,so as to explore the mechanism of acupuncture in treating allergic rhinitis.Methods Twenty-four healthy SD rats were randomly divided into blank,model,acupuncture,and sham acupuncture groups,with 6 rats in each group.The rat model of allergic rhinitis was established by using ovalbumin induction.The rats in the acupuncture group received bilateral acupuncture at the “Die E acupoint” with a depth of 15—20 mm,while the rats in the sham acupuncture group received only sham acupuncture(light and shallow acupunture of the skin at the “Die E acupoint”).Both interventions were performed once daily for a total of 6 days.Behavioral scores of rats in each group were recorded.Pathological changes of nasal mucosa were observed by H.E.staining.Serum contents of IgE,ovalbumin-specific IgE(OVA-sIgE),interferon(IFN)-γ,interleukin(IL)-4,IL-10 and IL-17 were measured by ELISA and the protein expression levels of T-bet,GATA-3,TLR4,MyD88 and NF-κB p65 in the nasal mucosa were detected by Western blot.Results After modeling,compared with the blank group,rats in the model group showed increased behavioral scores,serum IgE,OVA-sIgE,IL-4,and IL-17 contents,and nasal mucosal GATA-3,TLR4,MyD88,and NF-κB p65 protein expression levels(P<0.05),whereas the contents of serum IFN-γ,IL-10 and the protein expression level of T-bet in the nasal mucosa were decreased(P<0.05).Comparison between the EA and model groups showed that acupuncture intervention can decrease the behavioral scores of rats with allergic rhinitis,the contents of serum IgE,OVA-sIgE,IL-4,IL-17,and the protein expression levels of GATA-3,TLR4,MyD88,and NF-κB p65 in the nasal mucosa(P<0.05),and up-regulate the contents of serum IFN-γ,IL-10,and the nasal mucosal T-bet protein expression level.Sham acupuncture did not have a significant modulating effect on the above indicators.Inflammatory infiltration of nasal mucosa was seen in the model group and sham acupuncture,and the inflammatory reaction was milder in the acupuncture group.Conclusion Acupuncture at “Die E acupoint” can alleviate the symptoms of allergic rhinitis and suppress the inflammation of nasal mucosa in rats,which may be related to inhibiting the TLR4/MyD88/NF-κB signaling and balancing the levels of cytokines of Th1/Th2 and Treg/Th17,and T-bet/GATA-3.