Epigoitrin Reduces The Susceptibility To Influenza Virus

Objective: Epigoitrin is extracted in the main active composition of Isatis indigotica.In the current,Epigoitrin has rarely the antiviral effect to clarify research,which limits its development and application,and it causes that people can not find a suitable antiviral evaluation model in vivo and in vitro.Restraint stressed model in mice and CORT load A549 cells model have been successfully evaluated a variety of natural products and traditional Chinese medicine compound anti-influenza virus activity in our team.Therefore,restraint stress mouse model is adopted in this subject evaluation in accordance with the anti influenza virus effect of Epigoitrin.So does CORT load A549 cells model.All is for studying the antiviral mechanism of Epigoitrin,and contributing to the clinical application and development and beneficial theoretical and experimental basis.Methods:(1)The Study on the effect of Epigoitrin on the susceptibility of restraint stress mice to Influenza virus infection.Kunming mice were randomly divided into six groups,including normal,virus,stress+virus,oseltamivir+stress+virus(30 mg/kg/d;p.o.),Epigoitrin-L+ stress+virus(2.5 mg/kg/d;p.o.)and Epigoitrin-H+ stress+virus(5 mg/kg/d;p.o.)groups.Mice in each group were given corresponding doses of drugs by gavage for 7 consecutive days,while those in other groups were given the same amount of normal saline.On the day of administration,the mice in each group were subjected to stress load except the normal control group and the viral infection group.Mice were restrained in 50 mL ventilation restraint cage for 22 hours.During this period,all mice were banned from water and food.On the second day after the restraint,the mice were placed in a ether bottle under mild anesthesia except for the normal control group.Each mouse was given 35 ?L of PBS virus suspension(2×LD50)nasal drip infected with influenza virus.After 21 consecutive days of observation,themorbidity and mortality were recorded daily.If the mice show signs of morbiditysuch as loss of appetite,hunching,respiratory change,pleated hair,slow movement,weak perception,etc.(2)To explore the effects of Epigoitrin on the infection of influenza virus secondary pneumonia in restraint stress mice.Another batch of adaptive mice were randomly divided into six groups,including normal,virus,stress+virus,oseltamivir+stress+virus(30 mg/kg/d;p.o.),Epigoitrin-L+ stress+virus(2.5 mg/kg/d;p.o.)and Epigoitrin-H+ stress+virus(5 mg/kg/d;p.o.)groups.The mice in each group were given restraint stress load and influenza virus infection for 5th days.The mice were weighed and recorded,anesthetizedwith ethe,killed.the lung tissue was collected and weighed.some lung tissues were fixed for HE staining,and others were applied to the analysis of the protein expression of interleukin 1-? and tumor necrosis factor-? were detected by western blotting.(3)To explore the mechanism of antiviral susceptibility to influenza virus in Epigoitrin.The experiment was divided into groups and treatments.On the 5th day of infection,all mice were anesthetized and killed,and the lung tissues of mice were collected.Western blotting was used to detect the expression of IFN-?,IFITM3,Mfn2 and MAVSs in lung tissue.(4)CORT loaded A549 cell model was used to verify the antiviral effect of Epigoitrin on A549 cells.The appropriate dosage of Epigoitrin in A549 cells loaded with CORT was screened by MTT method.In order to verify the effect of Epigoitrin on the infection of A549 cells with CORT loading,the antiviral effect of Epigoitrin on A549 cells infected with influenza virus was studied.According to the needs of the experiment,the cells were randomly divided into the following six groups,including normal,virus,CORT,CORT+virus,Epigoitrin+virus,Epigoitrin+virus+CORT.The A549 cells in the corresponding group were preprotected for 2 hours,and then loaded with 100 ?M CORT for 48 hours.The A549 cells in the corresponding group were infected with 10 TCID50 influenza virus diluent for 1 hour,and then cultured for 8 hours.The collected cells were collected.The expression level of NP,Mfn2,MAVS,IFITM3,p-IRF3,NF-? B was detected.Results:(1)The results of the experiment showed that Epigoitrin could reduce the mortality and morbidity of mice infected with H1N1 influenza virus,and increase the survival rate of restraint stress loaded mice.It is suggested that Epigoitrin has a good protective effect on the mice infected with H1N1 influenza virus.(2)It can be seen by observing the weight of lung tissue,the appearance of HE staining section,and the expression of protein in lung tissue.The lung index of mice infected with H1N1 stress was decreased,the degree of pulmonary edema induced by influenza virus and the level of lung tissue injury werealleviated,and the expression of IL-1? and TNF-? protein in lung tissue was down-regulated.The inflammation of the diseased tissue was alleviated and the secondary pneumonia was improved.(3)The expression of IFN-? and IFITM3 in lung tissue of mice infected with influenza virus was up-regulated,the level of Mfn2 was down-regulated,the expression of IFN-? and IFITM3 was increased,and the effect of interferon was promoted.The results show that Epigoitrin plays a role in reducing the susceptibility of H1N1 by regulating the expression level of related proteins in the MAVS signaling pathway.(4)In A549 cell model loaded with CORT,Epigoitrin could decrease the expression of NP.Further,it was found that Epigoitrin could increase the expression of MAVS,down-regulate the expression of Mfn2,promote the phosphorylation of IRF-3,down-regulate NF-? B,and increase the expression of IFITM3 protein.Conclusions: Epigoitrin has obvious protective effect on mice infected with H1N1 influenza virus under restraint stress.Epigoitrin can reduce the expression of inflammatory factors in lung tissue of infected mice.The symptoms of secondary pneumonia were significantly improved by regulating the associated proteins in the MAVS signaling pathway.The same results were obtained by CORT loaded A549 cell model.