Data Sheet 1_The efficacy of ophiopogonanone B in treating the cough in mice infected with Mycoplasma pneumoniae.docx

IntroductionOphiopogonanone B is a potent component of Qinbai Qingfei-concentrated pills (Qinbai), a new traditional Chinese medicine developed by our hospital for the treatment of Mycoplasma pneumoniae pneumonia in children. We aim to study how ophiopogonanone B influences the expression of transient receptor potential anchor protein 1 (TRPA1), substance P (SP), and calcitonin gene-related peptide (CGRP) to treat coughing in MP-infected mice.
MethodsUltra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was used to detect ophiopogonanone B. Molecular docking of ophiopogonanone B with TRPA1 was performed using Autodock Vina 1.1.2, and subsequent visualization and analysis of docking outcomes were facilitated using Pymol 2.1 and Discovery Studio. For the evaluation of the pathological structure and morphology, lung tissue sections from mice were prepared for animal experiments and subjected to hematoxylin-eosin (HE) and Masson staining. The impact of ophiopogonanone B on the protein and mRNA expression levels of TRPA1, SP, and CGRP in mouse lung tissue was assessed using immunohistochemistry and real-time polymerase chain reaction (RT-PCR).
ResultsThe samples acquired through Biacore fishing, which were identified and analyzed by UPLC-Q-TOF-MS, confirmed the presence of ophiopogonanone B. This compound exhibited robust and specific binding affinity for TRPA1. Histological staining using HE and Masson techniques revealed that the lung tissue morphology and structure in the ophiopogonanone B-treated group closely mirrored those observed in the blank group. Subsequent immunohistochemistry and RT-PCR revealed a significant reduction (P < 0.01 or P < 0.05) in the proteins and mRNA expression levels of TRPA1, SP, and CGRP in the lung tissue of mice treated with high and medium doses of ophiopogonanone B.
ConclusionBy decreasing the expression of TRPA1, SP, and CGRP in the lung tissues of mice afflicted with coughing due to M. pneumoniae infection, ophiopogonanone B effectively alleviated post-infection cough symptoms.

引言
麦冬酮B（Ophiopogonanone B）是我院研发的新型中药制剂芩白清肺浓缩丸（Qinbai）的活性成分，该制剂用于治疗儿童肺炎支原体肺炎。本研究旨在探讨麦冬酮B通过调控瞬时受体电位锚蛋白1（TRPA1）、P物质（SP）及降钙素基因相关肽（CGRP）的表达，对肺炎支原体感染小鼠咳嗽症状的治疗作用。
方法
采用超高效液相色谱-四极杆飞行时间质谱联用技术（UPLC-Q-TOF-MS）检测麦冬酮B。使用Autodock Vina 1.1.2开展麦冬酮B与TRPA1的分子对接实验，并借助Pymol 2.1与Discovery Studio软件完成对接结果的可视化分析与数据处理。为评估肺组织病理结构与形态，本研究制备小鼠肺组织切片用于动物实验，并开展苏木精-伊红（HE）染色与马松（Masson）染色。采用免疫组化法与实时聚合酶链式反应（RT-PCR）检测麦冬酮B对小鼠肺组织中TRPA1、SP及CGRP的蛋白与mRNA表达水平的影响。
结果
经Biacore亲和捕获获取的样本经UPLC-Q-TOF-MS鉴定分析，确证含有麦冬酮B。该化合物对TRPA1展现出强效且特异性的结合亲和力。HE与马松染色结果显示，麦冬酮B给药组小鼠的肺组织形态与结构与空白组高度相近。后续免疫组化与RT-PCR检测结果表明，中、高剂量麦冬酮B给药组小鼠肺组织中TRPA1、SP及CGRP的蛋白与mRNA表达水平均显著降低（P < 0.01或P < 0.05）。
结论
麦冬酮B可通过下调肺炎支原体感染致咳嗽小鼠肺组织中TRPA1、SP及CGRP的表达，有效缓解感染后咳嗽症状。