The Study To The Function Of IL-1RA-PEP Fusion Protein And Its Curative Effect On Cerebral Ischemia-reperfusion Injury

Cerebral ischemic stroke is a cerebral disease caused by a disruption of blood circulation in the brain. Ischemia-reperfusion injury is a major damage form existing in the treatment of stroke, which has great harm and poor prognosis. There is no effective and safe drug to cure stroke at present.The inflammatory accompany the occurrence and development of ischemic cerebral injury. Interleukin-1(IL-1) is major inflammatory mediator which plays a crucial role in the secondary inflammation following the ischemia-reperfusion. Interleukin-1receptor antagonist(IL-1RA) is natural anti-inflammation cytokine, which can antagonize the signal transduction of IL-1, then block the synthesis and action of downstream inflammatory medium. The Study shows that IL-1RA has a certain therapeutic effects to cerebral ischemic injury, trauma injury, subarachnoid hemorrhage and other cerebral vascular disease. However these therapeutic effects are not remarkable owing to the blood brain barrier(BBB) restricts the effective access of IL-1RA to the site of cerebral injury an inflammation.In recent years, cell penetrating peptides(CPPs) develop a non-invasive method to transport macromolecules across cell membrane. It can carry molecules permeating some biomembranes, such as skin, BBB and nasal mucosa, to exert its biological effect.PEP-1 is an amphipathic molecule, a kind of CPPs, which has been applied to transport molecules into disparate tissues and organs. Therefore, the use of PEP-1 may provide a new clinical opportunity to IL-1RA, especially offer the impossible for IL-1RA to antagonize the inflammatory following cerebral ischemic reperfusion injury effectually.This study fuses PEP-1 with IL-1RA, constructing one fusion molecular. Firstly, we engineer the IL-1RA-PEP fusion gene, and achieve the effective expression in the prokaryotic system. The expression of IL-1RA-PEP fusion protein accounts for over30% of the total protein. We acquire the 95% purity of the fusion protein through the anion, cation exchange chromatography and gel filtration chromatography. The study of EL4 cell activity shows that IL-1RA-PEP can antagonize the biological activity of IL-1α, and its antagonistic ability is stronger than that of IL-1RA. Meanwhile, the immunofluorescence localization indicates that IL-1RA-PEP can penetrate the Ha Ca T cellular membrane, and mainly aggregate in the cytoplasm and nuclei.IL-1RA-PEP fusion protein intravenous inject into rats suffered from the middle cerebral artery occlusion, At the time point of 24 h, we analysis the IL-1RA-PEP’s permeating effect of the brain tissue and the inhibitory ability to cerebral inflammation.Results display that IL-1RA-PEP effectively penetrated the brain tissue following the ischemia-reperfusion, reduced the volume of cerebral infraction, lessened the brain edema, decreased the nerve defect score, diminished the inflammatory cytokines in the serum and cerebral tissue, abated the invasion of macrophages, and degraded the activated number of neutrophils in the brain.We analyzed the therapeutic mechanism of IL-1RA-PEP to the cerebral ischemia reperfusion injury preliminarily. The research shows that IL-1RA-PEP can significantly adjust NF- Κ B signal pathways, and by down-regulating of NF- Κ B signal pathways, IL-1RA-PEP inhibit the expression of inflammatory cytokine, the invasion and activation of inflammatory cells, which has significantly suppression effect to inflammation following the cerebral ischemia-reperfusion.In conclusion, ⑴ we engineered the IL-1RA-PEP fusion gene, and purified the fusion protein by inducing expression. ⑵ IL-1RA-PEP has a protective effect on the cerebral ischemic reperfusion, which can availably reduce cerebral infraction, decrease brain edema, degrade nerve defect, suppress the secondary inflammation in the brain tissue.⑶ the mechanism of IL-1RA-PEP exhibiting the treatment on the cerebral ischemic reperfusion injury is mainly through the regulation of NF-ΚB signal pathways.In summary, this study built a permeating membrane and anti-inflammatory bifunctional fusion molecular IL-1RA-PEP, laying the foundation for the research and development of novel drug on the treatment of cerebral ischemia-reperfusion injury.