中南药学

目的 基于肺炎支原体毒素CARDS TX S1亚单位介导的G蛋白偶联受体缓激肽受体B2 (BDKRB2)核糖基化,探究汉黄芩素缓解肺炎支原体肺炎咳嗽的机制。方法 CARDS TX S1蛋白结合在CM5芯片上,Biacore垂钓芩百清肺浓缩丸中的活性成分,超高效液相色谱-质谱联用(UPLC-Q-TOF-MS)技术对活性成分进行分析。运用Tripos Sybyl-X2.1.1和Discovery Studio 2016软件,进行药物分子与受体蛋白结合模式的可视化分析。将Balb/c小鼠随机分为空白组、模型组、阿奇霉素组、枸橼酸喷托维林组以及汉黄芩素高、中、低(1.4854×10^-4、7.427×10^-5、3.714×10^-5g/kg)剂量组,每组10只。除空白组外,其余各组建立肺炎支原体感染模型后,分别给小鼠灌胃相应药物7 d,末次给药结束后进行引咳实验,并处死小鼠取血液与肺组织。光学显微镜下观察各组小鼠肺组织苏木精-伊红(HE)染色、免疫组化后的肺组织变化;qRT-PCR法检测各组小鼠肺组织BDKRB2表达量。结果 Biacore垂钓得到的样品经UPLC-Q-TOF-MS鉴定分析为汉黄芩素。汉黄芩素与CARDS TX S1存在较强的特异性结合。与模型组相比,汉黄芩素各剂量组可显著降低肺炎支原体肺炎小鼠咳嗽次数,小鼠肺组织结构明显得到改善,结构清晰,组织形态规整;小鼠肺组织BDKRB2mRNA和蛋白表达极显著降低。结论 汉黄芩素通过降低肺组织中CARDS TX S1、BDKRB2的表达,减缓咳嗽症状,治疗肺炎支原体肺炎。

Objective To determine the mechanism of wogonin for Mycoplasma pneumoniaee-induced cough based on the ribosylation of G protein-coupled receptor BDKRB2 mediated by the CARDS TX S1subunit of Mycoplasma pneumoniae toxin.Methods The CARDS TX S1 protein was immobilized on a CM5 chip,and the active components of Qinbai Qingfei concentrated pills were screened by the Biacore fishing.Ultra-high performance liquid chromatography-quadrupole-time of flight-mass spectrometry(UPLC-Q-TOF-MS) was used to analyze the active ingredients.Tripos Sybyl-X2.1.1 and Discovery Studio 2016 software were used to visualize drug molecule-receptor protein binding modes.Balb/c mice were randomly divided into a black group,a model group,an azithromycin group,an pentoxyverine citrate group,and high,medium,and low doses of wogonin at 1.4854 × 10^-4,7.427 × 10^-5,and3.714 × 10^-5g/kg,with 10 mice in each group.Except for the blank group,Mycoplasma pneumoniae infection models were established in the other groups.The mice were administered corresponding drugs by gavage for 7 days.After the final dose,a cough induction test was conducted,and the blood and lung tissues were collected.The lung tissues were examined under a microscope after hematoxylin-eosin(HE) staining and immunohistochemistry.The expression levels of BDKRB2 in the lung tissue were detected by qRT-PCR.Results The samples obtained by Biacore fishing were identified as wogonin by UPLC-Q-TOF-MS.Wogonin had a strong specific binding to CARDS TX S1.Compared with the model group,each dose group of wogonin markedly reduced the number of coughs in mice with Mycoplasma pneumoniae.The lung tissue structure of mice in each wogonin group was obviously improved,with clear structure and regular tissue morphology.The expression of BDKRB2 mRNA and protein in the lung tissues of mice in each wogonin group was significantly decreased.Conclusion Wogonin alleviates cough symptoms and treats Mycoplasma pneumoniae by reducing the expression of CARDS TX S1 and BDKRB2 in the lung tissue.