基于网络药理学及实验验证探讨燥湿化痰泻肺方治疗重症肺炎的作用机制OA

Objective:To explore the mechanism by which Zaoshi Huatan Xiefei(燥湿化痰泻肺)Formula amelio-rates severe pneumonia induced by Klebsiella pneumoniae based on network pharmacology combined with animal experi-ments.Methods:The active compounds and corresponding targets of Zaoshi Huatan Xiefei Formula were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).Pneumonia-related targets were retrieved from GeneCards,DisGeNET,and OMIM.A protein-protein interaction(PPI)network of key tar-gets was constructed via STRING.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)path-way enrichment analyses were conducted via MetaScape.PDB and CB-Dock were used for molecular docking validation.A mouse model of severe pneumonia was established by infection with K.pneumoniae.Mice were treated by oral gavage of Zaoshi Huatan Xiefei Formula and intraperitoneal injection of ceftriaxone sodium.Mice were sacrificed on day 3,and then body weight and mortality were recorded.The lung wet/dry weight ratio(mg/mg)was calculated.Peripheral white blood cell and neutrophil counts were determined via routine blood analysis.Hematoxylin and eosin(H&E)staining was employed to observe histopathological changes in the lung tissue,and Masson's trichrome staining to assess collagen fiber deposition.The extent of pulmonary consolidation was evaluated by computed tomography(CT).The levels of inflamma-tory cytokines in the lung tissue and bronchoalveolar lavage fluid(BALF)were measured by enzyme-linked immunosor-bent assay.The proportion of neutrophils in the BALF was analyzed via flow cytometry.Immunohistochemistry was adopted to detect the expression of myeloperoxidase(MPO),neutrophil elastase(NE),and lymphocyte antigen 6 com-plex,locus G(Ly6G).The expression of neutrophil chemokines was determined by qRT-PCR,and the protein levels of phosphorylated(p)-Janus kinase 1(JAK1),p-JAK2,p-signal transducer and activator of transcription 3(STAT3),and p-STAT6 were assessed by Western blotting.Results:A total of 146 active compounds and 391 key targets of Zaoshi Huatan Xiefei Formula were identified for the treatment of pneumonia.GO enrichment analysis revealed that the potential targets were mainly involved in biological processes such as the positive regulation of cell migration,protein tyrosine ki-nase activity,and transcription regulator complex.KEGG pathway enrichment indicated that pneumonia-related signaling pathways were primarily concentrated in the JAK/STAT signaling pathway.Animal experiments demonstrated that com-pared with the normal control group,the model control group showed a continuous and significant decrease in body weight and an increase in mortality.Compared with the model control group,Zaoshi Huatan Xiefei Formula(9 g/kg and 18 g/kg)groups exhibited increased body weight,reduced mortality,thickened alveolar walls,destruction of alveolar structures,massive inflammatory cell infiltration in alveolar and interstitial regions,lung congestion and edema,increased lung injury and alveolitis scores(P<0.01),increased lung consolidation area,density,and collagen fiber deposition,ele-vated levels of interleukin(IL)-6 and tumor necrosis factor(TNF)-α in the lung tissue and BALF,increased expression of MPO,NE,Ly6G,CXCL2,and CCL9,and up-regulated protein levels of p-JAK1,p-JAK2,p-STAT3,and p-STAT6 in the lung tissue(P<0.05 or P<0.01).Compared with the model control group,Zaoshi Huatan Xiefei Formula(9 g/kg and 18 g/kg)groups exhibited improved alveolar structure,reduced inflammatory infiltration,lowered lung injury and al-veolitis scores(P<0.01),ameliorated pulmonary consolidation and collagen fiber deposition,declined levels of IL-6 and TNF-α in the lung tissue and BALF(P<0.05 or P<0.01),decreased proportion of neutrophils in BALF(P<0.01),re-duced expression of MPO,NE,Ly6G,CXCL2,and CCL9 in the lung tissue(P<0.05 or P<0.01),and down-regulated protein levels of p-JAK1,p-JAK2,p-STAT3,and p-STAT6 in the lung tissue(P<0.05 or P<0.01).Conclusion:Zaoshi Huatan Xiefei Formula can reduce neutrophil infiltration,inhibit inflammation,and ameliorate pathological conditions in the mouse model of severe pneumonia by inhibiting the JAK/STAT signaling pathway.