王不留行黄酮苷对脓毒症小鼠急性肺损伤保护作用及机制研究

Protective effect and mechanism of vaccarin on acute lung injury in sepsis mice

目的基于网络药理学和动物实验探究王不留行黄酮苷(VAC)对脓毒症小鼠急性肺损伤的保护作用机制。方法通过Swiss Target Prediction数据库收集VAC的潜在作用靶点;利用GeneCards数据库检索肺脓毒症相关的疾病靶点;运用Draw Venn Diagram软件构建VAC和疾病的共同靶点;利用STRING 11.5数据库和Cytoscape 3.10.0软件构建共同靶点蛋白质-蛋白质相互作用(PPI)网络;利用Metascape数据库进行基因本体(GO)富集分析和京都基因与基因组百科全书(KEGG)通路分析。采用ip给予脂多糖(LPS)构建脓毒症模型,造模同时给予VAC(1、5 mg·kg^(-1))或地塞米松干预,取各组小鼠肺组织及血清,苏木精-伊红(HE)、Masson及TUNEL染色观察肺组织形态变化、纤维化及细胞凋亡情况,ELISA法和实时荧光定量PCR(qRT-PCR)法分别检测血清和肺组织中炎症因子水平,免疫组织化学法和Western blotting法检测肺组织炎症通路相关蛋白表达。结果VAC和肺脓毒症共同靶点有44个;GO富集分析涉及生物过程(BP)823个条目、细胞组分(CC)52个条目和分子功能(MF)49个条目;KEGG分析筛选出癌症通路、PI3K-Akt、JAK-STAT信号通路等20条信号通路。验证实验结果显示,与对照组相比,模型组小鼠肺组织损伤且纤维化严重,肺脏指数显著增加(P<0.05),血清及肺组织中相关炎症因子表达升高(P<0.01、0.001)。与模型组相比,VAC及地塞米松组肺组织病理形态得到改善,纤维化程度减轻,肺脏指数显著降低(P<0.05),血清中肿瘤坏死因子(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素6(IL-6)水平、肺组织炎症蛋白表达量及细胞凋亡数量降低,PI3K、Akt蛋白表达升高(P<0.05、0.01、0.001)。动物实验结果与网络药理学结果一致。结论VAC对脓毒症小鼠急性肺损伤具有一定的保护作用,其机制可能与调控PI3K-Akt、NLRP3/TNF-α等通路抑制炎症的发展有关,为VAC抗炎作用机制的深入研究提供了依据。

Objective Based on network pharmacology and animal experiments to explore the protective mechanism of vaccarin(VAC)in acute lung injury of sepsis mice.Methods The potential targets of VAC and the disease targets related to pulmonary sepsis were collected through the Swiss Target Prediction database and the GeneCards database separately.Then,the common targets for VAC(1,5 mg·kg^(-1))and diseases were obtained using Draw Venn Diagram software.The STRING 11.5 database protein-protein interaction was used to construct PPI network and a drug target disease network was constructed using Cytoscape 3.10.0.Finally,Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis for key targets were conducted utilizing the Metascape database.A sepsis model was established by intraperitoneal injection of lipopolysaccharide(LPS)and treated with VAC or dexamethasone(DEX).Then,lung tissue and serum were collected from each group,and HE,Masson,and TUNEL staining were used to observe changes in lung tissue morphology,fibrosis area,and cell apoptosis.The levels of inflammatory factors in serum and lung tissue were detected by ELISA and qRT-PCR,respectively.The expression of inflammatory pathway related proteins in lung tissue was detected by immunohistochemistry and Western blotting.Results There were 44 common targets for VAC and pulmonary sepsis.A total of 924 GO items were obtained by GO enrichment analysis,including 823 for biological processes,52 for cellular composition,and 49 for molecular functions.KEGG analysis identified 20 signaling pathways,including cancer pathway,PI3K-Akt,and JAK-STAT signaling pathway.The animal experiment results showed that compared with the control group,the model group mice had severe lung tissue damage and fibrosis,lung index significantly increased(P<0.05),and the expression of inflammatory factor in serum and tissue was increased(P<0.01,0.001).While,compared with model group,VAC and DEX improved pathological morphology of lung tissue,reduced fibrosis and the expression levels of serum TNF-α,IL-1β,IL-6(P<0.05,0.01,0.001).The expression of inflammatory proteins in lung tissue,and the number of cell apoptosis decreased,while the expression of PI3K and AKT1 proteins increased(P<0.05,0.01,0.001).The results of animal experiments were consistent with the results of network pharmacology.Conclusion VAC have a certain protective effect on acute lung injury in sepsis mice,and its mechanism may be related to the regulation of PI3K-Akt and NLRP3/TNF-αpathways related to its occurrence and development,which provides a basis for the in-depth study of the anti-inflammation mechanism of VAC.