吴茱萸碱通过调控Notch通路抑制Skp2的表达对哮喘小鼠气道重塑和炎症反应的影响

Eff ects of evodialine on the airway remodeling and infl ammatory reactions in mice with asthma by regulating the Notch pathway to inhibit the expression of Skp2

目的 探究吴茱萸碱对哮喘小鼠的气道重塑和炎症反应的影响及其相关机制。方法 48只BALB/c小鼠按随机数字法分为正常对照组、模型组、吴茱萸碱组和吴茱萸碱处理的过表达Notch信号通路抑制剂Kyo T2的腺病毒载体干预组,每组12只。采用卵清蛋白(OVA)诱导建立小鼠哮喘模型,吴茱萸碱组和吴茱萸碱加Kyo T2腺病毒载体干预组以30 mg/kg的吴茱萸碱溶于生理盐水灌胃,其中,吴茱萸碱加Kyo T2腺病毒载体干预组经鼻滴入5×108pfu的Kyo T2腺病毒干预,其余组灌胃等量的生理盐水。末次激发24 h后,收集支气管肺泡灌洗液(BALF),进行细胞计数;ELISA法检测炎性因子TNF-α、IL-1β、IL-4和OVA特异性IgE和IgG1;过碘酸雪夫(PAS)染色法检测气道杯状细胞增生;Masson染色法观察肺组织胶原沉积现象;免疫组化法检测α-SMA的表达;Western blot法检测α-SMA、Hes1、Skp2蛋白的表达。结果 与对照组相比,模型组小鼠BALF中总细胞和嗜酸性粒细胞、巨噬细胞、淋巴细胞数均显著升高(P <0.05),TNF-α、IL-1β、IL-4因子水平上升(P <0.05),α-SMA、Hes1和Skp2的表达上调(P <0.05)。与模型组相比,吴茱萸碱组和吴茱萸碱加Kyo T2腺病毒载体干预组BALF中总细胞和嗜酸性粒细胞、巨噬细胞、淋巴细胞数显著降低(P <0.05),TNF-α、IL-1β、IL-4因子水平下调(P<0.05),α-SMA、Hes1和Skp2的表达降低(P <0.05),气道炎症得到明显改善;与吴茱萸碱组相比,吴茱萸碱加Kyo T2腺病毒载体干预组炎性因子显著降低,通路蛋白下调,气道炎症改善更为显著(P <0.05)。结论吴茱萸碱对哮喘小鼠气道重塑和炎症反应的改善,可能与Notch通路抑制Skp2的表达相关。

Objective To investigate the eff ects of evodialine on the airway remodeling and infl ammatory reactions in mice with asthma and its related mechanisms.Methods A total of 48 BALB/c mice were randomly divided into a normal control group,a model group,an evodialine group,and a target adenovirus vector intervention group with evodialine,which overexpressed the Notch signaling pathway inhibitor KyoT2,with 12 mice in each group.A mouse asthma model was induced by ovalbumin(OVA).The evodialine group and the evodialine+KyoT2(Notch signal inhibitor)adenovirus vector intervention group were given 30 mg/kg evodialine dissolved in normal saline by gavage.In the intervention group of evodiamine+KyoT2 adenovirus vector,5×108 pfu of KyoT2 adenovirus was instilled through the nose for intervention,and the other groups were intragastrically administered with the same amount of normal saline.Twenty-four hours after the last challenge,the bronchoalveolar lavage fluid(BALF)was collected for cell count,the inflammatory factors TNF-α,IL-1β,IL-4 and OVA specific IgE and IgG1 were detected by ELISA,the lung tissue histopathology changes were detected by HE staining,the airway goblet cell hyperplasia was detected by periodic acid-Schiff staining(PAS),the lung tissue collagen deposition was observed with the Masson staining method,the expression ofα-SMA was detected with the immunohistochemical method,and the protein expressions ofα-SMA,Hes1,Skp2 was detected by the Western blot method.Results Compared with the control group,the total number of BALF cells,eosinophils,macrophages,and lymphocytes in the model group increased signifi cantly(P<0.05),the levels of TNF-α,IL-1βand IL-4 factors increased(P<0.05),the expressions ofα-SMA,Hes1 and Skp2 were up-regulated(P<0.05),and the airway inflammation was significantly improved.Compared with the model group,the total number of BALF cells,eosinophils,macrophages,and lymphocytes in the evodialine group and the evodialine+KyoT2 adenoviral vector intervention group were significantly reduced(P<0.05),the levels of TNF-α,IL-1βand IL-4 factors were down-regulated(P<0.05),the expressions ofα-SMA,Hes1 and Skp2 were decreased(P<0.05),and the airway inflammation was improved significantly.Compared with the evodialine group,the inflammatory factors were significantly reduced,the pathway proteins were down-regulated,and the airway inflammation was improved more significantly in the evodialine+KyoT2 adenoviral vector intervention group(P<0.05).Conclusion Evodiamine can improve the airway remodeling and infl ammatory reactions in mice with asthma,which may be related to the inhibition of Skp2 expression by the Notch pathway.