| Objective:To explore the action mechanism of Maxing Shigan Decoction against Influenza A virus(IAV)based on the response effect of pulmonary macrophages to pulmonary epithelial cell-derived exosomes.Method:(1)Establishing the IAV-induced lung epithelial cell injury model: Injury of mouse lung epithelial cells(MLE-12)was induced by IAV,and cell activity was detected at different intervention time points using the CCK8 method in order to determine the optimal conditions for IAV-induced cell injury.Then,apoptosis was detected by flow cytometry,and the expression levels of inflammatory cytokines were measured by real-time fluorescence quantitative PCR(qRT-PCR)and Western blotting.(2)Damage mechanism of IAV-induced lung epithelial cell-derived exosomes(Exo)on macrophages: The MLE-12 cell-derived Exo were extracted by ultrahigh speed centrifugation,and were examined by Transmission Electron Microscopy,Nanoparticle Tracking Analysis(NTA)and Western blotting.Then,a co-culture system of MLE-12 cell-derived Exo and macrophages was established in order to detect the response effect of macrophages.Subsequently,high-throughput sequencing was performed to characterize the specific miRNA of IAV-induced MLE-12 cell-derived Exo and to predict the target genes and related signaling pathways.Finally,the selected miR-1249-5p was validated,and the expression levels of its target gene and related signaling pathway were measured by transfecting miR-1249-5p into macrophages.(3)Maxing Shigan Decoction inhibits the IAV-induced cascade cell injury effect by regulating miR-1249-5p: Firstly,the safe dose and therapeutic dose of Maxing Shigan Decoction serum against IAV-stimulated MLE-12 cells were detected using the CCK8 method.Secondly,taking the IAV-induced MLE-12 cell injury model as the intervention object,comparative experiments were carried out on the blank control group,the model group,the low-dose Maxing Shigan Decoction serum group,the high-dose Maxing Shigan Decoction serum group,and the Oseltamivir group.After intervention for 8h,the supernatant was collected from each group to extract Exo.The particle size distribution and concentration of Exo were detected by Nano Sight,and the expression level of miR-1249-5p in the Exo was measured by qRT-PCR.Finally,the Exo of each group were co-cultured with macrophages,and the expression level of miR-1249-5p in the macrophages was measured by qRT-PCR.The target sites of the Solute Carrier Family 4Member 1(SLC4A1)in the macrophages and the expressions of genes and important functional proteins in the nuclear factor kappa-B(NF-κB)pathway were detected by qRT-PCR and Western blotting.Results:(1)A stable IAV-induced lung epithelial cell injury model was established: According to CCK8 results,the cell activity of IAV intervention for 8h was most compliant with the needs of this study.Thus,the MLE-12 cells were intervened under this condition,and flow cytometry revealed a significantly increased apoptosis rate.The qRT-PCR and Western blotting results showed that the expression levels of IAV nuclear protein(NP),tumor necrosis factor-α(TNF-α)and Interleukin-6(IL-6)were significantly elevated.(2)The damage mechanism of IAV-induced pulmonary epithelial cell-derived Exo on macrophages: The Exo extracted by ultrahigh speed centrifugation were compliant with the Exo characterization.By fluorescence microscopy experiment showed that Exo could enter macrophages and promote the secretion of inflammatory factors by macrophages.The high throughput sequencing confirmed the existence of miR-1249-5p and therefore focused on the detection of its target gene SLC4A1 and the NF-κB pathway.It was found that macrophages were successfully transfected with miR-1249-5p mimetics and inhibitors.Western blotting showed that miR-1249-5p could inhibit the expression levels of the target gene SLC4A1 and the NF-κB pathway.(3)Maxing Shigan Decoction inhibits IAV-induced cascade cell damage by regulating miR-1249-5p: The CCK8 results showed that the 10% and 20% Maxing Shigan Decoction serum had no cytotoxicity and was able to inhibit IAV-induced MLE-12 cell injury.The Nano Sight results showed that there was no significant change in the Exo particle size among various groups,but the concentration varied obviously.The qRT-PCR results showed that the expression level of miR-1249-5p in IAV-stimulated MLE-12 cell-derived Exo was significantly reduced,whereas the expression level of miR-1249-5p in the high-dose and low-dose Maxing Shigan Decoction groups and Oseltamivir group was significantly elevated.The expression level of miR-1249-5p in co-cultured macrophages was consistent with the expression of MLE-12cell-derived Exo.The qRT-PCR and Western blotting results showed that the level of SLC4A1 in the macrophages and the gene and protein expressions of NF-κB pathway were significantly elevated in the model group,whereas those expressions of the high-dose and low-dose Maxing Shigan Decoction groups and Oseltamivir group were significantly reduced.Conclusions:The IAV-induced acute cell injury through the response effect of lung macrophages to MLE-12cell-derived Exo.Maxing Shigan Decoction can increase the transmission of miR-1249-5p in Exo to inhibit macrophages SLC4A1 and the NF-κB signaling pathway so as to improve the IAV-induced inflammatory response. |
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