枸杞多糖通过调控TLR9/AP-1信号通路对变应性鼻炎大鼠Th1/Th2细胞因子及鼻黏膜中嗜酸性粒细胞炎症的影响

Effects of lycium barbarum polysaccharide on Th1/Th2 cytokines and eosinophil inflammation in nasal mucosa of allergic rhinitis rats by regulating TLR9/AP-1 signaling pathway

目的探究枸杞多糖通过调控Toll样受体9/激活蛋白-1(TLR9/AP-1)信号通路对变应性鼻炎大鼠Th1/Th2细胞因子、鼻黏膜中嗜酸性粒细胞炎症的影响。方法选取60只SPF级SD雄性大鼠,随机分为正常(A)组,模型(B)组,糠酸莫米松(C)组,枸杞多糖(D)组,TLR9/AP-1信号通路抑制剂(E)组,TLR9/AP-1信号通路抑制剂联合枸杞多糖(F)组,每组各10只,对B、C、D、E、F组采用卵清蛋白致敏法建立变应性鼻炎模型,A组不建立该模型。建模成功后,对C组给予1喷/侧的糠酸莫米松喷鼻治疗,对D组灌胃100 mg/kg的枸杞多糖,对E组灌胃20 mg/kg AP-1抑制剂SP100030,对F组给予SP100030联合枸杞多糖,A、B组同期给予灌胃同体积生理盐水,对大鼠鼻部症状进行评分,HE染色法检测鼻黏膜组织病理形态及嗜酸性粒细胞并计数,ELISA法检测大鼠血清中Th1/Th2细胞因子水平,免疫印迹法检测鼻黏膜组织中TLR9/AP-1信号通路相关蛋白表达。结果与A组相比,B组鼻部症状评分、鼻黏膜中嗜酸性粒细胞数目、血清IL-4含量显著升高(P<0.05),血清中INF-γ含量显著降低(P<0.05);与B组比较,C、D两组鼻部症状评分、鼻黏膜中嗜酸性粒细胞数目、血清IL-4含量显著降低(P<0.05),血清中INF-γ含量显著升高(P<0.05),且D组比C组变化显著(P<0.05);A组大鼠鼻黏膜组织结构完整,B组鼻黏膜上皮结构紊乱且不完整,出血及嗜酸性粒细胞等炎性细胞浸润;与B组相比,C、D组病理状态明显改善,嗜酸性粒细胞明显减少;与A组比较,B组鼻黏膜组织中TLR9、AP-1蛋白表达显著升高(P<0.05),与B组比较,C、D组鼻黏膜组织中TLR9、AP-1蛋白表达显著降低(P<0.05),且D组比C组变化显著(P<0.05),而E组与D组相比无明显差异(P>0.05),F组比E组降低明显(P<0.05)。结论枸杞多糖可有效调控变应性鼻炎大鼠Th1/Th2细胞因子水平、减轻鼻黏膜中嗜酸性粒细胞炎症,其机制可能与抑制TLR9/AP-1信号通路有关。

Objective To explore the effect of lycium barbarum polysaccharide on Th1/Th2 cytokines and eosinophil inflammation in nasal mucosa of allergic rhinitis(AR)rats by regulating Toll-like receptor 9/activated protein-1(TLR9/AP-1)signaling pathway.Methods Sixty SPF male SD rats were randomly divided into normal(A)group,model(B)group,mometasone furoate(C)group,lycium barbarum polysaccharide(D)group,TLR9/AP-1 signaling pathway inhibitor(E)group and TLR9/AP-1 signaling pathway inhibitor combined lycium barbarum polysaccharide(F)group,with 10 rats in each group.AR model was established by ovalbumin sensitization method in all groups except group A.After successful establishment of AR model,one spray/side of mometasone furoate nasal spray was adopted intranasally in group C,100 mg/kg lycium barbarum polysaccharide,20 mg/kg AP-1 inhibitor SP100030,20 mg/kg SP100030+100 mg/kg lycium barbarum polysaccharide were administered intragastrically in group D,E and F respectively.In groups A and B,normal saline of the same volume was administered intragastrically.The nasal symptoms were scored,and HE staining was used to detect the pathological morphology of nasal mucosa with eosinophils count.Serum levels Th1/Th2 of cytokines were detected by enzyme-linked immuno sorbent assay.The expression of TLR9/AP-1 signaling pathway-related proteins in nasal mucosa was detected by western blot.Results Compared with group A,the nasal symptom score,the number of eosinophils in nasal mucosa and the serum level of interleukin(IL)-4 in group B were significantly increased with decreased serum level of interferon(INF)-γ(all P<0.05).Compared with group B,the score of nasal symptoms,the number of eosinophils in nasal mucosa and the serum level of IL-4 in groups C and D were significantly decreased with increased serum level of INF-γ(all P<0.05),and the changes in group D were more significant than those in group C(all P<0.05).The structure of nasal mucosa from group A kept intact,while the epithelial structure of nasal mucosa from group B was disordered and incomplete with bleeding and infiltration of inflammatory cells such as eosinophils.Compared with group B,the pathological statuses of groups C and D were significantly improved with decreased eosinophils.The protein expressions of TLR9 and AP-1 in nasal mucosa tissues of group B were significantly higher than those of group A(both P<0.05),and their expressions in groups C and D were significantly decreased compared with group B(all P<0.05),and the changes in group D were more significant than those in group C(P<0.05).The differences between the protein expressions of TLR9 and AP-1 of group E and group D were statistically insignificant(both P>0.05),and the expressions in group F were significantly lower than those in group E(both P<0.05).Conclusion Lycium barbarum polysaccharide can effectively regulate Th1/Th2 cytokine level and relieve eosinophil inflammation in nasal mucosa in rats with AR,and the mechanism may be related to the inhibition of TLR9/AP-1 signaling pathway.