硫化物-醌氧化还原酶促进缺血性脑损伤修复的机制研究

Selectively inducing alternative (M2) polarization of microglia/macrophages is a new therapeutic strategy to promote brain repair and functional recovery following ischemic stroke. Our previous study showed that the slow-releasing hydrogen sulfide (H2S) donor ADT-OH promoted M2 polarization of microglia/macrophages and brain repair by activating AMPK following cerebral ischemia. However, the molecular mechanism by which ADT-OH activates AMPK remains unclear so far. By acting as the enzyme that initiates sulfide oxidation, sulfide : quinone oxidoreductase (SQR) plays an essential role in the metabolism of the gaseous signaling molecular H2S. We propose in the project that SQR-mediated sulfide oxidation leads to mitochondrial uncoupling, which is the mechanism underlying AMPK activation by ADT-OH. To further investigate whether SQR is a novel therapeutic target for ischemic stroke and how SQR promotes brain repair and functional recovery following cerebral ischemia, we propose to use the cellular oxygen glucose-deprivation model as well as the gene engineered mice with specific deletion of SQR in microglia and macrophages. By using these approaches, we will investigate: 1) whether the promoting effects of ADT-OH on post-ischemic AMPK activation and M2 polarization of microglia/macrophages are dependent on SQR; 2) whether SQR acts through M2 polarization of microglia/macrophage to mediate the promoting effects of ADT-OH on brain repair and functional recovery following stroke. For the first time, the proposed project will show that SQR-mediated sulfide oxidation is novel mechanism to promote brain repair by inducing M2 polarization of microglia/macrophages following cerebral ischemia.

脑缺血后诱导小胶质/巨噬细胞M2极化是促进缺血性脑损伤修复的新策略。我们发现：缓释H2S供体ADT-OH通过激活AMPK促进脑缺血后小胶质/巨噬细胞M2极化及损伤修复。但ADT-OH激活AMPK的机制不明。硫化物-醌氧化还原酶（SQR）是气体信号分子H2S氧化代谢的关键酶。本课题提出并将在细胞模型证实：SQR对H2S氧化代谢导致线粒体解偶联是ADT-OH激活AMPK的关键分子机制。为了进一步表明SQR是促进缺血性脑损伤修复的新靶点并探讨作用机制，我们将利用细胞缺氧缺糖模型及小胶质/巨噬细胞特异性敲除SQR的基因工程鼠探讨：1）SQR介导ADT-OH脑缺血后促进小胶质/巨噬细胞AMPK活化及M2极化的作用；2）SQR介导ADT-OH通过M2极化机制促进脑缺血后血管新生及白质修复的作用。本课题首次提出：SQR介导的H2S氧化代谢是通过调控小胶质/巨噬细胞极化促进缺血性脑损伤修复的新机制。

前期研究发现：缓释H2S供体ADT-OH通过激活AMP激活的蛋白激酶（AMPK）促进脑缺血后小胶质/巨噬细胞M2极化及损伤修复。但ADT-OH激活AMPK的机制不明。硫化物-醌氧化还原酶（SQR）是气体信号分子H2S氧化代谢的关键酶。本课题的结果表明：SQR对H2S氧化代谢导致线粒体解偶联是ADT-OH激活AMPK的关键分子机制。我们发现：缓释供体ADT-OH释放的H2S被SQR氧化后驱动电子在线粒体复合物I的反向传递，从而产生线粒体氧自由基。线粒体氧自由基进而通过激活解偶联蛋白2导致H2S的胞内信号传导，如下游AMPK的激活。利用细胞缺血模型，我们进一步发现H2S供体ADT-OH通过SQR促进小胶质细胞AMPK活化及M2极化。本课题还首次制备了SQR条件性敲除鼠（Sqrfl/fl），并利用从Jackson Lab引进的Cx3cr1-cre小鼠，制备获得了小胶质/巨噬细胞特异性敲除SQR的基因工程鼠（Cx3cr1-cre:Sqrfl/fl）。利用Cx3cr1-cre:Sqrfl/fl小鼠，我们初步发现SQR介导H2S供体ADT-OH在脑缺血后促进血管新生及功能康复的作用。我们还初步发现H2S供体在脑缺血后促进白质损伤修复。综上，本课题的结果提示SQR介导的H2S氧化代谢是缺血性脑损伤修复的新机制。课题有望为脑卒中的治疗开拓新方向。