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  • Title: [Effect of carboxymethylated chitosan on apoptosis and expression of brain derived neurotrophic factor and glial cell line derived neurotrophic factor in oxidative stress induced Schwann cells in vitro].
    Author: He B, Tao H, Liu S.
    Journal: Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi; 2014 Dec; 28(12):1530-5. PubMed ID: 25826901.
    Abstract:
    OBJECTIVE: To 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 glial cell line derived neurotrophic factor (GDNF) in oxidative stress induced SCs. METHODS: Twenty-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 establish the apoptotic model. The cell counting kit 8 (CCK-8) and flow cytometry analysis were used to detect the cell viability 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 viability 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. RESULTS: The 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). CONCLUSION: CMCS 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.
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