目的 研究连翘苷对新生大鼠缺氧缺血性脑病（ hypoxic-ischemic encephalopathy，HIE）神经元凋亡的影响及其作用机制。方法 采用Rice-Vannucci法构建新生大鼠HIE模型，并将造模成功的72只新生大鼠随机分为模型组及连翘苷低、高剂量（ 25、50 mg/kg）组和连翘苷（ 50 mg/kg） ＋核因子E2相关因子2（ nuclear factor erythroid 2-related factor 2，Nrf2）抑制剂brusatol（ 0.4 mg/kg）组，另选取18只新生大鼠作为假手术组。连续给药20 d，末次干预12 h后，通过神经学评分评估大鼠神经行为学功能变化情况；苏木素-伊红（ HE）染色检测大鼠缺血侧海马组织病理损伤； TUNEL染色检测缺血侧海马神经元凋亡情况； ELISA法检测缺血侧海马组织炎性因子白细胞介素-6（ interleukin-6，IL-6）、IL-1β和肿瘤坏死因子-α（ tumor necrosis factor-α，TNF-α）水平；比色法检测缺血侧海马组织氧化应激指标超氧化物歧化酶（ superoxide dismutase，SOD）、过氧化氢酶（ catalase，CAT）活性和丙二醛（ malondialdehyde，MDA）水平； Western blotting检测缺血侧海马组织Nrf2/核因子-κB（ nuclear factor-κB，NF-κB）通路相关蛋白表达；免疫荧光检测缺血侧海马组织Nrf2与NF-κB的共定位情况。结果 与假手术组比较，模型组新生大鼠神经功能评分、神经元凋亡率及海马组织IL-1β、IL-6、TNF-α、MDA水平和细胞核NF-κB p65蛋白表达水平显著升高（ P＜0.05），海马组织SOD、CAT活性及细胞核Nrf2、细胞质NF-κB抑制蛋白α（ inhibitor α of NF-κB，IκBα）、血红素加氧酶-1（ heme oxygenase-1，HO-1）蛋白表达水平显著降低（ P＜0.05），Nrf2和NF-κB共定位荧光强度降低；与模型组比较，连翘苷各剂量组神经功能评分、神经元凋亡率及海马组织IL-1β、IL-6、TNF-α、MDA水平和细胞核NF-κB p65蛋白表达水平显著降低（ P＜0.05），海马组织SOD、CAT活性及细胞核Nrf2、细胞质IκBα、HO-1蛋白表达水平显著升高（ P＜0.05），Nrf2和NF-κB共定位荧光强度升高； Nrf2抑制剂可显著减弱连翘苷的上述作用（ P＜0.05）。结论 连翘苷可降低炎症反应和氧化应激，抑制神经元凋亡，减轻新生大鼠HIE的神经损伤，其作用机制可能与促进Nrf2的核内转移、抑制NF-κB通路有关。

Objective To investigate the effect and mechanism of phillyrin on neuronal apoptosis in neonatal rats with hypoxic-ischemic encephalopathy (HIE).Methods Rice Vannucci method was used to construct a neonatal rat model of HIE, and 72 successfully modeled neonatal rats were stochastically separated into model group, phillyrin low-, high-dose (25, 50 mg/kg) groups and phillyrin (50 mg/kg) + nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor brusatol (0.4 mg/kg) group. Another 18 newborn rats were regarded as sham group. Drugs were continuously given for 20 d, after 12 h of the last intervention, neurological scores were applied to evaluate changes in neurobehavioral function of rats; Hematoxylin-eosin (HE) staining was applied to detect pathological damage in ischemic hippocampus of rats; TUNEL staining was used to measure apoptosis of hippocampal neurons on ischemic side; ELISA method was used to detect the levels of inflammatory factors interleukin-6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) in ischemic hippocampal tissue; Colorimetric method was applied to detect the oxidative stress indicators of superoxide dismutase (SOD), catalase (CAT) activities and malondialdehyde (MDA) level in ischemic hippocampal tissue; Western blotting was applied to detect the expressions of Nrf2/nuclear factor-κB (NF-κB) pathway related proteins in ischemic hippocampus of rats; Co-localization of Nrf2 and NF-κB in ischemic hippocampal tissue was detected by immunofluorescence. Results Compared with sham group, the neurological function score, neuronal apoptosis rate, levels of IL-1β, IL-6, TNF-α and MDA, and nuclear NF-κB p65 protein expression level in hippocampal tissue of neonatal rats in model group were significantly increased (P < 0.05), activities of SOD, CAT and nuclear Nrf2, inhibitor α of NF-κB (IκBα) and heme oxygenase-1 (HO-1) protein expression levels in hippocampal tissue were significantly decreased (P < 0.05), the fluorescence intensity of Nrf2 and NF-κB colocalization was decreased; Compared with model group, the neurological function score, neuronal apoptosis rate, levels of IL-1β, IL-6, TNF-α and MDA, and nuclear NF-κB p65 protein expression level in hippocampal tissue in phillyrin groups were significantly decreased (P < 0.05), activities of SOD, CAT and nuclear Nrf2, IκBα and HO-1 protein expression levels in hippocampal tissue were significantly increased (P < 0.05), the fluorescence intensity of Nrf2 and NF-κB colocalization was increased; Nrf2 inhibitor was able to attenuate the aforementioned effects of phillyrin.Conclusion Phillyrin could reduce inflammation and oxidative stress, inhibit neuronal apoptosis, and alleviate nerve damage in neonatal rats with HIE. Its mechanism may be related to promoting nuclear metastasis of Nrf2 and inhibiting NF-κB pathway.

R285.5

河南省医学科技攻关计划项目（SB201904015)