Netrin-1 In Reactive Astrocytes Participates In Pathology After Traumatic Brain Injury Through NF-κB Canonical Pathway

Traumatic Brain Injury(TBI) caused by external mechanical damage, produces primary injury, followed by secondary injury which is induced by a series of organism biochemical reactions due to the primary injury. The areas surrounding lesions have gradually recovery and formed glial scar over a period of time. However, there is a certain functional decline at the lesion site after injury as the adult central nervous system lacks of the ability of the regeneration. Reduction of secondary injury deterioration as well as promotion of the damaged tissues rejuvenation facilitate the study of the mechanism of traumatic brain injury. The moment neurons damage occurs, astrocytes, through stimulated proliferation and dendrites increases, develope into reactive astrocytes with GFAP upregulation. Netrin-1, an axon guidance factor, belonging to laminin family, plays an important role in cell migration. NF-κb(nulear factor-κB), namely nuclear factor kappa B, is such a key factor in cell signalling pathway that its dimmer reactive sites are exposed after initiation and the dimmer translocate into nuclear to spark the downstream translation. Related researches proves that patients with rectal carcinoma are developed from inflammatory enteritis whose NF- kappa B activation and netrin-1 upregulation was positively correlated. The machanism between NF-κB and netrin-1 in the central nervous system is rarely reported. Especially when the adult central nerve system was injuried the variation of the netrin-1 expression is a moot point. Thus our study intends to evaluate the relationship between the netrin-1 expression and NF-κb canonical pathway in the rat brain traumatic injury model and cell scratch.Part One The relationship between Netrin-1 and NF- kappa B classic pathway in the reactive astrocytesObjective Through the establishment of rat traumatic brain injury model,the relationship between netrin-1 and NF-kappa B pathway in astrocyte was studied.Furthermore, their role in the repairment of nervous system was investigated.Methods With the rat model of brain injury, the expression netrin-1 and GFAP,and the quantity of nuclear NF-κB protein in injured cortex areas were detected by western blotting at 12 h, 1day, 2 day, 3day, 1 week, 2 week, 3 weeks, 5 weeks, 7 weeks after injury. Tissues injuried two weeks with the highest expression level of netrin-1 was used to study the morphological changes by immunofluorescence.Results We found that the expression of the GFAP and netrin-1 gradually increased by using western blotting and astrocytes around the lesion activated in the experimental group. Moreover, nuclear protein p65 arised within two weeks to reach the peak and then decreased. Tissue immunofluorescence staining of rat cortical tissue was detected at two weeks after injury. Astrocyte reactivite response increased and netrin-1 expressed positively. The nuclear translocation of p65 raised obviously and the netrin-1 staining was evident around the lesion.Conclusion In the rat TBI model netrin-1 expression has a certain relationship with the nuclear translocation of p65, which invovles in the repair of the central nervous system.PART TWO The role of NF- kappa B inhibitor on the migration of astrocytes in vitroObjective To study NF-κb inhibitor effects on astrocyte migration in vitro and investigate the role of netrin-1 and NF-κb in the injured astrocyte.Methods By establishing cell scrath model, the experimental group(PDTC, 100μM) and the control group were set and selected at 12 h, 48 h, 72 h after injury for the detection of cell migration under the microscope and morphological changes. Netrin-1 and GFAP protein expression, nuclear p65 protein regulation were analyzed by western blot.Results After injury, compared with the control group astrocytes around the little-clear lesion boundaries in the experimental group migrated slowed; while the control group migrated obviously, the borders gradually blurred, and cells growed well. Western Blotting results indicated the expression of GFAP and netrin-1 in the experimental group similarly resembled nuclear p65 protein expession.Conclusion NF-κb inhibitor(PDTC) is not conducive to the migration of astrocytes after injury or repairing scratches injury. And in the experimental group the netrin-1 protein, GFAP protein and p65 were in a weakening trend. Netrin-1 protein and NF-κb pathway have reference to central nervous system repairment, yet the sophisticated mechanism should be detected further.