内质网应激通路相关分子GRP78及CHOP在糖尿病脑病小鼠海马表达的变化

目的本研究通过观察内质网应激(endoplasmic reticulum stress,ER Stress)通路相关分子葡萄糖调节蛋白78(glucoseregulated protein78,GRP78)及CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein,CHOP)在糖尿病脑病小鼠海马CA1区表达的变化,探讨ER Stress在糖尿病脑病发生发展中的作用。方法 60只雄性昆明小鼠随机分为正常组(n=25)和糖尿病(diabetes mellitus,DM)组(n=35)。小鼠禁食12h后,按200mg/kg腹腔注射链脲佐菌素(streptozocin,STZ),3d后尾部非禁食血糖>15mmol/L者为DM模型复制成功。分别在STZ注射后1、4、8周应用免疫组织化学染色方法观察正常组与DM组小鼠海马CA1区GRP78及CHOP阳性细胞形态和数量的变化,并应用图像分析软件计数GRP78及CHOP阳性细胞。结果 ①GRP78免疫组织化学染色结果显示,注射STZ后1、4、8周DM组小鼠海马CA1区GRP78阳性细胞形态和数量与正常组相似。②CHOP免疫组织化学染色结果显示,注射STZ后1、4、8周正常组小鼠海马CA1区CHOP阳性细胞数量较少,染色浅;DM组小鼠海马CA1区CHOP阳性细胞数量较多,染色深。③注射STZ后1、4、8周,DM组小鼠海马CA1区GRP78阳性细胞数量(12.00±3.60,10.50±3.11,13.75±3.01)与正常组(10.33±2.34,8.88±1.89,7.00±3.20)相比差异无统计学意义(P=0.29,P=0.23,P=0.06);注射STZ后1、4、8周,DM组小鼠海马CA1区CHOP阳性细胞数量(45.12±10.27,32.88±6.58,20.19±3.54)比正常组(19.19±6.80,15.44±5.35,13.94±5.00)明显增加,经统计学分析差异均有统计学意义(P分别为0.000,0.000和0.009)。结论 ER Stress相关分子CHOP在DM小鼠海马CA1区表达比正常小鼠增加,说明糖尿病脑病小鼠海马组织内发生了ER Stress。ER Stress可能在糖尿病脑病神经元变性中发挥重要作用。

The Changes in Expression of Endoplasmic Reticulum Stress Related Moleculars GRP78 and CHOP in Hippocampus of Mice with Diabetic Encephalopathy

Objective To observe the changes in expression of glucose-regulating protein 78(GRP78) and CCAAT/enhancer-binding protein homologous protein(CHOP) in the hippocampal CA1 region of mice with diabetic encephalopathy, and to explore the important role of endoplasmic reticulum stress (ER Stress) in the pathogenesis of diabetic encephalopathy.
Methods Sixty male mice were divided into a normal control group(n=25) and a diabetes mellitus(DM) group(n=35). Streptozocin（STZ）was freshly prepared and injected at 200 mg/kg, i.p. into mice which had been fasted for 12 h. At 1 week, 4 weeks and 8 weeks after STZ administration, immunohistochemistry was performed with GRP78 and CHOP antibodies. The numbers of GRP78 and CHOP positive cells were measured by imaging analysis software. Data was expressed as mean±standard deviation(SD).
Results ① The result of GRP78 antibody immunohistochemical staining showed that the morphology and numbers of GRP78-positive cells in hippocampal CA1 region of DM group mice at 1 week, 4 weeks and 8 weeks after STZ injection were similar to those of normal control group mice. ② Immunohistochemical staining with CHOP antibody at 1 week, 4 weeks and 8 weeks after STZ injection showed that normal control group mice had few lightlystained CHOP positive cells in hippocampal CA1 region, while DM group mice had more darkly stained CHOP-positive cells in
hippocampalCA1 region. ③ At 1 week, 4 weeks and 8 weeks after STZ injection, the numbers of GRP78-positive cells in the hippocampal CA1 region of DM group mice（12.00±3.60, 10.50±3.11, 13.75±3.01） showed no significant difference(P=0.29, P=0.23, P=0.06) compared with those of normal control group mice(10.33±2.34, 8.88±1.89, 7.00±3.2). The numbers of CHOP-positive cells in the hippocampal CA1 region of DM group mice at 1 week, 4 weeks and 8 weeks after STZ injection(45.12±10.27, 32.88±6.58, 20.19±3.54) increased significantly(P=0.000, P=0.000, P=0.009) compared with those of normal control group mice（19.19±6.80, 15.44±5.35, 13.94±5.00）.
Conclusion The expression level of ER Stress related molecule CHOP in hippocampal CA1 region of diabetic encephalopathy mice was increased compared with that of normal mice. The hippocampus of diabetic encephalopathy mice underwent ER Stress. ER Stress might play an important role in the pathogenesis of neuronal degeneration of diabetic encephalopathy.