灯盏花素抑制大鼠液压冲击脑损伤内质网应激介导凋亡作用

Breviscapine attenuates endoplasmic reticulum-mediated apoptosis via modulating nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling pathway in rats following fluid percussion brain injury

目的探讨灯盏花素上调核因子E2相关因子2/血红素氧合酶-1(Nrf2/HO-1)通路抑制大鼠液压冲击脑损伤内质网应激介导凋亡的具体机制。方法72只成年雄性SD大鼠随机分为对照组、模型组和灯盏花素组。应用Dixon法制作颅脑损伤模型,造模后24 h用改良大鼠神经功能缺损评分(mNSS)、干湿重法、蛋白印迹法(Western blot)和原位缺口末端标记法(TUNEL)法分别评价动物神经功能障碍、脑组织含水量、内质网应激介导凋亡标志蛋白[葡萄糖调节蛋白78(GRP78)、环磷酸腺苷反应元件结合转录因子同源蛋白(CHOP)、半胱氨酸天冬氨酸蛋白酶(Caspase)-12]、Nrf2/HO-1通路关键蛋白[Nrf2、HO-1和醌氧化还原酶1(NQO-1)]表达以及神经细胞凋亡变化。组间比较差异性分析采用Student’s t检验。结果灯盏花素组造模后24 h大鼠mNSS评分[(8.12±1.76)分比(9.89±1.64)分,t=2.549,P<0.05]和脑组织含水量[(80.01±2.12)%比(83.88±5.74)%,t=2.187,P<0.05]均明显低于模型组。Western blot结果显示灯盏花素能够抑制GRP78(4.07±0.68比5.74±0.98,t=4.850,P<0.05)、CHOP(1.01±0.18比1.38±0.21,t=4.634,P<0.05)、Caspase-12(0.55±0.11比1.11±0.13,t=10.391,P<0.05)和Caspase-3(0.77±0.12比1.10±0.21,t=4.726,P<0.05)蛋白表达并减轻神经细胞凋亡(24.26±4.38比32.14±6.87,t=3.350,P<0.05),同时上调Nrf2(胞核)(0.21±0.04比0.15±0.03,t=4.157,P<0.05)、HO-1(0.31±0.05比0.21±0.05,t=5.941,P<0.05)和NQO-1(0.27±0.05比0.20±0.00,t=4.159,P<0.05)。结论灯盏花素通过激活Nrf2/HO-1通路抑制内质网应激介导凋亡发挥神经保护作用。

Objective To investigate the mechanism of breviscapine in attenuating endoplasmic reticulum stress(ERS)-mediated apoptosis via modulating nuclear factor erythroid 2-related factor 2/heme oxygenase-1(Nrf2/HO-1)pathway in rats following fluid percussion brain injury.Methods Totally,72 SD rats were randomly divided into control group,model group and brevidcapine group.The experimental models were established according to the Dixon’s method.Neurological deficits,brain water content,the expression of glucose regulated protein 78(GRP78),CHOP,cysteinyl aspartate-specific protease(Caspase)-12,HO-1,quinone oxidoreductase 1(NQO-1)and nucleus Nrf2 proteins as well as neural apoptosis were measured using modified neurological severity scores(mNSS),dry-wet method,Western blotting and terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)assay.Results Compared with model group,breviscapine significantly decreased mNSS scores[(8.12±1.76)vs.(9.89±1.64),t=2.549,P<0.05)and brain water content[(80.01±2.12)%vs.(83.88±5.74)%,t=2.187,P<0.05]at 24 h,significantly alleviated the expression of GRP78(4.07±0.68 vs.5.74±0.98,t=4.850,P<0.05),CHOP(1.01±0.18 vs.1.38±0.21,t=4.634,P<0.05),Caspase-12(0.55±0.11 vs.1.11±0.13,t=10.391,P<0.05)and Caspase-3(0.77±0.12 vs.1.10±0.21,t=4.726,P<0.05)proteins,and decreased apoptosis(24.26±4.38 vs.32.14±6.87,t=3.350,P<0.05).Moreover,breviscapine significantly increased the expression of nucleus Nrf2(0.21±0.04 vs.0.15±0.03,t=4.157,P<0.05),HO-1(0.31±0.05 vs.0.21±0.05,t=5.941,P<0.05)and NQO-1(0.27±0.05 vs.0.20±0.00,t=4.159,P<0.05)proteins.Conclusion Breviscapine provides neuroprotective effect of anti-ERS mediated apoptosis via modulating Nrf2/HO-1 signaling pathway in rats following fluid percussion brain injury.