The Underlying Mechanism Of Andrographolide For Suppressing Influenza Virus-induced Pneumonia

ObjectiveInfluenza viruses are a significant cause of respiratory illness and have been identified by the World Health Organization as a major global health problem.The excessive inflammatory response caused by influenza viruses is a leading cause of death.Andrographolide has been found to be effective in treating influenza virus infection,but its molecular mechanism is not yet fully understood.To address this gap in knowledge,our study aims to use transcriptome sequencing to analyze the signaling pathways and cytokines associated with andrographolide treatment of pneumonia.Our goal is to uncover the specific molecular mechanism by which andrographolide inhibits pneumonia,providing a theoretical basis for its clinical application.Methods1.A mice model of influenza virus infection was used to evaluate the effects of andrographolide on survival rate,body weight,pathological damage,and inflammatory factors in mice infected with influenza virus.2.Transcriptome sequencing technology was used to study changes in gene expression in the lung tissues of mice infected with influenza virus and treated with andrographolide,and to establish transcriptome expression profiles.The effect of andrographolide on the signaling pathway in influenza-infected mice was investigated by analyzing differentially expressed genes.The relevant signaling pathway protein changes were verified by Western Blot.Finally,the inhibitor assay was used to confirm that andrographolide inhibits influenza virus-induced pneumonia by regulating the PI3K/AKT signaling pathway.3.Transcriptome sequencing technology was used to investigate changes in gene expression in the lung tissues of normal mice treated with andrographolide,and to establish the transcriptome expression profile of mice.The effect of andrographolide on related genes in normal mice was investigated by analyzing differentially expressed genes.Then,using influenza virus-infected mice as a model,the function of chemokine CCL5 in influenza virus infection was revealed by RT-PCR,ELISA,flow cytometry,and immunohistochemistry.The effect of CCL5 on influenza virus replication and inflammatory cytokine secretion was investigated by si RNA interference assay.Finally,Ccl5^-/-mice were used to explore the biological role played by CCL5 in viral replication and inflammatory cytokine release.Results1.Andrographolide was found to increase survival rates,relative body weight,and decrease lung index in influenza virus-infected mice.It also showed a reduction in lung histopathological damage and inhibited the release of inflammatory cytokines.2.The study found that andrographolide activates the PI3K/AKT signaling pathway,which is associated with cell growth,apoptosis,pyroptosis,and inflammation in the body.When an AKT inhibitor was added,there was a significant increase in the level of inflammatory cytokines,histopathological damage in the mice’s lungs,and expression of key molecules associated with pyroptosis,such as cleaved-caspase3 and GSDME-N compared to the andrographolide group.3.Transcriptome sequencing revealed high expression of the Ccl5 gene,which is associated with inflammation,in normal mice given andrographolide.In influenza virus-infected mice,andrographolide increased the m RNA of Ccl5 and the protein expression level of CCL5 in the organism.Immunohistochemical experiments revealed that CCL5 was mainly secreted by mice lung epithelial cells.In addition,the secretion of CCL5 was significantly reduced by the addition of an AKT inhibitor,and it was determined that the secretion of CCL5 was regulated by the PI3K/AKT signaling pathway.CCL5 was found to inhibit the release of inflammatory cytokines caused by influenza virus and had no effect on viral replication by si RNA interference assay.Finally,the study found that the survival rate of Ccl5^-/-mice was lower than that of wild-type mice,suggesting that CCL5 plays a protective role in influenza virus infection.Conclusion1.Andrographolide protects mice against influenza virus infection.2.Andrographolide inhibited influenza virus-induced pneumonia by activating the PI3K/AKT signaling pathway,which resulted in reduced expression of cleaved-caspase3 and GSDME-N.Inhibition of the AKT signaling pathways can promote the release of inflammatory cytokines and pyroptosis,exacerbating lung histopathological damage.3.Andrographolide reduced influenza virus-induced inflammation by elevating the expression of CCL5.In an in vitro si RNA interference assay,CCL5 was found to inhibit the secretion of inflammatory cytokines.Andrographolide increased the expression of CCL5 in the epithelial cells of the lung,and the protective effect of CCL5 in influenza-infected mice was confirmed using Ccl5^-/-mice.