白花前胡经NF-κB/IL-6/PXR网络通路发挥对氨茶碱的减毒增效作用及相关机制研究

Aminophylline is the most commonly used anti-asthma drug, mainly metabolized by CYP3A4. When inflammation occurs, IL-6 and NF-κB can be activated, which influence the expression and activity of Cytochrome P450 CYP3A4, and eventually lead to the metabolism reduction and accumulation of theophylline in asthma inflammation, which would induce serious adverse reaction. The anti-inflammatory, anti-tussive and anti-asthma pharmacological effects of Peucedanum praeruptorum(bai-hua qian-hu) are closely related to the NF-κB-IL-6-inflammatory pathway. According to our previous results, bai-hua qian-hu can significantly up-regulate CYP3A4 expression via the pregnane X receptor- mediated pathway. Based on the above, the project aims to clarify that the co-administration of bai-hua qian-hu and theophylline can reverse the theophylline accumulation inhibition and toxicity through the interaction of NF-κB/IL-6-PXR pathway, and performs a synergistic and attenuated synergistic antiasthmatic effect. We are observing the effect of bai-hua qian-hu active compounds on CYP3A4 expression through NF-κB-PXR signaling pathway by using over-expression of NF-κB cells model with wild type or loss of PXR expression. Qianhu is administered after IL-6 incubation on PXR wild type and PXR cell model to find the transcriptional regulation of IL-6-PXR-drug metabolize enzymes by bai-hua qian-hu. Co-administration aminophylline with bai-hua qian-hu on asthmatic animal model is to investigate the pharmacokinetics characteristics and pharmacodynamics effects of asthma of aminophylline. Accordingly, the study is to find the detoxification efficiency and synergism mechanism when theophylline is co- administered with bai-hua qian-hu, and to illustrate the detoxification efficiency and synergism mechanism of bai-hua qian-hu co-treatment with theophylline in asthma through the cytokines-PXR signaling pathway.

氨茶碱是临床最常用的平喘药，主要经CYP3A4代谢。但在炎症状态下，NF-κB、IL-6过表达能抑制CYP3A4活性，导致氨茶碱因代谢抑制而造成蓄积毒性。中药白花前胡可通过抑制NF-κB、IL-6 等炎症相关因子发挥抗炎、镇咳、平喘作用；我们前期研究成果还表明，白花前胡活性成分可通过PXR等核受体诱导CYP3A4的表达及活性。基于此，我们提出白花前胡经NF-κB/IL-6/PXR网络通路发挥对氨茶碱的减毒增效作用的科学假设。本项目建立BN哮喘大鼠动物模型，证明白花前胡活性成分与氨茶碱合用减毒增效的药动学与药效学行为，应用NF-κB高表达细胞系等体外模型观察在PXR 野生型或PXR表达缺失时白花前胡活性成分经IL-6/NF-κB/PXR网络信号通路对CYP3A影响。在分子、细胞和动物水平阐明白花前胡对氨茶碱的减毒增效作用及机制，最终为白花前胡与氨茶碱的临床合理联用提供科学证据。

本项目拟阐明白花前胡活性成分与茶碱合用可通过炎症因子-核受体PXR信号通路相互作用，从而发挥增效减毒协同平喘的作用。本项目建立大鼠哮喘模型，并采用白花前胡甲素（PA）、丙素（PC）及戊素（PE）分别与氨茶碱联合给药后对哮喘大鼠进行肺泡灌洗，采用吉姆萨染色法计数了肺内细胞总数、细胞分类及数量；HE染色观察联合给药后对哮喘大鼠肺部及气管炎症变化的影响，免疫组化法观察联合给药后哮喘大鼠肺部IL-6、NF-κB的表达情况；ELISA检测观察哮喘大鼠血浆中细胞因子 IL-6、NF-κB以及药物代谢酶 CYP3A1的酶的含量确定PA、PC及PE与氨茶碱联合给药后对氨茶碱治疗哮喘的药效学增效作用。采用 LC-MS/MS 质谱技术观察PA、PC及PE分别与氨茶碱联合给药后在哮喘大鼠体内对茶碱代谢清除的影响。同时采用Q-PCR、Western blotting检测对哮喘疾病状态下大鼠肝脏PXR、IL-6、NF-κB、CYP3A1、酶表达的变化，及检测PA、PC和PE单独给药对哮喘大鼠肝脏的PXR、IL-6、NF-κB、CYP3A1、MDR-1及P-gp表达的影响。本项目采用OVA和氢氧化铝凝胶联合致敏法建立大鼠模型，HE染色切片证明大鼠哮喘模型成功建立。联合给药组和氨茶碱单独给药组与模型组相比均能减少哮喘大鼠肺内细胞总数和嗜酸性粒细胞数量。PA、PC及PE与氨茶碱联合给药比单用氨茶碱组能显著降低哮喘大鼠体内嗜酸性粒细胞、IL-6和NF-κB。哮喘大鼠体内PXR及CYP3A1的mRNA及蛋白表达水平较空白组显著升高，PA、PC单用组较模型组PXR及CYP3A1显著降低。提示哮喘发生时大鼠体内PXR及CYP3A1表达水平增加。课题现有研究结果表明，白花前胡活性成分PA、PC及PE与氨茶碱合用比单用氨茶碱具有更为显著抑制哮喘症状及炎症因子IL-6和NF-κB的作用，且发现在哮喘发生时核受体及药物代谢酶的表达显著升高，但基于IL-6、PXR调控药物代谢酶CYP3A4表达及哮喘时PXR、CYP3A4表达变化特征的分子作用机制研究尚在进行中。本课题的实施可为白花前胡作为氨茶碱治疗哮喘的增效减毒剂，阐明哮喘时药物代谢酶CYP4A4及PXR核受体的变化特征提供理论依据。