山楂酸调节AMPK/TXNIP降低血管内皮ROS水平改善糖尿病大血管病变的作用及机制研究

Diabetic macrovascular disease is one of the leading causes of death and disability in patients with DM. It is a variant of "xiaoke ", and the basic pathogenesis is blood stasis. Endothelial dysfunction is an important starting factor for DM macroangiopathy. Our previous studies confirmed that maslinic acid (MA) activated autophagy by AMPK/mTOR to protect islet beta cells. The literature suggests that MA can bind to AMPK to protect the vascular endothelium, and the mechanism is unknown. TXNIP is a new target for the treatment of early DM and vascular lesions and is regulated by AMPK. In this study, high glucose induced HUVEC cell injury, apoptosis, morphogenesis, cell ROS, NO levels, AMPK inhibitors acting on cells and siRNA gene knockout AMPKα two ways, observe AMPK-regulated TXNIP and related NLRP3, The expression of IL-1β, IL-6, ET-1 and caspase-3 was used to investigate the protective mechanism of MA on vascular endothelial function. Animal vascular pathology, ultrastructural morphology and hemodynamic parameters were used to study the mechanism of vasospasm and the active ingredients of blood circulation and phlegm-removing drugs to improve the mechanism of DM macroangiopathy. The results are in accordance with traditional Chinese medicine theories and provide theoretical basis for drug development. experimental basis.

糖尿病（DM）大血管病变是DM患者致死致残的主要原因之一，属中医消渴之变证，基本病机为血瘀。血管内皮功能损伤是DM大血管病变重要的始动因素。我们前期研究证实，山楂酸（MA）通过AMPK/mTOR激活自噬保护胰岛β细胞。文献提示MA可与AMPK结合保护血管内皮，机制未明。TXNIP是新发现的治疗早期DM及血管病变的新靶点，受AMPK调控。本课题以高糖诱导HUVEC细胞损伤，检测细胞凋亡、形态，细胞ROS、NO等水平，AMPK抑制剂作用于细胞和siRNA基因敲除AMPKα两种方式，观察AMPK调控的TXNIP及相关的NLRP3、IL-1β、IL-6、ET-1、caspase-3表达，探讨MA对血管内皮功能的保护机制。动物水平通过血管病理和超微结构形态学、血流动力学指标研究血管血瘀发生机制和活血化瘀药有效成分改善DM大血管病变的作用机理，佐证中医理论，为药物研发提供理论基础和实验依据。

糖尿病（DM）大血管病变是DM患者致死致残的主要原因之一，属中医消渴之变证，基本病机为血瘀，高血糖诱发的炎症和内皮损伤是其始动因素。山楂酸（MA）具有抗炎活性，但是否能抑制高血糖引起的内皮损伤尚不清楚。我们运用高脂饲料喂养GK大鼠造模诱导糖尿病早期血管内皮损伤，在前期课题研究的基础上，我们发现山楂酸可以降低空腹血糖，光镜和电镜下均发现可以减轻大鼠血管内皮损伤，B超发现，一定程度上MA可降低胸主动脉RI和PI从而改善血流动力学，可以抑制组织TNF-α、IL-6、TXNIP mRNA表达水平，抑制组织TXNIP蛋白表达；细胞水平上，以高糖诱导人脐静脉内皮细胞（HUVEC）损伤，发现30uM MA抑制33.3mmol/L浓度高糖诱导的 HUVECs凋亡，可减轻高糖对细胞ROS水平的升高和NO合成的抑制，并减少TNF-α、IL-6和IL-1β炎症因子的产生。虽然MA处理不影响与TXNIP表达相关的AMPKα或Erk1/2的激活，但它在表观遗传上降低了TXNIP mRNA的稳定性，从而下调其表达水平。其机制可能是MA促进RNA去甲基化酶ALKBH5招募到TXNIP mRNA，进而增强HUVECs中n6 -甲基腺苷（m6A）去甲基化。本课题通过探讨MA对血管内皮功能的保护机制，有望为解释活血化瘀药有效成分改善DM大血管病变的作用机理，为药物研发提供一定的中医理论基础和实验依据。