Dynamic Change Of Key Molecules IKKs In The TLRs/MyD88 Signaling Pathway Of Rana Dybowskii Under The Stress Of Aeromonas Hydrophila

In recent years,the number of global amphibian populations has dropped sharply,presumably due to the habitat destruction of amphibians,environmental pollution and infectious diseases.Amphibians are highly susceptible to environmental factors due to their high skin permeability.Especially in the environment,pathogenic microorganisms have become one of the important environmental pressures for amphibians to survive.During the long-term evolution of amphibians,natural immune defense systems have been established to protect against harmful factors such as environmental pathogenic microorganisms.Mammalian studies have reported that Toll-like receptors(TLRs)play an important role in innate immune defense,and TLRs signaling pathways induce downstream immune effectors to participate in immune responses through MyD88-dependent pathways and in-dependent pathways.As a key molecule in the TLR pathway,IKKs are involved in the activation of downstream nuclear transcription factors in the MyD88-dependent pathway and the in-dependent pathway.At present,the role of IKKs in the TLR signaling pathway in amphibians is still unclear.Rana dybowskii,a dominant amphibious species in northeastern China,was studied.The frog-like susceptibility strain Aeromonas hydrophila(Ah)was used as a stimulator to investigate the stress of Aeromonas hydrophila.The key molecules IKKs(IKKa,IKK? and TBK1)in the TLRs/MyD88 pathway of Rana dybowskii participate in the changes and regularity of immune response,and provide a theoretical basis for further understanding of amphibious innate immunity.In this study,the Rana dybowskii was divided into Ah infection group,LB(negative control)group and LPS(positive control)group,lml of Aeromonas hydrophila suspension(1×107 cfu/ml)and LB liquid culture were intraperitoneally injected.Base and LPS solution(1 mg/ml).Fluorescence quantitative PCR was used to detect the expression of IKKa,IKK? and TBK1 mRNA in kidney,liver,lung and spleen of Rana dybowskii.The results showed that the expression of IKKa,IKK? and TBK1 mRNA increased at 4-72 h after Ah infection,and the change trend of LPS group was the same as that of Ah group,and IKKa and IKK? were more significant than TBK1 mRNA.The expression levels of IKKa,IKK? and TBK1 in kidney,liver,lung and spleen were tissue-specific,especially in the liver.Western Blot showed that the protein level of IKKa was up-regulated in 72 hours,Ah group in 4.4 times(p<0.05),and LPS group in 5.87 times(p<0.01).The results of IHC staining of hepatocytes showed that the number of IHC-positive cells in liver tissue increased,IKKa and IKKp were concentrated in the cytoplasm.In summary,during the infection of Aeromonas hydrophila,IKKa,IKK? and TBK1 are involved in the immune response,and the MyD88-dependent pathway is the main factor,and the MyD88 in-dependent pathway is supplemented.