Study On The Effect Of NF-κB Signaling Pathway Activator/inhibitor To DEV Proliferation

Duck enteritis virus（DEV）,belongs to the herpesvirus family,is the causative agent of duck virus enteritis（DVE）,which poses a serious threat to the economic development of the aquaculture industry.The NF-κB signaling pathway plays an important role in pathogen infection,immune response and inflammatory response,and it’s will affect the transcription of various inflammatory mediators,such as TNF-a,IL-1β,IL-8 and other adhesion factors.Our laboratory has studied the effect of DEV on NF-κB.Based on the fact,this study use NF-κB pathway activators and inhibitors to further explore the effect of NF-κB pathway on DEV proliferation,in order to provide a basis for the pathogenesis,data and new ideas for the prevention and control of DEV.1.Establishment the real-time RT-PCR for detection of NF-κB1 gene:According to the the nucleotide sequence of NF-κB1 gene sequence,using duck embryo fibroblast mRNA extraction sample as template,designed a specific amplification primers to establish a real-time quantitative PCR assay for NF-κB1 gene.Then the curve was tested for specificity,reproducibility and sensitivity.The results showed that the curve had high specificity and It had a good linear relationship in the range of 1.79*10⁷¹.79*10¹¹copies/μL template.The lowest detection limit was 1.79 copy numbers of viral nucleic acid.So this method had high sensitivity.The standard curve equation of the established RT-PCR method was y=-3.12x+44.086,and the amplification efficiency was 109.2%.In three repeated experiment cases,the coefficient of variation was less than 0.2%,showing the characteristics of good repeatability.These results indicate that the NF-κB1 gene fluorescence quantitative PCR assay was successfully established.2.Effect of activator LPS/inhibitor SN50 on cell proliferation:Activator LPS and inhibitor SN50 were prepared at 3 different concentrations（ie 2μg/mL,4μg/mL,6μg/mL and 25μg/mL）,DEF cells treated with LPS/SN50,then added CCK-8 after 12h,24h and 48h and tested the OD.The results showed that different concentrations of LPS and SN50 were obtained.The survival rates of DEF cells in the three different time periods were not different from the normal DEF,which suggesting that LPS and SN50 have little effect on the proliferation of DEF cells.3.Effect of activator LPS/inhibitor SN50 on NF-κB pathway:DEF cells were treated with the above three concentrations of activator LPS.The cells and supernatants were collected at 12 h,24 h,36 h,48 h,60 h,72 h,84 h,96 h,108 h and 120 h after treatment.The mRNA content of NF-κB1 gene was detected by real-time PCR.The secretion levels of five cytokines were detected by ELISA.The results showed that all different concentrations of LPS can activate the transcription level of NF-κB1 gene in DEF cells,which can increase the transcription level of NF-κB1 gene.The concentration of NF-κB1 gene was significantly increased by LPS at 4μg/mL（P<0.01）.The secretion levels of five cytokines in DEF cells were not significantly affected,and the secretion changes were irregular.The above three different concentrations of inhibitor SN50 were used to treat DEF cells.The mRNA levels of NF-κB1 gene and the secretion levels of five cytokines in DEF cells were detected as above.The results showed that different concentrations of SN50 had different levels of NF-κB1 gene transcription in DEF cells.The effect of 50μg/mL and75μg/mL SN50 treatment can effectively reduce the transcription level of NF-κB1 gene in DEF cells,which is significantly different from the normal group（P<0.01）.The concentration of SN50 at 25μg/mL is 48h.The NF-κB1 gene transcription level was significantly increased after treatment with the normal group（P<0.05）.There was no significant change in the cytokine secretion level of DEF cells treated with different concentrations of SN50.4.Effect of activator LPS/inhibitor SN50 on DEV proliferation:DEF cells were treated with the above three concentrations of activator LPS and DEV.The cells and supernatants were collected at 12 h,24 h,36 h,48 h,60 h,72 h,84 h,96 h,108 h and 120 h after treatment.The mRNA levels of NF-κB1 and DEV-NP genes and the secretion levels of five cytokines in DEF cells were detected as above.The results showed that the transcription levels of NF-κB1 gene in DEF cells were detected by different concentrations of LPS.Different effects,2 Lg/mL concentration of LPS can reduce the transcription level of NF-κB1gene in DEV-infected cells,while LPS at 4μg/mL and 6μg/mL can increase the transcription of NF-κB1 gene at most time points.The level of DEV-NP gene transcription was gradually increased after 84 hours.Trend;different concentrations of LPS treatment had no significant effect on the secretion level of cytokines in DEV-infected cells.It is suggested that LPS will promote DEV proliferation in the late stage of infection.The above three different concentrations of inhibitor SN50 were used to treat and inoculate DEV,and then the mRNA levels of NF-κB1 gene and DEV-NP gene and the secretion levels of five cytokines were detected.The results showed:concentration of 25μg/mL and 75μg/mL.The SN50 treatment,in addition to some time points,can effectively reduce the transcription level of NF-κB1 gene in DEV-infected cells,which is significantly different from the infection control group（P<0.01）.The concentration of SN50 at 50μg/mL can be obtained at all time points.The effective reduction of NF-κB1 gene transcription level was significantly different from the infection control group（P<0.01）.Different concentrations of SN50 treatment were effective in reducing the transcription level of DEV-NP gene;different concentrations of LPS treatment were responsible for DEV infection of DEF cytokine secretion.The level has no significant effect.It is suggested that SN50 inhibits DEV proliferation through the NF-κB pathway.In summary,this study successfully established a RT-PCR assay for duck-derived NF-κB1 gene.Different concentrations of activator LPS can promote the transcription level of NF-κB1 gene in normal DEF cells,while SN50 can effectively reduce the transcription level of NF-κB1 gene at high concentrations（50μg/mL and 75μg/mL）.LPS promotes DEV proliferation in the late stage of DEV infection,while the inhibitor SN50 can effectively inhibit DEV proliferation and is completed by the NF-κB pathway.