RSC96 proliferation and migration induced by Etifoxine through up-regulating CELSR2 expression / 中华显微外科杂志

ABSTRACT

Objective:To observe the effects of Etifoxine on proliferation and migration of RSC96 (Schwann cells of rat) and its potential molecular mechanisms.Methods:From March, 2020 to October, 2020, cultured RSC96 were treated with 20 μmol/L Etifoxine and saline respectively for 48 h. Cell proliferation was tested by EdU assay using Cell-Light EdU DNA Cell Proliferation Kit and the capability of migration was determined by wound healing assay and a transwell system. To investigate the effects of Etifoxine on CELSR2 protein expression, after treated with different concentrations of Etifoxine at 0-20 μmol/L for 48 hours, cells were subject to Western blot analysis to verify the expression of CELSR2 protein. To explore whether CELSR2 would be a potential target of Etifoxine, siRNA targeting CELSR2 and control siRNA groups were transfected into 20 μmol/L Etifoxine-treated RSC96 using Lipo3000. Again, the cell proliferation and migration of were investigated after 48 hours with the same procedures. The two-tailed Mann-Whitney U test was employed in statistical assessment.Results:EdU results showed a significant higher percentage of Edu-positive (proliferating) cells in the 20 μmol/L Etifoxine-treated group than the control group[(36.30±3.09)% vs (19.40±2.50)%, P<0.05]. Transwell migration assay demonstrated that the number of 20 μmol/L Etifoxine-treated RSC96 which migrated through the transwell membrane was higher than saline group, with significant statistical difference [(132.30±6.77) vs(65.33±7.37), P<0.05]. The percentage of reduction of wound area measured at 24 hours and 36 hours after the scratch also showed the similar results [(30.67±2.16)% vs (23.00±2.61)%; (86.00±2.19)% vs (49.67±2.81)%, respectively, P<0.05]. Besides, with increase of the concentration of etifoxine, the expression of CELSR2 showed an trend of increase in RSC96 ( P<0.05), but no significant statistical difference was found between 10 μmol/L and 20 μmol/L groups ( P>0.05). Interestingly, the rate of cell proliferation, the number of migrating cells and the percentage of wound area reduction of RSC96 in which were treated by Etifoxine and transfected with CELSR2 siRNA were significantly decreased compared with the control siRNA treatment ( P<0.05). Conclusion:Etifoxine could promote proliferation and migration of RSC96. Upregulation of CELSR2 protein expression in RSC96 is associated with the Etifoxine-induced enhancement of cell proliferation and migration.