Neuritin对脑损伤皮层神经元凋亡及GRP78/CHOP通路的影响

Effects of neuritin on apoptosis of cortical neurons and GRP78/CHOP pathway after traumatic brain injury

目的探讨神经营养因子(neuritin)在大鼠脑损伤(TBI)中的神经保护作用及对内质网应激相关因子-葡萄糖调节蛋白78(GRP78)/增强子结合同源蛋白(CHOP)通路的影响。方法将45只大鼠随机分为假手术组(Sham组)、模型组(TBI组)、脑损伤+神经营养因子组(TBI+neuritin组),每组15只。除Sham组外,其余各组均采用改良Feeney自由落体法建立大鼠TBI模型;TBI+neuritin组在模型建立20 min内经侧脑室注射100μL neuritin,sham组和TBI组经侧脑室注射100μL生理盐水。各组大鼠于手术3 d后,用苏木精-伊红(HE)进行模型鉴定;用改良神经功能损伤评分(mNSS)法评估神经功能受损程度;用伊文思蓝染色法检测血脑屏障破坏程度;用免疫组化染色检测脑皮层组织GRP78、CHOP、半胱氨酸天冬氨酸蛋白酶12(caspase-12)表达;原位末端标记法(TUNEL)检测脑皮层组织神经元细胞凋亡率,蛋白免疫印迹(Western blot)法检测脑皮层组织GRP78、CHOP、caspase-12、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(cleaved caspase3)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)表达。结果与Sham组比较,TBI组及TBI+neuritin组mNSS评分、伊文思蓝渗出、GRP78染色表达、神经细胞凋亡、促凋亡蛋白cleaved caspase3、Bax、内质网应激相关分子GRP78、CHOP及caspase-12阳性表达及蛋白表达均升高,抗凋亡蛋白Bcl-2表达降低,差异均有统计学意义(P<0.05);与TBI组比较,TBI+neuritin组mNSS评分改善,伊文思蓝渗出、GRP78染色表达、神经细胞凋亡、促凋亡蛋白cleaved caspase3、Bax、内质网应激相关分子GRP78、CHOP及caspase-12阳性表达及蛋白表达均减少,抗凋亡蛋白Bcl-2表达升高,差异均有统计学意义(P<0.05)。结论Neuritin可降低CHOP、GRP78、caspase-12表达,抑制内质网应激反应,减轻血脑屏障破坏程度,降低脑皮层组织中神经元细胞凋亡,对TBI模型大鼠有神经保护作用。

Objective To investigate the neuroprotective effect of neuritin on traumatic brain injury(TBI)in rats and its effect on endoplasmic reticulum stress-related factor glucose regulated protein 78(GRP78)/CCAAT enhancer binding protein homologous protein(CHOP)pathway.Methods A total of 45 rats were randomly divided into sham operation group(Sham group),model group(TBI group),traumatic brain injury+neurotrophic factor group(TBI+neuritin group),with 15 rats in each group.In addition to sham group,the TBI model of rats was established by modified Feeney free fall method;in the TBI+neuritin group,100μL of neuritin was injected into the lateral ventricle within 20 minutes after the establishment of the model,and in the sham group and the TBI group,100μL of normal saline was injected into the lateral ventricle.Three days after the operation,the modified neurological impairment score(mNSS)scoring method was used to evaluate the degree of neurological impairment;the degree of pathological damage and nerve cell damage was measured by hematoxylin eosin(HE)and toluidine blue(TB)staining;terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)method was used to detect the apoptosis rate of neurons.GRP78,CHOP and cysteine aspartate protease12(caspase-12)in cerebral cortex tissue were detected by immunohistochemical staining;and the expressions of GRP78,CHOP,caspase-12,cleaved caspase3,B-cell lymphoma-2(Bcl-2),Bcl-2 related X protein(Bax)proteins in cortical tissues were detected by Western blot.Results Compared with the Sham group,mNSS score,Evans blue exudation,GRP78 staining expression,neuronal apoptosis,pro-apoptotic protein cleaved caspase3,Bax,endoplasmic reticulum stress-related molecule GRP78,CHOP and caspase-12 positive expression and protein expression in TBI group and TBI+neuritin group were increased(P<0.05),and anti-apoptotic protein Bcl-2 expression was decreased(P<0.05).Compared with the TBI group,mNSS score,Evans blue exudation,GRP78 staining expression,neuronal apoptosis,pro-apoptotic protein cleaved caspase3,Bax,endoplasmic reticulum stress-related molecules GRP78,CHOP and caspase-12 positive expression and protein expression in TBI+neuritin group were decreased(P<0.05),and anti-apoptotic protein Bcl-2 expression was increased(P<0.05).Conclusion Neuritin could reduce the expressions of CHOP,GRP78 and caspase-12,inhibit the stress response of endoplasmic reticulum,reduce the apoptosis of neurons in cerebral cortex,and had neuroprotective effect on TBI model rats.