Pharmacological effects and mechanism of Maxing Shigan Decoction in the treatment of influenza A viral pneumonia

Ethnopharmacology relevance Maxing Shigan Decoction (MXSGD), derived from the traditional Chinese medicine (TCM) classic Shang Han Lun , is recognized as an effective TCM herbal formula for treating respiratory diseases, although its precise molecular mechanisms against influenza A virus (IAV) have not yet been fully elucidated. Aim of the study This study aimed to clarify the pharmacological effects of MXSGD on IAV-infected pneumonia mouse models, with a particular focus on its regulatory mechanism through miR-1260. Materials and methods The chemical composition of MXSGD was analyzed using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). After establishing IAV-infected mouse models, the pathological changes in lung tissues were observed by hematoxylin-eosin (HE) staining; the pulmonary epithelial cells and macrophages in lung tissues were examined through immunological assays; and the expression levels of miR-1260 and its target genes in lung tissues were evaluated by RT-qPCR and Western blot analysis. A co-culture system of MLE-12 and RAW246.7 cells was constructed as the research model. Then, the intracellular miR-1260 expression was detected by RT-qPCR, and the Sema3A expression was examined by immunofluorescence. The macrophages were further transfected with miR-1260 mimic, inhibitor, and Sema3A siRNA to determine whether MXSGD exerts its therapeutic effects through the miR-1260-targeted Sema3A-mediated PI3K/AKT/mTOR signaling pathway. Subsequently, alveolar macrophage-depleted mouse models were established using CL2MBP, and were infected with IAV. To clarify the role of macrophages in the therapeutic mechanism of MXSGD against IAV, the body weight and organ indices of mice were monitored and recorded; the total protein content and levels of inflammatory factors in the bronchoalveolar lavage fluid (BALF) were measured; the histopathological changes were observed by HE staining; the pulmonary epithelial cells and macrophages in lung tissues were examined through immunological assays; and the expressions of miR-1260 and its target genes in lung tissues were evaluated by RT-qPCR and Western blot analysis. Results MXSGD was found to alleviate IAV-induced pathological injury and suppress macrophage recruitment in mouse lung tissues, while downregulating the viral load and levels of inflammatory factors. The attenuation of macrophage recruitment was closely associated with pulmonary epithelial cells, potentially mediated through the miR-1260-targeted Sema3A-regulated PI3K/AKT/mTOR signaling pathway. In cellular experiments, both cell types in the co-culture system were restored following MXSGD treatment, accompanied by lowered intracellular viral load and levels of inflammatory factors. Additionally, macrophage apoptosis and miR-1260 expression were significantly reduced, whereas the Sema3A fluorescence intensity in macrophages was markedly increased. By transfecting miR-1260 mimic, inhibitor, and Sema3A siRNA into co-cultured macrophages, the therapeutic effects of MXSGD were further confirmed to be associated with the miR-1260/Sema3A-mediated PI3K/AKT/mTOR pathway. In IAV-infected models established after pulmonary macrophage depletion, the therapeutic efficacy of MXSGD was partially weakened following elimination of alveolar macrophages. In general, above in vivo findings are consistent with the cellular experimental results, collectively supporting the involvement of the miR-1260-targeted Sema3A-mediated PI3K/AKT/mTOR signaling pathway in the therapeutic mechanism of MXSGD. Conclusions MXSGD was demonstrated to regulate the PI3K/AKT/mTOR pathway through the macrophage miR-1260/Sema3A axis, through which excessive inflammatory responses in IAV-induced pneumonia were effectively suppressed. The findings provide a novel therapeutic target for antiviral treatment in TCM.