Expression Change Of NF-κB And Its Related Immunologic Factors In Organization Of Rana Dybowskii Under RGV Stress

In the long-term evolution, the amphibians formed a immune defense system to address a variety of microorganisms. Nuclear transcription factor kappa B (NF-kappa B) are an important class of model molecules involved in the body’s innate immune and also a bridge to connected innate and acquired immunity. To study the molecular expression patterns and the expression of effector protein under pathogenic microorganisms stress has a huge role in immunology.In this study, we selected Rana dybowskii which is amphibians representatives species as subject investigated, Rana grylio virus (RGV) as pathogenic microorganisms infected healthy adult R.dybowskii. We found abdominal organs by the anatomical lesions to confirm the frog iridescent virus infection. Selecting the frog skin, liver, spleen and blood tissue after the injection0,0.5,1,2,4,8,12,16,24,48and72hours as the experimental material. In accordance the High-throughput sequencing results with the laboratory, design NF-κB1, the NF-κB2, I-kappa B, TNF-alpha, VCAM, SOD and iNOS primers, using SYBR Green I quantitative PCR technique, we detected NF-κB1, the NF-κB2, I-kappa B, TNF-alpha, VCAM, SOD and iNOS molecule mRNA expression levels over the time, analysised the dynamic expression pattern, in order to explore if the NF-κB involving in immune response after RGV infected, and the expression pattern of reaction with RGV.The results showed that after designing primers and optimization of reaction conditions and reaction system, we successfully found quantitative PCR of the mRNA of NF-κB1, the NF κB2, I-kappa B, TNF-alpha, VCAM, SOD and iNOS molecule in R.dybowskii skin, liver, spleen and blood. Using this method, the melting curve figure showing single specific peak, not existed specific amplification and primer-dimers, illustrated the optimal conditions provide a reliable quantitative method for the further study R.dybowskii skin, liver, spleen and blood gene expression after RGV stress. After RGV stress, the NF-κB1, the NF-κB2and I-kappa B molecule mRNA expression showed the law change in the R.dybowskii skin. After0.5h, NF-κB1and NF-κB2in the three organizations have raised trend. Respectively after stimulation the2h (in the liver) and4h (in the skin) quickly raised to the peak of NF-κB1and NF-κB2. NF-κB1and NF-κB2showed slow upward trend in the spleen, respectively, in16h (NF-κB2) and24h (NF-κB1) has the peak. After the peak, NF-κB1and NF-κB2starts to rise again. And on the8h (in the liver) and12hours (the skin) down to the initial level, on the72h to reach the initial level in the spleen. By early stimulation, I-kappa B immediately reduced to the expression levels. Its raised the point in time that is substantially identical with the NF-B up regulation time point. There were significant differences between the experimental group and control group (P<0.05).R·dybowskii with RGV stress for the experimental group and R·dybowskii give the NF-kappa B inhibitor for the control group. To detected the expression of immune-related factor TNF-alpha, VCAM, SOD and iNOS molecules in the blood. The results show that the gene expression of the experimental group has significantly increase compared with the control group. After stress48hours, TNF-alpha expression increased of the most significant, is five times that of the control group. VCAM were significantly increased at each time point. Where in the expression amount of2hours to60times that of the control group. On96hours recovery to normal levels. SOD in test points the expression levels of increased significantly, the highest expression level up to100times that of the control group. iNOS expression was increased significantly by RGV stress1,2,12,48hours. There were significant differences between the experimental group and control group (P<0.05).Conclusion:Under RGV stress, the NF-κB expression of Rana dybowskii decreased first, then increased, and then decreased trend. I-κB expression were basically the same with the NF-κB, and expression increased point is slightly of backwardness NF-κB. Proved that under RGV stress NF-κB signaling pathway of Rana dybowskii is activated. Compared with NF-κB inhibitor group, the expression of immune factors in the experimental group was significantly higher. Proof that NF-κB molecules can regulate transcription and translation process of TNF-a, VCAM, SOD and iNOS, thus participate in the immune defense reaction of Rana dybowskii.