氢气对深低温停循环脑保护作用及其与miR-29作用的分子机制研究

Deep hypothermic circulatory arrest (DHCA), which was accompanied by cerebral ischemia-reperfusion injury, was restricted in the application in cardiovascular surgery and neurosurgery as the significant damage in central nerve. Research related to the postoperative brain protection after DHCA has become the hotspot in recent years. It was reported that the hydrogen ( H2 ) could reduce oxidative stress, decrease inflammatory responses and protect neural cells from apoptosis. The H2 also showed protective effects on tissue and cells suffering from stress. Data indicated miR-29 was directly involved in the regulation of apoptosis and differentiation. Our preliminary studies reported that H2 showed its activity in improving neural cell injury in DHCA by inhibition cell apoptosis, which may be related to its effects on the expression of miR-29 in rat brain, but the underlying mechanism is still not clear. Based on our previous results, this work is intends to establish DHCA animal model and cell hypoxia reoxygenation model, by using molecular biology techniques. Our study was aimed to explore the mechanisms of miR-29 in regulating brain injury in DHCA, and furthermore, to observe the regulatory effects of H2 on miR-29. Our study may provide evidence on the interpretation of molecular mechanisms in brain protection of H2, and further support the idea of clinical application of H2 in cerebral protection when DHCA was performed.

深低温停循环（DHCA）术后脑保护成为当前研究的热点和难点之一。氢气（H2）具有减轻氧化应激和炎症反应以及抗凋亡的作用，对应激状态下的组织细胞具有一定的保护作用。miR-29直接参与细胞凋亡和分化因子的调控，在细胞的增殖和凋亡中起到重要作用。申请人前期研究发现H2能够有效减轻DHCA脑组织损伤、抑制神经细胞凋亡，可能与其能够调节miR-29的表达有关。在前期研究基础之上，本课题拟建立DHCA 动物模型和脑细胞缺氧复氧模型，运用分子生物学技术，以miR-29为切入点，探索miR-29在DHCA脑细胞损伤中的机制，深入观测H2对miR-29表达的调控作用，阐释H2脑保护作用的分子机制，为H2临床应用于DHCA提供实验依据。

深低温停循环（DHCA）技术是复杂心脑血管疾病外科治疗的重要辅助手段之一，该技术为心脑血管外科医生带来便利的同时，也会产生严重的早期和晚期中枢神经系统并发症，严重影响患者的生活和生存质量。近年来，大量研究表明氢气（H2）具有不仅具有急性抗氧化效应还具有慢性基因表达调控效应，对许多疾病模型中的组织细胞都具有保护性作用。我们之前的研究证明了氢气对DHCA大鼠的脑保护作用。MiR-29家族直接参与细胞凋亡和分化因子的调控，在细胞的增殖和凋亡中起到重要作用。在前期研究的基础上，我们发现氢气能够呈剂量依赖性减轻HT22细胞损伤，且能够增加低温糖氧剥夺/再灌注（OGD/R）诱导的HT22细胞内miR-29s的表达；将miR-29s类似物转染至受损脑细胞后，脑细胞损伤显著减轻：活细胞数目增加，活性氧（ROS）含量减少，线粒体膜电位（MMP）下降减轻，氧化应激相关蛋白Keap1和凋亡相关蛋白PUMA、BAX表达下降，抗氧化应激相关蛋白Nrf2表达增加；此外，富氢盐水能够显著上调DHCA大鼠海马组织内的miR-29s表达；在大鼠海马组织注入miR-29s合成物后，DHCA大鼠脑损伤显著减轻：海马细胞凋亡减少，海马CA1区细胞组织病理学特征改善，丙二醛（MDA）含量和一氧化氮合成酶（NOS）活性下降，超氧化物歧化酶（SOD）活性增加，炎性因子如肿瘤坏死因子-α（TNF-α）以及白介素-1β（IL-1β）降低，抗炎因子白介素-10（IL-10）增加，氧化应激相关蛋白Keap1和凋亡相关蛋白Caspase-3、PUMA、BAX表达下降，抗氧化应激相关蛋白Nrf2表达增加；双荧光素酶报告分析实验结果还表明miR-29s能够直接靶定促凋亡蛋白PUMA，并抑制其表达。总之，上述结果表明氢气的脑保护效应和其能够上调miR-29s的表达有关，其下游的作用靶点之一为PUMA。