【作者】 刘丹；

【导师】 杜建玲；

【作者基本信息】 大连医科大学 ， 内科学， 2017， 博士

【摘要】 背景:糖尿病心肌病(DCM)发病机制复杂,涉及心肌细胞线粒体功能异常、心肌能量代谢异常、心肌微血管病变、胰岛素抵抗等,而上述病理生理过程均与氧化应激有关,氧化应激为DCM发生发展的中心环节。因目前缺乏明确的DCM临床诊断标准,早期诊断困难,成为防治难点。为此,本研究对近10年于我院住院的2型糖尿病合并心肌损伤患者进行临床特点分析,以期为DCM的早期临床诊断和防治提供借鉴。硒蛋白S(Sel S)为一种糖调节蛋白,参与氧化应激、炎症和内质网应激反应,可以保护血管内皮细胞抵抗氧化应激诱导的损伤,而Sel S对糖尿病心肌损伤是否具有抗氧化保护作用尚未见报道。因此,本研究通过体外观察高糖诱导大鼠H9C2心肌细胞氧化损伤与Sel S的关系,进一步于体内诱导大鼠心肌Sel S高表达探讨Sel S在糖尿病心肌氧化损伤中的作用,为防治DCM提供新策略。方法:选择符合2型糖尿病心肌损伤诊断标准的病例作为病例组(n=23,IG);性别、年龄、病程、血糖水平与IG相匹配的2型糖尿病患者作为糖尿病对照组(n=46,CG)。IG以射血分数(LVEF)50%为界分为收缩功能障碍组(n=10,CDG)(LVEF<50%)及收缩功能正常组(n=13,CNG)(LVEF≥50%)。比较组间一般临床资料和超声心动图相关指标。体外分别应用5.5mmol/L葡萄糖(5.5Glu组)、33mmol/L葡萄糖(33Glu组)、50mmol/L葡萄糖(50Glu组)培养大鼠H9C2心肌细胞24小时后,倒置显微镜观察细胞形态变化,Annexin-V/PI双染流式细胞仪分析细胞凋亡率,比色法测定细胞内丙二醛(MDA)含量、超氧化物歧化酶(SOD)活力和谷胱甘肽(GSH)含量,Western Blot检测不同浓度葡萄糖干预后Sel S蛋白表达情况。8周龄的健康SD大鼠适应性喂养1周后,随机分为普通饲料喂养的正常对照组(NC)和高脂高糖喂养组,4周后高脂高糖喂养组大鼠链脲佐菌素40mg/kg单次腹腔注射诱发糖尿病(DM),随后继续高糖高脂喂养19周制备DM心肌损伤大鼠模型。第25周开始转染,根据后期转染条件不同将糖尿病大鼠随机分为Sel S未转染组(DM)、Sel S转染组(DM-SMMV)和空质粒转染组(DM-CMMV)。利用超声微泡基因转染技术,将含pc DNA3.1质粒或含pc DNA3.1-Sel S质粒的微泡液按不同组别进行尾静脉注射联合心前区照射进行转染。每5天转染1次,共三次,最后一次转染结束第5天处死大鼠,收集心脏组织进行HE染色、比色法测定心肌组织MDA含量、SOD活力和GSH含量、Western Blot检测Sel S蛋白表达。结果:CG、CNG和CDG三组E/A均值低于正常水平1,CDG的E/A低于CG(P<0.05)。LVEF:CDG(36.40±7.01)(%)比CNG(59.15±1.77)(%)及CG(59.59±3.75)(%)降低(P<0.05)。左心室内径缩短率(LVFS):CDG(21.55±2.75)(%)比CNG(33.58±0.89)(%)及CG(33.80±1.49)(%)降低(P<0.05)。室间隔(IVS)厚度:CDG(11.60±1.78 mm)比CNG(9.92±1.32 mm)及CG(9.52±1.64mm)增加(P<0.05)。CNG(207.62±72.66 mg/dl)和CDG(200.10±75.75 mg/dl)甘油三酯(TG)水平高于CG(156.41±69.04 mg/dl)(P<0.05)。多分类logistic回归分析显示,病程(OR=1.339,95%CI 1.032～1.737,P=0.028)为糖尿病心肌收缩功能障碍发生的独立危险因素,TG为糖尿病心肌损伤发生的独立危险因素(OR=1.012,95%CI 1.002～1.022,P=0.018)。Spearman相关性分析显示,LVEF与IVS厚度(r=–0.391,P=0.001)和糖尿病病程(r=–0.261,P=0.030)呈负相关。不同浓度葡萄糖培养大鼠H9C2心肌细胞后,倒置相差显微镜下,5.5Glu组细胞呈长梭形,单层排列整齐,极性生长;33Glu组细胞排列紊乱、出现皱缩细胞;50Glu组细胞形态不规则,排列紊乱,极性消失,皱缩细胞增多。细胞凋亡率:33Glu组(4.85±1.17)(%)和50Glu组(10.56±3.40)(%)较5.5Glu组(2.38±0.75)(%)增加(P<0.05)。MDA含量:33Glu组(18.19±0.92 nmol/mg protein)和50Glu组(18.86±1.05 nmol/mg protein)较5.5Glu组(12.08±1.47 nmol/mg protein)升高(P<0.05)。SOD活力:33Glu组(22.26±4.27 U/mg protein)和50Glu组(14.30±4.04 U/mg protein)较5.5Glu组(43.48±15.83 U/mg protein)降低(P<0.05)。GSH含量:33Glu组(12.1±0.8μmol/g prot)和50Glu组(6.62±0.58μmol/g prot)较5.5Glu组(51.1±2.8μmol/g prot)降低(P<0.05)。Sel S蛋白表达:与5.5Glu组(0.027±0.0005)比较,25Glu组(0.05±0.005)和33Glu组(0.052±0.009)Sel S表达升高(P<0.05);与12.5Glu组(0.034±0.005)比较,25Glu组和33Glu组Sel S蛋白表达增高(P<0.05);与33Glu组比较,37.5Glu组和50Glu组Sel S蛋白表达降低(P<0.05)。糖尿病大鼠存在心肌病理改变及心脏重量指数增加。GSH含量比较如下:NC(10.41±0.87μmol/mg protein)>DM-SMMV(7.57±1.02μmol/mg protein)>DM(5.82±0.90μmol/mg protein)>DM-CMMV(5.56±0.87μmol/mg protein),组间差异显著(P<0.05)。SOD活力有如下趋势:NC(1.47±0.27 U/mg protein)>DM-SMMV(1.35±0.33 U/mg protein)>DM(1.25±0.33 U/mg protein)>DM-CMMV(1.22±0.31 U/mg protein),组间比较差异不显著(P>0.05)。MDA含量有如下趋势:NC(0.29±0.05 nmol/mg protein)<DM-SMMV(0.34±0.10nmol/mg protein)<DM(0.37±0.12 nmol/mg protein)<DM-CMMV(0.39±0.06nmol/mg protein),组间比较差异不显著(P>0.05)。结论:1.T2DM患者可以出现不同程度的舒张功能异常,且随着病程延长,舒张功能障碍加重并出现收缩功能障碍,因此,寻找DM心肌损伤的早期、有效的防治措施具有重要临床意义。2.高糖可诱导H9C2心肌细胞发生氧化应激损伤,且这种作用与Sel S有关。3.高表达的Sel S可抗氧化保护糖尿病大鼠抵抗氧化应激诱导的心肌损伤,提示调节Sel S表达可能是防治DCM的新途径。更多还原

【Abstract】 Background: The pathogenesis of diabetic cardiomyopathy(DCM)is complicated,including myocardial mitochondrial dysfunction,myocardial energy metabolism abnormality,microvascular disease,as well as insulin resistance.However,all the above pathophysiological processes associate with oxidative stress,indicating a central role of oxidative stress in DCM.Lack of clear clinical diagnostic criteria of DCM makes it difficult to diagnose DCM in the early stage and subsequently to prevent and treat.Thus,this study aims to compare the data between type 2 diabetes mellitus(T2DM)patients with or without myocardial injury,and to investigate the clinical indicators for myocardial injury induced by T2 DM,in order to provide clinical evidences for the early diagnosis and prevention of DCM.Selenoprotein S(Sel S),identified as glucose-regulated protein,plays roles in the regulation of oxidative stress,inflammation and endoplasmic reticulum stress.Sel S has been proved to protect vascular endothelial cells from oxidative stress induced injury,however,whether Sel S can exert anti-oxidative protective effect on diabetic myocardial injury is unclear.Thus,this study aims to study the association of Sel S with oxidative stress induced injury in H9C2 myocardial cells in vitro,and further analyze the effect of Sel S on oxidative stress induced myocardial injury in diabetic rats in vivo,to explore the potential of Sel S in preventing and treating DCM.Methods: This study included T2 DM patients with myocardial injury identified as illness group(IG,n=23),T2 DM patients with sex,age,diabetic duration,fasting plasma goucose matched with patients in IG group were assigned to the control group(CG,n=46).Patients in IG were subdivided into contraction dysfunction group with left ventricular ejection fraction(LVEF)<50%(CDG,n=10)and contraction normal group with LVEF≥50%(CNG,n=13).Clinical,laboratory and echocardiographic data were collected and analyzed.H9C2 cells were treated with DMEM media containing different concentration of glucose,including 5.5mmol/L glucose(5.5Glu),33mmol/L glucose(33Glu)and50mmol/L glucose(50Glu).After cells cultured for 24 hours,cell morphology were observed with inverted phase contrastmicroscope,cell apoptosis was analyzed through flow cytometry,the MDA content,SOD activity and GSH content were detected via colorimetric method,as well as the detection of Sel S expression through Western Blot.8-week old Sprague-Dawley(SD)rats were randomly divided into normal control group(NC)and high fat and high sugar diet group.After feeding with respective diet for one month,rats were injected intraperitoneally with STZ with the dose of 40mg/kg to induce diabetes mellitus(DM).Then the diabetic rats were randomly divided into three groups,including Sel S untransfected group(DM),Sel S transfected group(DM-SMMV),empty vector transfected group(DM-CMMV),followed by feeding with respective diet for another 19 weeks to construct diabetic myocardial injury rat model.After diabetic myocardial injury rat model were made,transfection was performed through the injection of ultrasound microbubble containing pc DNA3.1plasmid or pc DNA3.1-Sel S plasmid via tail vein,and through irradiating the precordial region via transfecting therapeutic apparatus simultaneously.At last,rats were killed and heart tissue were collected for HE staining,as well as the detection of MDA content,SOD activity and GSH content via colorimetric method and Sel S expression through Western Blot.Results: The E/A ratios were below the normal level at 1 in CG,CNG and CDG groups,and there was a significant difference in E/A ratio between CDG and CG(0.79±0.13 vs 0.92±0.10,P<0.05).The LVEF of CDG(36.40±7.01)(%)were significantly decreased in comparison with CNG(59.15±1.77)(%)and CG(59.59±3.75)(%)(P<0.05).The left ventricular fraction shortening(LVFS)of CDG(21.55±2.75)(%)were significantly decreased in comparison with CNG(33.58±0.89)(%)and CG(33.80±1.49)(%)(P<0.05).Whereas,the thickness of interventribular septal(IVS)was increased in CDG(11.60±1.78 mm)in comparison with CNG(9.92±1.32mm)and CG(9.52±1.64 mm)(P<0.05).Multivariate logistic regression showed that duration of diabetes(OR=1.339,95% CI 1.032-1.737,P=0.028)was an independent risk factor associated with systolic dysfunction caused by T2 DM.The triglyceride level was found to be an independent factor significantly associated with myocardial injury caused by T2DM(OR=1.012,95% CI 1.002–1.022,P=0.018).The LVEF was significantly correlated with the IVS thickness(r = –0.391,P = 0.001)and diabetic duration(r = –0.261,P = 0.030).Cell apoptosis was significantly increased in 33 Glu group(4.85±1.17)(%)and50Glu group(10.56±3.40)(%),compared with 5.5Glu group(2.38±0.75)(%)(P<0.05).MDA content in 33 Glu group(18.19±0.92 nmol/mg protein)and 50 Glu group(18.86±1.05 nmol/mg protein)was higher than in 5.5Glu group(12.08±1.47nmol/mg protein)(P<0.05).SOD activity in 33 Glu group(22.26±4.27 U/mg protein)and 50 Glu group(14.30±4.04 U/mg protein)was lower than in 5.5Glu group(43.48±15.83 U/mg protein)(P<0.05).GSH content in 33 Glu group(48.60±2.50 μmol/g prot)and 50 Glu group(12.10±0.80μmol/g prot)was significantly decreased compared with 5.5Glu group(51.10±2.80μmol/g prot)(P<0.05).Sel S protein level was increased in 25 Glu group(0.05±0.005)and 33 Glu group(0.052±0.009)compared with 5.5Glu group(0.02±0.000)(P<0.05);Sel S protein level was decreased in37.5Glu group(0.033±0.005)and 50 Glu group(0.03±0.003)compared with 33 Glu group(0.02±0.000)(P<0.05).GSH was decreased in DM(5.82±0.90 μmol/mg protein),DM-SMMV(7.57±1.02 μmol/mg protein)and DM-CMMV(5.56±0.87 μmol/mg protein)compared with NC(10.41 ± 0.87 μmol/mg protein)(P<0.05).And it was increased in DM-SMMV compared with DM and DM-CMMV.SOD activity showed the same tendency but with no statistical difference(P>0.05).MDA showed the inverse tendency but with no statistical difference(P>0.05).Conclusions: Patients with T2 DM may have diastolic dysfunction to some degree,and systolic dysfunction may occurred as the prolonged duration of diabetes.Thus,exploring effective strategy for the prevention and treatment of diabetic myocardial indury in the early stage may have an important clinical significance.High glucose could induce rat H9C2 cell oxidative injury in vitro,and this effect is associated with Sel S;Overexpression of Sel S could protect myocardium from oxidative stress induced injury in diabetic rats in vivo,which incates the modulation of Sel S expression may provide a new strategy for the prevention and treatment of DCM.更多还原