去甲斑蝥素通过Nrf2通路保护新生大鼠缺氧缺血性脑损伤的机制

Mechanism of norcantharidin in protecting the neonatal rats with hypoxic-ischemic brain damage through Nrf2 pathway

目的探究去甲斑蝥素(norcantharidin,NCTD)预处理介导核因子E2相关因子2(nuclear factor erythroid-2 related factor 2,Nrf2)通路对新生大鼠缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)的保护作用。方法60只新生幼鼠分为假手术组、模型组、低剂量组、中剂量组和高剂量组,每组12只。用不同剂量NCTD预处理低、中、高剂量组幼鼠,假手术组和模型组给予等体积的生理盐水灌胃。除假手术组外,其余各组均构建缺氧缺血性脑损伤模型,造模成功后,分别检测各组大鼠神经功能评分、脑积水量、神经细胞凋亡率、氧化应激指标[丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-P_(x))]、炎症因子指标[白细胞介素-6(interleukin-6,IL-6)、白细胞介素-10(interleukin-10,IL-10)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)]、Nrf2、血红素氧合酶1(heme oxygenase-1,HO-1)和醌氧化还原酶1[NAD(P)H:quinone oxidoreductase 1,NQO1]蛋白的表达水平。结果与假手术组比较,各组缺氧缺血幼鼠神经功能评分均显著下降(P<0.05),与模型组相比,低、中和高剂量组随着NCTD剂量增加,幼鼠神经功能评分显著改善(P<0.05)。与假手术组相比,各组缺氧缺血幼鼠脑积水量和神经细胞凋亡率显著增加(P<0.05),与模型组相比,低、中和高剂量组随着NCTD剂量增加,幼鼠脑积水量和神经细胞凋亡率随之降低(P<0.05)。与假手术组相比,各组缺氧缺血幼鼠氧化应激指标MAD含量显著上升,SOD和GSH-P_(x)含量显著下降(P<0.05),与模型组相比,低、中和高剂量组随着NCTD剂量增加,MDA含量随之下降,SOD和GSH-P_(x)含量随之上升(P<0.05)。与假手术组相比,各组缺氧缺血幼鼠炎症因子指标IL-6和TNF-α水平显著上升,IL-10水平显著降低(P<0.05),与模型组相比,低、中和高剂量组随着NCTD剂量增加,IL-6和TNF-α水平随之降低,IL-10水平随之升高(P<0.05),与假手术组相比,各组幼鼠脑组织内Nrf2、HO-1和NQO1蛋白的表达水平显著降低(P<0.05),而与模型组相比,NCTD预处理的低、中和高剂量组幼鼠脑组织内Nrf2、HO-1和NQO1蛋白的表达显著上升,且呈剂量依赖性(P<0.05)。结论NCTD通过激活Nrf2信号通路,抑制炎症反应和氧化应激反应,对新生大鼠缺氧缺血性脑损伤有较好的脑保护作用。

Objective To explore the protective effect of norcantharidin(NCTD)preconditioning mediation of nuclear factor erythroid-2 related factor 2(Nrf2)pathway in neonatal rats with hypoxic-ischemic brain damage(HIBD).Methods 60 neonatal rats were divided into sham operation group,model group,low-dose group,medium-dose group and high-dose group,with 12 in each group.Different doses of NCTD were used to pretreat young rats in low-dose group,medium-dose group and high-dose group.The sham operation group and the model group were given the same dose of normal saline intragastrically.Except for the sham operation group,the other groups all were constructed hypoxic-ischemic brain damage models.After the model was successfully established,the neurological function score,hydrocephalus volume,nerve cell apoptosis rate,and oxidative stress indicators[malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione peroxidase(GSH-P_(x))],inflammatory factors[interleukin-6(IL-6),interleukin-10(IL-10)and tumor necrosis factor-α(TNF-α)]and protein expressions of Nrf2,heme oxygenase 1(HO-1)and NAD(P)H:quinone oxidoreductase 1(NQO1)were detected in each group.Results Compared with the sham operation group,the neurological function score of each group of hypoxic-ischemic rats was significantly decreased(P<0.05).Compared with the model group,the neurological function score of young rats was significantly improved with the increase of NCTD dose in the low-dose,medium-dose and high-dose groups(P<0.05).The hydrocephalus volume and the apoptosis rate of nerve cells in each group of hypoxic-ischemic young rats were significantly increased compared to the sham operation group(P<0.05).As the dose of NCTD increased,the hydrocephalus volume and apoptosis rate of nerve cells in young rats were decreased in the low-dose,medium-dose and high-dose groups compared to the model group(P<0.05).The oxidative stress indicator MAD content of hypoxic-ischemic young rats in the other groups was risen significantly while the contents of SOD and GSH-P_(x)were reduced significantly compared with those in the sham operation group(P<0.05).Compared with the model group,as the dose of NCTD increased,the content of MDA was decreased while the contents of SOD and GSH-P_(x)were increased in the low-dose,medium-dose and high-dose groups(P<0.05).The levels of inflammatory factors IL-6 and TNF-αof each group of hypoxic-ischemic young rats were significantly higher while the level of IL-10 was significantly lower compared to the sham operation group(P<0.05).Compared with the model group,the levels of IL-6 and TNF-αin the low-dose,medium-dose and high-dose groups were decreased with the increase of NCTD dose while the level of IL-10 was increased with the increase of NCTD dose(P<0.05).The protein expressions of Nrf2,HO-1 and NQO1 in the brain tissues of rats were significantly down-regulated in the other groups compared to the sham operation group(P<0.05),but were significantly up-regulated in the low-dose,medium-dose and high-dose groups pretreated by NCTD compared to the model group in a dose dependent manner(P<0.05).Conclusion NCTD can inhibit inflammatory response and oxidative stress response by activating the Nrf2 signaling pathway,and has a good brain protection effect on neonatal rats with hypoxic-ischemic brain damage.