胰岛素样生长因子-1对新生大鼠神经细胞氧化损伤保护作用的研究

Protective effects of IGF-1 on cortical nerve cells of neonatal rats under oxidative stress

摘要:

目的 探讨氧化应激状态下，胰岛素样生长因子-1（IGF-1）对新生大鼠神经细胞氧化损伤的保护作用。方法 原代培养新生大鼠大脑皮层神经元、少突胶质细胞及星形胶质细胞，不同浓度H2O2（0~60 μM）诱导氧化应激细胞模型，LDH 法检测各种神经细胞损伤程度，MTT 法检测各种神经细胞活力；不同浓度H2O2（0~80 μM）诱导氧化应激神经元细胞模型，Western blot 检测IGF-1（25 ng/mL）施加前后神经元细胞内Akt 的磷酸化水平。结果 与未经H2O2 处理组相比，不同浓度H2O2 处理细胞24 h 后，大脑皮层神经元、少突胶质细胞及星形胶质细胞损伤程度均有升高、细胞活力均有降低；但神经元变化更为显著，与少突胶质细胞和星形胶质细胞相比，差异有统计学意义（均P<0.01）；不同浓度的H2O2 处理大脑皮层神经元5 min 后，相比于未经H2O2 处理组，可见Akt 磷酸化水平呈H2O2 浓度依赖性降低（均P<0.01），而在上述处理基础上加入25 ng/mLIGF-1 后，IGF-1 能够逆转低浓度H2O2 导致的神经元细胞Akt 磷酸化，与未经H2O2 处理组比较，差异无统计学意义（P>0.05），但是对高浓度H2O2 导致的Akt 磷酸化作用无明显效果，其磷酸化水平均低于未经H2O2 处理组（均P<0.01）；经不同浓度的H2O2 处理1 h 后，再加入25 ng/mL 的IGF-1，IGF-1 处理前后Akt 磷酸化水平均已恢复至未经H2O2 处理时的水平（均P>0.05）。结论 大脑皮层神经元对H2O2 诱导的氧化应激损伤较其他神经细胞敏感；IGF-1 对皮层神经元氧化应激损伤具有保护作用。

Abstract:

Objective To investigate the protective effects of insulin-like growth factor-1 (IGF-1) on the nerve cells of neonatal rats under oxidative stress. Methods Primary cortical neurons, oligodendrocytes, and astrocytes from newborn rats were cultured. An oxidative stress model was established with different concentrations of H2O2 (0-60 μmol/L); the degree of damage of nerve cells was evaluated by lactate dehydrogenase assay, and the viability of nerve cells was tested by MTT assay. An oxidative stress model was established with different concentration of H2O2 (0-80 μmol/L). Expression of Akt/p-Akt (Ser473) in neurons was measured by Western blot before and after IGF-1 (25 ng/mL) administration. Results Compared with those not treated with H2O2, the cortical neurons, oligodendrocytes, and astrocytes treated with different concentrations of H2O2 for 24 hours showed increased damage and decreased cell viability; compared with oligodendrocytes and astrocytes, neurons showed significantly more changes (P<0.01). Compared with those not treated with H2O2, the cortical neurons treated with different concentrations of H2O2 for 5 minutes showed a significant decrease in p-Akt (Ser473) level (P<0.01), which was dependent on the concentration of H2O2. For the neurons treated with low-concentration H2O2, the addition of IGF-1 could reverse the inhibition of Akt phosphorylation, eliminating the difference in p-Akt level compared with the neurons not treated with H2O2, (P>0.05); however, it had no significant effect on the inhibition of Akt phosphorylation by high-concentration H2O2, and the treated neurons still had a lower p-Akt level than untreated neurons (P<0.01 for all). For the cortical neurons that had been treated with different concentration of H2O2 for 1 hour, the addition of IGF-1 (25 ng/mL) could eliminate thedifference in p-Akt level between the treated neurons and untreated neurons (P>0.05). Conclusions Cortical neurons are more sensitive to oxidative stress induced by H2O2 than other nerve cells. IGF-1 has protective effects on cortical nerve cells under oxidative stress.