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摘要：
目的 探讨核转录因子红系2相关因子2（nuclear factor‑erythroid 2‑related factor 2, Nrf2）信号通路在硫化氢（hydrogen sulfide, H2S）减轻新生鼠缺氧缺血性脑病（hypoxic ischemic encephalopathy, HIE）中的作用。 方法 36只7日龄C57BL野生型（wild type, WT）小鼠和36只7日龄Nrf2基因敲除（knock out, KO）小鼠分别按随机数字表法分为野生型对照组（WT‑Con组）、Nrf2基因敲除对照组（KO‑Con组）、野生型模型组（WT‑HI组）、Nrf2基因敲除模型组（KO‑HI组）、野生型模型+NaHS组（WT‑NaHS组）和Nrf2基因敲除模型+NaHS组（KO‑NaHS组），每组12只。结扎一侧颈总动脉后缺氧环境中处理2 h复制小鼠HIE模型，腹腔注射3 mg/kg NaHS干预。缺氧缺血（hypoxic and ischemic, HI）后24 h，收集小鼠全脑组织，应用H‑E染色检测神经元损伤情况，JC‑1法检测线粒体膜电位（mitochondrial membrane potential, MMP），酶标仪检测活性氧（reactive oxygen species, ROS）释放，Clark氧电极检测呼吸率（respiration3/respiration4, R3/R4），生物发光法检测ATP含量，Western blot检测B淋巴细胞瘤‑2蛋白 （B‑cell lymphoma‑2, Bcl‑2）、Bcl‑2 相关 X 蛋白（Bcl‑2‑Associated X, Bax）、血红素氧合酶（heme oxygen, HO）‑1、硫氧还蛋白（thioredoxin, Trx）‑1、甘油醛‑3‑磷酸脱氢酶（glyceraldehyde‑3‑phosphate dehydrogenase, GAPDH）、Nrf2和组蛋白（Histone）3。分别在HI后4周和6周，应用Y迷宫检测小鼠学习记忆能力。 结果 与WT‑Con组比较：WT‑HI组小鼠存活神经元明显减少（P<0.05）；Bax、Nrf2、HO‑1和Trx‑1表达增加（P<0.05），Bcl‑2表达减少（P<0.05）；MMP、线粒体呼吸率（R3/R4）和ATP水平降低（P<0.05），ROS释放增加（P<0.05）；交替次数百分比和新异臂停留时间减少（P<0.05）。与WT‑HI组比较：WT‑NaHS组小鼠存活神经元增加（P<0.05）；Bax表达减少（P<0.05），Bcl‑2、Nrf2、HO‑1和Trx‑1表达增加（P<0.05）；MMP、线粒体呼吸率和ATP水平增加（P<0.05），ROS释放减少（P<0.05）；交替次数百分比和新异臂停留时间增加（P<0.05）。与WT+NaHS组比较：KO+NaHS组存活神经元明显减少（P<0.05）；Bax表达增加（P<0.05），Bcl‑2、Nrf2、HO‑1和Trx‑1表达减少（P<0.05）；MMP、线粒体呼吸率和ATP水平降低（P<0.05），ROS释放增加（P<0.05）；交替次数百分比和新异臂停留时间减少（P<0.05）。 结论 NaHS通过激活Nrf2信号通路发挥对HI导致的学习记忆和神经元存活的保护作用。
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Abstract: Objective To investigate the role of the nuclear factor erythroid 2‑related factor 2 (Nrf2) signaling pathway on alleviated hypoxic ischemic encephalopathy (HIE) by hydrogen sulfide in neonatal mice. Methods According to the random number table method, 36 C57BL wild type (WT) mice and 36 Nrf2 knocking‑out (KO) mice, aged seven days, were randomly divided into six groups (n=12): a WT control (WT‑Con) group, a WT model (WT‑HI) group, a WT model+NaHS (WT‑NaHS) group, a Nrf2 knocking‑out control (KO‑Con) group, a Nrf2 knocking‑out model (KO‑HI) group and a Nrf2 knocking‑out model+NaHS (KO‑NaHS) group. After ligation of the carotid artery, the mice were treated in a hypoxic environment for 2 h to simulate a model of HIE in rats, followed by intraperitoneal administration of 3 mg/kg NaHS. Then, 24 h after hypoxic and ischemia (HI) , the brain tissues were collected, and the neuronal injury was detected by hematoxylin‑eosin (H‑E) staining. The mitochondrial membrane potential (MMP) was detected by JC‑1 method. The release of reactive oxygen species (ROS) was detected by a microplate reader. The mitochondrial respiration rate (R3/R4) was measured using a Clark‑type electrode. The ATP content was detected by bioluminescence. The levels of B‑cell lymphoma‑2 (Bcl‑2), Bcl‑2‑associated X (Bax), heme oxygen (HO)‑1, thioredoxin (Trx)‑1, glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH), Nrf2 and Histone3 were detected by Western blot. Then, 4 and 6 weeks after HI, the Y‑maze was applied to examine the learning and memory abilities of the mice. Results Compared with the WT‑Con group, mice in the WT‑HI group presented remarkable decreases in the number of surviving neurons (P<0.05)； increases in the levels of Bax, Nrf2, HO‑1 and Trx‑1 (P<0.05), decreases in the level of Bcl‑2 (P<0.05)； decreases in the levels of MMP, R3/R4 and ATP content (P<0.05), increases in ROS release (P<0.05); and decreases in the percentage of alternations and the residence time of the new arms (P<0.05). Compared with the WT‑HI group, the WT‑NaHS group presented significant increase in the number of surviving neurons (P<0.05); decrease in the level of Bax (P<0.05), increases in the levels of Bcl‑2, Nrf2, HO‑1 and Trx‑1 (P<0.05); increases in the MMP, R3/R4 and ATP content (P<0.05), decreases in ROS release (P<0.05); and increase in the percentage of alternations and the residence time of the new arms (P<0.05). Compared with the WT‑NaHS group, the KO+NaHs group presented remarkable decreases in the number of surviving neurons (P<0.05); increases in the level of Bax (P<0.05), decreases in the levels of Bcl‑2, Nrf2, HO‑1 and Trx‑1 (P<0.05); decreases in MMP, R3/R4 and ATP content (P<0.05), increase in ROS release (P<0.05); and decrease in the percentage of alternations and the residence time of the new arms (P<0.05) Conclusions NaHS exerts a regulatory effect on learning and memory and neuronal survival resulting from HI through activation of the Nrf2 signaling pathway.
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