β-羟丁酸抗硝基化干预糖尿病心脏微血管病变的机制

The typical feature of diabetic micro-cardiovascular disease is proliferation of vascular basement membrane, the pathological base of which is accumulation of collagen IV. The persistent nitration caused by oxidative stress is its core pathogenesis, which has also been proved by our previous study. The endogenous small molecule β-hydroxybutyrate has good effects of anti-oxidation and anti-nitration, to act mainly in cardiovascular system. The distinctness of β-hydroxybutyrate from conventional antioxidants is regulating gene expression. It is reported that β-hydroxybutyrate can reverse fibrosis, but the mechanism is not clarified. In this study, cardiac microvascular endothelial cells that play a major pathological role are adopted to perform experiments in vivo and in vitro. On the basis that nitration leads to accumulation of collagen IV, the effect target of β-hydroxybutyrate in regulating the generation and degradation balance of collagen IV is investigated. In the aspects of the binding activity of Smad3 to collagen IV gene promoter and NF-κB to MMP-9 gene promoter, the binding activity of Smad3 to the cofactor p300 or HDAC3 and NF-κB to the cofactor p300 or HDAC3, and the enzyme activity of p300, HDAC3 and MMP-9, the effect target and mechanism of β-hydroxybutyrate in regulating collagen IV metabolism balance by antagonizing nitration is systematically studied in the gene regulation level, to provide new thinking of the prevention and treatment of diabetic micro-cardiovascular disease.

血管基底膜增生是糖尿病心脏微血管病变的典型特征，病理基础为IV型胶原积聚。氧化应激导致的持续硝基化是核心病机，这也被我们前期研究所证实。内源性小分子β-羟丁酸具有良好的抗氧化、抗硝基化作用，主要在心血管系统起效。β-羟丁酸不同于传统抗氧化剂的独特性是调控基因表达。有报道，β-羟丁酸可逆转纤维化，但机制未明。本项目拟采用起主要病理作用的心脏微血管内皮细胞进行体内、外实验，首先在明确硝基化导致IV型胶原积聚的基础上，探寻β-羟丁酸调控IV型胶原来源和去路平衡的作用靶点，在基因调控水平，从Smad3与IV型胶原，和NF-κB与MMP-9基因启动子的结合活性；Smad3与共因子p300或HDAC3，和NF-κB与共因子p300或HDAC3的结合活性；p300、HDAC3和MMP-9酶活性方面，系统研究β-羟丁酸抗硝基化调控IV型胶原代谢平衡的作用靶点和机制，为糖尿病心脏微血管病变的防治提供新思路。

血管基底膜增生是糖尿病心脏微血管病变的典型特征，病理基础为IV 型胶原积聚。内源性小分子β -羟丁酸具有良好的心血管保护作用，其不同于传统抗氧化剂的独特性是调控基因表达。有研究提示，β -羟丁酸可逆转纤维化，但机制未明。本研究采用起主要病理作用的心脏微血管内皮细胞进行体内、外实验，分别从IV 型胶原来源和去路两方面的基因表达调控，探索β -羟丁酸的保护作用机制。1.首先发现了β-羟丁酸可提高FOXO3a及SOD的含量，抑制硝基化损伤，起到拮抗糖尿病心脏微血管病变的作用。相关实验结果已整理成文，待发表。2.首次发现了β-羟丁酸呈剂量依赖性的引起独特的组蛋白修饰，并促进NF-κB p65启动MMP-9基因的表达，增加IV型胶原的去路，起到拮抗糖尿病微血管病变IV型胶原积聚的作用。相关实验结果已整理成文，待投稿。3. 探索了β-羟丁酸对糖尿病及其并发症保护作用的浓度范围和作用机制，为β-羟丁酸及其盐对糖尿病并发症，尤其是微血管纤维化中的应用及其拓展应用，提供了重要的实验数据支持。相关内容申报专利1项。本研究首先初步揭示了β-羟丁酸调控基因表达，拮抗糖尿病微血管纤维化的分子机制和作用靶点，为进一步探索其直接引起组蛋白、关键调控分子等的修饰，发挥其保护作用的分子机制，打下了坚实的实验基础。..