本研究以肝克颗粒入血成分为基础,结合网络药理学,探讨其干预急性肺损伤(ALI)的潜在作用机制。分子对接,和动物实验验证。将甘克颗粒大鼠的血液进入成分导入SwissTargetPrediction平台以预测药物靶点,和ALI相关目标从疾病数据库中收集.十字路口被采取了,并构建了蛋白质-蛋白质相互作用(PPI)网络来筛选核心靶标,其次是基因本体论(GO)功能和京都百科全书基因和侏儒(KEGG)途径富集分析。A"血液进入成分-目标-途径-疾病"网络构建,并根据其拓扑参数筛选疾病干预的核心成分。分子对接用于预测核心组分与关键靶标的结合能力。通过脂多糖(LPS)诱导的ALI小鼠模型验证了肝克颗粒干预ALI的关键靶点。通过PPI拓扑参数分析,STAT3、SRC、HSP90AA1,MAPK3,HRAS,获得与ALI相关的MAPK1。GO功能分析显示主要与ERK1和ERK2级联反应有关,炎症反应,和对LPS的反应。KEGG分析表明,MAPK的富集途径主要为,中性粒细胞胞外捕获(NET)的形成,等等。六个核心成分(精神分裂症B,五味子,besigomsin,harpagoside,同位素鸟苷元,和trachelanthamine)通过基于拓扑参数分析的血液进入成分-目标-途径-疾病"网络进行过滤。分子对接结果表明,颗粒中含量最高的6种核心成分和Tectoridin与关键靶标MAPK3、SRC、MAPK1和STAT3。体内实验结果表明,与模型组相比,甘克颗粒能有效减轻LPS诱导的小鼠肺部组织病理学损伤,降低炎性浸润百分率。总蛋白质含量,一氧化氮(NO)水平,髓过氧化物酶(MPO)含量,肿瘤坏死因子-α(TNF-α),γ干扰素(IFN-γ),白细胞介素-1β(IL-1β),白细胞介素-6(IL-6),血管内皮生长因子(VEGF),支气管肺泡灌洗液(BALF)中趋化因子(C-X-C基序)配体1(CXCL1)减少,和淋巴细胞抗原6G(Ly6G)的表达水平,瓜氨酸化组蛋白3(Cit-H3),和磷酸化蛋白SRC,ERK1/2和STAT3在肺组织中显著下调。总之,肝克颗粒能有效抑制LPS诱导的ALI炎症反应,保护肺组织,调节炎症因子的释放,并抑制中性粒细胞浸润和NET形成,其作用机制可能与抑制SRC/ERK1/2/STAT3信号通路的激活有关。
This study aims to explore the potential mechanism of action in the intervention of acute lung injury(ALI) based on the blood entry components of Ganke Granules in rats and in conjunction with network pharmacology, molecular docking, and animal experimental validation. The blood entry components of Ganke Granules in rats were imported into the SwissTargetPrediction platform to predict drug targets, and ALI-related targets were collected from the disease database. Intersections were taken, and protein-protein interaction(PPI) networks were constructed to screen the core targets, followed by Gene Ontology(GO) functional and Kyoto encyclopedia of genes and gnomes(KEGG) pathway enrichment analyses. A "blood entry components-target-pathway-disease" network was constructed, and the core components for disease intervention based on their topological parameters were screened. Molecular docking was used to predict the binding ability of the core components to key targets. The key targets of Ganke Granules in the intervention of ALI were verified by the lipopolysaccharide(LPS)-induced ALI mouse model. Through PPI topological parameter analysis, the top six key targets of STAT3, SRC, HSP90AA1, MAPK3, HRAS, and MAPK1 related to ALI were obtained. GO functional analysis showed that it was mainly related to ERK1 and ERK2 cascade, inflammatory response, and response to LPS. KEGG analysis showed that the main enrichment pathways were MAPK, neutrophil extracellular trap(NET) formation, and so on. Six core components(schizantherin B, schisandrin, besigomsin, harpagoside, isotectorigenin, and trachelanthamine) were filtered out by the "blood entry components-target-pathway-disease" network based on the analysis of topological parameters. Molecular docking results showed that the six core components and Tectoridin with the highest content in the granules had a high affinity with the key targets of MAPK3, SRC, MAPK1, and STAT3. In vivo experiment results showed that compared with the model group, Ganke Granules could effectively alleviate LPS-induced histopathological injury in the lungs of mice and reduce the percentage of inflammatory infiltration. The total protein content, nitric oxide(NO) level, myeloperoxidase(MPO) content, tumor necrosis factor-α(TNF-α), gamma interferon(IFN-γ), interleukin-1β(IL-1β), interleukin-6(IL-6), vascular endothelial growth factor(VEGF), and chemokine(C-X-C motif) ligand 1(CXCL1) chemokines in bronchoalveolar lavage fluid(BALF) were decreased, and the expression levels of lymphocyte antigen 6G(Ly6G), citrullinated histones 3(Cit-H3), and phosphorylated proteins SRC, ERK1/2, and STAT3 in lung tissue were significantly down-regulated. In conclusion, Ganke Granules could effectively inhibit the inflammatory response of ALI induced by LPS, protect lung tissue, regulate the release of inflammatory factors, and inhibit neutrophil infiltration and NET formation, and the mechanism of action may be related to inhibiting the activation of SRC/ERK1/2/STAT3 signaling pathway.