In Repairment Of Secondary Nerve Injury Of Experimental Intracerebral Hemorrhage Via A Netrin-1-mimetic Peptide

Intracerebral hemorrhage(ICH)is a type of primary non-traumatic intracranial hemorrhage.The release of blood and blood breakdown products may damage and destroy the brain parenchyma,leading to incapacity and even life-threatening to patients.The surgical treatment can be used to effectively remove hematoma,but there is still a lack of effective treatment for neuronal injury of secondary brain injury caused by blood breakdown products.How to reduce neuronal injury plays crucial role in the recovery of patients with intracerebral hemorrhage.Netrin-1 is a secretory axon guidance cue which can regulate neural development and injury repair.Clinical studies have been found that the level of Netrin-1 in patients with intracerebral hemorrhage gradually increases in the process of rehabilitation.It is suggesting that Netrin-1 may be associated with the functional recovery after intracerebral hemorrhage.In experimental intracerebral hemorrhage models,intracerebral injection of Netrin-1 has also been demonstrated to reduce receptor-mediated neuronal apoptosis.At present,one of the limiting factors for the clinical application of Netrin-1 is that Netrin-1 is also a potential carcinogenic factor.Therefore,further study and design of functional peptides derived from Netrin-1 is meaningful for the development of the neuroprotective effects of Netrin-1.According to the crystal structure analysis,the EGF3 domain of Netrin-1 is the key domain binding with deleted in colorectal cancer(DCC)of FN5 domain to form a stable ligand and receptor complex.We firstly confirmed Netrin-1’s EGF3 domain specific binding with DCC’s FN5 domain by GST pull down.The Netrin-1 without the EGF3 functional domain(407-443 amino acids)could not bind to its receptor DCC.Further,FITC-labeled Pep EGF3,which derived from Netrin-1’s EGF3 domian,can specifically bind to its receptor DCC.The Pep EGF3 also can activate Netrin-1 signaling pathways,including FAK(focal adhesion kinase),SRC(Src family kinase)and ERK(extracellular signal regulated kinase)signaling.In order to optimize the minimum functional fragment of Netrin-1-derived peptides,Pep EGF3 is truncated into two peptides,Pep E1(residures 407-422)and Pep E2(residures 423-433).Western blotting assay showed that the synthesized Pep E1 and Pep E2 interacted with DCC and activated FAK and SFK signaling pathways.However,Pep E2 still activated ERK signaling pathway,but not Pep E1.Next,in order to investigate the repair effect of Pep E1 and E2,neuronal injury models mediated with hemin in NLT cells(Gn RH-expressing neuronal cells)and primary neurons to simulate secondary brain injury after intracerebral hemorrhage in vitro.The results showed that the expression of Netrin-1 incresed and reached the peak after 6h when cells incubated with Hemin.Subsequently,different concentrations of Pep E1 and E2 were co-incubated containing hemin cells.Cell viability assay and cell survival assay showed that Pep E1 activated the phosphorylation of FAK and SFK dependent on DCC.The cell survival rate increased from 50% to 60-76%.These results suggested that Netrin-1-derived Pep E1 had a neuroprotective effect on hemin mediated neuronal damage and death in vitro.No significant improvement of Pep E2 was observed,which may be related to activation of ERK phosphorylation and promotion of cell necrosis after hemin treatment.Finally,We developed a intracerebral hemorrhage model of mice through injection of Ⅶtype collagenase into murine right striatum.The expression of Netrin-1 protein in the cortex of mice was upregulated and reached the peak 2 days after intracerebral hemorrhage.We found that intraperitoneal injection of transmembrane peptide(TAT)sequence labeled E1 effectively crossed the blood-brain barrier,increased the survival rate of ICH modle mice,significantly improved the asymmetric behavior of the left forelimb of ICH modle mice.Meanwhile,Pep E1 can effectively reduce the volume of striatal hematoma and reduce neuronal damage by activating FAK and Akt signaling pathwaysIn summary,we found that a functional Pep E1 which derived from Netrin-1 specifically bound receptor DCC and activated downstream FAK and SFK signaling pathways,but did not activate ERK phosphorylation.Pep E1 protect neurons from hemin-induced toxicity and improve functional recovery through reduced apoptosis in an experimental murine model of intracerebral hemorrhage.These results provide a safer and more reliable candidate peptide for the treatment of ICH.