基于网络药理学方法、分子对接技术及动物实验验证探讨通塞颗粒治疗慢性阻塞性肺疾病急性加重期的作用机制

目的：基于网络药理学方法、分子对接技术和动物实验验证探讨通塞颗粒治疗慢性阻塞性肺疾病急性加重期（AECOPD） 的作用机制。方法：①预测分析。通过中药系统药理学分析平台筛选通塞颗粒的活性成分及其对应靶点；利用人类基因（Genecards）、在线人类孟德尔遗传数据系统（OMIM） 等数据库检索AECOPD 疾病基因；绘制韦恩图并获取药物-疾病共同靶点；构建通塞颗粒活性成分-靶点网络图和蛋白质相互作用网络图，根据网络关系筛选核心靶点；借助R 语言软件进行基因本体（GO） 富集分析和京都基因与基因组百科全书（KEGG） 通路富集分析，使用AutoDockTool 1.5.6 软件对通塞颗粒关键活性成分和核心靶点进行分子对接。②实验验证。随机将50 只SD 大鼠分为对照组、模型组、中药组、西药组、联合组各10 只。除对照组外，其余各组均构建AECOPD 大鼠模型，并于急性加重前2 d 及其后4 d 予药物灌胃治疗。治疗结束后检测各组大鼠的肺功能指标[0.3 s 用力呼气容积（FEV0.3）、用力肺活量（FVC）、0.3 s 用力呼气容积与用力肺活量的比值（FEV0.3/FVC）、呼气流量峰值（PEF） ]，检测血清肿瘤坏死因子（TNF） -α、白细胞介素（IL） -17含量，通过苏木精-伊红染色法（HE） 观察气道与肺组织的病理改变，采用免疫印迹法和酶联免疫吸附测定法初步验证网络药理学的预测结果。结果：通塞颗粒治疗AECOPD 的关键活性成分为槲皮素、芹黄素、木犀草素、山奈酚、β-谷甾醇，主要作用于肿瘤抑制蛋白53（TP53）、丝裂原活化蛋白激酶3（MAPK3） 等核心靶点。GO 富集分析和KEGG 通路富集分析结果表明，通塞颗粒治疗AECOPD 与TNF、IL-17 等信号通路干预氧化应激、减少细胞凋亡、抑制炎症反应有关。分子对接结果显示，通塞颗粒的关键活性成分与治疗AECOPD的核心靶点均有较好的结合活性。动物实验研究结果显示，模型组FVC、FEV0.3、FEV0.3/FVC、PEF 值均较对照组降低（P＜0.05）。中药组、西药组以及联合组的FVC、FEV0.3、PEF 值均较模型组升高（P＜0.05）；联合组FEV0.3/FVC 值较模型组升高（P＜0.05）。中药组FVC、FEV0.3、FEV0.3/FVC、PEF 值与西药组比较，差异均无统计学意义（P＞0.05）。联合组FVC、PEF 值均较中药组及西药组升高（P＜0.05）；联合组FEV0.3 较西药组升高（P＜0.05）。模型组血清TNF-α、IL-17 含量均高于对照组（P＜0.05）；中药组、西药组及联合组的TNF-α、IL-17 含量均低于模型组（P＜0.05）；联合组血清TNF-α、IL-17 含量均低于中药组、西药组（P＜0.05）。模型组肺组织磷酸化P65/非磷酸化P65、磷酸化AKT/非磷酸化AKT、磷酸化MAPK1/3/非磷酸化MAPK1/3 和非磷酸化TP53/GAPDH 蛋白表达含量均高于对照组（P＜0.05）；中药组、西药组以及联合组上述蛋白表达含量均低于模型组（P＜0.05）；联合组上述蛋白表达含量均低于中药组、西药组（P＜0.05）。结论：通塞颗粒治疗AECOPD 具有多成分、多靶点、多通路、整体调节的作用特点，为后续深入研究提供了数据支持。

Mechanism of Action of Tongsai Granules in the Treatment of Acute Exacerbation ofCOPD Based on Network Pharmacology，Molecular Docking Technology，and Experi⁃mental Validation in Rats

Abstract：Objective：To explore the mechanism of action of Tongsai Granules (TSG) in the treatment ofacute exacerbation of chronic obstructive pulmonary disease (AECOPD) based on network pharmacology，molecular docking technology， and animal experiments. Methods： ① Predictive analysis. The activeingredients and corresponding targets of TSG were screened by the Traditional Chinese Medicine SystemsPharmacology Database and Analysis Platform. AECOPD-related genes were retrieved by GeneCards，OMIM， and other databases. Venn diagrams were drawn to obtain intersecting drug-disease targets；active ingredient of TSG-intersecting target network and a protein-protein interaction network wasconstructed and core targets were screened out based on network relationships；R programming languagesoftware was used for Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes andGenomes (KEGG) enrichment analysis. Molecular docking of key active ingredients of TSG and core targetswas performed by using AutoDockTool 1.5.6 software. ②Experimental verification. Fifty rats were randomlydivided into control group, model group, Chinese medicine group, western medicine group, and combinationgroup, with 10 rats in each group. The AECOPD rat model was constructed in all groups except the controlgroup. And received drug gavage 2 days before and 4 days after the acute exacerbation. After treatment，the lung function indicators，including forced expiratory volume in 0.3 seconds (FEV0.3)，force vital capacity(FVC)，the ratio of forced expiratory volume in 0.3 seconds to forced vital capacity (FEV0.3 /FVC)，and peakexpiratory flow (PEF)， were measured in each group. The contents of tumor necrosis factor (TNF) - α andinterleukin (IL) - 7 in serum were measured； the pathological changes in airway and lung tissues wereobserved by using hematoxylin-eosin (HE) staining and the network pharmacological prediction results werevalidated by western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). Results： In thetreatment of AECOPD，the key active ingredients of TSG were quercetin，apigenin，luteolin，kaempferol，and β - sitosterol， which mainly act on tumor suppressor protein 53 (TP53)， mitogen-activated proteinkinase 3 (MAPK3)， and other core targets. GO and KEGG analyses showed that in AECOPD treatment，TSG was related to TNF， IL-17， and other signaling pathways related to intervening oxidative stress，reducing apoptosis， and inhibiting inflammation. Molecular docking analysis showed that the keyingredients of TSG had good binding activity to core targets for the treatment of AECOPD. The results ofanimal experimental research showed that the FVC， FEV0.3， FEV0.3 / FVC， and PEF values in the modelgroup were all lower than those in the control group (P＜0.05). The FVC， FEV0.3， and PEF values in theChinese medicine group， western medicine group， and combination group were all higher than those inthe model group (P＜0.05). The FEV0.3/FVC value in the combination group was higher than that in the modelgroup (P＜0.05). There were no significant differences in FVC， FEV0.3， FEV0.3 / FVC， and PEF valuesbetween the Chinese medicine group and the western medicine group (P＞0.05). The FVC and PEF values inthe combination group were higher than those in the Chinese medicine group and the western medicinegroup (P＜0.05)； the FEV0.3 in the combination group was higher than that in the western medicine group(P＜0.05). The levels of TNF-α and IL-17 in serum in the model group were significantly higher than thosein the control group (P＜0.05). The levels of TNF-α and IL-17 in the Chinese medicine group，the westernmedicine group，and the combination group were all significantly lower than those in the model group (P＜0.05). The levels of TNF - α and IL-17 in the serum in the combination group were lower than those in theChinese medicine group and the western medicine group (P＜0.05). The protein expression levels of P-P65/P65，P-AKT/AKT，P-MAPK1/3/MAPK1/3，and TP53/GAPDH in the lung tissue in the model group werehigher than those in the control group (P＜0.05). The expression levels of the above proteins in the Chinesemedicine group，the western medicine group，and the combination group were all lower than those in themodel group (P＜0.05). The expression levels of the above proteins in the combination group were lowerthan those in the Chinese medicine group and the western medicine group (P＜0.05). Conclusion：TSG hasthe characteristics of multiple components，multiple targets，multiple pathways，and general regulation inthe treatment of AECOPD，providing data support for further in-depth research.