亚低温状态下冷诱导RNA结合蛋白调节氧化还原系统对海马神经元的保护作用

Cold inducible RNA-binding protein inhibits hippocampal neuronal apoptosis under hypothermia by regulating redox system

本文旨在探讨冷诱导RNA结合蛋白(cold inducible RNA-binding protein,CIRP)是否通过抑制氧自由基的生成而起到抑制神经元凋亡的作用。采用体外分离培养的原代大鼠海马神经元,随机分为5组:常温对照组(37℃)、空病毒感染组、CIRP过表达组、CIRP干扰组、亚低温32℃处理组。将后四组移入32℃含有5%CO2培养箱内培养。采用Annexin V-FITC/PI标记后用流式细胞术对海马神经元进行细胞凋亡检测、Western blot检测CIRP的表达,同时采用ELISA方法进行相关氧化还原指标(T-AOC,GSH-Px,SOD,MDA)检测。Western blot及流式凋亡检测结果显示:与常温对照组相比,单纯亚低温处理组及CIRP过表达组海马神经元中CIRP的表达量明显增加(P<0.01),神经元的凋亡率明显下降;而干扰CIRP的表达后,与单纯亚低温处理组相比,细胞凋亡的数目则明显增加(P<0.05)。相关氧化还原指标检测结果显示:亚低温32℃处理组、CIRP过表达组与常温对照组、CIRP干扰组、空病毒感染组相比均有显著差异(P<0.05或P<0.01)。以上结果表明:亚低温处理通过上调CIRP的表达,抑制细胞内氧自由基的生成,从而直接或间接地抑制了氧自由基诱导的神经元凋亡,进而起到海马神经元的保护作用。

In this study, we intend to confirm our hypothesis that cold inducible RNA-binding protein （CIRP） can inhibit neuronal apoptosis through suppressing the formation of oxygen free radicals under hypothermia. Primary rat hippocampal neurons were isolated and cultured in vitro, and were divided into five groups： （1） normal control group （37 oC）, （2） cells infected by empty viral vector group, （3） CIRP over-expressed group, （4） CIRP knock-down group, and （5） hypothermia control group. Cells in groups 2–5 were cultured under 32 oC, 5% CO2. Apoptosis of hippocampal neurons were detected by Annexin V-FITC/PI staining and flow cytometry; Expression of CIRP was determined by Western blot; Redox-related parameters （T-AOC, GSH-Px, SOD, MDA） were detected by ELISA kits. Results showed that CIRP expression levels were significantly increased （P 〈 0.01） and the apoptotic rates were significantly decreased （P 〈 0.01） in hypothermia control group and CIRP over-expressed group when compared with normal control group. On the other hand, the apoptotic rate was significantly increased （P 〈 0.05） in CIRP knock-down group compared with that in hypothermia control group. The levels of redox parameters in hypothermia control group and CIRP over-expressed group were significantly changed in comparison with those in normal control group, CIRP knock-down group and empty viral vector infected group, respectively （P 〈 0.05 or P 〈 0.01）. These results suggest that up-regulation of CIRP by hypothermia treatment can protect the neuron from apoptosis through suppressing the formation of oxygen free radicals.