山蜡梅叶颗粒对LPS诱导RAW264.7细胞炎症反应及变应性鼻炎小鼠炎症的影响

Effects of Folium Chimonanthi Nitentis Granules on the Inflammation of RAW264.7 Cells Induced by LPS and the Inflammation in Mice with Allergic Rhinitis

目的:探讨山蜡梅叶颗粒对脂多糖(LPS)诱导RAW264.7细胞炎症反应的抑制作用及卵清蛋白(OVA)诱导小鼠的变应性鼻炎(Allergic Rhinitis, AR)的治疗作用。方法:以LPS与IFN-γ联合刺激RAW264.7细胞诱导炎症模型。采用MTT法比较细胞活力;采用比色法(Griess)比较细胞上清液中一氧化氮含量的变化;采用ELISA法比较细胞上清液中肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)含量的变化。将SPF级BALB/c小鼠随机分成正常对照组、模型对照组、鼻炎灵片0.5 g/kg组、山蜡梅叶颗粒3.9、7.8、15.6 g/kg组。除正常对照组外,其余各组利用OVA建立小鼠变应性鼻炎模型。造模期间观察小鼠变应性鼻炎行为学指标,给药结束后取材,使用吉姆萨染色测定小鼠全血中嗜酸粒细胞的数目;HE染色比较小鼠鼻黏膜的形态结构差异;ELISA法检测小鼠血清中免疫球蛋白IgE含量及小鼠鼻腔灌洗液中TNF-α及IL-6的含量。结果:在LPS诱导RAW264.7细胞的炎症反应试验中,与空白对照组比较,模型对照组RAW264.7细胞NO含量,炎症因子TNF-α和IL-6含量显著升高(P<0.01);与模型对照组相比,山蜡梅叶颗粒0.25、0.5、1 mg/mL组均能显著抑制NO分泌,降低TNF-α和IL-6的含量(P<0.01)。在OVA诱导的小鼠变应性鼻炎的研究中,与正常对照组比较,模型对照组小鼠行为学评分、嗜酸性粒细胞数、血清中IgE含量、鼻腔灌洗液中TNF-α和IL-6含量明显升高(P<0.05或P<0.01);与模型对照组相比,山蜡梅叶颗粒各剂量组给药后小鼠行为学评分、嗜酸性粒细胞数、血清中IgE含量、鼻腔灌洗液中TNF-α和IL-6含量明显降低(P<0.05或P<0.01),明显改善OVA模型小鼠鼻黏膜充血、水肿、炎细胞浸润状态。结论:山蜡梅叶颗粒对变应性鼻炎小鼠的炎症反应起到了明显的抑制作用,且这种作用可能与其抑制鼻炎发生过程中促炎因子的释放有关。

Objective:The purpose of the research was to investigate the inhibitory effect of folium chimonanthi nitentis granules on lipopolysaccharides(LPS)-induced inflammation in RAW264.7 cells and the therapeutic effect on ovalbumin(OVA)-induced allergic rhinitis(AR)in mice.Methods:LPS and IFN-γwere used to induce inflammation model in RAW264.7 cells.MTT assay was used to determine the cell viability and Griess assay was used to detect the Nitric Oxide(NO)level in the cell culture supernatant.Enzyme-linked immunosorbent assay(ELISA)was applied to test the levels of Tumor Necrosis Factor-α(TNF-α)and Interleukin-6(IL-6)in the cell culture supernatant.SPFgrade BALB/c mice were randomly divided into the blank control group,the model group,the Biyanling tablet 0.475 g/kg group,and folium chimonanthi nitentis granules groups of 3.9 g/kg,7.8 g/kg and 15.6 g/kg doses.Except for control group,Ovalbumin(OVA)was used in remaining groups to establish a mice model of AR.Behavioral indicators of AR in mice were detected during modeling.After the last administration,samples were collected,and the number of eosinophils in the whole blood of mice were determined by Giemsa staining.Meanwhile,the morphological and structural differences of the nasal mucosa of mice in different groups were observed by HE staining.The serum content of IgE and the expressions of inflammatory factors TNF-αand IL-6 in the nasal lavage fluid of mice were detected by ELISA method.Results:In the LPS-induced inflammatory response experiment of RAW264.7 cells,compared to the blank control group,the NO content and the levels of inflammatory factor TNF-αand IL-6 were significantly increased in the model group(P<0.01).Compared with the model group,excessive expression of NO and the levels of TNF-αand IL-6 were significantly reduced in all doses of folium chimonanthi nitentis granules groups.In the study of OVA-induced AR in mice,compared with blank control group,the behavioral scores,eosinophil counts,the serum levels of IgE,TNF-αand IL-6 in nasal lavage fluid were significantly increased in the model group(P<0.05 or P<0.01).Compared with the model group,the behavioral scores,eosinophil count,the serum levels of IgE,TNF-αand IL-6 in nasal lavage fluid were significantly decreased in each dose group of folium chimonanthi nitentis granules(P<0.05 or P<0.01).In addition,the congestion,swelling and inflammatory cells infiltration of nasal mucosa was improved in OVA-induced AR mice by the granules.Conclusion:Folium chimonanthi nitentis granules has significantly inhibitory effect on the inflammatory response of mice with AR,which may be related to the inhibition of the expression of pro-inflammatory factors in the development of AR.