硫化氢-缺氧诱导因子-1α-自噬信号通路在心肌缺血损伤中的作用和机制

Myocardial ischemia is a popular clinical pathological process. Severe ischemia may result in patients death. Myocardial ischemia is closely correlated with pathological autophagy, while the mechanisms are largely unknown. Recent papers showed that hypoxia inducible factor - 1α (HIF-1α) contributed to autophagy under hypoxia. Hydrogen sulfide (H2S) has multiple biological functions. But the effects and mechanisms of H2S on autophagy regulation are largely elusive. We found that exogenous H2S at a physiological concentration decreased HIF-1α protein levels and HIF-1α transcriptional activity. Meanwhile our pilot work showed that exogenous H2S downregulated the levels of protein markers for autophagy. Thus, we speculated that H2S - HIF-1α – autophagy signal pathway is involved in myocardial ischemia injury. To confirm our hypothesis, by establishing rat myocardial ischemia model and cell model of hypoxia, using molecular biology techniques such as luciferase assay, gene overexpression and gene silence, we hope to elucidate changes of this pathway in myocardial autophagy induced by ischemia and hypoxia. Our work would give a further explanation of the mechanisms by which H2S regulates myocardial ischemia injury. We would pave the way for further elucidation of preventions and therapies to myocardial ischemia injury.

心肌缺血是临床常见病理过程，严重时可导致病人死亡。其发生与异常自噬有关，具体机制不详。一般认为，缺氧诱导因子-1α（HIF-1α）表达增加是细胞缺氧时自噬发生的关键因素。硫化氢（H2S）具有广泛生物学作用。但是H2S对心肌细胞缺血自噬的影响和机制迄今仍未阐明。本课题组发现，生理浓度的外源性H2S可抑制缺氧状态下HIF-1α蛋白表达和转录活性。预实验观察到，外源性H2S可下调缺血心肌自噬蛋白标志物表达。据此，我们推测“硫化氢-缺氧诱导因子-1α-自噬信号通路参与心肌缺血损伤的发生”。为验证该假说，本课题拟复制大鼠心肌缺血和细胞缺氧模型，采用荧光素酶报道基因检测、基因过表达、RNA干扰、流式细胞仪等技术，从器官、细胞及分子水平，观察心肌缺血和缺氧时该通路的变化和外源性H2S的影响，从新的视觉进一步阐明H2S调节缺血性心肌损伤的分子机制，为缺血性心肌病的防治提供新思路和新靶点。

硫化氢（H2S）及其生成关键酶胱硫醚β-裂解酶（Cystathionine β-synthase, CBS）与细胞生长的关系是最近研究的热点问题，有关文献报道各不相同。我们通过建立野生型和突变型CBS稳转细胞系观察内源性CBS/H2S对于HEK29细胞生长的作用。结果表明，与空载组相比，野生型CBS组和突变型CBS组H2S生成率均升高，其中野生型CBS组变化最为明显；细胞活力检测结果显示，野生型CBS组细胞活力高于空载组，而突变型CBS组细胞活力明显低于野生型CBS组；细胞生长曲线提示第3-5天野生型CBS组细胞数目明显高于空载组。这些结果暗示内源性CBS/H2S对于细胞生长具有促进作用。同时，我们以硫氢化钠为供体观察了H2S对于药物敏感性的调节作用。硫氢化钠本身对肝癌细胞活力没有影响，但显著增强了As2O3对细胞活力的抑制作用。 As2O3降低了CBS和CSE的表达，并降低了H2S的产生。siRNA介导的CBS或CSE表达降低消除了As2O3对细胞的活力抑制。硫氢化钠增加了As2O3在细胞内的积累。As2O3刺激了多个药物外输蛋白的表达，包括ABCB1, ABCA1, ABCG8和MPR1。实验进一步证明As2O3降低了ABCB1的巯基化水平，而硫化氢增加了ABCB1的巯基化。上述结果表明H2S可能通过增加ABCB1的巯基化抑制其活性，从而增加As2O3在细胞内的积累，促进其对细胞的杀伤作用。1