原花青素B2对H2O2诱导的星形胶质细胞氧化损伤和凋亡的保护作用及机制研究

Protective Effect and Mechanism of Proanthocyanidin B2 Against H2O2-induced Oxidative Damage and Apoptosis of Astrocytes

关键词:  原花青素B2  星形胶质细胞  H₂O₂  氧化损伤  细胞凋亡  Akt/Stat3  Nrf2/HO-1

Key words:  proanthocyanidin B2  astrocytes  H₂O₂  oxidative damage  cell apoptosis  Akt/Stat3  Nrf2/HO-1

摘要:

目的 探讨原花青素B2(proanthocyanidin B2，PC-B2)对过氧化氢(H₂O₂)诱导的小鼠星形胶质细胞(astrocytes，AS)氧化损伤和凋亡的保护作用及其机制。方法 利用C57BL/6新生小鼠(1~3 d)分离、培养AS，通过筛选的H₂O₂和PC-B2最佳作用浓度分为正常组、正常+PC-B2组(100 μg·mL^-1 PC-B2处理24 h)、H₂O₂模型组(200 μmol·L^-1 H₂O₂处理24 h)、PC-B2组(200 μmol·L^-1 H₂O₂与100 μg·mL^-1 PC-B2共同处理24 h)；CCK-8法检测各组细胞存活率，LDH法进行细胞毒性检测；ABTS和DPPH法检测PC-B2的抗氧化能力；ELISA试剂盒检测各组细胞中MDA含量以及SOD、CAT和GSH-Px活力；TUNEL染色法检测各组细胞凋亡情况；RT-PCR和Western blotting分别检测AS中Bax、Bcl-2、Caspase-3、Akt/Stat3、p-Akt、p-Stat3、Nrf2/HO-1的mRNA和蛋白表达水平。结果 PC-B2能够明显增强细胞活力，抑制AS凋亡。并且与H₂O₂模型组相比，PC-B2干预能够显著降低AS中LDH、MDA含量，提高SOD、CAT和GSH-Px活力，抑制Bax、Caspase-3的mRNA和蛋白表达，上调Akt/Stat3、Bcl-2、Nrf2/HO-1的mRNA和蛋白表达。结论 PC-B2能够通过Akt/Stat3和Nrf2/HO-1途径增强AS抗氧化能力，减轻H₂O₂诱导的AS氧化损伤和凋亡。

Abstract:

OBJECTIVE To investigate the protective effect proanthocyanidin B2(PC-B2) on oxidative damage and apoptosis of mouse astrocytes(AS) induced by hydrogen peroxide(H₂O₂) and its mechanism. METHODS AS were isolated and cultured from neonatal C57BL/6 mice(1-3 d). The optimal concentration of H₂O₂ and PC-B2 was divided into four groups: normal group, normal+PC-B2 group(100 μg·mL^-1 PC-B2 treated for 24 h), H₂O₂ model group(200 μmol·L^-1 H₂O₂ treated for 24 h), PC-B2 group(200 μmol·L^-1 H₂O₂ and 100 μg·mL^-1 PC-B2 treated for 24 h). The cell viability of each group was detected by CCK-8 method. Cytotoxicity was detected by LDH method. The antioxidant capacity was detected by ABTS and DPPH. The content of MDA and the activity of SOD, CAT and GSH-Px were detected by ELISA kit. Detection of apoptosis in each group was done by TUNEL staining. The mRNA and protein expression levels of Bax, Bcl-2, Caspase-3, Akt/Stat3, p-Akt, p-Stat3 and Nrf2/HO-1 in AS were detected by RT-PCR and Western blotting, respectively. RESULTS PC-B2 could significantly enhance cell viability and inhibit AS apoptosis. Compared with the H₂O₂ model group, PC-B2 intervention could significantly reduce the content of LDH and MDA in AS, and increase the activity of SOD, CAT and GSH-Px. PC-B2 intervention could inhibit the mRNA and protein expression of Bax and Caspase-3, and up-regulate the mRNA and protein expression of Akt/Stat3, Bcl-2, Nrf2/HO-1. CONCLUSION PC-B2 can enhance the antioxidant capacity of AS through Akt/Stat3 and Nrf2/HO-1 pathways, therefore reduce H₂O₂-induced AS oxidative damage and apoptosis.