高压氧对缺氧缺血性脑损伤新生大鼠脑内β-连接素活化的影响

Effects of hyperbaric oxygen on the activation of β-catenin in hypoxic ischemic neonate rats

目的探讨高压氧（HBO）治疗后缺氧缺血性脑损伤（HIBD）新生大鼠神经干细胞增殖与β-连接素（β-catenin）蛋白的关系。方法共选取7日龄SD新生夫鼠180只，将其分为正常对照组（对照组）、模型组及HBO组。采用Rice法制作HIBD模型，HBO组于造模后3h内给予HBO治疗，治疗压力为2个绝对大气压，稳压60min，每日治疗1次，连续治疗7d；模型组制模后不给予任何处理。于HBO治疗后第3、21小时，第3、5、7及14天时采用巢蛋白／β-catenin免疫荧光双标法动态检测各组大鼠侧脑室室管膜下区巢蛋白、β-catenin蛋白表达，并对p—eatenin与巢蛋白的荧光强度进行相关性分析；采用Westernblot法检测各组大鼠左侧脑组织β-catenin总蛋白与核蛋白的表达情况。结果免疫荧光双标结果显示HBO治疗后第21小时时，HBO纠与模型组神经干细胞内β-catenin蛋白表达开始增强；HBO治疗后第5天时，HBO组β-catenin蛋白表达达到较高水平并显苫高丁模型组（P〈0．01）；HBO治疗后第14天时，HBO组β-catenin蛋白表达显著减少；HBO组巢货门表达于HBO治疗后第21小时时开始增强，于治疗后第7天时达到较高水平，显著高于模型组（P〈0．05）。另外本研究还发现HBO组β-catenin蛋白与巢蛋白荧光强度具有直线相关性（r^2=0．66，P〈0．01）；Western blot结果显示HBO组β-catenin总蛋白及核蛋白均于HBO治疗后第5天时达到较高水平，并显著高于模型组（P〈0．05）。结论HBO治疗可促进HIBD新生大鼠神经于细胞增殖，其治疗机制可能与促进β-catenin蛋白活化有关。

Objective To explore the relationship between the proliferation of neural stem cells （NSCs） and the expression of β-catenin protein in neonate rats with hypoxic isehemie brain damage （HIBD） after hyperbaric oxygen （HBO） therapy. Methods One hundred and eighty Sprague-Dawley rats aged 7 days were randomly divided into a normal control group （CON）, a HIBD model group and a HBO treatment group. The HIBD model was induced using Rice＇s method. Beginning 3h after the HIBD, HBO was administered to the HBO treatment group at 2 atmospheres for 60 rain, once daily for 7 days. The HIBD model group was not given any treatment. The expression of nestin/β-catenin protein in the subventricular zone of the ischemic brain was double-stained for immunofluoreseence and analyzed by eonfocal scanning microscopy dynamically at 3 hours, 21 hours, and then on the 3rd, 5th, 7th and 14th day of HBO therapy. The expression of whole cell β-catenin and nuclear β-catenin protein in the left brain were also examined by Western blotting at these 6 time points. Linear correlation was used to analyze the correlation between β-eatenin and nestin protein. Results The expression of β-catenin protein in NSCs increased initially at the 21st hour after HBO therapy in the model group and the HBO group as compared with the normal control group. β-catenin protein in the model group reached a higher level, though there was no significant difference between model group and the HBO group. At the 5th day of HBO therapy β-catenin protein in the HBO group had reached a significantly higher level than in the model group. At the 14th day the average expression of β-catenin in the HBO group began to decrease. The expression of nestin protein began to increase 21 hours after HBO therapy began, and it peaked at the 7th day of HBO therapy and then decreased. In the HBO group the increase in nestin protein was linearly correlated with that of β-catenin protein. The whole cell β-eatenin protein and β-eatenin uucleie protein readings increased initially by the 21st hour of HBO therapy and by the 5th day were significantly higher than the levels in the model group. Conclusion HBO treatment is capable of stimulating the proliferation of NSCs in HIBD neonate rats. The proliferation of NSCs is correlated with the activation of β-catenin protein.