神经球蛋白对慢性高眼压小鼠视网膜神经节细胞损伤的保护作用

背景 神经球蛋白(Ngb)是2000年发现的球蛋白,可以在缺氧或氧化应激的环境中保护细胞.Ngb在视网膜神经元中呈高表达,提示视网膜很可能是Ngb发挥其功能的重要场所. 目的 研究Ngb对小鼠高眼压所致视网膜神经节细胞(RGCs)损伤的保护作用及作用机制. 方法 本研究分为体外实验和体内实验两部分.将体外纯化培养的成年野生型(WT)小鼠和本实验室培育的Ngb转基因(Ngb-Tg)新生小鼠RGCs用不同浓度的谷氨酸作用3d后,以dead/live双染试剂盒染色计算RGCs的存活率,比较Ngb对不同浓度谷氨酸环境下培养的RGCs存活率的影响.将聚苯乙烯荧光微球注入WT和Ngb-Tg小鼠前房内以制备小鼠慢性高眼压模型,将小鼠分为WT小鼠对照组(n=18)、Ngb-Tg小鼠对照组(n=18)、WT小鼠微球单次注射组(n=38)、Ngb-Tg小鼠微球单次注射组(n=38)、WT小鼠微球2次注射组(n=6)及Ngb-Tg小鼠微球2次注射组(n=6).另设WT小鼠PBS注射组(n=6)、Ngb-Tg小鼠PBS注射组(n=6)观察PBS前房注射对眼压的影响.分别于前房注射微球后即刻(对照组),3d及1、4、8周处死小鼠,用实时荧光定量PCR、Western blot和免疫组织化学法定量分析Ngb mRNA及其蛋白在视网膜中的表达变化和RGCs的存活率,并对比两种小鼠视网膜中过氧化物和ATP表达水平. 结果 5.0、7.5、10.0 mmol/L谷氨酸处理后Ngb小鼠RGCs存活率均明显高于WT小鼠,差异均有统计学意义(t=2.810、3.020、3.110,P＜0.01).WT小鼠微球单次注射组及Ngb-Tg小鼠微球单次注射组小鼠眼压均明显高于WT小鼠对照组及WT小鼠PBS注射组,高眼压状态持续至4周,2次微球注射后眼压复升,高眼压可维持至8周.WT小鼠微球单次注射组第3天视网膜中Ngb含量明显上调,而Ngb-Tg小鼠视网膜中Ngb呈持续高表达.与Ngb-Tg小鼠相比,WT小鼠RGCs凋亡率在前房注射微球后第1、4、8周均逐渐升高,差异均有统计学意义(P＜0.05).1周时,Ngb-Tg小鼠微球单次注射组视网膜中DHE含量明显低于WT小鼠微球单次注射组(t=3.212,P=0.008),而ATP的含量则明显高于WT小鼠微球单次注射组(t=2.864,P＜0.01). 结论 Ngb可能是青光眼损伤的内源性神经保护因子,对高眼压所致RGCs损伤有保护作用,其机制可能是通过降低氧化应激和改善线粒体功能实现的.

Protective effect of neuroglobin on retinal ganglion cell in glaucoma mice model

Background Neuroglobin （Ngb）is a newly discovered member of globin superfamily. It is thought to regulate cell survival under hypoxia or oxidative stress condition. Ngb is expressed at a high level in retinal neuron,suggesting that retina may be one of important functional sites of Ngb. Objective The aim of this study was to investigate the protective role of endogenous Ngb on retina ganglion cells （RGCs） following chronic high intraoeular pressure（ IOP） in mice and the underlying mechanisms. Methods This study included the in vitro and in vivo experiment. RGCs derived from adult C57BL/6J wild type（WT） mice and Ngb-transgenic（Ngb-Tg） mice which cultivated by our laboratory were incubated with 5.0,7.5,10.0 mmol/L glutamic acid for 3 days. RGCs survival rate was calculated for the ration of dead and survival cells using a double labeling kit to evaluate the influence of Ngb on RGCs survival rate in the addition of glutamic acid. Chronic ocular hypertension models were established by injection of fluorescent microballon（MB） （ 10 p~m） into the anterior chamber of WT mice and Ngb-Tg mice, The mice were divided into WT control group （ n = 18 ） , Ngb-Tg control group （ n = 30） , WT＋MB single injection group （ n = 38） , Ngb- Tg＋MB single injection group（ rt = 38）, WT＋MB twice injection group（ n- 6）and Ngb-Tg ＋ twice injection group （n = 6）. In addition, WT＋PBS injection group （n = 6） and Ngb-Tg＋PBS injection group （n = 6 ） were designed as negative controis to identify if it can affect IOP or not. The mice were sacrificed on 0 day（control group） ,3 days and 1,4,8 weeks followed the M B anterior chamber injection. Real-time PCR,Western blot and immunoytochemistry were used respectively for the analysis of the expressions of Ngb mRNA and protein in mouse retina, and the survival rates of RGCs were compared between the two types mice. Dihydroethidium （DHE） in retina was detected after cardiac perfusion and ATP level in mouse retina homogenate was analyzed. Results The RGCs survival rate was significantly higher in Ngb mice compared with WT mice in 5.0,7.5 and 10.0 mmol/L glutamic acid treated groups （t=2. 810,3. 020,3. ll0,P〈0.01 ）. IOP of WT＋MB single injection group and Ngb-Tg＋MB single injection group were elevated in comparison with the WT control group and WT＋PBS injection group and the high IOP remained for 4 weeks. Ngb level was raised in the WT＋MB single injection group on the third day following injection, but the Ngb concentration remained a high level in the Ngb-Tg mice during the period of the experiment duration. The RGCs apoptosis rate was elevated both in the WT mice and Ngb-Tg mice 1,4,8 weeks after injection of MB, however, the cell apoptosis rate was higher in WT mice than that of Ngb-Tg mice（P〈0.05）. DHE content in retina in the Ngb-Tg＋ MB single injection group was significantly lower than that in the WT ＋ MB single injection group （t = 3. 212, P= 0. 008 ） , and ATP content in retina was elevated in the Ngb-Tg＋MB single injection group compared with WT＋MB single injection group（t = 2. 864, P〈0.01 ）. Conclusions It is suggested that Ngb might be a neuroprotective molecules against RGCs death by decreasing oxidative stress and improving mitochondria function.