摘要
为探讨升降平喘止咳丸干预支气管哮喘的作用机制,该研究采用蛋白质组学技术分析其对卵清蛋白(OVA)诱导支气管哮喘大鼠的关键蛋白和分子机制,并对核心差异蛋白进行实验验证。选取60只SPF级大鼠,分为空白组、造模组,用OVA和氢氧化铝混合溶液腹腔注射和雾化激发的方法造模,造模成功后,分为空白组,模型组,阳性对照组,升降平喘止咳丸低、中、高剂量组,每组10只,对各组大鼠进行一般状态的观察;苏木素-伊红(HE)染色观察肺组织病理变化;PAS染色观察黏液分泌、基底膜的破坏和增厚;通过蛋白质组学分析差异表达蛋白和分子机制。酶联免疫吸附测定(ELISA)检测肺泡灌洗液(BALF)中细胞因子的含量。免疫荧光定位杯状细胞的位置和核因子κB(NF-κB)发生核转位,同时用蛋白免疫印迹(Western blot)验证通路和关键蛋白表达水平。与模型组比较,升降平喘止咳丸干预后喘息伴哮鸣音、咳嗽次数减少,毛发光泽度好转,精神状态改善,活动量增加,呼吸频次趋于平稳,对外界的刺激反应度及灵活度提高,体质量呈增长趋势;病理染色发现,干预后哮喘大鼠炎症浸润减轻,气道平滑肌增厚减轻,黏液分泌减少,基底膜的破坏改善,炎症评分降低,PAS评分降低;ELISA结果显示,升降平喘止咳丸高剂量组白细胞介素-4(IL-4)、白细胞介素-5(IL-5)、白细胞介素-13(IL-13)、白细胞介素-17(IL-17)的表达水平下降,干扰素(INF)-γ表达水平上升;免疫荧光结果显示,升降平喘止咳丸高剂量组杯状细胞减少,黏液分泌减少,核内的磷酸化NF-κB(p-NF-κB)荧光减弱,部分分布由核转至胞质中。Western blot结果表明,升降平喘止咳丸高剂量组下调组织蛋白酶S(CTSS)、黏蛋白5AC(MUC5AC)、p-NF-κB/NF-κB蛋白表达,上调低亲和力免疫球蛋白γFc区受体Ⅱ-b(FCGR2B)蛋白表达。综上,升降平喘止咳丸通过抑制NF-κB通路减轻OVA诱导的气道炎症,杯状细胞化生及黏液分泌,可能与下调CTSS,上调FCGR2B蛋白表达,进而调节Th1/Th2/Th17细胞免疫平衡有关。
To explore the mechanism of action of Shengjiang Pingchuan Zhike Pills in the treatment of bronchial asthma,this study used proteomics technology to analyze the key proteins and molecular mechanisms of the ovalbumin(OVA)-induced bronchial asthma in rats and experimentally verified the core differential proteins.Sixty specific-pathogen free(SPF)rats were selected and divided into a blank group and a model group.The model was established by intraperitoneal injection and nebulization of OVA and aluminum hydroxide mixed solution.After successful modeling,the rats were divided into a blank group,a model group,a positive control group,and low-dose,medium-dose,and high-dose groups of Shengjiang Pingchuan Zhike Pills,with 10 rats in each group.The general conditions of the rats in each group were observed.The pathological changes of lung tissue were observed by hematoxylin-eosin(HE)staining,and mucus secretion,as well as basement membrane destruction and thickening were examined by periodic acid-Schiff(PAS)staining.Differentially expressed proteins and molecular mechanisms were analyzed by proteomics.The content of cytokines in bronchoalveolar lavage fluid(BALF)was verified by enzyme-linked immunosorbent assay(ELISA).The location of goblet cells and nuclear translocation of nuclear factor-κB(NF-κB)were measured by immunofluorescence,and the pathways and expression level of key proteins were verified by Western blot.Compared with those of the model group,after the intervention of Shengjiang Pingchuan Zhike Pills,the number of wheezing,stridor,and coughing times decreased;the hair luster and mental state improved;the activity increased;the breathing frequency tended to be stable;the response to external stimuli and flexibility increased,and the body weight showed an increasing trend.Pathological staining showed that after the intervention,the inflammatory infiltration of asthma rats was reduced;the thickening of airway smooth muscle was alleviated;the mucus secretion was decreased;the destruction of the basement membrane was ameliorated,and the inflammatory score and PAS score were decreased.ELISA results indicated that the expression level of interleukin-4(IL-4),interleukin-5(IL-5),interleukin-13(IL-13),and interleukin-17(IL-17)in the high-dose group decreased,and the expression level of interferon(INF)-γincreased.Immunofluorescence results showed that the number of goblet cells and mucus secretion in the high-dose group decreased;the fluorescence of phospho-NF-κB(p-NF-κB)in the nucleus weakened,and some were distributed from the nucleus to the cytoplasm.Western blot results showed that the high-dose group exhibited the down-regulated expression of cathepsin S(CTSS),mucin 5AC(MUC5AC),and p-NF-κB/NF-κB proteins and the up-regulated expression of low-affinity immunoglobulinγFc region receptorⅡ-b(FCGR2B)protein.In conclusion,Shengjiang Pingchuan Zhike Pills may alleviate OVA-induced airway inflammation,goblet cell metaplasia,and mucus secretion by inhibiting the NF-κB pathway,which may be related to the down-regulation of CTSS and up-regulation of FCGR2B protein expression,thereby regulating the immune balance of Th1/Th2/Th17 cells.
作者
吴桐
何腾飞
王成娟
白敏
赵思超
鲁宝堂
闫文瑞
张常喜
WU Tong;HE Teng-fei;WANG Cheng-juan;BAI Min;ZHAO Si-chao;LU Bao-tang;YAN Wen-rui;ZHANG Chang-xi(College of Traditional Chinese Medicine,Ningxia Medical University,Yinchuan 750004,China;Key Laboratory of Ningxia Ethnomedicine Modernization,Ministry of Education,Yinchuan 750004,China;Ningxia Hui Autonomous Region Hospital of Traditional Chinese Medicine,Yinchuan 750021,China)
出处
《中国中药杂志》
北大核心
2025年第22期6461-6469,共9页
China Journal of Chinese Materia Medica
基金
宁夏回族自治区科技领军人才培养项目(2023GKLRLX18)
自治区重点研发项目(2024BEG02023)
宁夏自然科学基金项目(2024AAC03730)。
