Indirubin Reduces Influenza Susceptibility By Regulating MAVS Pathway

Aim:Influenza,as a highly contagious disease caused by influenza virus,poses a health threat to humans.Isatis indigotica has a long history in treating virus infection and related symptoms in China.Indirubin,a natural bisindole alkaloid,is a marker chemical constituent of Isatis indigotica.Nevertheless,the research associated with the antiviral effect and mechanism of indirubin is not satisfactory,which might be due to lack appropriate in vivo and in vitro model.In fact,apart from viral factors,host factors are also important during the process of influenza virus infection.The establishment of a susceptibility model associated with host factors is useful in assessing the antiviral activity of traditional Chinese medicine and natural products.Our group has previously utilized restraint stress to disrupt type I IFN secretion and increase the susceptibility to H1N1 influenza virus.Based on this model,we have successfully evaluated the anti-influenza efficacy of several traditional Chinese medicines or natural products,such as apple polyphenols,Sarcandra glabra extract,and ReDuNing injection.Further studies found that corticosterone-induced immune suppression was the underlying mechanism of increased susceptibility to H1N1influenza virus.Therefore,we employed restraint-stressed mice model and CORT-loaded A549 cell model to evaluate the antiviral effect of indirubin and explore its antiviral mechanism,aiming to reveal the antiviral activity and mechanism of indirubin and provide a scientific basis for the development of natural products with anti-influenza virus effects.Methods:In the first animal experiment,the effect of indirubin on susceptibility to influenza virus in restraint-stressed mice was evaluated.Mice were distributed at random to six groups:Normal,Virus,Stress+Virus,Oseltamivir+Stress+Virus?30mg/kg/d oseltamivir?,and two indirubin groups?5 or 2.5 mg/kg/d indirubin+Stress+Virus?,which are described as Indirubin-H+Stress+Virus and Indirubin-L+Stress+Virus respectively.Oseltamivir and indirubin were administered orally to mice for 7 continuous days,while mice in groups were taken water orally.On the first day of administration,mice except those in Normal and Virus groups were restricted in ventilated plastic centrifuge tubes of 50 mL for 22 h.On the 3^rd day,mice were anesthetized using diethyl ether vapor and immediately infected with influenza A virus suspension(2×LD₅₀)in PBS of 35?L.Subsequently,the daily changes of mice in survival,body weight,typical influenza symptoms,such as hunched back,ruffled fur,altered respiration and unresponsiveness,were observed and recorded for 21 days or until death.Consequently,the data of morbidity and survival rate were calculated.The second animal experiment was also conducted to evaluate the effect of indirubin on pneumonia caused by influenza virus in restraint-stressed mice.Mice were treated by the same grouping,administration,stress and viral infection as the first experiment.However,five days post virus infection,the weight of mice was recorded.Subsequently,mice were anesthetized using ether vapor and sacrificed to harvest lungs for histopathologic examination and western blotting analysis of inflammation mediators.The lungs were weighted and lung index was counted in accordance with the formula:Lung index?mg/g?=lung weight/body weight.The third animal experiment was conducted to investigate the effect and mechanism of indirubin on type I IFN secretion in restraint-stressed mice.Mice were distributed at random to four groups:Normal,Virus,Stress+virus and Indirubin+Stress+Virus?5 mg/kg/d indirubin?.The following treatment was the same as the second experiment.The lung tissues were collected to determine the protein expressions of IFN-?,IFITM3 and some proteins involving in mitochondrial antiviral signaling?MAVS?pathway.Finally,the effect and mechanism of indirubin on susceptibility to influenza virus were investigated in CORT-loaded A549 cells.In the in vitro experiment,cells were randomly divided into four groups:Normal,Virus,CORT+Virus,Indirubin+CORT+Virus.Cells were seeded onto cell culture dish in 8×10⁴ cells/mL for experiments.After one day,the cells were cultured with CORT?100?M?or CORT?100?M?plus indirubin?10?M?for 48 h and were subsequently overlaid with H1N1 influenza virus of 10 TCID₅₀ for 45 min at 4°C,then PBS were used to clean cells for two times and serum-free DMEM was added.Eight hours post influenza virus infection,the cells were collected for the determination of the expressions of proteins involving in MAVS pathway.In order to further study the effect of indirubin on MAVS pathway,the effect of indirubin on mitochondrial function and morphology of CORT-loaded A549 cells infected with influenza virus was investigated.In addition,molecular docking,siRNA and other techniques were employed to search for the protein that indirubin targets to regulate MAVS pathway.Results:Indirubin exerted a protective effect on restraint-stressed mice infected with influenza with lower mortality,higher survival rate and less typical influenza symptoms.Indirubin reduced the lung index,pulmonary edema and lung injury in restraint-stressed mice infected with influenza virus.Meanwhile,indirubin reduced the expression of TNF-?,IL-1?,IL-6 and increase the production of IL-10.These results indicated that indirubin alleviated pneumonia associated with influenza infection and decreased the susceptibility to influenza virus in restraint-stressed mice.In restraint-stressed mice model,indirubin reduced the expression of NP protein and increased the protein expression of IFN-?and IFITM3 in lung tissue.Further study found that indirubin regulated proteins related to MAVS pathway to exert the antiviral effect.In CORT-loaded A549 cells model,indirubin was also found to promote the expression of IFN-?and IFITM3 by regulating the proteins related to MAVS pathway.Indirubin could alleviate the mitochondrial damage in CORT-loaded A549 cells infected with influenza virus.The results of molecular docking and siRNA experiments showed that STING may be the target of indirubin to enhance the MAVS pathway.Conclusion:Our study indicated that indirubin reduced H1N1 susceptibility in restraint-stressed mice.Indirubin could reduce the expression of inflammation mediators and alleviate pneumonia associated with influenza infection in restraint-stressed mice.Moreover,indirubin regulated the expression of IFN-?and IFITM3 via MAVS pathway.In CORT-loaded A549 cells model,indirubin promoted the production of IFN-?and IFITM3 by recovering MAVS pathway and maintaining mitochondrial function affected by CORT.Moreover,indirubin was found to regulate IFN-?secretion via MAVS pathway through targeting STING.