Expression Of IL-8 In Chinese Fire-bellied Newts During Limb Regeneration

Background: Limb regeneration after injury has always been a difficulty and research point in medicine.The immune system and its regulatory mechanisms play a key role in wound healing and regeneration.An amphibian newt which has a unique and powerful regenerative capacity is a prefect regeneration model.Macrophages are critical for the early limb regeneration,but which signal molecule initiate macrophages to recruite to the wound is still unknown.Interleukin-8(IL-8)is a cytokine that strongly chemotaxis to macrophages and plays a key role in the inflammatory response.IL-8 is also involved in cell migration,angiogenesis,mitosis and other life activities.However,whether IL-8 plays a role in wound healing and regeneration has poorly reported.Methods and Results: In this study,the amputation model of newt was firstly established.the primers were designed according to the transcriptome sequencing.The open reading frame(ORF)of newt IL-8 was firstly obtained by Polymerase Chain Reaction(PCR)and encoded306 bp,which can encode 101 amino acids.By constructing the prokaryotic expression vector p ET32a/IL-8,the IL-8 fusion protein about 29 k D was successfully induced to express.Subsequently,using the purified fusion protein,we prepared the IL-8 polyclonal antibody and antibody specificity was detected by ELISA,cell transfection and Western Blot.Quantitative Real-time PCR(QRT-PCR)and in situ hybridization were used to analyze the expression of IL-8 in m RNA level at different time points during limb regeneration.We found that IL-8m RNA was up-regulated in the early stage of newt limb regeneration,the expression was gradually decreased and returned to nomal level until the 14dpa(day post amputation).Subsequently,IL-8 polyclonal antibody was used to observe the expression and distribution of IL-8 at the protein level at different time points of limb regeneration.Western Blot results showed that the expression of IL-8 protein continued to increase and peaked at 3dpa after amputated,and the expression of IL-8 protein gradually recovered to the normal baseline level after 14 dpa regeneration.A large number of IL-8 positive cells were detected near the amputation site by fluorescence immunohistochemical staining in the early limb regeneration.Conclusions: This study demonstrated that IL-8 is significantly up-regulated in the early stages during limb regeneration,suggesting that IL-8 may be involved in the early process of limb regeneration.This results provided a basis for further revealing the function of IL-8 in limb regeneration and explaining its related mechanism of action.