氢对脓毒症相关性脑病小鼠海马A1型星形胶质细胞活化的影响

Effect of hydrogen on activation of A1 astrocytes in hippocampus of mice with sepsis-associated encephalopathy

目的评价氢对脓毒症相关性脑病(SAE)小鼠海马A1型星形胶质细胞活化的影响。方法清洁级健康雄性C57BL/6J小鼠164只,6~8周龄,体重20~25 g。采用随机数字表法分为4组(n=41):假手术组(Sham组)、假手术+氢气组(Sham+H2组)、SAE组和SAE+氢气组(SAE+H2组)。采用盲肠穿孔结扎法制备SAE模型。Sham+H2组和SAE+H2组分别于术后1和6 h时吸入2%氢气1 h。每组取20只小鼠,观察术后7 d生存率。于术后12 h处死其余小鼠,取脑组织,光镜下观察海马CA1区病理学结果,计算异常神经元比率;TUNEL法确定神经元凋亡率;采用免疫荧光染色法检测海马胶原纤维酸性蛋白(GFAP)与补体C3共表达情况;采用Western blot法测定海马C3表达水平,采用ELISA法检测海马TNF-α、IL-6、高迁移率族蛋白1(HMGB1)含量;于术后7 d,每组取6只小鼠行Y迷宫新异臂探索实验。结果与Sham组比较,SAE组术后7 d生存率降低,海马CA1区异常神经元比率和神经元凋亡率升高,TNF-α、IL-6、HMGB1含量升高,C3表达上调,GFAP/C3共表达细胞计数增多,新异臂探索时间缩短,偏好指数降低(P<0.05)。与SAE组比较,SAE+H2组术后7 d生存率升高,海马CA1区异常神经元比率和神经元凋亡率降低,TNF-α、IL-6、HMGB1含量降低,C3表达下调,GFAP/C3共表达细胞计数减少,新异臂探索时间延长,偏好指数升高(P<0.05)。Sham组和Sham+H2组各项指标比较差异无统计学意义(P>0.05)。结论氢改善脓毒症小鼠SAE的机制可能与抑制海马A1型星形胶质细胞活化有关。

Objective To evaluate the effect of hydrogen on activation of A1 astrocytes in the hippocampus of mice with sepsis-associated encephalopathy(SAE).Methods A total of 164 clean-grade healthy male C57BL/6J mice,aged 6-8 weeks,weighing 20-25 g,were divided into 4 groups(n=41 each)using a random number table method:sham operation group(group Sham),sham operation plus hydrogen group(group Sham+H2),group SAE and SAE plus hydrogen group(group SAE+H2).The SAE model was established by cecal ligation and perforation.Group Sham+H2 and group SAE+H2 inhaled 2%hydrogen starting from 1 and 6 h after operation,respectively.Twenty mice in each group were selected to observe the 7-day survival rate after operation.The remaining mice were sacrificed at 12 h after operation,and brain tissues were removed for examination of the pathological changes in hippocampal CA1 region(with a light microscope)and for determination of the apoptosis in neurons(by TUNEL),co-expression of hippocampal glial fibrillary acidic protein(GFAP)and complement C3(by immunofluorescence staining),expression of A1 astrocyte marker C3(by Western blot),and contents of tumor necrosis factor-alpha(TNF-α),interleukin-6(IL-6)and high-mobility group box 1 protein(HMGB1)(by enzyme-linked immunosorbent assay).The abnormal cell ratio and apoptosis rate were calculated.Six mice in each group were selected at 7 days after operation to perform Y-Maze paradigm.Results Compared with group Sham,the 7-day survival rate after operation was significantly decreased,the abnormal cell ratio and apoptosis rate of hippocampal neurons were increased,the contents of TNF-α,IL-6 and HMGB1 were increased,the expression of C3 was up-regulated,the number of cells coexpressing GFAP and C3 was increased,the exploration time spent in the novel arm in Y-Maze paradigm was shortened,and the preference index was decreased in group SAE(P<0.05).Compared with group SAE,the 7-day survival rate after operation was significantly increased,the abnormal cell ratio and apoptosis rate of hippocampal neurons were decreased,the contents of TNF-α,IL-6 and HMGB1 were decreased,the expression of C3 was down-regulated,the number of cells coexpressing GFAP and C3 was decreased,the exploration time spent in the novel arm in Y-Maze paradigm was prolonged,and the preference index was increased in group SAE+H2(P<0.05).There was no significant difference in each parameter mentioned above between Sham group and Sham+H2 group(P>0.05).Conclusion The mechanism by which hydrogen improves SAE may be related to inhibiting activation of A1 type astrocytes in mice.