粒细胞集落刺激因子对缺氧缺血性脑损伤新生大鼠内质网应激的影响

Effect of granulocyte-colony stimulating factor on endoplasmic reticulum stress in neonatal rats after hypoxic-ischemic brain damage

目的探讨粒细胞集落刺激因子(G-CSF)对缺氧缺血性脑损伤(HIBD)新生大鼠的神经保护作用及其对内质网应激的影响。方法54只7日龄新生SD大鼠采用随机数字表法分为3组:假手术组(Sham组)、缺氧缺血模型组(HIBD组)及给药组(G-CSF组),每组18只,采用改良Rice法建立HIBD模型,G-CSF组造模后1 h腹腔注射50μg/kg G-CSF,Sham组及HIBD组注射相应剂量9 g/L盐水。各组分别于造模后24 h采用2,3,5-氯化三苯基四氮唑(TTC)染色法检测脑梗死大小,苏木精-伊红(HE)染色观察大脑病理变化,脱氧核糖核苷酸未端转移酶介导的缺口末端标记法(TUNEL)观察大脑皮质及海马CA1区神经细胞凋亡情况,采用免疫荧光检测各组大鼠大脑皮质葡萄糖调节蛋白78(GRP78)表达情况,采用Western blot检测GRP78、CCAAT/增强子结合蛋白(CHOP)、半胱氨酸天冬氨酸蛋白酶12(Caspase-12)内质网应激相关蛋白表达情况。结果造模后24 h,HE染色示Sham组未见明显神经元损伤表现,HIBD组可见脑组织结构改变明显,G-CSF组病理改变较HIBD组减轻。HIBD组梗死面积[(25.40±5.15)%]显著高于Sham组[(0.31±0.15)%]及G-CSF组[(16.36±4.97)%],差异均有统计学意义(均P<0.05)。HIBD组免疫荧光GRP78阳性表达[(49.38±10.06)%]显著高于Sham组[(9.12±4.50)%]及G-CSF组[(30.61±6.35)%],差异均有统计学意义(均P<0.05)。HIBD组海马CA1区及皮质凋亡细胞比例[(44.84±11.54)%、(48.23±14.07)%]均明显高于G-CSF组[(17.87±7.20)%、(26.18±9.96)%],均显著低于HIBD组,差异均有统计学意义(均P<0.05)。HIBD组GRP78、CHOP、Caspase-12相对表达水平(0.63±0.24、0.72±0.21、0.68±0.25)均显著高于Sham组(0.20±0.08、0.28±0.08、0.23±0.07)及G-CSF组(0.39±0.13、0.51±0.18、0.48±0.16),差异均有统计学意义(均P<0.05)。结论腹腔注射G-CSF对HIBD新生大鼠有神经保护作用,可能通过抑制内质网应激减少神经细胞凋亡。

Objective To evaluate the protective effect of granulocyte-colony stimulating factor(G-CSF)on neonatal rats after hypoxic-ischemic brain damage(HIBD)and its effect on endoplasmic reticulum(ER)stress.Methods According to the random number table,a total of 54 Sprague-Dawley(SD)rats aged 7 days were divided into 3 groups(18 rats in each group):Sham group,HIBD group and G-CSF group,and the improved Rice method was used to establish a neonatal rat model of HIBD.A dose of 50μg/kg of G-CSF was administered intraperitoneally 1 hour after HIBD(G-CSF group),while the rats in HIBD group and Sham group received saline only.At 24 hours of HIBD,pups were euthanized to quantify brain infarct volume by using 2,3,5-Triphenyltetrazolium chloride.Hematoxylin-Eosin(HE)staining was used to observe the changes of brain structure.Neuronal cell death was determined by using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL).Then the expressions of glucose-regulated protein 78(GRP78),cysteinyl aspartate specific proteinase 12(Caspase-12),CCAAT/enhancer binding-protein homologous protein(CHOP)were assessed by Western blot and immunofluorescence staining.Results Twenty-four hours after operation,HE staining showed that no significant neuronal damage was observed in Sham group.The brain tissue structure of rats in the HIBD group was significantly damaged,while some improvement was observed in the G-CSF group.The infarction volume in HIBD group[(25.40±5.15)%]increased compared with that in the Sham group[(0.31±0.15)%]and the G-CSF group[(16.36±4.97)%],and the differences were statistically significant(all P<0.05).There was increased positive expression of GRP78 protein in HIBD group,compared with that in the Sham group and the G-CSF group[(49.38±10.06)%vs.(9.12±4.50)%,(30.61±6.35)%],and the differences were statistically significant(all P<0.05).The percentage of apoptosis in the hippocampal CA1 region and conex in HIBD group[(44.84±11.54)%,(48.23±14.07)%]were higher than those in the G-CSF group[(17.87±7.20)%,(26.18±9.96)%],and the differences were statistically significant(all P<0.05).The expression of GRP78,CHOP and Caspase-12 in the HIBD group(0.63±0.24,0.72±0.21,0.68±0.25)were higher than those in the Sham group(0.20±0.08,0.28±0.08,0.23±0.07),and the G-CSF group(0.39±0.13,0.51±0.18,0.48±0.16),and the differences were statistically significant(all P<0.05).Conclusions G-CSF exerts neuroprotective effect on the neonatal rats after HIBD.G-CSF may be an effective treatment of HIBD by reducing ER stress-induced neuronal apoptosis.