Objective

2

To investigate the active ingredients and mechanism of Ganlu Xiaodu Dan in the treatment of influenza A virus infection based on network pharmacology，and to verify it by animal experiments.

Methods

2

Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP）database were used to obtain and screen the active ingredients，and TCMSP and Swiss Target Prediction database were used to predict the potential targets.Comparative Toxicogenomics Database （CTD），Disease Gene Network（DisGeNET）database，GeneCards database and Online Mendelian Inheritance in Man（OMIM）database were used to obtain the disease targets of influenza A virus infection，and the potential targets of drug prediction were intersected with the disease targets to obtain the intersection targets.The drug-ingredient-target network was constructed by using Cytoscape3.9.1 and topological attribute analysis was performed to predict the main active ingredients of traditional Chinese medicine.Protein-protein interaction（PPI）network was mapped using Search Tool for the Retrieval of Interaction Gene/Proteins （STRING）database and Cytoscape3.9.1 to obtain key targets. Gene Ontology（GO）function and Kyoto Encyclopedia of Genes and Genomes（KEGG）path enrichment were analyzed using Metascape database.C57BL/6J mice were divided into blank group，model group，traditional Chinese medicine group and oseltamivir group，with 5 mice in each group.Mice infected with influenza A virus PR8 nasal drops were constructed to establish the infection model.The traditional Chinese medicine group was given Ganlu Xiaodu Dan with gavage administration and the control group was given oseltamivir phosphate with gavage administration.V bThe lung index and lung tissue pathology were observed.The expression levels of Phosphatidylinositol 3-kinase（PI3K）and Protein kinase B （AKT）gene were detected by quantitative reverse transcription PCR（RT-qPCR），and the expression levels of IL-6，IL-1β and TNF-α were detected by enzyme linked immunosorbent assay（ELISA）.

Results

2

A total of 125 compo

unds were selected as candidate effective ingredients，and 286 potential targets，2223 disease targets associated with influenza A virus and 61 intersection targets were obtained.Ten key components，including quercetin，kaonaphthol，baicalein，luteolin，baicalein，puerarin，acacia，isorhamnetin，dehydrodiisoeugenol and naringenin，were obtained by drug-ingredient-target network.The PPI network obtained 10 key targets including TNF，IL-6，IL-1β，AKT1，TP53，JUN，HIF1A，PTGS2，MMP9 and ESR1.A total of 54 items were obtained by GO functional enrichment analysis，including 20 biological processes，14 cell components，and 20 molecular functions.A total of 20 items were obtained by KEGG pathway enrichment analysis，including AGE-RAGE signaling pathway，cancer signaling pathway，relaxin signaling pathway，and PI3K-Akt signaling pathway.Compared with the blank group，the lung index in the model group was higher（

P

<

0.05）.Compared with the model group，the lung index of the Chinese medicine group and the control group was lower（

P

<

0.05）.In the model group，interstitial edema of lung tissue，infiltration of a large number of lymphocytes and monocytes，obvious widening of alveolar wall and alveolar fusion were observed.The interstitial edema of lung tissue，the infiltration of lymphocytes and monocytes，the widening of alveolar wall and partial alveolar fusion were reduced in the Chinese medicine group and the control group.Compared with the blank group，the expression of PI3K and AKT mRNA in the lung tissue of the model group was higher（

P

<

0.05）.Compared with model group，PI3K mRNA and AKT mRNA expression were lower in TCM group（

P

<

0.05），and PI3K mRNA expression was lower in control group（

P

<

0.05）.Compared with blank group，the levels of IL-6，IL-1β and TNF-α in lung tissue of model group were higher（

P

<

0.05）.Compared with model group，the levels of IL-6，IL-1β and TNF-α in lung tissue of TCM group were lower（

P

<

0.05），while the leve

ls of IL-6 and IL-1β in lung tissue of control group were lower（

P

<

0.05）.

Conclusion

2

Ganlu Xiaodu Dan can improve lung injury by inhibiting inflammatory response in mice infected with influenza A virus，and the mechanism may be related to inhibiting the over-activation of PI3K/Akt signaling pathway.