DNA甲基化修饰调控CBS表达在高同型半胱氨酸加剧脑缺血后血脑屏障损伤中的作用及机制研究

Hyperhomocysteinemia (HHcy) was reported to associated was a high risk of poor functional outcome, mortality and hemorrhage transformation after acute ischemic stroke (AIS). However, the underlying mechanisms remain unclear. The blood–brain barrier (BBB) dysfunction was the pathophysiological of hemorrhage transformation after AIS. From our preliminary study, we found that the BBB dysfunction was significant increased after acute ischemic in mice with HHcy, which suggest HHcy may aggravate BBB dysfunction after ischemic. We further found the expression of cystathionine β-synthase (CBS) and its metabolites hydrogen sulfide (H2S) were decreased in Mouse brain endothelial cell line (bEnd.3) intervention by Hcy combined with oxygen and glucose depravation (OGD). DNA methyltransferase (DNMT) inhibition can reversed the decrease of CBS induced by Hcy and OGD, which suggest CBS expression mediated by Hcy through DNA Methylation. Therefore, we come up with a scientific hypothesis that HHcy increased promoter DNA methylation and transcriptional repression of CBS in brain endothelial cell, decreased expression of CBS as well as its metabolites such as H2S, taurine and GSH, then aggravate the BBB dysfunction after ischemic. The aim of the current project is to detect the mechanisms of the aggravated BBB dysfunction by HHcy after ischemic via cellular biology, molecular biology and experiment animals. The project hopes to reveal the mechanisms of the high risk of poor functional outcome, and hemorrhage transformation after acute ischemic stroke (AIS) by HHcy and may provide evidence of the mechanisms and potential therapeutic targets of BBB dysfunction after ischemic.

高同型半胱氨酸血症(HHcy)增加缺血性卒中患者不良预后风险，且与梗死后出血转化相关，然而机制仍未阐明。血脑屏障(BBB)损伤是出血转化的重要病理基础，我们前期研究发现HHcy小鼠在脑缺血后急性期BBB损伤明显增加，且在小鼠bEND3细胞中，同型半胱氨酸(Hcy)和氧糖剥夺(OGD)共同处理后胱硫醚β-合成酶（CBS）表达降低，其下游硫化氢(H2S)生成减少，而甲基化转移酶抑制剂能逆转CBS下调。因此，我们提出假设：HHcy加剧脑缺血后BBB损伤，其机制可能与CBS启动子甲基化增加引起的CBS转录下调，其下游H2S、还原型谷胱甘肽、牛磺酸等保护分子生成减少有关。本项目拟借助分子生物学、细胞生物学和实验动物等，探讨HHcy加剧脑缺血后BBB损伤的机制，有助于深化HHcy增加脑缺血后出血转化及不良预后机制的认识，且为脑缺血后BBB损伤的病理机制及干预提供新思路和靶点。

高同型半胱氨酸血症(HHcy)是缺血性卒中的危险因数，我们前期研究发现在缺血性卒中患者中，基线同型半胱氨酸(Hcy)水平增高显著增加患者住院期间死亡和不良预后风险，但确切的机制不清楚。本研究中我们通过蛋氨酸饮食喂养成功构建小鼠HHcy模型并开展相关研究。采用TTC染色、烘干称重法及小鼠头颅MRI检查，我们发现与单独MCAO组相比，HHcy小鼠在脑缺血再灌注24小时后缺血侧梗死体积明显增大，脑缺血再灌注48小时后缺血侧脑水肿量增高。行为学试验表明，与单独MCAO相比，HHcy小鼠在脑缺血再灌注24小时后Bederson 评分显著增高，而Grip Strength评分降低，表明HHcy通过增加缺血后梗死体积和脑水肿从而加重缺血后脑损伤。与单独MCAO组相比，HHcy小鼠在脑缺血再灌注30分钟后缺血区Evans blue渗透率明显增高，缺血再灌注24小时后缺血区occludin蛋白表达明显降低，表明HHcy能够显著加剧脑缺血后血脑屏障(BBB)损伤。与假手术组相比，普通小鼠和HHcy小鼠在脑缺血再灌注24小时后缺血区胱硫醚-β-合成酶(CBS)蛋白表达明显降低，予以CBS激活剂S-腺苷甲硫氨酸(SAM)能够显著提高缺血区occludin蛋白表达，表明CBS途径参与HHcy加剧脑缺血后BBB损伤。在小鼠脑微血管内皮细胞(bEND3)模型中进一步验证了上述结果，我们发现与OGD组相比，Hcy联合OGD显著降低occludin蛋白的表达，且CBS激活剂SAM能够提高occludin蛋白的表达。最后，在临床研究部分，我们发现缺血性卒中患者基线Hcy水平增高显著增加患者住院期间肺部感染发生的风险。研究结果有助于深化HHcy增加脑缺血后脑损伤机制的认识，为脑缺血后BBB损伤的病理机制及干预提供新思路。