| Background: Influenza viruses are the major pathogen causing viral pneumonia infections.Seasonal influenza,caused by influenza viruses,infects about one billion people yearly.The "cytokine storm" in the body is one of the important causes of death after influenza virus infection.The "cytokine storm" is a life-threatening systemic inflammatory syndrome,which can cause multiple organ dysfunction and extremely high mortality.Therefore,controlling the "cytokine storm" progress in the body is crucial.Our previous studies have shown that the inhibitory oligonucleotide MS19 has the effect of inhibiting inflammation.However,single nucleotide drugs are easily degraded by nucleases and are unstable in circulation.This study investigated whether the delivery of inhibitory oligonucleotide MS19 by using nanocarrier PEI-PLA to enhance its antiinflammatory effect,and observed whether PEI-PLA-MS19 could alleviate lung injury and prolong survival in mice with viral pneumonia.Thus,this study provided a new idea for treating the excessive inflammatory reaction induced by pathogen infection.Method: In this study,hydrophilic PEI was coated with hydrophobic PLA,and then nanoparticles PEI-PLA were self-assembled by the nano-precipitation method,and then PEI-PLA-MS19 was prepared by the charge attraction method.The nano-materials were characterized by dynamic particle size scattering and transmission electron microscopy,and the nano-delivery vehicles with appropriate dimensions and strong stability were screened and prepared.After preparing PEI-PLA-MS19,we observed the effect of delivering MS19 into RAW264.7 cells by PEI-PLA nanocarrier under a confocal microscope,and the presence and distribution characteristics of PEI-PLAMS19 in mice were also observed by in vivo imaging of small animals.Moreover,the level of influenza virus-induced IL-6 production treated by PEI-PLA-MS19 in RAW264.7 cells was detected by ELISA.In vivo experiments,we evaluated the therapeutic effect of PEI-PLA-MS19 on mice with viral pneumonia,which mainly included observing the survival time of the mice,assessing the pathological changes of lung tissues after the HE staining,and detecting the levels of inflammatory factors IL-6 and TNF-α in bronchoalveolar lavage fluid by ELISA.In addition,the proportion of macrophages and neutrophils in the mouse lung and spleen was measured by flow cytometry.Finally,the differential expression genes in the RAW264.7 cells treated with MS19 against influenza virus infection were analyzed by RNA-Seq and validated by Western Blot.Research result: PEI-PLA nanoparticles have good stability.The nanoparticle size was about 220 nm,and the Zata potential was about 21.2 m V.Then,PEI-PLA-MS19 was obtained by charge adsorption force,its size was about 295 nm,and the Zata potential was about-8.2 m V.The confocal results showed that PEI-PLA nanoparticles could effectively promote MS19 to enter cells.Meanwhile,the imaging results of small animals in vivo showed that PEI-PLA-MS19 had a longer retention time and a broad distribution range in mice than MS19 only.ELISA results showed that PEI-PLA-MS19 significantly reduced the level of IL-6 secretion by RAW264.7 cells stimulated by the influenza virus.Notably,our in vivo results showed that PEI-PLA-MS19 could effectively prolong the survival time of mice with viral pneumonia,alleviate the pathological changes in the lungs of mice with viral pneumonia,and reduce the levels of IL-6 and TNF-α in the alveolar lavage fluid of mice.Furthermore,PEI-PLA-MS19 effectively inhibited the neutrophil infiltration in the mouse lung and spleen,and increased the proportion of M2-type macrophages in mouse lung tissue.In addition,RNA-Seq and Western Blot results showed that PEI-PLA-MS19 promoted the expression of Adcy4 to inhibit inflammatory signaling in RAW264.7 cells.Conclusion: 1.PEI-PLA-MS19 copolymer is successfully constructed with an appropriate size and good stability;2.PEI-PLA nanocarrier can effectively deliver MS19 into cells and improve the stability and durability of MS19 in mice;3.PEI-PLA-MS19 inhibits influenza virus-induced inflammatory response in vitro and in vivo and has a remarkable treatment effect on mouse viral pneumonia. |
