The Mechanisms Of PRV Activating NF-?B Signaling Pathway In Trigeminal Ganglion Cells

Pseudorabies?PR,also known as Aujeszky's disease,AD?,caused by Pseudorabies virus?PRV?,is an acute infectious disease in a variety of domestic and wild animals and one of the main infectious diseases of the pig industry,causing its huge economic losses every year.Pseudorabies virus?PRV?belongs to the family Herpesviridae,subfamily Alphaherpesvirinae.During the latent infection period of PRV,no virus particles can be detected in the animal body and no detoxification is performed.However,under the stimulation of corresponding stress factors,latent infected PRV can be activated to regenerate infectious virus particles,causing disease in animals and detoxification outwards.The establishment,maintenance and activation mechanisms of PRV latent infection are unknown,and the main obstacle to the control and eradication of pseudorabies is due to PRV latent infection.Existing reports showed that inoculation with PRV on pigs in vitro can lead to the activation of ganglion cells which is mainly manifested by changes in morphology,secretion of inflammatory factors and cell proliferation.The NF-?B is the key signaling pathway for Ganglion cells to secrete inflammatory factors induced by pathogen infection.The virus activates the NF-?B signaling pathway and then the activation of NF-?B initiates the expression of many inflammatory factors,which in turn activates the host's immune system.In terms of cytokines,the virus is caused by infection of immune cells.Cell death leads to the release of many cytokines,such as IL-1,IL-6 and IL-8 as well as TNF-?,INF-?and INF-?.Through which signal pathway PRV activates TG cells,the molecular mechanism remains unclear.In order to further help to better solve the above problems,this research carried out the investigation of PRV activation of the NF-?B signaling pathway in TG cells.The relevant research contents are as follows:1.PRV infection of mouse trigeminal ganglion cellsIn this study,a combination of trypsin,DNase,trypsin and collagenase was used to digest adult mouse trigeminal ganglion to prepare TG cells,which were cultured for 1 to 3days to adhere to the wall,5 to 6 days to grow and mature,and TG cells were stained blue by Nissl,On the 7th to 8th day,age and death begin,and all died on the 11th to 12th day.Identify through separation,culture and observation,the normal morphology of mouse TG cells is that the individual is larger,with many synaptic-like structures and synaptic-like morphology at the edge,but the shape and size of the synapses are different,The edges of the cells are irregular gear-shaped,the nucleus is generally in the center of the cell.The senescence process of TG cells is that the surrounding cells gradually become brighter and smaller,the color of the nucleus becomes darker,Eventually the synapse breaks and disappears,and the cell becomes round and brighter and die.The results of the study show that:using this culture method,mouse TG cells can be successfully isolated and cultured,and they grow well and have stable morphology,but they cannot be digested,passaged and frozen,and can only be cultured in the primary generation.After TG cells are infected by PRV,And it was observed that TG cells can have corresponding cytopathic effects?CPE?in the later stage of PRV infection.The study found that:CPE began to appear 48 h after PRV inoculation,and it began to die within 72 h.Compared with normal TG cells,the death time of the inoculated group was 2?3 days earlier,while the Verocells in the control group showed CPE 24 h after inoculation.Almost all cells died within 48 h,while the CPE and death of TG cells were later.It was about 36 hours later,and the lesion time was longer.This may be related to the self-latency and copy protection mechanism of TG cells,which is consistent with the expected results of the experiment.The beginning of the lesion is the breakage and disappearance of the prominent structure of the TG cell,the cell volume shrinks,the nucleus becomes larger,the nucleus becomes darker,and the position is shifted.The shape of the cell gradually becomes rounded,and then the periphery of the cell becomes brighter and gradually die.Using indirect cellular immunofluorescence technology,Use two types of fluorescence to observe the same location,and then the two fluorescence pictures were merged.The two fluorescence images showed the same position.The corresponding fluorescence was detected at the 24h and 48h respectively.The fluorescence at 48 h after inoculation was brighter than at 24 h,indicating that after PRV infects TG cells,PRV can quickly enter TG cells,and can quickly replicate and proliferate in TG cells.Furthermore,the effect of PRV on the growth of TG cells was detected by detecting the content of 5-bromodeoxyuridine?BrdU?in the TG cell culture medium.The study found that the BrdU content in the control group with inactivated PRV solution continued during the growth period.After reaching a peak value of 7.85 pg/mL,it gradually decreased.The time for BrdU content to reach the peak value was 156 h,which was the 6th to 7th day of TG cell culture,which was consistent with the mature period of TG cells.The BrdU content of the experimental group vaccinated with PRV solution immediately decreased after 120 h,and the decrease was large.At the 12 h after exposure to PRV,the detection value of the experimental group suddenly dropped from 6.67 pg/m L to 2.81 pg/mL,Indicating that the division speed of TG cells slowed down rapidly at this time,and it may even stop dividing.When reaching144 h,the BrdU content dropped to a minimum,and at the same time it began to increase rapidly.The reason may be that the TG cells were exposed to With the destruction of PRV,the permeability of TG cells increases or ruptures,and BrdU is released to the outside of the cell successively,which causes BrdU to increase in the later stage of PRV infection.In the late period of the control group?when the cells died on the 8th to 11th day?,the BrdU content rebounded and increased.2.The effect of PRV on the NF-?B pathway of trigeminal ganglion cellsinoculation with PRV After Mouse TG cells were cultured to the 5th day,And design a controlled experiment,take samples at 3 h,6 h,9 h,12 h,24 h,36 h,48 h,60 h,72 h after infection,using interleukin IL-Valukine^TMELISA method to detect changes in the levels of IL-1 and IL-6.Both IL-1 and IL-6 are downstream factors that activate the NF-?B signaling pathway,especially IL-1 is the pathway.As a marker inflammatory factor,the activation of the NF-?B signaling pathway can be inferred by measuring the content of IL-1.The experimental results show that the content of IL-1 begins to increase significantly after 36hours of inoculation,The IL-1 content reached a peak value of 10.55 pg/m L at 60 h of infection,and the content of IL-6 in the experimental group reached a peak of 165.109 pg/mL within 24 hours,and the content of IL-1 and IL-6 in the control group was basically unchanged.At the same time,Vero was also designed.Comparing cell inoculation experiments,the IL-1 content of Vero cells reached a peak of 9.161 pg/mL 24 hours after inoculation,indicating that the IL-1 response of TG cells after PRV infection was slower,about 36 h later,which may be due to The latent mechanism of PRV virus in neuronal cells is related.IL-1 is a cytoinflammatory factor.The time when IL-1 content reaches its peak coincides with the time when cells start to undergo a lot of apoptosis.Experiments show that PRV can activate the NF-?B signaling pathway of TG cells in the late stage of infection,and can regulate cell apoptosis by activating this pathway.By synthesizing MyD88 and TRIF siRNA,and then transfecting the si RNA into the cultured mouse TG cells by lipofection,respectively,the MyD88 and TRIF genes were silenced,and an empty transfection group was designed for control.After transfection,Start receiving PRV 24 to 36 hours after cell transfection,and take samples at 3 h,6 h,9 h,12 h,24h,36 h,48 h,60 h,and 72 h after inoculation,until most of the cells in the inoculated cell die.After that,the IL-1 Valukine^TMELISA method was used to detect the change in IL-1 content,and the silencing effect was compared with the peak value.The silencing effect of MyD88gene was 21.67%,18.60%and 39.52%,respectively,and the silencing effect of TRIF gene was 9.55%and respectively.5.86%.The simultaneous silencing experiment of MyD88 gene and TRIF gene was further carried out,and the double silencing effect was 27.12%and33.08%.The experimental results show that the silencing effect of MyD88 gene is better than that of TRIF gene.The average silencing effect is 18.89%higher,indicating that MyD88 is the way to activate the NF-?B pathway,and the activation of the NF-?B pathway is through the MyD88 activation pathway.To activate.3.In vivo verification of research on the effect of PRV on cellular NF-?B pathwayTwelve adult mice were selected,the experimental group and the control group were six respectively.The experimental group received 10?L of live PRV solution into the brain at one time,while the control group was injected with 10?L of PRV neuron cell culture solution into the brain,and the brain tissue was exposed to poison The brain tissues of 3 mice were taken at 24 h and 48 h later,and the changes in the levels of IL-1 in the brain tissues were measured.The experimental results showed that the levels of IL-1 in the brain tissues of mice in the control group did not change much.The content of IL-1 in brain tissue was significantly increased.Take samples according to the above method for fluorescence quantitative PCR detection experiment.Make 3 replicate holes for each sample,and calculate the relative expression level of each sample's mRNA according to formula 2^-??ct.The result of 0 h is used as the reference,and the control group is 24 h Transcription was up-regulated by 2.35 times,the control group was up-regulated by 4.78 times at 48 h,the inoculation group was up-regulated by 5.82 times and 11.61 times at 24 h and 48 h,respectively.The content of p65 transcription factor in the inoculated group was significantly increased,indicating that PRV has a positive effect on p65 after infection of TG cells.The production of transcription factors played a direct role.Sampling was carried out according to the above method for Western Blot experiment,the sample amount of the sample to be tested was 10?L,and the amount of total protein was20-50?g/well.The chemiluminescence map was obtained through WB experiment,and then the photoshop software was used to read out each blot.Average gray value,calculate the relative content of p65 expressed protein according to the gray value ratio relationship between the internal reference protein and p65 expressed protein,and the p65 protein gray value/internal reference protein gray value at 24 h and 48 h in the PRV group The ratio of the values is significantly lower than that of the control group and the untreated group.The ratios are 1.386 at 0 h,1.381 at 24 h in the control group,1.379 at 48 h in the control group,1.277at 24 h in the experimental group,The experimental group was 1.141 at 48 h,and the slope of the curve in the exposure group changed significantly,indicating that the expression of NF-?B p65 protein in the exposure group was significantly increased.The results of in vivo validation experiments in mice are consistent with in vitro experiments,further verifying the reliability of the results of this study.In summary,after PRV infects TG cells,it can quickly enter TG cells to proliferate,and can make TG cells produce corresponding CPE,and at the same time,it has a strong inhibitory effect on the growth of TG cells.This research progress has confirmed the TG The pathological changes of cells are directly related to PRV infection.PRV can activate the NF-?B signaling pathway at the later stage of infection of TG cells,and the NF-?B pathway activation pathway is achieved through the MyD88 pathway.This research can not only lay the foundation for the in-depth elucidation of the pathogenic and immune mechanism of PRV,but also Further revealing the latent infection of porcine pseudorabies virus and its pathogenic mechanism provides new clues,and can also provide reference for the revealing of similar scientific problems with other herpes viruses.