Effects Of Autophagy Regulators On The Degree Of JEV Infection And Inflammatory Response In The Brain Of Mice Infected With JE Virus

Japanese encephalitis(JE)is a mosquito-borne zoonotic infectious disease caused by Japanese encephalitis virus(JEV)infection.JEV infection mainly causes inflammatory diseases of the central nervous system,and causes damage to brain tissue.Autophagy is a lysosomal degradation process,a highly conservative process that maintains homeostasis,and plays an important role in viral infection and cellular immunity.Although in vitro experiments have shown that inhibition of the autophagy pathway can effectively reduce JEV replication and protect cells from damage.However,the interaction between the virus and many signaling molecules in the autophagy pathway is very complicated.It is particularly important to further study the effect of autophagy pathways on the JEV infection and host response in vivo.At the same time,autophagy regulating drugs are better used in treatment of Japanese encephalitis(JE)provides an effective basis.In this study,we established an animal model of JEV infection in mice,and mice injected with autophagy-regulating drugs including inducers Rapamycin(Rapa),early inhibitor Wortmannin(Wort)and late inhibitor Chloroquine(CQ).The mice were grouped as follows: JEV + Rapa group,JEV + Wort group,JEV + CQ group,JEV group,Control group,Rapa group,Wort group and CQ group.The mice were administered for 10 days and the mice were reared for 20 days.We observed the clinical symptoms of mice treated with autophagy-regulating drugs and calculated the survival rate of mice.Pathological techniques are used to observe the extent of brain damage in mice.Molecular biology techniques are used to detect the JEV load and the secretion of inflammatory factors in mice.The relationship between autophagy pathway and virus infection and host response in mice was further studied.The main research contents of the paper were as follows:1.Effects of autophagy-regulating drugs on clinical symptoms in miceDuring the rearing period of the mice,observation and statistics showed that the duration of symptoms in the JEV + Rapa group was 5-20 days,and obvious standing hair,arch back and motor dysfunction appeared.The duration of symptoms in the JEV group was 5-12 days,and obvious neurological symptoms appeared.The mice in the JEV + Wort group only experienced mild depression on 7-10 days,and then returned to normal.No obvious neurological symptoms in other groups of mice.The incidence of mice in the JEV + Rapa group was 65.5%.The incidence of mice in the JEV group was 32.7%.And the incidence of mice in the JEV + Wort group and in the JEV + CQ group both the lower rate.The other groups of mice were free of disease and death.It was initially found that the autophagy inhibitors Wort and CQ can slow the occurrence of neurological symptoms in infected JEV mice.2.Effects of autophagy regulating drugs on the degree of JEV infection in brain tissue of miceFirst,TEM was used to observe the emergence of autophagolysosomes and mitochondrial damage in the brain tissue of mice in the JEV group.The presence of autophagolysosomes and severe mitochondrial damage in the brain tissue of mice in the JEV + Rapa group.But no autophagic vesicles or lysosomes were observed and mitochondria were slightly damaged in the JEV + Wort group.Autophagic vesicles of bilayer membranes were formed,but mitochondria were slightly damaged in the brain tissue of the mice in the JEV + CQ group.At the same time,Immunofluorescence technique was used to observe the co-localization of autophagy protein LC3 and JEV-E protein in some neurons in the brain tissue of mice in the JEV group and in a large number of neurons in the brain tissue of the mice in the JEV + Rapa group.There was no significant colocalization was observed in the brain tissues of other groups of mice.This result indicates that autophagy inhibitors attenuate the interaction between autophagy and viral infection,and attenuate the damage of subcellular structure levels in brain tissue of mice.We used RNAscope and IHC staining technique to observe the distribution of JEV in mice brain.JEV in the brain tissue of mice in the JEV group was mainly distributed in the cerebral cortex and thalamus.JEV in mice of the JEV + Rapa group was mainly distributed in the cerebral cortex,olfactory nodules,thalamus,midbrain,Pontine and medulla.And JEV was mainly distributed in the cerebral cortex in the brain tissues of JEV + Wort group and JEV + CQ group.JEV antigen distribution was not detected in other groups of mice.Real-time quantitative PCR(Q-PCR)technology detected that the viral load of mice in the JEV + Rapa group and the JEV group was significantly higher than that in the other groups(** P <0.01).These results further indicate that autophagy inhibitors Wort and CQ can weaken the interaction between autophagy pathway and viral infection,and attenuate the degree of JEV infection in brain tissue of mice.3.Effects of autophagy-regulating drugs on encephalitis in miceJEV infection mainly causes a strong inflammatory reaction in the brain.Observation of pathological changes revealed that vascular inflammation and microglia proliferation occurred in the brain tissue of mice in the JEV group and JEV + Rapa group.There was no obvious vascular inflammatory response,but had microglia proliferation occurred in the brain tissue of mice JEV + Wort grop and JEV + CQ group.Real-time quantitative PCR(Q-PCR)was used to detect the secretion of inflammatory factors IL-6,IL-1β,and TNF-α in brain tissues of mice in the JEV group and JEV + Rapa group respectively higher than other groups at 10 days(** P <0.01).At the same time,Western blot technology was used to assess the regulation of PI3 K / AKT and PERK pathways on downstream signals.The expression levels of PI3 K,PAKT,PJNK,and P65 proteins in the brain tissue of mice in the JEV and JEV + Rapa groups showed an upward trend(* P <0.05),PERK had no obvious upward trend.According to the above results,it can be seen that JEV infection can cause the activation of immune pathways.Autophagy inhibitors reduced the degree of inflammatory response in brain tissues of mice by inhibiting the activation of the PI3 K / AKT / NF-кB pathway.This result weaken the degree of inflammatory response in the brain tissue and plays a protective role in the brain tissue.