The Effect Of Rho/ROCK Pathway Inhibition On Cultured Astrocytes From Rat Spinal Cord

Objective:To study the effect of Rho/ROCK pathway inhibition by Y27632, a special ROCK inhibitor, on spinal cord astrocytic proliferation in vitro.Methods:Primary cultured rat spinal astrocytes were randomly divided into two groups:control group and Y27632group (treated by DMEM/F12containing10%FBS with10μM Y27632or with distilled water for control group). At different time points (0h、6h、12h、24h、48h), the numbers of cells were counted using a hemocytometer, and cell cycle analysis was performed by flow cytometry with prodium iodine (PI) staining. The expression of cyclin D1、cyclinE、CDK4、P27and p-ERK were evaluated by western blot assay. Brdu incorporation experiments were done to confirm the involvement of ERK signaling pathway in astrocyte cell cycle progression.Results:Compared with control group, the percentage of astrocytes in S phase,as well as the number of astroytes were significantly increased in Y27632group at6h and12h (P<0.05). Meanwhile, the expression of cyclin D1, CDK4, cyclinE and p-ERK1/2were up-regulated in Y27632groups at different time points investigated compared with the control group. Brdu incorporation was higher in Y27632groups than that in control groups (P<0.05), which was suppressed when U0126, a inhibitor of ERK, was used with Y27632.Conclusion:Rho/ROCK pathway inhibitor Y27632can remarkably promote proliferation and cell cycle progression of spinal cord astrocyte, in which the ERK signaling cascade was involved.