锌对高脂饮食诱导的小鼠肥胖相关性心肌病的保护作用及机制研究

发布时间：2018-05-30 13:10

本文选题：锌 + 肥胖相关性心肌病　；参考：《吉林大学》2017年博士论文

【摘要】：背景:目前肥胖人群在世界范围内迅速增加,而肥胖所引起的并发症正在威胁着人类的健康,尤其是肥胖相关性心肌病。氧化应激和炎症反应在肥胖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。既往研究提示锌可以预防糖尿病并发症如糖尿病心肌病和肾病以及主动脉病变。但不同浓度的锌,如低锌,正常锌和补锌对肥胖相关性心肌病有无影响,以及肥胖性相关性心肌病的发病机制尚未有报道。目的:本研究旨在探讨应用锌处理肥胖小鼠,是否具有预防和/或延缓肥胖诱导的心血管的病理改变的发展进程。方法:实验一:4周龄雄性C57BL/6J小鼠给予正常饮食(normal diet,ND)或者高脂饮食(high fat diet,HFD)。同时给予不同浓度锌:低锌(ZD,每4057千卡含有10mg Zn);正常锌(ZN,每4057千卡含有30mg Zn);和补锌(ZS,每4057千卡含有90mg Zn)处理3个月,6个月。在3个月(3M)和6个月(6M)时间点时,行动脉血压和小动物心脏彩超检测;同时对心肌炎症反应(TNF-α,IL-6,CD68),纤维化(CTGF,TGF-β),氧化应激(3-NT,4-HNE),心肌肥厚(ANP和BNP及β-MHC)应用免疫印迹或者实时聚合酶链反应检测;应用天狼星红染色和WGA染色检测纤维化及心肌肥厚;应用免疫印迹技术检测CARD9,BCL10和p-p38MAPK,p38MAPK;应用免疫共沉淀技术检测CARD9/BCL10复合物。应用免疫印迹和实时聚合酶链反应检测MT;实验二:应用p38MAPK抑制剂(SB203580)处理高脂饮食同时给予低锌和正常锌喂养的雄性C57BL/6J小鼠3个月,分组如下:正常饮食组(Ctrl),高脂饮食/正常锌(HFD/ZN);高脂饮食/正常锌/SB处理组(HFD/ZN/SB);高脂饮食/低锌(HFD/ZD),高脂饮食/低锌/SB处理组(HFD/ZD/SB)SB剂量为1mg/kg体重隔日皮下注射3个月。检测实验一指标。细胞实验:应用2个月鼠龄的C57BL/6J小鼠及MT-KO小鼠及129S1小鼠(MT-KO背景鼠)分离原代心肌细胞,应用100μmmol/L(μM)棕榈酸处理48小时。同时给予2μM TPEN(锌的螯合剂)及50μM氯化锌处理。根据实验目的,应用针对p38 MAPK或者BCL10的小干扰RNA(specific small interfering RNA,si RNA)去减弱此两种基因的表达。应用免疫印迹法检测CARD9,BCL10和p-p38MAPK,p38MAPK,3-NT,4-HNE,BNP等蛋白的表达水平。实验四:17周龄的B6.BKS(D)-Leprdb/J(db/db)转基因鼠给予低脂饮食处理6个月。低脂饮食处理同时给予低锌,正常锌和补锌饮食。在3个月时,将一部分补锌饮食处理的小鼠转换为正常锌处理三个月。分组情况:WT:C57BL/6J小鼠(n=7);肥胖正常锌处理:(db/db-ZN,n=6);肥胖低锌处理:(db/db-ZD,n=7);肥胖补锌处理:(db/db-ZS,n=4);肥胖转换正常锌处理:(db/db-ZS-N,n=4);结果:研究发现补锌和p38MAPK抑制剂均能有效的防止肥胖引起的小鼠心肌炎症、纤维化、氧化损伤、心肌肥厚和心功能不全。低锌可以加重上述表现。同时,CARD9/BCL10/p38 MAPK在肥胖小鼠心肌中明显上调,缺锌加重而补锌可以减少其表达。免疫共沉淀提示CARD9/BCL10形成复合物发挥作用。p38MAPK抑制剂可以几乎完全阻断正常锌和低锌饮食处理的肥胖小鼠心肌炎症及心肌肥厚;原代成年小鼠心肌细胞培养实验中应用BCL10 si RNA减弱BCL10的表达,可导致心肌细胞中p38 MAPK明显下调。给予p38 MAPK抑制剂(SB203580)处理肥胖小鼠及原代心肌细胞应用p38αMAPK si RNA均未影响BCL10和CARD9表达。MT在肥胖小鼠心肌中3个月上调,但6个月明显下调,缺锌减少其表达,补锌明显增加其表达。细胞实验提示MT-KO小鼠心肌中补锌对CARD9/BCL10/p38 MAPK的抑制作用消失。锌可以改善db/db转基因鼠心功能不全。结论:在肥胖小鼠中,补锌能够显著预防肥胖诱导的心功能紊乱、炎症反应、氧化应激损伤、纤维化和心肌肥厚。而锌对肥胖相关性心肌病的的保护作用的机制可能是通过抑制CARD9/BCL10/p38 MAPK通路,而抑制此通路主要是通过上调MT而实现的。
[Abstract]:Background: obese people are rapidly increasing worldwide, and the complications caused by obesity are threatening human health, especially obesity related cardiomyopathy. Oxidative stress and inflammation play an important role in the occurrence and development of obesity complications. Oxidative stress refers to the excessive activity in cells and tissues. Previous studies suggest that zinc can prevent diabetic complications such as diabetic cardiomyopathy and kidney disease, as well as aorta lesions. But zinc, such as zinc, and zinc and zinc, have no effect on obesity related cardiomyopathy, as well as zinc, zinc and zinc. The pathogenesis of obesity related cardiomyopathy has not been reported. Objective: the purpose of this study was to explore the development process of the application of zinc treatment in obese mice to prevent and / or delay the development of cardiovascular pathological changes induced by obesity. Methods: experimental one: 4 weeks old male C57BL/6J mice were given normal diet (normal diet, ND) or high fat diet (HI GH fat diet, HFD). Give different concentrations of zinc at the same time: low zinc (ZD, 10mg Zn per 4 million 57 thousand cards); normal zinc (ZN, 30mg Zn for every 4 million 57 thousand cards); and zinc (ZS, 4 million 57 thousand cards containing 90mg) for 3 months, 6 months. Pulse blood pressure and small animal heart color Doppler detection at 3 months and 6 months; and at the same time against myocarditis TNF- alpha, IL-6, CD68), fibrosis (CTGF, TGF- beta), oxidative stress (3-NT, 4-HNE), myocardial hypertrophy (ANP and BNP and beta -MHC) were detected by Western blot or real-time polymerase chain reaction; Sirius red staining and WGA staining were used to detect fibrosis and myocardial hypertrophy, and immunoblotting was used to detect CARD9, immunoblotting and immunoblotting. CARD9/BCL10 complex was detected by coprecipitation. MT was detected by immunoblotting and real-time polymerase chain reaction; experiment two: the male C57BL/6J mice fed with low zinc and normal zinc were treated with p38MAPK inhibitor (SB203580) for 3 months with low zinc and normal zinc. The group was grouped as follows: normal diet group (Ctrl), high fat diet / normal zinc (HFD/ZN); high fat drink. Food / normal zinc /SB treatment group (HFD/ZN/SB); high fat diet / low zinc (HFD/ZD), high fat diet / low zinc /SB treatment group (HFD/ZD/SB) SB dose for 3 months subcutaneous injection of 1mg/kg weight. Test one index. Cell experiment: the isolation of primary cardiomyocytes from C57BL/6J mice of 2 months old mice and MT-KO mice and 129S1 mice (MT-KO background mice) should be used. At the same time, 100 mmol/L (mu M) palmitic acid was treated for 48 hours. 2 mu M TPEN (zinc chelating agent) and 50 mu M zinc chloride were treated. According to the experimental purpose, the small interference RNA (specific small interfering) was applied to reduce the expression of the two genes. The expression level of PK, 3-NT, 4-HNE, BNP and other proteins. Experiment four: 17 weeks old B6.BKS (D) -Leprdb/J (db/db) transgenic mice were treated with low fat diet for 6 months. Low fat diet was given to low zinc, normal zinc and zinc supplementation diet. At 3 months, a part of the zinc diet treated mice were converted to normal zinc for three months. Group cases: WT:C5 7BL/6J mice (n=7); obese normal zinc treatment: (db/db-ZN, n=6); obese and low zinc treatment: (db/db-ZD, n=7); obese zinc treatment: (db/db-ZS, n=4); obesity converted to normal zinc treatment: (db/db-ZS-N, n=4); results: the study found that zinc and p38MAPK inhibitor could effectively prevent myocarditis, fibrosis, oxidative damage, and myocardial fertilizer induced by obesity. Low zinc can aggravate the above performance. At the same time, CARD9/BCL10/p38 MAPK is obviously up-regulated in the myocardium of obese mice, zinc deficiency is aggravated and zinc supplementation can reduce its expression. Immunoprecipitation suggests that CARD9/BCL10 is formed to play a role in.P38MAPK inhibitors, which can almost completely block the obesity of normal zinc and low zinc diet. The use of BCL10 Si RNA to reduce the expression of BCL10 in the primary adult mouse cardiomyocytes can lead to the obvious downregulation of p38 MAPK in the cardiomyocytes of the primary adult mice. The application of p38 MAPK inhibitor (SB203580) to the treatment of obese mice and the primary cardiomyocytes by p38 alpha MAPK Si In fat mice, the myocardium was up-regulated for 3 months, but the expression of zinc deficiency reduced its expression in 6 months, and zinc increased its expression obviously. The inhibitory effect of zinc supplementation to CARD9/BCL10/p38 MAPK in the myocardium of MT-KO mice disappeared. Zinc could improve the cardiac dysfunction of db/db transgenic mice. Conclusion: zinc supplementation in obese mice can prevent obesity significantly. Induced cardiac dysfunction, inflammatory response, oxidative stress injury, fibrosis and myocardial hypertrophy. The mechanism of zinc protection for obesity related cardiomyopathy may be through inhibition of the CARD9/BCL10/p38 MAPK pathway, and the inhibition of this pathway is mainly achieved by up regulation of MT.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R589.2;R542.2

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