Apelin减轻糖尿病缺血心肌易损性的作用及机制：改善心肌胰岛素敏感性

Insulin resistance is an independent risk factor in diabetes myocardial injure, oxidative stress is one of the main reason of insulin resistance. As a newly discovered adipokine, Apelin and its receptor APJ are widely distributed in various tissues including myocardium. Recent studies show that Apelin could improve insulin sensitivity, Apelin have the cardiovascular activity and can be degradation by angiotensin converting enzyme. Our most recently studies have shown that Apelin could inhibit oxidative stress, given Apelin can significantly improve insulin sensitivity in liver, but its role in diabetic myocardial injury and mechanism are still unclear. Thus, we propose the hypothesis that Apelin could reduce myocardial injury by improves insulin sensitivity and inhibits oxidative stress. To this end, we will use cell line, primary cell culture, insulin resistance, animal models and patients tissue, by receptor antagonist, Si-RNA, drugs blocking, real-time PCR, Western Blot and other means, to investigate the effects of Apelin in insulin resistance-induced myocardial injury and to identify the mechanism responsible for inhibite oxidative stress and restore insulin sensitivity. From the new viewpoint of Apelin, our results will depth study the role of improve insulin resistance in myocardial protection, and provide new therapeutic approach and experimental evidence for the treatment of Type 2 diabetes and its cardiovascular complications.

胰岛素抵抗是糖尿病心肌损伤的独立危险因素，氧化应激是胰岛素抵抗形成的主要原因。脂肪因子Apelin广泛分布于机体多种组织包括心脏中，新近发现Apelin可改善胰岛素敏感性，具有心血管活性并可被血管紧张素转换酶所降解。我们近期研究显示Apelin可抑制氧化应激，给予Apelin可显著改善肝细胞的胰岛素敏感性，但其在糖尿病心肌损伤中的作用和机制尚待阐明。为此，我们将利用细胞系、原代细胞培养、胰岛素抵抗动物模型和临床患者标本，应用受体拮抗剂、药物阻断、Western blot、real-time PCR等多种方法，从分子、细胞、组织及整体水平，明确apelin在糖尿病胰岛素抵抗时的变化，探讨其抑制氧化应激、改善心肌胰岛素敏感性与减轻心肌损伤易感性的关系，阐明其作用机制。本项目将从Apelin这个新视点研究改善胰岛素敏感性在心肌损伤保护中的作用，为糖尿病心血管并发症的治疗提供新的思路和治疗靶点。

目的：糖尿病为危害人类健康的重大疾病，胰岛素抵抗是导致糖尿病及其并发症形成的关键病理生理基础。脂肪因子Apelin广泛分布于机体多种组织包括心脏中，本研究从Apelin深入研究其在胰岛素抵抗和心血管保护中的机制及作用。方法：① 收集在我院行口服葡萄糖耐量实验（OGTT）患者的空腹和餐后2h血清，根据临床症状和生化检测指标诊断为2 型糖尿病。其中2 型糖尿病患者131例，健康人血清样品54例。生化仪分析仪检测生化指标，ELISA方法分批检测其中Apelin、ROS、TNF-α等水平变化。我院病理科收集符合上述标准的糖尿病患者心肌组织。检测标本中的Apelin及糖原含量。② 成年雄性ob/ob小鼠，结扎左冠状动脉造心肌梗死模型，结扎前30min 腹腔注射20 nmol/kg的Apelin，检测各组间Apelin、ROS、TNF-α，心肌细胞凋亡及心肌梗死面积。③ TNF-α诱导鼠心肌H9c2细胞系建立胰岛素抵抗心肌细胞模型，C57BL/6小鼠背部皮下埋置TNF-α（8 μg/kg•day）渗透压缓释泵建立胰岛素抵抗动物模型。④ 用Apelin处理胰岛素抵抗细胞模型，通过蒽酮法检测糖原水平。⑤ H9c2细胞模型4 nmol/L的apelin及Apelin-si-RNA预处理、胰岛素抵抗小鼠模型注射20nmol/kg的F13A 预处理30 min 后加入Apelin。⑥ ROS来源应用各个途径的抑制剂研究Apelin抑制ROS生成的主要途径。结果：① 2型糖尿病患者HOMA-IR指数评估患者呈现胰岛素抵抗。患者血清Apelin水平也明显高于健康人群，伴有炎性因子TNF-α和ROS含量的显著增高。②胰岛素抵抗ob/ob小鼠中检测上述指标。结果和临床研究相一致。③ TNF-α诱导细胞出现胰岛素抵抗的现象，Apelin在mRNA和蛋白水平表达增加。④ Apelin处理抑制ob/ob和胰岛素抵抗C57/BL6小鼠心肌细胞凋亡，缩小缺血后梗死面积。⑤ ROS产生酶体的抑制剂结果显示， NOS是TNF-α诱导H9c2产生ROS的主要来源，Apelin可以显著抑制TNF-α和缺氧诱导的iNOS蛋白表达。⑥ Apelin处理后胰岛素抵抗心肌细胞和小鼠心脏组织JNK 磷酸化水平降低，IRS1 Ser307 磷酸化水平下降，AKT、GSK磷酸化水平上升。结论：研究提示Apelin通过抑制iNO