Baicalin Exerts Anti-influenza Effects By Inhibiting Pyroptosis In Lung Epithelial Cells Via Caspase-3/GSDME Pathway

BackgroundInfluenza A is a common and serious respiratory disease with high morbidity and mortality.Given the viral resistance and antigenic drift,influenza vaccines must be reformulated annually to keep pace with the anticipated circulating strains.An increasing number of scientists have been focusing on the research of host-targeted antiviral drugs in order to break through the bottleneck of few targets and easy drug resistance faced by virus-targeted drug development.Traditional Chinese medicine has broad application prospects in antiviral infection,and it is expected to develop broad-spectrum antiviral drugs,but the application of Chinese medicine active ingredients is facing the dilemma of complex components and unclear mechanisms.Among them,baicalin,as the main active ingredient of Scutellaria baicalensis with the function of"entering the lung meridian and clearing the upper heat",has been reported to have certain effect in antiviral,but its mechanism is still unclear.Therefore,we used RNA-seq gene expression differential analysis to explore the pathogenic mechanism of H1N1 and the antiviral mechanism of baicalin.Content（1）The main cell types infected by H1N1.Explore the types of cells mainly infected by H1N1 virus by co-stain of M1 protein and major parenchymal and immune cells in mouse lung tissue.（2）Pathogenic mechanism of H1N1.RNA-seq analysis to enrich the main signaling pathways activated by H1N1 infection in mice;explore the specific mechanisms of H1N1 activation related pathways.（3）The effect of baicalin on pyroptosis caused by H1N1.Explore the effect of baicalin on caspase-3 activation induced by H1N1 in vivo and in vitro,and investigate the effect of baicalin on pyroptosis and pyroptosis pathway activation induced by H1N1.（4）The pharmacological effects of baicalin in anti-influenza.Investigate the effect of baicalin on mouse weight loss,death,and cytokine storms induced by H1N1;explore the effect of baicalin on H1N1 replication and RNA virus receptors RIG-I/MDA5 pathway;investigate the effect of baicalin on lung tissue damage induced by H1N1 in mice;study the effect of baicalin on the activity of the virus itself.（5）Synergistic effect of baicalin and oseltamivir.Explore the synergistic effect of baicalin and oseltamivir on anti-influenza in the late stage of influenza infection in mice.Methods:（1）The main cell types infected by H1N1 were investigated by co-staining H1N1M1 protein with lung epithelial cells（CC10+:clara cells,SPC+:type II alveolar epithelial cells,PDPN+:type I alveolar epithelial cells）and immune cells（F4/80+:macrophages,Ly6G+:neutrophils）.（2）The main signaling pathways activated after H1N1 infection were investigated by transcriptome sequencing in mice,and focus on the study of the programmed cell death pathway.The type of programmed cell death induced by H1N1 was determined based on characteristics such as cell morphology,cell membrane integrity,and lactate dehydrogenase release.Western blot was used to determine the activation of pyroptosis-related pathways and to explore the specific mechanism of H1N1-induced pyroptosis in lung epithelial cells.（3）The effect of baicalin on the changes in programmed cell death genes induced by H1N1 was explored using transcriptome sequencing.Flow cytometry and immunofluorescence were used to further demonstrate that baicalin can reduce programmed cell death induced by H1N1.Western blot was used to determine the effect of baicalin on caspase-3/GSDME pathway activation.（4）The in vitro anti-influenza effect of baicalin was analyzed using the MDCK infected with 100 TCID₅₀ H1N1,and the effect of baicalin on related cytokines was analyzed using the A549 cell infected with H1N1.The in vivo anti-influenza effect of baicalin was evaluated using the BALB/c mouse infected with 4 LD₅₀H1N1,by analyzing changes in mouse body weight and survival rate.The effect of baicalin on the levels of related cytokines and lung tissue lesions in vivo was also analyzed using the BALB/c mouse infected with 2 LD50 H1N1.（5）Give baicalin intraperitoneal injection two days before mice were infected with H1N1,and give oseltamivir gastric administration on the fifth day when obvious influenza symptoms appeared after H1N1 infection,to explore the preventive effect of baicalin and the synergistic effect of oseltamivir against influenza.Results:（1）H1N1 mainly infects clara cells and type II alveolar epithelial cells.（2）H1N1 induces lung epithelial cell pyroptosis mainly through the caspase-3/GSDME pathway.H1N1 infection activates the programmed cell death-related pathway in mice;H1N1 mainly induces pyroptosis in lung epithelial cells through the caspase-3/GSDME pathway.GSDME^-/-mice have a certain resistance to influenza（survival rate increased by 33%）.（3）Baicalin can inhibit pyroptosis induced by H1N1 via inhibiting caspase-3activation.Baicalin can reduce H1N1-induced programmed cell death（reducing the proportion of Annexin V⁺PI⁺cells by 7.37%）;Baicalin can inhibit caspase-3 activation in vivo and in vitro,and can inhibit H1N1-induced pyroptosis in lung epithelial cells（reducing bubble-like cell ratio by 40%）;baicalin can inhibit the activation of the caspase-3/GSDME pathway.（4）Baicalin has anti-influenza effects in vivo and in vitro.Baicalin can improve the growth status of mice,reduce weight loss caused by H1N1,and increase mouse survival rate（high dose by 41.3%,low dose by 36.3%）.It also reduces cytokines induced by H1N1（IL-1α,TNF-α,IL-1β,IL-5,IL-9,IL-10）,and improves lung tissue lesions in mice.Baicalin intervention can increase cell survival rate and reduce H1N1replication in cells.（4）Baicalin can synergize with oseltamivir’s antiviral effects.Baicalin’s preventive intervention can synergize with oseltamivir’s efficacy in the late stage of influenza,further improving the growth status of mice and increasing mouse survival rate（by 20%）.Conclusions:Given the fact that existing vaccines and antiviral drugs are highly prone to developing resistance,this study focuses on the mechanism of influenza infection and the mechanism of baicalin’s anti-influenza effects.Building on previous studies that showed that influenza viruses mainly infect type II alveolar epithelial cells,this study further confirmed that H1N1 is more likely to invade clara cells;For the first time,it was found that H1N1 mainly induces pyroptosis of alveolar epithelial cells through the caspase-3/GSDME pathway,and inhibition of the pyroptosis pathway can improve cell and mouse survival rates;For the first time,it was found that baicalin can inhibit caspase-3 activation and inhibit pyroptosis caused by H1N1;It was confirmed that baicalin pre-intervention can synergize with oseltamivir in the late stage of influenza infection,improve the growth status of mice,and significantly improve the survival rate of mice.