牛磺酸对缺氧/复氧性脑损伤斑马鱼幼鱼脑组织葡萄糖调节蛋白78、生长停滞与 DNA 损伤可诱导蛋白34表达的影响

摘要

Objective To investigate the expression of endoplasmic reticulum stress - related factors, glucose - regulated protein 78(GRP78)and growth arrest and DNA damage - inducible protein 34( GADD34)and neuronal apoptosis in the brain of zebrafish larvae after hypoxia/ reoxygenation brain injury,and the neuroprotective role of Taurine. Methods The 5 day post - fertilization zebrafish larvae were randomly assigned to 3 groups:control group, hypoxia/ reoxygenation model group(model group)and Taurine treatment group(Taurine group). According to the dif-ferent time points for observation,each group was subdivided into 5 subgroups(1 h,3 h,6 h,24 h,48 h)with 100 ze-brafish larvae each. The pathological changes in the brain tissues and cell apoptosis were detected by fluorescence ter-minal deoxynucleotidyltransferase - mediated dUTP nick end - labeling( TUNEL). The expression of GRP78 and GADD34 mRNA in the brain of zebrafish larvae were detected by real - time quantitative reverse transcription PCR (qRT - PCR). The changes in GRP78 and GADD34 protein were detected by Western blot. Results (1)TUNEL:apoptosis index(AI)was increased after hypoxia/ reoxygenation,and reached the peak at 3 h in model group,the AI in Taurine group was decreased compared with that in the model group at the same time point(1 h:22. 83 ± 1. 80 vs 30. 18 ± 1. 81,3 h:23. 22 ± 2. 46 vs 42. 97 ± 4. 01,6 h:16. 80 ± 1. 69 vs 22. 97 ± 1. 91,all P ﹤ 0. 05).(2)qRT -PCR:the expression of GRP78 and GADD34 mRNA was increased at 1 h after hypoxia/ reoxygenation,and reached the peak at 3 h in the model group,the expression in Taurine group was decreased compared with that in the model group at the same time point(GRP78 mRNA:1 h:2. 35 ± 0. 13 vs 5. 36 ± 0. 35,3 h:3. 08 ± 0. 33 vs 4. 27 ± 0. 52,6 h:1. 57 ± 0. 12 vs 3. 00 ± 0. 13,all P ﹤ 0. 05;GADD34 mRNA:1 h:5. 14 ± 0. 55 vs 7. 45 ± 0. 67,3 h:2. 79 ± 0. 58 vs 5. 83 ± 0. 51,6 h:1. 79 ± 0. 22 vs 3. 67 ± 0. 30,all P ﹤ 0. 05).(3)Western blot:the expression of GRP78 and GADD34 pro-tein was increased at 1 h,reached the peak at 6 h,but it was decreased in Taurine group(GRP78 protein:1 h:1. 12 ± 0. 11 vs 1. 37 ± 0. 13,3 h:0. 79 ± 0. 11 vs 1. 25 ± 0. 10,6 h:0. 55 ± 0. 10 vs 1. 52 ± 0. 14,all P ﹤ 0. 05;GADD34 pro-tein:1 h:0. 92 ± 0. 11 vs 1. 11 ± 0. 13,3 h:0. 96 ± 0. 11 vs 1. 52 ± 0. 09,6 h:0. 76 ± 0. 05 vs 1. 89 ± 0. 06,all P ﹤0. 05).(4)The expression of GRP78 and GADD34 protein was positively correlated with AI in the model group(r =0. 53,0. 56 respectively,all P ﹤ 0. 05). Conclusion One of neuroprotective mechanisms of Taurine against hypoxia/reoxygenation brain injury may down - regulate GRP78 and GADD34 expression.%目的：观察牛磺酸对缺氧/复氧性脑损伤斑马鱼幼鱼脑组织中内质网应激相关因子葡萄糖调节蛋白78（GRP78）、生长停滞与 DNA 损伤可诱导蛋白34（GADD34）表达及神经细胞凋亡的影响，探讨其脑保护机制。方法将受精后5 d 的斑马鱼幼鱼随机分为对照组、缺氧/复氧模型组（模型组）以及牛磺酸组，每组按造模后不同观察时间点又分为1 h、3 h、6 h、24 h、48 h 5个亚组，每亚组100条。采用荧光原位末端标记法（TUNEL）检测各时间点脑组织细胞凋亡情况，实时荧光定量反转录 PCR 法（qRT-PCR）检测各时间点 GRP78、GADD34 mRNA 水平，采用 Western blot 法检测 GRP78、GADD34蛋白水平。结果1. TUNEL：缺氧/复氧后脑组织细胞凋亡指数（AI）增加，模型组3 h 达高峰，牛磺酸组同时间点低于模型组（1 h：22.83±1.80比30.18±1.81，3 h：23.22±2.46比42.97±4.01，6 h：16.80±1.69比22.97±1.91，P 均﹤0.05）。2. qRT-PCR：GRP78及 GADD34 mRNA 表达在缺氧/复氧后1 h 即上调，模型组1 h 达高峰，同时间点牛磺酸组低于模型组（GRP78 mRNA：1 h：2.35±0.13比5.36±0.35，3 h：3.08±0.33比4.27±0.52，6 h：1.57±0.12比3.00±0.13，P 均﹤0.05；GADD34 mRNA：1 h：5.14±0.55比7.45±0.67，3 h：2.79±0.58比5.83±0.51，6 h：1.79±0.22比3.67±0.30，P 均﹤0.05）。3. Western blot：GRP78及 GADD34蛋白在缺氧/复氧后1 h 表达上调，模型组6 h 达高峰，牛磺酸组亦低于模型组（GRP78蛋白：1 h：1.12±0.11比1.37±0.13，3 h：0.79±0.11比1.25±0.10，6 h：0.55±0.10比1.52±0.14，P 均﹤0.05；GADD34蛋白：1 h：0.92±0.11比1.11±0.13，3 h：0.96±0.11比1.52±0.09，6 h：0.76±0.05比1.89±0.06，P 均﹤0.05）。4.模型组幼鱼脑组织 GRP78、GADD34蛋白表达均与 AI 呈正相关（r =0.53、0.56，P 均﹤0.05）。结论牛磺酸可能通过下调 GRP78蛋白及 GADD34蛋白表达，减少神经细胞凋亡，缓解斑马鱼幼鱼缺氧/复氧性脑损伤。