Objective To investigate the effect of carboxymethylated chitosan (CMCS) on the proliferation, cell cycle, and secretion of neurotrophic factors in cultured Schwann cells (SCs). Methods SCs were obtained from sciatic nerves of 20 Sprague Dawley rats (3-5 days old; male or female; weighing, 25-30 g) and cultured in vitro, SCs were identified and purified by immunofluorescence against S-100. The cell counting kit 8 (CCK-8) assay was used to determine the proliferation of SCs. The SCs were divided into 4 groups: 50 μg/mL CMCS (group B), 100 μg/mL CMCS (group C), 200 μg/mL CMCS (group D), and the same amount of PBS (group A) were added. The flow cytometry was used to analyze the cell cycle of SCs; the real-time quantitative PCR and Western blot analysis were used to detect the levels of never growth factor (NGF) and ciliary neurotrophic factor (CNTF) in cultured SCs induced by CMCS. Results The purity of cultured SCs was more than 90% by immunofluorescence against S-100; the CCK-8 results indicated that CMCS in concentrations of 10-1 000 μg/mL could promote the proliferation of SCs, especially in concentrations of 200 and 500 μg/mL (P lt; 0.01), but no significant difference was found between 200 and 500 μg/mL (P gt; 0.05). CMCS at a concentration of 200 μg/mL for 24 hours induced the highest proliferation, showing significant difference when compared with that at 0 hour (P lt; 0.01). The percentage of cells in phase S and the proliferation index were significantly higher in groups B, C, and D than in group A (P lt; 0.05), in groups C and D than in group B (P lt; 0.05); and there was no significant difference between group C and group D (P gt; 0.05). Real-time quantitative PCR and Western blot results showed that the levels of NGF and CNTF in groups B, C, and D were significantly higher than those in group A (P lt; 0.05), especially in group D. Conclusion CMCS can stimulate the proliferation, and induce the synthesis of neurotrophic factors in cultured SCs.
ObjectiveTo investigate the protective effects of carboxymethylated chitosan (CMCS) on oxidative stress induced apoptosis of Schwann cells (SCs), and the expressions of brain derived neurotrophic factor (BDNF) and gl ial cell line derived neurotrophic factor (GDNF) in oxidative stress induced SCs. MethodsTwenty-four 3-5 days old Sprague Dawley rats (weighing 25-30 g, male or female) were involved in this study. The bilateral sciatic nerves of rats were harvested and SCs were isolated and cultured in vitro. The purity of SCs was identified by immunofluorescence staining of S-100. SCs were treated with different concentrations of hydrogen peroxide (H2O2, 0.01, 0.10, and 1.00 mmol/L) for 3, 6, 12, and 24 hours to establ ish the apoptotic model. The cell counting kit 8 (CCK-8) and flow cytometry analysis were used to detect the cell viabil ity and apoptosis induced by H2O2, and the optimal concentration and time for the apoptotic model of SCs were determined. The 2nd passage SCs were divided into 5 groups and were treated with PBS (control), with 1.00 mmol/L H2O2, with 1.00 mmol/L H2O2+50 μg/mL CMCS, with 1.00 mmol/L H2O2+100 μg/mL CMCS, and with 1.00 mmol/L H2O2+200 μg/mL CMCS, respectively. After cultured for 24 hours, the cell viabil ity was assessed by CCK-8, cell apoptosis was detected by flow cytometry analysis, the expressions of mRNA and protein of BDNF and GDNF were detected by real-time quantitative PCR and Western blot. ResultsThe immunofluorescence staining of S-100 indicated the positive rate was more than 95%. CCK-8 and flow cytometry results showed that H2O2 can inhibit the proliferation of SCs and induce the SCs apoptosis with dose dependent manner, the effect was the most significant at 1.00 mmol/L H2O2 for 24 hours; after addition of CMCS, SCs exhibited the increased proliferation and decreased apoptosis in a dose dependent manner. Real-time quantitative PCR and Western blot analysis showed that 1.00 mmol/L H2O2 can significantly inhibit BDNF and GDNF expression in SCs when compared with control group (P<0.05), 50-200 μg/mL CMCS can reverse the oxidative stress-induced BDNF and GDNF expression in SCs in a dose dependent manner, showing significant difference compared with control group and 1.00 mmol/L H2O2 induced group (P<0.05). There were significant differences among different CMCS treated groups (P<0.05). ConclusionCMCS has the protective stress on oxidative stress induced apoptosis of SCs, and may promote the BDNF and GDNF expressions of neurotrophic factors in oxidative stress induced SCs.