胱硫醚β-合酶硝基化导致血浆同型半胱氨酸水平升高的机制及在衰老血管功能障碍中的作用

An essential characteristic of the aging process is the development of hyperhomocysteinemia, rendering the vasculature susceptible to cardiovascular events. However, the underlying mechanisms still remain unclear. Our previous studies have demonstrated that the activity of cystathionine β-synthase (CBS), the key enzyme in homocystein metabolism, decreased in aged rats, and anti-nitration treatment would reverse this change. It was notable that in aged individuals the nitrative stress was enhanced significantly, nevertheless the role of this phenomenon plays in age-related hyperhomocysteinemia still remains to be determined. In this study, we will utilize both in vivo and in vitro models to demonstrate the mechanism involved in enhanced nitrative stress and reduced CBS activity in age-related hyperhomocysteinemia and vascular dysfunction. Vascular function was determined using vascular rings assay, CBS activity was determined using ninhydrin assay, plasma Hcy was determined using enzyme-linked-immunoassay (ELISA), nitrated CBS was determined using immuno-precipitation，and the possible nitrated amonine acid residues of CBS were assayed using mass spectrometry. Furthermore, aiming on these targets, we will try different anti-nitration approaches to restore the declined CBS activity and vascular dysfunction with aging. Based on these studies, we hope to identify the possibility and effectivity of attenuating age-related vascular dysfunction through suppression of CBS nitration, and finally provide a new strategy to improve vascular health for the aged.

随年龄增加而升高的血浆同型半胱氨酸(Hcy)水平在衰老血管功能下降的病理过程中具有重要作用。本课题组前期研究发现，衰老时降解Hcy的关键酶胱硫醚β-合酶（CBS）活性下降，抗硝基化干预可提高其活性，但机制不清。本课题拟在前期研究基础上，以衰老引起的硝化应激增强为切入点，采用血管环灌流法研究血管舒、缩功能，茚三酮显色法检测CBS酶活性，ELISA法检测血浆Hcy水平, 免疫沉淀法检测硝基化CBS含量，应用质谱法分析CBS的硝基化修饰位点，探讨CBS硝基化修饰所导致的酶活性下降在促进血浆Hcy升高、加剧衰老血管功能障碍中的作用；采用清除ONOO－或抑制其生成来源，探讨抑制CBS硝基化修饰可否提高其酶活性，进而降低血浆Hcy水平、改善衰老血管功能。本课题拟从分子、组织和整体水平探讨通过抗CBS硝基化失活途径来降低衰老血浆Hcy水平，进而改善衰老血管功能的可能性及意义，为血管衰老的防治提供新靶点。

研究背景：老年人心脑血管疾病高发，但机制未明。高同型半胱氨酸血症（HHcy）是心脑血管疾病的一个重要危险因素，其发生机制复杂，尚无有效的防治手段。研究表明老年时硝化应激水平升高，可能与胱硫醚β-合酶(CBS)的活性降低、从而加速HHcy进程有关，但是其中的具体机制仍需进一步探讨。 研究目的：探讨老年时硝化应激增强致CBS活性降低及血浆Hcy水平升高的机制，以及在老年血管舒张功能障碍和内皮损伤中的作用。研究方法：本研究选用自然衰老大鼠模型，对照组和实验组分别给予生理盐水和过氧亚硝基清除剂FeTMPyP。采用大/微血管环、Elisa、免疫组织化学染色、蛋白质免疫印迹及免疫沉淀等方法，观察： 增龄和FeTMPyP对大鼠 (1) 血管舒张功能和内皮结构；(2) 血浆Hcy的浓度；(3) 肝脏CBS蛋白表达和活性；(4) 肝脏过氧亚硝基水平；(5) 肝脏硝基化CBS含量的影响。同时运用体外基因突变的方法，构建包含突变CBS基因的重组质粒，利用转染细胞实验分析Hcy诱导的CBS硝基化位点。研究结果：大鼠随月龄增加，Hcy水平升高，阻力血管舒张功能障碍，内皮损伤；老年大鼠肝脏中Hcy代谢关键酶CBS的活性降低，硝基化CBS增多；给予抗硝基化治疗可以减少CBS的硝基化，增加CBS 活性、降低血浆Hcy水平，降低老年大鼠阻力血管舒张功能障碍和内皮损伤。Hcy （500 μmol/L, 24h）增强了野生型CBS（WT）和突变型CBS（Y233A、Y302A、Y308A和Y484A）硝基化程度，但是没有提高突变型CBS（Y163A、Y223A、Y381A和Y518A）的硝基化程度。给予Hcy刺激后，野生型CBS活性降低。与野生型CBS相比，突变型CBS（Y163A、Y223A、Y381A和Y518A）的活性均有所升高。研究结论：随增龄出现的硝化应激水平升高使CBS 的酪氨酸残基（Y163、Y223、Y381和Y518）发生了硝基化修饰，导致CBS活性降低，对Hcy分解代谢不足，引起Hcy在体内蓄积，进而导致老年阻力血管的舒张功能障碍和内皮损伤。本研究为揭示老年时高同型半胱氨酸血症的发病机制提供了新的思路，为防治血管衰老提供了新的靶点和研究依据。