内质网应激蛋白GRP78在脑出血后星形胶质细胞炎性活化中的作用及机制

Role and mechanism of endoplasmic reticulum stress protein GRP78 in inflammatory activation of astro⁃cytes after intracerebral hemorrhage

目的探讨内质网应激蛋白葡萄糖调节蛋白78(GRP78)在脑出血(ICH)后星形胶质细胞炎性活化中的作用及机制。方法CD1小鼠通过自体全血注射诱导ICH,使用GRP78靶向抑制剂HA15。(1)将小鼠随机分为7组:假手术及ICH后3、6、12、24、48和72 h。采用Western blot检测各组血肿周围区域GRP78的表达。(2)将小鼠随机分为3组:假手术、ICH+载体(PBS)、ICH+HA15,在ICH后24、72 h评估神经行为测试和脑水含量;在ICH后24 h使用TUNEL法评估神经元凋亡,Western blot分析ER应激的经典下游标志物Caspase 12和CHOP蛋白表达。结果(1)与假手术组相比,脑出血小鼠血肿周围区域中GRP78水平在ICH后3 h开始上升,24 h达到最高(P<0.05)。(2)在ICH后24和72 h,与ICH+PBS组相比,ICH+HA组小鼠神经功能缺损显著改善(P<0.05),并且基底神经节和皮质的脑水肿显著降低(P<0.05)。(3)HA治疗后血肿周围区域星形胶质细胞、GFAP+GRP78+细胞、TUNEL阳性神经元明显减少(P<0.05)。(4)HA治疗后Caspase 12和CHOP蛋白表达减弱,ER应激明显抑制。结论ER应激蛋白GRP78通过介导星形胶质细胞炎性活化参与ICH后神经损伤。HA15可能对ICH后神经元存活产生积极影响。

Objective To investigate the role and mechanism of endoplasmic reticulum stress protein glu⁃cose regulatory protein 78(GRP78)in the inflammatory activation of astrocytes after intracerebral hemorrhage(ICH).Methods ICH was induced by autologous whole blood injection in CD1 mice,and GRP78 targeted inhibitor ha15 was used.Forty⁃two prepared mice were randomly divided into 7 groups:sham operation group and groups of 3,6,12,24,48 and 72 hours after ICH.The expression of GRP78 in the area around hematoma was detected by Western blot.Another group of prepared 36 mice were randomly divided into 3 groups:sham operation,ICH+car⁃rier(PBS),ICH+ha15.Neurobehavioral tests and brain water content were evaluated at 24 h and 72 h after ICH.At 24 h after ICH,TUNEL method was used to evaluate neuronal apoptosis,and Western blot was performed to analyze the expression of caspase 12 and chop protein,the classical downstream markers of ER stress.Results Compared with the sham operation group,the level of GRP78 in the area around the hematoma began to rise at 3 h after ICH and reached the highest at 24 h(P<0.05).At 24 h and 72 h after ICH,compared with ICH+PBS group,the neurological deficit in ICH+HA group was significantly improved(P<0.05),and the brain edema in the basal ganglia and cortex was significantly reduced(P<0.05).After HA treatment,astrocytes,GFAP+GRP78+cells and TUNEL positive neurons in the area around the hematoma decreased significantly(P<0.05).After HA treatment,the expression of caspase 12 and chop protein decreased,and ER stress was significantly inhibited.Conclusion ER stress protein GRP78 participates in nerve injury after ICH by mediating the inflammatory activa⁃tion of astrocytes.HA15 may have a positive effect on neuronal survival after ICH.