Signaling Pathway Mechanism Of Deoxynivalenol-induced Autophagy And Inflammatory Factors To Promote PCV2 Infection

Deoxynivalenol(DON)is one of the most widely distributed mycotoxins.It is ubiquitous in human food and animal feed crops and often causes serious food safety problems.DON has toxic effects on both humans and animals.Its effects include loss of appetite,weight loss,metabolic disorders,etc.It also affects the immune system of humans and animals.Porcine circovirus(PCV)is a single-strand circular,non-enveloped DNA virus,belonging to the genus Circovirus,and is currently the smallest animal virus.PCV is considered to be the main pathogen of porcine circovirus-associated disease(PCVAD),which is widespread all over the world and causes huge losses to the global pig industry.Among them,the clinical symptoms caused by porcine circovirus type 2(PCV2)are more complicated and often lead to a variety of diseases,such as weaned piglet multiple system failure syndrome(PMWS),porcine dermatitis nephrotic syndrome(PDNS),piglet respiratory syndrome(PRDC)and pig reproductive disorders.The clinical manifestations of PCV2 in pig farms around the world are not completely consistent,because in addition to the complex symptoms of the virus itself,a series of other factors are also closely related to the production of PCVAD,such as local feeding and management,whether it is accompanied by other toxin mixed infections,the nutritional status of the pigs,and immune stimulation.Both DON and PCV2 infection can cause host immune dysfunction,which can cause tissue or organ inflammation by regulating the expression of inflammatory cytokines such as IL-1β and IL-6 at the transcriptional level.Autophagy is a self-protection mechanism by which eukaryotes degrade damaged organelles or misassembled proteins through lysosomes.Eukaryotic cells wrap and close the phagosomes into double-layered vesicles by forming a bowl-shaped envelope structure and transported to the lysosome for degradation.Under normal circumstances,the body uses autophagy as a defense mechanism to resist viral infections.However,through a long-term evolutionary process,some viruses have evolved a variety of strategies to inhibit or immune evade host cell autophagy,and even use the autophagy mechanism to promote virus self-replication.The previous research of our group showed that DON can induce PCV2 replication in vitro,and the different pollution degree of DON in pig feed may partly explain why the PCV2 infection situation and severity of the disease are different in each pig farm.However,the signal pathway mechanism that DON induces autophagy and inflammatory factors and promotes PCV2 replication remains to be studied in depth.This thesis takes DON,PCV2 and PK-15 cells as the main research objects,starting from the autophagy-related pathway MAPK signaling pathway,PI3K/AKT/mTOR signaling pathway,and the inflammatory cytokines IL-1β and IL-6,using Western Blot,QPCR and IFA methods to study the signal pathway mechanism of DON-induced autophagy and inflammatory cytokines,and promote PCV2 infection.On the one hand,it helps to clarify the pathogenesis of porcine circovirus,and on the other hand,it provides some scientific basis for DON pollution to induce diseases in agriculture and animal husbandry production.Experiment Ⅰ,Study on the signaling pathway mechanism of deoxynivalenol inducing autophagyDeoxynivalenol(DON)is the trichothecenes mycotoxin with the widest range of pollution.Autophagy is a protective metabolic mechanism by which eukaryotes degrade long-lived proteins and break down damaged organelles.The results of this group’s previous experiments show that DON can induce autophagy in PK-15 cells at low doses,but the signal pathway mechanism of autophagy induced by DON needs to be further studied.In this experiment,the expression level of autophagy-related proteins was determined by DON treatment for different time,and the relationship of DON to PK-15 cell autophagy was explored.Then,by measuring the expression levels of autophagy pathway proteins under different DON treatment time,the effect of DON on the signal pathway was studied.Finally,by using specific kinase inhibitors to inhibit the activity of different kinases in the signal pathway,the role of the autophagy in DON-induced autophagy was studied.The results show that 1μg/m L DON can affect the expression of key autophagy proteins such as LC3-II,ATG5,beclin1 and P62,and the change in expression level is significantly correlated with the time of action of DON.It shows that DON can cause a complete autophagy response in PK-15 cells at this dose.At the same time,DON enhances autophagy by inhibiting the activity of AKT and mTOR in PK-15 cells.MHY1485 treatment can reverse the decrease of mTOR phosphorylation level caused by DON,and at the same time can reverse the autophagy response induced by DON,further indicating that mTOR signaling pathway plays an important role in DON-induced autophagy of PK-15.In addition,DON can activate the MAPK signaling pathway.U0126,SP600125,and SB203580 can independently inhibit the phosphorylation of ERK1/2,JNK1/2,and p38 in the MAPK signaling pathway,and inhibit the autophagy response induced by DON.It proves that DON also induces autophagy by activating the MAPK signaling pathway.At the same time,U0126,SP600125,and SB203580 can also increase the decrease in mTOR phosphorylation caused by DON,indicating that DON first activates the MAPK signaling pathway and then inhibits the activity of mTOR,which ultimately induces autophagy.Experiment Ⅱ,Study on the mechanism of autophagy signaling pathway that deoxynivalenol promotes PCV2 replicationThere is a complex relationship between autophagy and virus replication.Studies have shown that some viruses can use autophagy to promote their own replication.The results of the previous chapter show that DON can induce autophagy by activating the MAPK signaling pathway and inhibiting the PI3K-AKT-mTOR signaling pathway.This test mainly explores whether the MAPK signaling pathway and the PI3K/AKT/mTOR signaling pathway are related to DON’s promotion of PCV2 replication.Through the drug-specific inhibition of the activity of different kinases in the signaling pathway,the PCV2 virus virulence,Cap protein expression,viral DNA replication number and virus-positive cell infection number changes were detected to explore the DON-induced autophagy signaling pathway for PCV2 virus replication The impact.The test results show that the treatment of DON can promote the replication of PCV2.The addition of MHY1485 can reverse the decrease of PI3K-AKT-mTOR signal pathway activity caused by DON,and it can also inhibit the promotion of PCV2 replication caused by DON.The addition of U0126,SP600125 and SB203580 can inhibit the phosphorylation of ERK,JNK and p38 in the MAPK pathway,respectively,and can also reduce the replication of PCV2 caused by DON.It shows that the promotion of PCV2 replication depends on the PI3K/AKT/mTOR signaling pathway and the enhancement of autophagy caused by the MAPK signaling pathway,and DON promotes PCV2 replication by inhibiting the PI3K-AKT-mTOR signaling pathway and activating the MAPK signaling pathway.Experiment Ⅲ,The combined effect of deoxynivalenol and PCV2 on inflammatory cytokinesBoth DON poisoning and PCV2 infection can invade the host’s immune system and affect the host’s natural immune function.Inflammatory cytokines IL-1β and IL-6 are important regulators in the process of natural immune response,which participate in the inflammatory response and enhance immune-mediated tissue damage.The test results in the previous chapter show that DON promotes PCV2 replication and infection by activating the MAPK signaling pathway.This experiment mainly explores whether the MAPK signaling pathway is involved in the combined action of DON and PCV2 to induce the expression of IL-1β and IL-6 at the transcriptional level.Fluorescence quantitative PCR was used to detect the expression of IL-1β and IL-6 mRNA in PK-15 cells after DON and PCV2 act alone or in combination.The specific pharmacological antagonists U0126,SP600125 and SB203580 inhibit the activities of ERK,JNK and p38 in the MAPK signaling pathway,respectively,and detect the expression of IL-1β and IL-6 mRNA in PK-15 cells.To explore the effect of MAPK signaling pathway on the combined effect of DON and PCV2 to induce the expression of IL-1β and IL-6 at the transcriptional level.The test results showed that PK-15 cells treated with DON and PCV2 alone can induce the expression of IL-1β and IL-6 mRNA,and it is time-and dose-dependent.The combined effect of DON and PCV2 has an additive effect on inducing the expression of IL-1β and IL-6 mRNA.The addition of U0126 and SB203580 can inhibit the expression of IL-1β and IL-6 mRNA induced by DON,but SP600125 has no effect.The addition of U0126,SP600125 and SB203580 can not only inhibit the expression of IL-1β and IL-6 mRNA induced by PCV2 alone,but also inhibit the expression of IL-1β and IL-6 mRNA induced by the combined action of DON and PCV2.It shows that the expression of IL-1β and IL-6mRNA induced by DON depends on the ERK and p38 MAPK signal pathways,while the expression of IL-1β and IL-6 mRNA induced by PCV2 depends on the ERK,JNK and p38 MAPK signal pathways.