姜黄素衍生物C66对1型糖尿病小鼠心血管系统并发症的保护作用

发布时间：2018-10-31 15:08

【摘要】：背景：氧化应激和炎症反应在糖尿病并发症的发生和发展中扮演着重要的角色。氧化应激是指在细胞和组织中过度产生的活性氧和氮自由基(ROS和RNS)或者内源性抗氧化基因的活性下调而引起的不平衡。临床研究显示，外源性给予糖尿病患者单剂或者两到三种抗氧剂均无明显预防并发症的效果。因此，上调内源性、多种抗氧化剂来预防糖尿病心血管并发症可能是一个更好的方法。我们发现，C66作为姜黄素的一种衍生物能够有效的抑制高血糖引起的炎症反应和巨噬细胞浸润，进而显著的预防糖尿病诱导的JNK活性增加而引起的大鼠的肾脏损伤。那么，是否C66作为一个有效的抗氧化剂能够通过抑制JNK的活性和上调内源性抗氧化剂的表达及功能，进而预防糖尿病心血管并发症尚未有报导。目的：本研究旨在探讨应用C66处理糖尿病小鼠，是否具有预防和/或延缓糖尿病诱导的心血管的病理改变的发展进程。方法：雄性C57BL/6J小鼠应用单次腹腔注射链脲霉素造成糖尿病小鼠模型。糖尿病和年龄性别一致的对照组小鼠均随机分为3组，给予溶剂、C66或者JNKi（sp600125）处理。这三种处理方法均采用剂量为5mg/kg体重隔日灌胃达3个月。血压和心功能分别在应用链脲霉素诱导糖尿病前（0M）和小鼠处死时（3M）检测；心肌炎症反应(CD68, MCP-1),纤维化(CTGF, TGF-β1),氧化应激（3-NT,MDA）,内质网功能紊乱(GRP78，IRE1，ATF4，CHOP, Caspase-12)和凋亡（BAX/BCL-2， Caspase-3， TUNEL）通过天狼星红染色、免疫印迹、末端脱氧核糖核酸转移酶介导的dUTP切口末端标记技术、硫代巴比土酸法等方法检测。同时，我们主要应用免疫组织化学染色和实时聚合酶链反应等方法进行小鼠主动脉被用于检测炎症反应(PAI-1，TNF-α，和p-JNK)、氧化应激(3-NT)、纤维化(天狼星红染色和肥大细胞染色)、细胞凋亡(TUNEL)和增殖(PCNA)和Nrf2的表达和功能以及MT的表达。结果：研究发现C66和JNKi均能有效的防止1型糖尿病引起的小鼠心肌炎症、纤维化、氧化损伤、内质网功能紊乱和凋亡。C66上调NFE2-相关因子2（Nrf2）的表达及其转录活性，增加Nrf2的磷酸化作用及其下游抗氧化因子的表达。并且，C66能够恢复糖尿病小鼠的金属硫蛋白(MT)将近对照组水平。C66和JNKi通过抑制JNK磷酸化作用，，可以显著预防糖尿病诱导的小鼠主动脉炎症反应、氧化和氮化损伤、细胞凋亡、增殖和纤维化的增加。此外，在正常和糖尿病的条件下，通过抑制JNK磷酸化作用，C66和JNKi显著增加了主动脉Nrf2的表达和转录功能(增加Nrf2下游基因的表达)，并且恢复了糖尿病小鼠MT的表达。结论：在糖尿病小鼠中，应用C66处理能够显著预防糖尿病诱导的心功能紊乱、内质网应激；心肌和主动脉的炎症反应、氧化应激损伤、纤维化和细胞凋亡。而C66对糖尿病的心血管的保护作用的机制可能是通过抑制JNK的活性，同时上调内源性抗氧化基因(Nrf2和MT)的表达和转录功能。
[Abstract]:Background: oxidative stress and inflammation play an important role in the occurrence and development of diabetic complications. Oxidative stress refers to the imbalance caused by excessive production of reactive oxygen species and nitrogen free radicals (ROS and RNS) or down-regulation of endogenous antioxidant genes in cells and tissues. Clinical studies showed that exogenous administration of single or two to three antioxidants did not significantly prevent complications in diabetic patients. Therefore, upregulating endogenous, multiple antioxidants to prevent diabetic cardiovascular complications may be a better way. We found that C66 as a derivative of curcumin can effectively inhibit hyperglycemia induced inflammation and macrophage infiltration and thus significantly prevent diabetic induced increase in JNK activity in rats kidney damage. Whether C66 as an effective antioxidant can prevent diabetic cardiovascular complications by inhibiting the activity of JNK and up-regulating the expression and function of endogenous antioxidants has not been reported. Objective: to investigate whether the treatment of C66 in diabetic mice can prevent and / or delay the progression of diabetic induced cardiovascular pathological changes. Methods: male C57BL/6J mice were induced by single intraperitoneal injection of streptozotocin. Diabetic and age-matched control mice were randomly divided into 3 groups, treated with solvent, C66 or JNKi (sp600125). All of the three treatments were treated with 5mg/kg for 3 months by gavage every other day. The blood pressure and heart function were measured before streptozotocin induced diabetes (0 M) and mice were killed (3 M). Myocardial inflammatory response (CD68, MCP-1), fibrosis (CTGF, TGF- 尾 1), oxidative stress (3-NTN MDA), endoplasmic reticulum dysfunction (GRP78,IRE1,ATF4,CHOP, Caspase-12) and apoptosis (BAX/BCL-2, Caspase-3,) TUNEL) was detected by Sirius red staining, Western blot, terminal deoxyribonucleic acid mediated dUTP Nick end labeling and thiobarbituric acid method. At the same time, we mainly used immunohistochemical staining and real-time polymerase chain reaction to detect inflammatory response (PAI-1,TNF- 伪, and p-JNK), oxidative stress (3-NT) in mouse aorta. Fibrosis (Sirius red staining and mast cell staining), the expression and function of apoptotic (TUNEL) and proliferative (PCNA) and Nrf2, and the expression of MT. Results: both C66 and JNKi could effectively prevent myocarditis, fibrosis, oxidative injury, endoplasmic reticulum dysfunction and apoptosis induced by type 1 diabetes in mice. C66 upregulated the expression of NFE2- related factor 2 (Nrf2) and its transcriptional activity. The phosphorylation of Nrf2 and the expression of antioxidants downstream were increased. In addition, C66 can restore the level of metallothionein (MT) in diabetic mice near the control level. C66 and JNKi can significantly prevent diabetic induced aortic inflammation, oxidation and nitridation injury by inhibiting the phosphorylation of JNK. Increased apoptosis, proliferation and fibrosis. In addition, under normal and diabetic conditions, by inhibiting JNK phosphorylation, C66 and JNKi significantly increased the expression and transcription of Nrf2 in aorta (increased the expression of downstream Nrf2 gene) and restored the expression of MT in diabetic mice. Conclusion: in diabetic mice, C66 treatment can significantly prevent diabetic induced cardiac dysfunction, endoplasmic reticulum stress, myocardial and aortic inflammation, oxidative stress injury, fibrosis and apoptosis. The protective mechanism of C66 on diabetic cardiovascular may be by inhibiting the activity of JNK and upregulating the expression and transcription of endogenous antioxidant genes (Nrf2 and MT).
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.2

【共引文献】