GRP78蛋白N-Hcy修饰介导PI3K/Akt信号通路在心肌缺血再灌注损伤中的作用及机制研究

Homocysteine (Hcy) plays an important role in ischemic heart disease, but the mechanism is unknown. GRP78 play a key role in the endogenous endoplasmic reticulum protection pathway of myocardial I / R injury. The literature shows that homocysteine metabolism produces homocysteine thiolactone. The protein N-homocysteinylation is an important mechanism of homocysteine toxicity; The protective effect of GRP78 is achieved by membrane translocation increased interaction with PI3K and activation of Akt. GRP78 membrane translocation is critical for the realization of myocardial protection. Our preliminary results show that methionine at positions 28 and 569 of GRP78 in cardiomyocytes can be significantly modified by Hcy; treatment with Hcy can be aggravated Myocardial I/R injury and reduce the interaction between GRP78 and PI3K. It is hypothesized that GRP78 protein N-homocysteinylation can inhibit membrane translocation, reduce interaction with PI3K, and aggravate myocardial I/R injury. The project used mass spectrometry, immunoblotting, co-immunoprecipitation and other techniques to detect the effect of Hcy on myocardial injury and GRP78 function; the N-homocysteinylation sites of GRP78 and the endogenous protection of this modification the effect of action, elucidates a new mechanism that Hcy triggers ischemic heart disease, providing new targets for the development of ideal drugs for the treatment of Hcy-cardiac diseases.

Hcy在缺血性心脏病中扮演着重要角色，但机制未明。GRP78是心肌I/R损伤内源内质网保护通路中的关键一环。文献显示Hcy代谢产生同型半胱氨酸硫内酯导致蛋白质的N-Hcy修饰是Hcy毒性的重要机制；GRP78是通过膜转位增加与PI3K互作并激活Akt得以实现，GRP78膜转位对心肌保护作用的实现极为关键。预实验结果显示：心肌细胞内GRP78第28和569位的蛋氨酸可显著被Hcy修饰；Hcy处理可加剧心肌I/R损伤，降低GRP78与PI3K的互作。据此假设：GRP78的N-Hcy修饰可抑制膜转位，降低与PI3K的互作，加剧心肌I/R损伤。为证此假设，本项目采用质谱分析、免疫印迹、免疫共沉淀等技术检测Hcy对心肌损伤和GRP78功能的影响；明确GRP78的N-Hcy修饰位点以及此修饰对心肌内源保护作用的影响，阐明Hcy引发缺血性心脏病的新机制，为研发治疗Hcy-心脏系统疾病的理想药物提供新靶点

高同型半胱氨酸（Homocysteine, Hcy）是缺血性心脏病的独立高风险因子，Hcy水平与缺血心脏病发病率、心肌梗死面积大小呈正向关联，而与患者预后呈反向关联。虽然目前认为半胱氨酸发生自氧化是导致缺血性心脏病的首要因素，但另有研究表明：Hcy发生自氧化却不会导致缺血性心脏病，因为用叶酸显著降低Hcy水平对缺血性心脏病并没有确切的改善效果。这提示Hcy在缺血性心脏病中作用的分子机制还有待确认。Hcy代谢产生同型半胱氨酸硫内酯(HTL)并进一步导致蛋白质的N-Hcy修饰是Hcy产生毒性的重要机制。内源内质网信号通路在保护心脏缺血再灌注损伤中起到重要作用，而GRP78又是内质网信号通路发挥心肌保护作用的关键一环。通过本项目的实施，我们发现：.1)通过检测不同时期急性心肌梗死（AMI）患者血清Hcy水平，结果显示，在AMI比人样本中Hcy水平上调，并随着心功能分级（NYHN stages）而升高。基于血清Hcy的 ROC 曲线， 可以很好区分normal v.s AMI, 和 AMI 1 v.s AMI 2。.2)通过体外AC16，H2C9和HL1 cells细胞培养，给与Hcy处理或缺血再灌注（IR）处理，PI/Annexin-V 染色流式检测结果显示，Hcy和IR都可以显著促进细胞凋亡。.3)质谱检测心肌细胞内的GRP78的Hcy修饰位点，结果显示心肌细胞内GRP78的第28和569位点的蛋氨酸（K）可以被Hcy显著修饰修饰。.4)在AC16细胞中，敲低GRP78并给与Hcy处理后，PI/Annexin-V 染色流式检测结果显示，敲低GRP78可以加剧Hcy诱导的细胞凋亡。.5)IR 动物模型中血清Hcy含量显著升高，而高Hcy动物模型的心脏功能受损伤，且GPR78 Hcy 修饰水平升高。.6)Hcy 通过影响细胞凋亡通过 GPR78/PI3K 信号通路.通过本项目的资助，分别在中国老年学杂志、江苏医药等杂志发表论文各一篇，还有一篇SCI文章准备投稿