Study On TLR2 Mediated Activation Of NF-κB Signaling Pathway To Regulate The Inflammatory Cytokines Expression During Porcine Parvovirus Infection

Porcine Parvovirus(PPV)is one of the most important pathogens causing sow reproductive failure and abortion.PPV is mainly transmitted through respiratory tract,gastrointestinal tract and placenta,and all kinds of poisonous pigs can spread the disease.PPV infection is a worldwide epidemic and is prevalent in many pig farms in China,which seriously affects the development of pig industry.Therefore,it has always been a hot topic in pig research.The current research on porcine parvovirus vaccine mainly concentrated on the detection method.The related research reports the infection mechanism of less mainly concentrated on the invasion process of the virus.The virus infection and host response to few reports seriously restrict the development of new vaccines and new drugs.Therefore,it is very urgent to develop the interaction mechanism of PPV and host cells to research and develop the antiviral drugs and effective vaccines of PPV.Toll like receptor(TLRs)is a transmembrane signal transduction receptor that can recognize specific microbial pathogens.Our preliminary studies show that porcine parvovirus infection caused a series of alteration in the expression profile of inflammatory cytokines in PK-15 cells,including IL-6 and NF-κB.Therefore,we speculate that PPV may regulate the expression of inflammatory cytokines through the NF-κB signaling pathway.This experiment combined with the role of NF-κB pathway in the virus infection,and carried out the related research on the relationship between the two.First of all,the experiment confirmed that PPV activated NF-κB signaling pathway after infection of PK-15 cells.Secondly,PPV was activated by TLR2 receptor.At last,it confirmed TLR2 mediated PPV NS1 activation NF-κB signaling pathway.The results of the specific test are as follows:1.In order to confirm whether the NF-κB signaling pathway was activated when PPV infected cells,the Western blot technique was used to detect the expression of each protein in the NF-κB whole pathway after PPV infected PK-15 cells.PPV infected PK-15 cells were tested at 0,3,6,12,24,36,48 and 60 h,respectively.The results showed that the expression level of IκBα increased at 3 and 6 h after PPV infection,12 h decreased,24 h increased,and then showed a downward trend,indicating that IκBα was degraded.The results of the detection of p65 phosphorylation protein(p-p65)showed that the total p-p65 expression after PPV infection was to the peak at 3 h,then the expression decreased,and the p-p65 expression in the cytoplasm decreased after PPV infection,and the expression of p-p65 in the nucleus was not obvious after the virus infection 3 and 6 h,and the 24 h reached the peak,followed by a decline.The experiment further used indirect immunofluorescence to observe the situation of p65 transposition into the nucleus.After PPV(MOI=1)infected PK-15 cells 36 h,the cells were fixed and incubated for one and two resistance.Under the fluorescence microscope,the majority of p65 in the PPV infected group was expressed in the nucleus,while the p65 in the uninfected group was still in the cytoplasm,indicating the PPV infection promotes the nucleation of p65.Then the expression of other proteins(My D88,IRAK1,TRAF6,TAK1)in the NF-κB signaling pathway was detected.The test results showed that the expression of My D88 in the cell decreased after PPV infection PK-15 cells,and the 24 h expression increased,and then decreased.The expression level of IRAK1 showed a downward trend before infection of 12 h,and increased after 24 h.The expression level of TRAF6 increased after PPV infection,and reached a peak at 6 h,then decreased gradually.The expression level increased at 24 h and then decreased.The expression level of TAK1 increased after PPV infection,reaching a peak value of 24 h,followed by a downward trend.These results indicate that PPV activated NF-κB signaling pathway through a series of cascade reactions after infecting PK-15 cells.2.The expression of TLR2,NF-κB and IL-6 increased after PPV infection at the m RNA level in the early stage of the laboratory.In order to further verify whether PPV activates NF-κB signaling pathway through TLR2,Western blot technology was used to detect TLR2 protein expression at different time points after PPV infection.The results showed that the protein expression of TLR2 after stimulation of PPV increased,12 h reached the peak value,and then decreased,indicating that PPV transmits signal through TLR2.Subsequently,two pairs of specific si RNA primers(si RNA210 and si RNA662)of TLR2 were designed,and the best working concentration of si RNA was 150 pmol after pre-test.When PPV(MOI=1)infected PK-15 cells transfected with si RNA,the cell samples were collected at 24,36 and 48 h respectively.The expression of TLR2 was detected by real-time PCR and Western blot.The results showed that compared with the untransfected si RNA test group,the expression of TLR2 in the test group transfected with si RNA decreased.The changes in m RNA expression of NF-κB and IL-6 were further detected at 24,36 and 48 h,and the expression of NF-κB and IL-6 were inhibited in varying degrees.These results confirm that PPV regulates the expression of IL-6 inflammatory factors through the activation of the NF-κB signaling pathway through the TLR2 receptor.3.The recombinant expression plasmid of PPV NS1 was successfully constructed in the early stage of the laboratory.After transfecting 293 T cells,the results showed that the expression of NF-κB and IL-6 were increased.In order to further verify whether PPV NS1 recombinant expression plasmid induced inflammatory response through TLR2,we transfected p EGFP-N1-NS1 into two cell lines of PK-15 cell and 293 T cell line 5 h,then transfected TLR2 si RNA,and cultured continuously 24,36 and 48 h to collect cell samples.The results of real-time PCR detection showed that the expression of NF-κB in PK-15 cells and 293 T cells was inhibited,and the expression of NF-κB in si RNA210 interference group decreased by 2.5 times and about 1 times than that in the recombinant plasmid group respectively.The expression of NF-κB in si RNA662 interference group decreased by 1.2 times and 6 times than that in the recombinant plasmid group respectively.two kinds of expression of IL-6 in cells decreased,si RNA210 interference group compared the recombinant plasmid group expression is decreased by 7 times and 1.5 times,two cell lines in the si RNA662 interference group compared the recombinant plasmid group expression is down nearly 1 times.This experiment confirms that PPV can activate NF-κB signaling pathway.Si RNA technology confirmed that PPV increased the expression of NF-κB and IL-6 through TLR2 infected PK-15 cells.It also confirmed that TLR2 mediated PPV NS1 transfection cells promoted the up regulation of the expression of NF-κB.Therefore,this study successfully confirmed that PPV activates the NF-κB signaling pathway through TLR2 infected PK-15 cells and then regulates the inflammatory response.