Netrin-1Signal Mediates NO-induced The Migration Of Glial Cells

Astrocytes and microglia are the major componet in the central nervous system. Thesecells occur widely to construct the complex neural network by migration. Cell migration isregulated by a variety of factors, including cell signaling system, the cytoskeleton andvarious molecules involved in the formation of focal adhesions. Cell migration takes animportant position in a variety of physiological and pathological phenomena, such asneurogenesis, nerve regeneration, injury healing, immunity, infection and cancer metastasis.Netrins belong to axon guidance factor family, including netrin-1,3,4and G-netrin inmammalian. Netrin-1is a highly conserved member, pilays an important role in attractingor repelling the cell migration, guide the axon growth. In response to nerve injury andpathophysiological process, NO plays a key regulatory role in the inflammatory cascade,especially in the inflammatory reaction and the signal transduction after inflammation. Inthe central nervous system NO as a neurotransmitter, regulating synaptic plastcity, isinvolved in long-term potentiation effect. Whether cell migration and expression ofnetrin-1and its receptors DCC and UNC5B were regulated by NO has not been studieduntil now, therefore, study of NO influencing on cell migration and netrin-1signaling inastrocytes and microglia after nervous injury would be helpful to further explore the glialcell migration mechanism and provide new ideas for clinical treatment of neurologicalinflammatory diseases.Objective: To investigate the NO influence the migration of astrocytes and microglia,as well as the expression of netrin-1and its receptors DCC and UNC5B, and explore themechanism of cell migration and the function of NO and netrin-1in nerve inflammation.Methods:(1) Purified cultures of astrocytes and microglia were prepared fromneonatal rats, based on the differential properties of developmental time-course andcellular adhesions.(2) To determine the SNP (NO donor) concentration of the bestadministration by MTT， we established astrocytes and microglia scratch model, set thecontrol group (0h group) and the experimental groups, the experimental groups were given50μmol/L SNP (sodium nitroprusside), observed at different times respectively.(3) Astorcytes and microglia were treated with final concentration50μmol/L SNP for differenttimes respectively (0h,6h,12h,24h,48h,72h). Samples were collected, NO3-,NO2-concentration was detected in culture medium through Griess reagent. Expression ofnetrin-1after treatment was examined by Western Blotting and Immunocytochemistry.(4)Normal control group, SNP+Hb group, SNP group were cultured for24h, the mRNAexpression of netrin-1, DCC, UNC5B in astrocytes and microglia of three groups wasdetected by real time PCR.Results:(1) Astrocytes and microglia were successfully isolated by mechanicalshaking and differential adhesion method, purity>95%.(2)Adequate concentration of NOdonor sodium nitroprusside was identified by MTT. Scratch model results showed that themigration of astrocytes and microglia in experimental groups was faster than that of thecontrol group. NO detection of cell culture medium at different times found that NO2-concentration increased significantly after treatment, indicating that in the experimentalgroup NO was mainly originated from the exogenous SNP.(3)Results of western blottingand immunocytochemistry showed that the expression of netrin-1increased significantlyafter SNP treatment (P<0.01).(4)The results of real time PCR showed that compared withthe control group, the mRNA expression of netrin-1、DCC、UNC5B was upregulated inSNP group but had no statistical change in SNP+Hb group.Conclusion: NO can promote the migration of astrocytes and microglia and increasethe expression of netrin-1, indicating that NO can promote the migration of astrocytes andmicroglia via netrin-1signalling. The result suggests NO and Netrin-1played a role innerve injury and inflammation.