NLRP3炎症小体对糖尿病性心肌病的影响及其机制研究
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摘要
研究背景
糖尿病性心肌病(diabetic cardiomyopathy, DCM)是由糖尿病引起的,并以心肌重构和心功能下降为特征,最终导致心力衰竭的心肌病变。糖尿病状态下,高糖刺激活性氧(reactive oxygen species, ROS)生成增多被认为是DCM发生和发展的重要原因。过多的ROS刺激多种炎症因子表达升高,如核因子kB(nuclear factor kappa-light-chain-enhancer of activated B cells, NF-kB)、硫氧还蛋白相互作用蛋白(thioredoxin interacting/inhibiting protein, TXNIP)及炎症小体。既往研究表明,炎症小体与胰岛素抵抗、2型糖尿病及糖尿病并发症关系密切。但目前炎症小体在DCM中的作用及其调控机制尚未见报道。
炎症小体是一类分布于细胞胞浆中的蛋白复合体,它可以感受细胞内多种与免疫及死亡相关的信号刺激,参与细胞功能调控。目前已发现多个炎症小体家族成员,主要包括：NLRs (nucleotide binding oligomerization domain-like receptors)家族及AIM2(absent in melanoma2)家族。NLRP3(NOD-like receptor protein3)是NLRs家族中的一员。当接收到相关信号刺激后,NLRP3将与含CARD结构域的凋亡相关颗粒样蛋白(apoptosis-associated speck-like protein containing CARD, ASC)及含半胱氨酸的天冬氨酸蛋白水解酶1(cysteinyl aspartate specific proteinase-1, caspase-1)组成炎症复合体,促进caspase-1前体(pro-caspase-1)的自我剪切,形成具有活性的caspase-1p20/p10复合物,最终促进白介素1β (interleukin1β, IL-1β)前体蛋白pro-IL-1β水解成具有活性的IL-1p。既往研究表明,IL-1p在心肌细胞凋亡中具有重要作用。
NLRP3炎症小体的异常激活通常表现在两个方面：一方面,NLRP3表达上调；另一方面,NLRP3炎症小体形成复合物,促进caspase-1及IL-1p等效应分子的产生。在巨噬细胞中,NF-kB可促进NLRP3表达上调。在HEK293T细胞中,TXNIP可促进NLRP3炎症复合物的形成,最终促进caspase-1及IL-1β等效应分子的产生。但是在高糖刺激的心肌细胞中,NF-kB及TXNIP是否与NLRP3炎症小体的激活相关,目前尚未见报道。
近期研究显示,caspase-1除了可以促进IL-1β成熟外,还可以促进细胞程序性死亡,这一过程被称为“细胞焦亡(pyroptosis)"。细胞焦亡是一种与炎症相关,依赖于caspase-1的程序性死亡方式。焦亡的细胞同时具备细胞凋亡及细胞坏死的部分形态特征。这主要表现在：细胞焦亡存在DNA片段化损伤,并在脱氧核糖核苷酸末端转移酶介导的粘性末端标记检测(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, TUNEL)中呈现阳性；细胞焦亡时胞膜完整性消失,胞内炎症物质会通过膜上的孔隙释放到细胞间质,胞外物质会从孔隙渗入胞浆,促进细胞器及细胞浆肿胀,最终导致细胞破裂。因此,不能通过完整细胞膜而只能通过形成孔隙的胞膜的染料可将活细胞与焦亡细胞区分开,如EthD-Ⅲ染料,它可将焦亡细胞染成阳性,但不能将正常细胞染色。焦亡现象最初是在巨噬细胞及树突细胞等髓系细胞受到病原体刺激后观察到的。近期研究显示,一些非病原体也可刺激非髓系细胞出现焦亡现象。但是,高糖刺激的心肌细胞是否发生焦亡,目前未见报道。
在糖尿病小鼠及大鼠模型中,心肌组织的电镜结果显示大量心肌细胞呈现与焦亡细胞类似的形态特征,如线粒体肿胀空泡化、肌纤维紊乱溶解、细胞肿胀、及细胞膜模糊不清等。此外,我们在预实验中发现细胞焦亡关键因子caspase-1在DCM大鼠模型心肌组织中表达升高。据此,我们提出假设：糖尿病状态下,心肌细胞ROS生成增多。ROS通过NF-kB及TXNIP促进NLRP3炎症小体过度激活。NLRP3炎症小体的异常激活通过促进心肌间质炎症反应、心肌细胞焦亡及心肌纤维化等途径导致心脏结构和功能异常。
研究目的
(1)建立2型DCM模型,探究NLRP3炎症小体在DCM病程不同阶段的表达水平；
(2)利用NLRP3-miRNA'慢病毒对DCM大鼠进行体内干预,探究NLRP3炎症小体在DCM中的作用；
(3)探究NLRP3炎症小体影响DCM发生发展的相关机制。
研究方法
2型糖尿病大鼠模型
将120只5周龄雄性Sprague-Dawley (SD)大鼠随机分为4组(每组30只)：对照组(control组)、糖尿病组(DM组)、糖尿病+空载体慢病毒组(DM+vehicle组)及糖尿病+NLRP3-miRNA慢病毒组(DM+NLRP3-miRNA组)。Control组给予基础饮食,主要成分为：5%脂质,不添加胆固醇。其余3组给予高脂饮食喂养,主要成分为：16%脂质及0.25%胆固醇。4周后4组大鼠均行腹腔葡萄糖耐量实验(Intraperitoneal glucose tolerance test, IPGTT)及腹腔胰岛素耐量实验(Intraperitoneal insulin tolerance test, IPITT)。DM组、DM+vehicle组及DM+NLRP3-miRNA组出现胰岛素抵抗的大鼠接受单次腹腔STZ注射,剂量为35mg/kgo1周后,检测4组大鼠的空腹血糖(BG)及胰岛素(insulin, INS),计算胰岛素敏感指数(ISI), ISI=In[(INS×BG)"1]。DM组、DM+vehicle组及DM+NLRP3-miRNA组大鼠连续两次空腹血糖≥11.1mmol/L,且存在胰岛素抵抗则认为2型糖尿病造模成功。
糖尿病性心肌病模型
糖尿病大鼠造模成功后,继续给予高脂喂养。至STZ注射后8周,检测大鼠心功能,糖尿病大鼠出现左室舒张功能障碍则提示开始进展为DCM。至STZ注射后16周,检测大鼠心脏结构及功能,糖尿病大鼠心脏组织呈现心肌间质纤维化及左室肥厚的,并出现左室收缩及舒张功能障碍,则提示DCM模型已经成功建立。
基因沉默技术
根据RNAi干扰技术的原则,设计4对针对大鼠NLRP3基因的miRNA片段,并分别构建4对pcDNA6.2-GW/EmGFP-NLRP3-miRNA质粒,经筛选后,选择沉默效率最高的质粒,随后构建pDONR221-EmGFP-NLRP3-miRNA重组穿梭质粒,最终构建pLenti6.3—EmGFP-NLRP3-miRNA慢病毒载体。
慢病毒干预
在STZ腹腔注射8周后,分别给予DM+vehicle组及DM+NLRP3-miRNA组慢病毒空载体(vehicle)或NLRP3-miRNA慢病毒(NLRP3-miRNA)干预8周。病毒干预8周后,对DM+vehicle组及DM+NLRP3-miRNA组全部大鼠实施安乐死,随机抽取部分control及DM组大鼠(每组10只)实施安乐死。立即心脏取材,行冰冻切片检测心肌病毒转染效率。
NLRP3炎症小体表达趋势
Control组及DM组大鼠,分别在STZ注射后的第0、4、8、12、16及20周时取材,每组取3只,然后进行相关指标的检测。
血清学检测
待实验结束时,大鼠饥饿过夜,抽取外周静脉血,分别检测血清中血糖(blood glucose)、甘油三酯(triglyceride, TG)及总胆固醇(total cholesterol,TC)含量。
酶联免疫吸附试验(Enzyme Linked Immunosorbent Assay, ELISA)
利用ELISA测定大鼠外周血血清中胰岛素的水平。
心脏彩超
大鼠经10%水合氯醛麻醉后,行经胸心脏彩超,收集LVEDd、LVEF、F/S、 E/A及E’/A’等数据评价大鼠左室功能。
组织学检测
大鼠实施安乐死后,立即心脏取材,随后称重、测量心脏大小并留取图像。于乳头肌水平横切心脏,制成石蜡切片,于乳头肌平面连续切片,行苏木精-伊红(hematoxylin-eosinstaining, H&E)染色后留取图像。
心肌纤维化
对左室心肌组织行Masson染色、天狼猩红染色,使用图像分析软件对染色结果进行分析,评价左室心肌纤维化程度。
透谢电镜
大鼠实施安乐死后,立即取出心脏,于左室壁取1mm3小组织块,立即放入戊二醛固定液中,为透射电镜下观察左室心肌超微结构做准备。
免疫组织化学
利用免疫组织化学的方法,观察caspase-1及IL-1β在组织中的分布。
实时定量RT-PCR
取新鲜左室心肌组织,利用实时定量RT-PCR的方法检测大鼠左室心肌中NLRP3, ASC, caspase-1及IL-1βmRNA表达水平。
Western blot
取新鲜左室心肌组织,利用Western blot方法检测大鼠左室心肌中TXNIP、 NF-kB、NLRP3、ASC、pro-caspase-1、 caspase-1及IL-1β的表达。
TUNEL检测
利用TUNEL检测大鼠左室组织中,心肌细胞核出现DNA损伤的情况。
细胞培养及干预
培养大鼠H9c2心肌细胞系,分为3组：基础糖培养组(control组)、中等浓度糖刺激组(median glucose, MG组)及高浓度糖刺激组(high glucose, HG组)。在进行干预前,利用无血清基础糖培养基进行饥饿处理。随后给予含不同浓度葡萄糖的培养基进行不同时间梯度的干预。在ROS抑制实验中,我们选择N-乙酰半胱氨酸(N-acetylcysteine, NAC)作为抑制剂对心肌细胞进行预处理。
心肌细胞病毒转染
利用vehicle或NLRP3-miRNA慢病毒对心肌细胞进行干预,以探究NLRP3在心肌细胞中的作用及调控机制。
免疫荧光
利用免疫荧光技术观察caspase-1在心肌细胞中的分布。
Caspase-1活性检测
利用caspase-1活性检测试剂盒检测高糖刺激下caspase-1的活性。
ROS检测
利用DCFH-DA检测高糖刺激下心肌细胞ROS的产生。细胞焦亡检测
利用TUNEL检测心肌细胞核DNA损伤,利用EthD-III染色监测心肌细胞胞膜损伤,利用calcein-AM检测细胞活性。
数据分析
利用SPSS v18.0处理数据,连续变量以均数±标准误来表示。利用单因素方差分析(one-way ANOVA)和Tukey-Kramer post hoc检验比较多组间的连续变量。利用独立样本t检验比较两组间的连续变量。当p<0.05时认为有统计学意义。
研究结果高脂饮食4周可诱导大鼠出现胰岛素抵抗
IPGTT结果显示,与基础饮食相比,高脂喂养大鼠4周后,在IPGTT的Omin、30min、60min、120min时血糖值均明显升高(p<0.05～p<0.01)；血糖曲线下面积(AUC)明显增大(p<0.01)。IPITT实验也呈现类似结果(p<0.05～p<0.01)。
NLRP3炎症小体及IL-1β在糖尿病性心肌病中表达增高
与对照组相比,糖尿病大鼠在STZ注射后第4或第8周起即出现NLRP3、 ASC、caspase-1及IL-1β表达升高(p<0.01)。此外,NLRP3炎症小体各组分及IL-1β的mRNA水平多在STZ注射后第8周时达到顶峰,并持续高表达至第20周(p<0.01)。与control组相比,NLRP3、ASC、pro-caspase-1、活化的caspase-1(caspase-1p20)及活化的IL-1β (IL-1β p17)在DM组中表达升高,且多在STZ注射后第8周达到顶峰,并持续高表达至第20周(p<0.01)。
NLRP3基因沉默不能改善糖尿病引起的代谢异常
在DM组大鼠中,血糖、血脂及胰岛素水平均显著高于control组(p<0.01),胰岛素敏感指数显著低于control组(p<0.01)。利用NLRP3-miRNA进行体内干预后,并不能改善糖尿病引起的代谢异常。基因干预能有效抑制心肌组织NLRP3表达
与vehicle干预组相比,NLRP3-miRNA干预后,心肌组织中NLRP3的mRNA及蛋白水平明显降低(p<0.01)。此外,心肌组织中caspase-1和IL-1β的表达也明显降低(p<0.01)。免疫组化结果显示,在糖尿病大鼠的心肌组织中,caspase-1主要分布在细胞核周围,IL-1β主要呈弥散分布(p<0.01)。NLRP3基因沉默可改善DCM左室功能异常
与control组比较,DM组的LVEF、FS、E/A及E’/A’均降低(p<0.01),LVEDd增大(p<0.01)。将NLRP3基因沉默后,糖尿病引起的LVEF、FS、E/A及E’/A’降低均明显增加(p<0.05-p<0.01), LVED明显降低(p<0.01)。
NLRP3基因沉默改善DCM心肌重构
与control组比较,DM组心肌重构的特点是：心肌细胞DNA损伤增多,心肌细胞超微结构受损,1型及3型胶原表达增加,1型及3型胶原比例增高,间质纤维化增多,心脏向心性肥厚,心身比增大等。
与vehicle组相比,NLRP3基因沉默可明显改善DCM心肌重构,主要表现为：心肌细胞DNA损伤减少,1型及3型胶原表达降低,1型及3型胶原比例降低,间质纤维化减少,身心比降低等。
高糖刺激心肌细胞NLRP3炎症小体及L-1β表达增高
与control组相比,MG或HG刺激24至48小时,可引起H9c2心肌细胞NLRP3, ASC, caspase-1及IL-1β的mRNA表达升高,且此升高呈现糖浓度依赖性(p<0.01)。MG或HG刺激心肌细胞24至48小时,NLRP3炎症小体各组分及IL-1βp17的表达较control组均升高(p<0.01)。HG刺激时,NLRP3、ASC及IL-1β p17在36小时表达量最高,并持续到48小时(p<0.05)。据此,在后续的实验中,我们选择HG为刺激条件,36小时为刺激时间。与基础培养基相比,与MG、HG渗透压匹配的甘露醇并不能引起NLRP3炎症小体及IL-1p表达升高。
高糖刺激诱导H9c2细胞caspase-1表达升高及细胞焦亡
与control及MG组比较,HG组中的caspase-1p20表达明显增高(p<0.05～p<0.01)。免疫荧光结果显示,MG及HG可促进caspase-1在心肌细胞胞质中聚集,同时我们还发现,与control组相比,心肌细胞核DNA的损伤及心肌细胞膜的破坏随着caspase-1表达升高而加重(p<0.01)。
NLRP3在高糖引起的细胞焦亡中发挥重要的作用
我们利用NLRP3-miRNA抑制心肌细胞内NLRP3的表达,探究NLRP3在心肌焦亡中的作用。在确保病毒转染效率达80%以上的前提下,NLRP3-miRNA能够有效抑制靶基因mRNA及蛋白的表达(p<0.01)。随着NLRP3蛋白的抑制,高糖刺激下caspase-1及IL-1β的活性也明显受到抑制(p<0.05-p<0.01)。与此同时,高糖刺激下心肌细胞核DNA损伤及细胞膜受损较之前有明显的改善(p<0.05)。
NF-kB及TXNIP在ROS刺激NLRP3激活中发挥重要作用
与control组相比,MG及HG组H9c2细胞ROS生成增多(p<0.01)。同时,MG及HG组NF-kB p65磷酸化及TXNIP表达较control组增多(p<0.01)。利用NAC预处理细胞能够明显减少细胞内ROS产生(p<0.01)。随着ROS的生成减少, NF-kB p65磷酸化及TXNIP表达减少,同时NLRP3、ASC、pro-caspase-1、caspase p20及IL-1βp17的表达降低(p<0.01)。研究结论
(1)糖尿病状态下,心肌细胞焦亡及心肌纤维化促进心脏结构和功能异常；
(2)在糖尿病早期,心肌组织NLRP3炎症小体即表达升高；
(3)利用NLRP3-miRNA慢病毒实施体内干预后,心肌组织NLRP3的表达被有效抑制；
(4)抑制心肌组织NLRP3表达后,糖尿病引起的心肌结构和功能的异常明显改善；
(5)高糖刺激可诱导心肌细胞ROS生成增多,ROS可促进NLRP3炎症小体表达升高,增高的caspase-1可促进心肌细胞出现细胞焦亡的特征；
(6) NF-kB及TXNIP可能介导了ROS促进NLRP3炎症小体表达增多。
研究背景
糖尿病性心肌病(diabetic cardiomyopathy, DCM)是以舒张功能显著受损和收缩功能轻度下降为特征的心肌病变,是糖尿病病人的主要死亡原因之一。既往研究表明,炎症反应在DCM中发挥着重要作用。因此,对DCM中炎症反应的相关调控机制进行深入研究并寻找相关治疗策略具有重要意义。
促炎因子白介素1β (interleukin-1β, IL-1β)在DCM的发病过程中起到非常重要的作用。既往研究表明,IL-1β的激活主要受炎症小体(inflammasome)调控。炎症小体是位于细胞内的一种多聚蛋白复合体,其家族成员包括：NLRs (nucleotide binding oligomerization domain-like receptors)家族及AIM2(absent in melanoma2)家族。NLRP3(NOD like receptor protein3, NLRP3)是NLRs亚组中的一员。NLRP3炎症小体由NLRP3,含CARD结构域的凋亡相关颗粒样蛋白(apoptosis-associated speck-like protein containing CARD, ASC)和含半胱氨酸的天冬氨酸蛋白水解酶(?)(cysteinyl aspartate specific proteinase-1, caspase-1)组成。近来有研究表明,NLRP3炎症小体在糖尿病、糖尿病肾病及糖尿病视网膜病变等疾病的炎症反应中起到非常重要的作用。此外,在高糖诱导的氧化应激反应中,NLRP3炎症小体通过与硫氧还蛋白相互作用蛋白(thioredoxin interacting/inhibiting protein, TXNIP)直接结合而被激活,继而促发下游的炎症反应。我们前期的实验结果表明,NLRP3炎症小体通过促进炎症反应、心肌损伤及心肌纤维化而加速DCM的发生和发展。既往研究表明丝裂原活化蛋白激酶(mitogen-activatedproteinkinases, MAPKs)在DCM的炎症反应中发挥着重要作用。
瑞舒伐他汀是一种HMG-CoA还原酶抑制剂。研究显示,瑞舒伐他汀除了具有调脂作用外,还具有抗炎、抗氧化及逆转心肌重构的作用。在心肌肥厚、心肌梗死及实验性自身免疫性心肌炎的动物模型中,瑞舒伐他汀的心脏保护作用已经得到证实。但是,具有抗炎作用的瑞舒伐他汀能否影响DCM中异常表达的NLRP3炎症小体及MAPKs通路,目前尚未见报道。瑞舒伐他汀能否通过NLRP3炎症小体及MAPKs通路改善心肌重构,目前尚未见报道。
据此,本课题拟构建DCM大鼠模型,并研究瑞舒伐他汀在DCM发生、发展中的作用,探讨瑞舒伐他汀是否通过调控NLRP3炎症小体及MAPKs通路发挥心肌保护作用,进一步研究NLRP3炎症小体在DCM中潜在临床价值。
研究目的
(1)建立DCM大鼠模型,探讨RSV在DCM进展中的作用；
(2)探讨NLRP3炎症小体及MAPKs通路在RSV对DCM大鼠心肌保护过程中的作用；
(3)探讨RSV抑制NLRP3炎症小体的可能机制。
研究方法
2型糖尿病大鼠模型
选择105只健康雄性Sprague-Dawley (SD)大鼠(山东省中医药大学实验动物中心),平均体重为100-120g。将大鼠随机分为7组：对照组(control组),高脂饮食组(HF组),糖尿病组(DM组),高脂饮食+瑞舒伐他汀10mg/kg组(HF+RSV10mg/kg组),高脂饮食+瑞舒伐他汀15mg/kg组(HF+RSV15mg/kg组),糖尿病+瑞舒伐他汀10mg/kg组(DM+RSV10mg/kg组),糖尿病+瑞舒伐他汀15mg/kg组(DM+RSV15mg/kg组)。Control组给予基础饲料喂养,主要成分为：5%脂质,不添加胆固醇。其余6组给予高脂饮食喂养,主要成分为：16%脂质,0.25%胆固醇。4周后7组大鼠均行腹腔葡萄糖耐量实验(IPGTT)及腹腔胰岛素耐量实验(IPITT)。DM组、DM+RSV10mg/kg组及DM+RSV15mg/kg组出现胰岛素抵抗的大鼠接受单次腹腔STZ注射,剂量为35mg/kg。1周后,检测DM组、DM+RSV lOmg/kg组及DM+RSV15mg/kg组大鼠的空腹血糖(BG)及胰岛素(INS),计算胰岛素敏感指数(ISI), ISI=In[INS×BG)-1]连续两次空腹血糖≥11.1mmol/L,且存在胰岛素抵抗则认为2型糖尿病造模成功。在行STZ腹腔注射8周后,HF+RSV10mg/kg组、HF+RSV15mg/kg、DM+RSV10mg/kg组及DM+RSV15mg/kg分别给予瑞舒伐他汀干预8周。瑞舒伐他汀干预8周后,7组大鼠均实施安乐死。
糖尿病性心肌病模型
糖尿病大鼠造模成功后,继续给予高脂喂养。至STZ注射后8周,检测大鼠心功能,糖尿病大鼠出现左室舒张功能障碍则提示开始进展为DCM。至STZ注射后16周,检测大鼠心脏结构及功能,糖尿病大鼠心脏组织呈现心肌间质纤维化及左室肥厚的,并出现左室收缩及舒张功能障碍,则提示DCM模型已经成功建立。基因沉默技术
根据RNAi干扰技术的原则,设计4对针对大鼠NLRP3基因的miRNA片段,并分别构建4对pcDNA6.2-GW/EmGFP-NLRP3-miRNA质粒,经筛选后,选择沉默效率最高的质粒,随后构建pDONR221-EmGFP-NLRP3-miRNA重组穿梭质粒,最终构建pLenti6.3-EmGFP-NLRP3-miRNA慢病毒载体。
大鼠体内转染慢病毒载体
选择90只健康雄性SD大鼠(山东省中医药大学实验动物中心),平均体重为100-120g。将大鼠随机分为6组：对照组+空载体组(control+vehicle组),糖尿病组+空载体组(DM+vehicle组),糖尿病+瑞舒伐他汀15mg/kg+空载体组(DM+RSV15mg/kg+vehicle组),对照组+NLRP3-miRNA慢病毒干扰组(control+NLRP3-miRNA组),糖尿病组+NLRP3-miRNA慢病毒干扰组(DM+NLRP3-miRNA组),糖尿病+瑞舒伐他汀15mg/kg+NLRP3-miRNA慢病毒干扰组(DM+RSV15mg/kg+NLRP3-miRNA组)。于STZ注射8周后,分别给予6组大鼠vehicle或NLRP3-miRNA慢病毒行体内干预,同时还需给予DM+RSV15mg/kg+vehicle组及DM+RSV15mg/kg+NLRP3-miRNA组大鼠瑞舒伐他汀干预,慢病毒及瑞舒伐他汀的干预时间均为8周,慢病毒注射剂量为1×108TU/只。干预8周后给予大鼠安乐死,心脏取材后行冰冻切片,检测心肌内病毒转染效率。
血清学指标检测
于实验结束时,行血清学检测。大鼠禁食禁水12小时后,取外周静脉血。检测血糖(blood glucose, BG)、血清甘油三酯(triglyceride, TG)、总胆固醇(total cholesterol, TC)、胰岛素(INS),并计算胰岛素敏感指数(ISI)。
心脏彩超
大鼠经10%水合氯醛麻醉后,行经胸心脏彩超,收集LVEF、F/S、E/A以及E’/A’等数据评价大鼠左室功能。
心肌纤维化
对左室心肌组织行Masson染色、天狼猩红染色,使用图像分析软件对染色结果进行分析,评价左室心肌纤维化程度。
透谢电镜
大鼠行安乐死后,立即心脏取材,于左室壁上取1mm3小组织块,立即放入戊二醛固定液中,为透射电镜下观察左室心肌超微结构做准备。
实时定量PCR
取新鲜左室心肌组织,通过实时定量荧光PCR技术检测TXNIP、NLRP3、 ASC、caspase-1、IL-1β、collagen I及collagen III的mRNA相对表达量。Western blot检测
取新鲜左室心肌组织,提取蛋白,利用Western blot检测心肌组织内TXNIIP、 NLRP3、ASC、活化与非活化的caspase-1、活化的IL-1β及磷酸化和非磷酸化的ERK1/2、p38. JNK的蛋白表达量。
统计学分析
利用SPSS v18.0处理数据,连续变量以均数±标准误来表示。利用单因素方差分析和Tukey-Kramer post hoc检验比较多组间的连续型变量。利用独立样本t检验比较两组间的连续变量。当p<0.05时认为有统计学意义。
研究结果
高脂饮食4周可诱导大鼠出现胰岛素抵抗
IPGTT结果显示,与基础饮食相比,高脂喂养大鼠4周后,在IPGTT的0min、30min、60min、120min的血糖值均明显升高(p<0.05～p<0.01)；血糖曲线下面积(AUC)明显增大(p<0.01)。IPITT的结果显示,与基础饮食相比,高脂喂养大鼠4周后,在IPITT的Omin、30min、60min、120min的血糖值均明显升高(p<0.05～p<0.01)；血糖曲线下面积(AUC)明显增大(p<0.01)。
瑞舒伐他汀对糖尿病大鼠血糖及胰岛素水平无明显影响
与Control及HF组大鼠相比,DM组大鼠表现出高血糖、高血脂、低胰岛素水平及胰岛素敏感性指数下降等特点(p<0.01)。与Control组比较,HF也存在代谢异常的情况(p<0.05～p<0.01)。
与未治疗组相比,10mg/kg及15mg/kg瑞舒伐他汀均能降低DM及HF组的胆固醇水平(p<0.05),且15mg/kg瑞舒伐他汀降脂效果优于10mg/kg (p<0.05)。与未用药组相比,10mg/kg或15mg/kg的瑞舒伐他汀均未能逆转DM组及HF组出现的血糖、甘油三酯及胰岛素异常。
瑞舒伐他汀改善糖尿病引起的左室功能异常
DM组的LVEF、FS、E/A及E’/A’均低于Control组,HF组的E/A及E’/A’均低于Control组(p<0.05～p<0.01)。
与未治疗组相比,15mg/kg瑞舒伐他汀可以提高DM组的LVEF、E/A及E’/A’(p<0.05)。10mg/kg瑞舒伐他汀可提高DM组的E'/A'(p<0.05)。
瑞舒伐他汀改善糖尿病引起的心肌重构
与Control组相比,DM组及HF组的心身比(heart weight to body weight, HW/BW)均增加(p<0.05～p<0.01)。糖尿病诱导心肌间质纤维化,促进胶原I及胶原Ⅲ的(?)nRNA表达增加,促进胶原Ⅰ/Ⅲ的增大。与Control组相比,HF组也呈现心肌纤维化增多、胶原(?)nRNA表达增加的现象(p<0.05-p<0.01)。Control组大鼠左室心肌组织透射电镜结果显示：心肌纤维成对称性分布,Z线有序排列在肌节周围,线粒体规则分布在肌纤维附近。DM组电镜结果显示：心肌超微结构混乱无序,部分区域心肌纤维溶解,Z线退化,线粒体肿胀并出现空泡化,线粒体聚集、融合、内部线粒体嵴混乱。HF组电镜结果显示左室心肌组织超微结构亦出现破坏,但破坏程度较DM组轻。
与未治疗组相比,15mg/kg瑞舒伐他汀可降低DM组的HW/BW、左室纤维化面积、胶原Ⅰ/Ⅲ的表达量及比例的异常(p<0.01)。10mg/kg瑞舒伐他汀仅能改善心肌纤维化及胶原Ⅰ的表达异常(p<0.05～p<0.01)。但与未治疗组相比,瑞舒伐他汀未能有效改善HF组HW/BW及胶原Ⅰ及Ⅲ的表达量及比例异常。电镜结果显示,经瑞舒伐他汀干预后,DM组及HF组的左心室心肌组织超微结构较未治疗组有明显改善。
瑞舒伐他汀抑制炎症小体激活
我们检测不同组别大鼠左室心肌组织中TXNIP、NLRP3、ASC、未活化及活化的caspase-1(pro-caspase-1, caspase-1p20)及IL-1β (IL-1β, IL-1β p17)的mRNA及蛋白的表达水平。DM组中TXNIP、NLRP3、ASC、caspase-1及IL-1p的mRNA表达水平均高于control组(p<0.01)。DM组TXNIP、NLRP3、ASC、pro-caspase-1、 caspase-1p20及IL-1β p17蛋白表达水平均高于control组(p<0.05～p<0.01)。与control组相比,HF组的TXNIP、NLRP3炎症小体及IL-1β的mRNA及蛋白表达量明显增高(p<0.05～p<0.01)。
与未治疗组比较,15mg/kg瑞舒伐他汀可降低糖尿病引起的TXNIP、NLRP3炎症小体及IL-1β的mRNA表达(p<0.05-p<0.01)。10mg/kg瑞舒伐他汀也可明显降低TXNIP及IL-1β的mRNA表达水平。与未治疗组比较,瑞舒伐他汀降低了HF组TXNIP、NLRP3、ASC及IL-1p的mRNA表达水平(p<0.05～p<0.01)。与未治疗组相比,15mg/kg瑞舒伐他汀降低糖尿病引起的TXNIP.NLRP3炎症小体、ⅠL-1βp17蛋白表达升高(p<0.05-p<0.01).10mg/kg瑞舒伐他汀只降低糖尿病引起的pro-caspase-1蛋白表达升高(p<0.05)。此外,15mg/kg瑞舒伐他汀的对TXNIP、 ASC、pro-caspase-1、caspase-1p20及IL-1β p17蛋白表达的抑制效果优于10mg/kg(p<0.01)。对HF组进行相同干预,观察到类似结果。
瑞舒伐他汀抑制MAPK信号通路
我们检测不同组别大鼠左室心肌组织中磷酸化及未磷酸化的ERK1/2, p38及JNK的表达水平。与对照组相比,DM及HF组中磷酸化的ERK1/2, p38及JNK表达升高(p<0.05-p<0.01)。与未治疗组比较,10mg/kg及15mg/kg均能降低DM组磷酸化的ERK1/2,p38及JNK(p<0.05-p<0.01)。此外,15mg/kg瑞舒伐他汀抑制磷酸化的ERK1/2的作用优于10mg/kg瑞舒伐他汀。在瑞舒伐他汀干预的HF组中也发现了类似结果(p<0.05～p<0.01)。
检测体内传染后NLRP3沉默效率
行NLRP3-miRNA慢病毒体内转染8周后,检测心肌组织转染效率。与转染空载体组相比,转染NLRP3-miRNA'慢病毒组可明显抑制大鼠心肌组织中NLRP3的mRNA及蛋白水平的表达。此外,随着NLRP3表达的降低,IL-1β p17的表达也降低(p<0.05～p<0.01)。
瑞舒伐他汀通过抑制NLRP3改善糖尿病引起的心功能损害
进行8周的病毒干预后,与空载体组相比,NLRP3-miRNA干预组能显著改善糖尿病引起的LVEF、FS、E/A及E’/A’降低(p<0.05)。此外,与空载体组相比,NLRP3-miRNA干预组能显著提高DM+RSV15mg/kg组的E/A及E'/A'(p<0.05)。
在空载体转染的糖尿病大鼠中,给予瑞舒伐他汀干预能明显改善心脏的收缩及舒张功能(p<0.05)。但与空载体组相比,NLRP3-miRNA干预组中瑞舒伐他汀的这种心脏保护作用并不明显。
瑞舒伐他汀通过抑制NLRP3减轻心肌损伤
与空载体组相比,NLRP3-miRNA可以降低DM组的心身比、间质纤维化、胶原Ⅰ及Ⅲ的(?)nRNA表达及比例(p<0.05-p<0.01)。NLRP3-miRNA还能显著改善心肌超微结构的损伤,包括逆转降解的Z线、紊乱的心肌纤维、肿胀的线粒体及积聚的脂肪微粒。与空载体联合15mg/kg瑞舒伐他汀相比,NLRP3-miRNA联合15mg/kg瑞舒伐他汀能更显著地改善糖尿病引起的心脏结构和功能的改变(p<0.05～p<0.01)。
瑞舒伐他汀的心肌保护作用在空载体干预组中非常明显,但这种保护作用在NLRP3-miRNA干预组中并不明显。
研究结论
(1) NLRP3炎症小体介导了瑞舒伐他汀对DCM的心脏保护作用；MAPKs通路可能参与了瑞舒伐他汀对DCM的心脏保护作用；
(2)长期给予瑞舒伐他汀干预可剂量依赖性地改善糖尿病性心肌病左室重构及收缩和舒张功能障碍；
(3)瑞舒伐他汀的心肌保护作用包括：抑制心肌炎症反应；减轻心肌细胞的肌纤维紊乱、Z线降解、线粒体肿胀融合及脂质沉积等超微结构的破坏；抑制胶原纤维的产生及沉积；
(4) TXNIP可能介导了瑞舒伐他汀抑制NLRP3炎症小体的作用。
研究背景
原发性扩张型心肌病(idiopathic dilated cardiomyopathy, IDCM)是一种病因不明,以心肌受累为基础,以心室扩张及收缩功能受损为特点的疾病。IDCM是继冠状动脉疾病及高血压疾病之后,引起心功能衰竭的第三大常见疾病。临床资料显示, IDCM患者通常需要反复住院治疗,部分患者预后不良。流行病学资料显示,IDCM的治疗已经成为公共卫生医疗系统的负担。因此,寻找与IDCM预后相关、并能提供干预预警信号及临床治疗靶点的因子,已经成为目前研究的热点。
虽然IDCM的病因不明,但研究显示病毒感染、炎症、自身免疫异常及基因突变均能促进IDCM的发生及发展。其中,炎症反应在IDCM的病理生理过程中发挥重要作用。白介素1β (interleukin-1β, IL-1β)是一种重要的促炎因子,其在多种心血管疾病中均表达上调。研究显示,IL-1β的成熟主要受炎症小体调控。炎症小体是一种蛋白复合物,主要位于细胞质内,其家族成员主要包括NLRs (nucleotide binding oligomerization domain-like receptors)家族及AIM2(absent in melanoma2)家族。NLRP3(NOD like receptor protein3)是NLRs亚组中的一员。NLRP3炎症小体由NLRP3、含CARD结构域的凋亡相关颗粒样蛋白(apoptosis-associated speck-like protein containing CARD, ASC)及含半胱氨酸的天冬氨酸蛋白水解酶1(cysteinyl aspartate specific proteinase-1, caspase-1)组成。当NLRP3炎症小体在相关信号的刺激下形成复合物之后,pro-caspase-1将会自我剪切,最终具有活性的caspase-1p10/p20蛋白,活化的caspase-1又可促进IL-1β从非活性的前体pro-IL-1β转化成具有活性的IL-1β。
外周血单核淋巴细胞(peripheral blood mononuclear cells, PBMCs)是血液循环中NLRP3炎症小体的主要来源。在系统性红斑狼疮、风湿性关节炎及系统性硬化等疾病中,PBMCs中NLRP3炎症小体的异常表达在疾病病程中起到重要的作用。近期的研究显示,NLRP3炎症小体在心肌缺血再灌注、急性心肌梗死及心衰等动物模型中表达上调,并促进心肌炎症损伤及纤维化,最终导致心功能异常。我们的前期结果表明,NLRP3炎症小体在糖尿病性心肌病中发挥重要作用。这些研究提NLRP3炎症小体的异常激活与心血管疾病密切相关,PBMCs中NLRP3炎症小体的异常激活在疾病的发展过程中具有重大意义。但目前,IDCM患者PBMCs中NLRP3炎症小体的表达尚未见报道。
据此,本实验将检测IDCM患者PBMCs中NLRP3炎症小体的表达水平,并探究IDCM患者体内NLRP3炎症小体表达水平与患者预后的相关性。
研究目的
(1)探究NLRP3炎症小体在IDCM患者PBMCs中的表达水平；
(2)探究NLRP3炎症小体与IDCM患者临床指标的相关性；
(3)探究NLRP3炎症小体在IDCM患者预后评估中的价值。
研究方法
研究对象
连续收录就诊于山东大学齐鲁医院的IDCM患者54人(IDCM组),收录健康志愿者20人(Control组)。IDCM患者同时患有以下疾病者不能纳入实验：活动期的心肌炎、冠脉疾病、心脏瓣膜病、高血压、急性或慢性炎症性疾病、糖尿病、甲状腺功能异常、痛风及类风湿性关节炎。在患者入院及健康志愿者入组时开始收集临床资料,主要包括：年龄、性别、血压、12导联心电图(12-lead electrocardiography, ECG)、心脏彩超检查结果、纽约心功能分级(New York Heart Association, NYHA)、血常规检查结果、血清N末端B型脑钠肽(N-terminal pro-brain natriuretic peptide, NT-pro BNP)检查结果等。IDCM患者住院期间均得到规范治疗。本实验设计已通过山东大学齐鲁医院伦理委员会审核。
PBMCs分离和纯化
收集研究对象的外周血,并分离出血清及PBMCs,分别存于-80℃备用。实时定量PCR
利用实时定量方法检测研究对象PBMCs中NLRP3、ASC、caspase-1及IL-1β的mRNA表达水平。
流式细胞术
分别利用固定剂和打孔剂处理PBMCs,随后分别使用NLRP3特异性一抗及带有FITC的二抗孵育PBMCs,处理完毕后利用流式细胞术检测PBMCs胞浆中NLRP3的蛋白表达水平。
Western blot
从研究对象PBMCs中提取蛋白,利用western blot方法检测ASC、pro-caspase-1及活化的caspase-1的蛋白表达。
酶联免疫反应(Enzyme-linked immunosobent assay, ELISA)
利用ELISA检测研究对象外周血血清中IL-1β的含量。随访
IDCM患者从山东大学齐鲁医院出院后,将患者因心功能恶化而再入院定为终点事件,对患者进行为期6个月的随访,并记录IDCM患者的再入院的时间。
统计分析
SPSS18.0用于统计分析PASS用于效能计算。连续变量以均数±标准差的形式表示,分类变量以百分数形式表示。t检验用于两组连续变量间统计分析。方差分析用于多组连续变量间统计分析。卡方检验用于分类变量统计分析。偏相关分析用于目标因子与临床指标相关性的统计分析。Cox回归分析用于IDCM患者6个月内再入院的影响因素的统计分析。Kaplan-Meier法用于2组IDCM患者6个月再入院率的统计分析,并绘制时间-效应曲线Log-rank检验用于比较2组IDCM患者6个月再入院率的比较。双侧p<0.05时认为有统计学意义。
研究结果
研究对象基本信息
IDCM组及Control组的临床资料如表1所示。在所研究的参数中,仅左室射血分数(left ventricular ejection farction, LVEF)(p<0.001)、NT-proBNP(p<0.001)及外周血单核细胞计数(p=0.008)在IDCM组及control组中存在统计学差异。
NLRP3炎症小体及IL-1β在IDCM患者PBMCs中表达上调
入院24小时内,在未进行治疗干预前,IDCM组PBMCs中NLRP3炎症小体及IL-1β mRNA表达水平较control组明显升高(3.50±2.02vs.0.90±0.27,3.54±1.90vs.0.80±0.22,3.82±2.79vs.0.88±0.11,3.96士2.21vs.0.73±0.25,p<0.001)。在IDCM组中,出院时NLRP3.ASC.caspase-1及IL-1β的mRNA水平较入院时降低(1.74±1.20vs.3.50±2.02,1.81±1.17vs.3.54±1.90,2.19±1.53vs.3.82±2.79,2.20±1.23vs.3.96±2.21,p<0.001),但仍高于control组(1.74±1.20vs.0.90±0.27,p<0.05;1.81±1.17vs.0.80±0.22,p<0.01；2.19±1.53vs.0.88±0.11,p<0.01;2.20±1.23vs.0.73±0.25,p<0.05).
入院24小时内,在未进行治疗干预前,IDCM组PBMCs中NLRP3.ASC. pro-caspase-1.caspase-1蛋白表达水平及血清中IL-1p蛋白表达水平较control组明显升高(p<0.01)。在IDCM组中,出院时NLRP3.ASC.caspase-1及IL-1p的蛋白水平较入院时均降低(p<0.01),但仍高于control组(p<0.01)。在IDCM组中,PBMCs中pro-caspase-1蛋白表达在入院时和出院时的表达水平未见明显差异。
IDCM患者NLRP3炎症小体的表达水平与临床参数相关
入院时,剔除了年龄、性别等影响因素后,IDCM组PBMCs中NLRP3的mRNA水平与LVEF(r=-0.520,p<0.05).单核细胞数(r=0.613,p<0.05)及NT-pro BNP水平(r==0.514,p<0.05)相关。IDCM组PBMCs中ASC的mRNA水平仅与LVEF(r=-0.334,p<0.05)及单核细胞数(r=0.592,p<0.05)相关。
NLRP3mRNA表达水平是IDCM患者6个月内再入院的独立危险因素
将IDCM患者出院时的LVEF及NLRP3.ASC.pro-caspase-1及IL-1β的mRNA表达水平纳入Cox多因素回归分析模型,结果显示：LVEF(RR:0.001,95%置信区间：0.000-0.128,p<0.001),NLRP3mRNA水平(RR:1.643,95%置信区间：1.193-2.264,p=0.002),IL-1p mRNA水平(RR：1.512,95%置信区间：1.131-2.031,p=0.032)是IDCM患者6个月内再入院的独立危险因素。
NLRP3mRNA表达水平与IDCM预后相关
在IDCM患者中,出院时NLRP3mRNA表达量低的患者再入院的风险低于NLRP3mRNA表达水平高的患者(p<0.05)。
研究结论
(1) NLRP3炎症小体在IDCM患者PBMCs中表达上调；
(2) IDCM患者NLRP3及ASC mRNA表达水平与心功能相关；
(3) IDCM患者中,NLRP3mRNA高表达可提示患者半年再入院风险增加。
Background
Diabetic cardiomyopathy (DCM), an important complication of diabetes, is characterized by consistent diastolic dysfunction and ventricular mass. In addition, DCM is a common cause of heart failure. In the state of diabetes, hyperglycemia-induced reactive oxygen species (ROS) generation is considered to be responsible for progression and development of DCM. The increased ROS could induce a number of cytokine and inflammatory factors, such as nuclear factor-kB (NF-kB), thioredoxin interacting/inhibiting protein (TXNIP), and inflammasome. Although inflammasome was shown to be involved in the pathogenic mechanisms of type2diabetes and its complications, the potential role and regulatory mechanism of inflammasome in DCM has remained largely unexplored.
Inflammasomes are multi-protein platforms that interact with various immune and cell death pathways. Different inflammasomes have been identified, including nucleotide-binding oligomerization domain-like receptors (NLRs) and absent in melanoma2(AIM2). NLRP3, the most extensively studied NLRs, forms a complexes comprised of the apoptosis associated speck like protein (ASC), and the serine protease caspase-1. Upon activation, NLRP3forms a complex with its adaptor ASC, which facilitates the autocatalytic activation of pro-caspase-1and the formation of an active caspase-1p10/20tetramer. The activated caspase-1can process pro-IL-1β into its mature form, which is important in cardiomyocyte apoptosis.
The activation of NLRP3inflammasome consist of two characteristics, including the the up-regulation of NLRP3and the production of caspase-1and IL-1β. Previous research showed that NF-κB could induce NLRP3expression. Thioredoxin interacting/inhibiting protein (TXNIP) can bind NLRP3directly and lead to NLRP3inflammasome assembly in HEK293T cells. However, little is known about whether NF-κB and TXNIP participate in the regulation of NLRP3in hyperglycemia-treated cardiomyocyte.
In addition to resulting in the maturation of IL-1β, activated caspase-1can induce a distinct form of programmed cell death called "pyroptosis". Pyroptosis, a highly inflammatory form of cell death, is dependent on caspase-1activity. The morphology of pyroptosis shares the unique characteristics with both apoptosis and necrosis. As in apoptotic cell, pyroptotic cells incur DNA damage and become positive in the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. As necrosis, pyroptosis results in pore formation in the cell membrane, release of pro-inflammatory cytosolic content, and cell lysis. Therefore, membrane impermeant dyes such as EthD-III stain pyroptotic cells by entering through the pores, but do not stain apoptotic cells. Pyroptosis is initially described in macrophages and dendritic cells infected with different pathogens. Recent studies showed that pyroptosis could also occur in non-myeloid cells induced by non-infectious stimuli. However, it is not clear whether pyroptosis participates in hyperglycemia-induced cardiomyocyte death.
Electron microscopy studies of myocardium in diabetic mice and rats showed that the majority of dying cells had swollen fibril and mitochondria, which are the characteristics of cell swelling and lysis in pyroptosis. Activated caspase-1, the executor caspase of pyroptosis, is found to be elevated in DCM in a rat model. Thus, we hypothesized that pyroptosis, regulated by NLRP3inflammsome, might participate in the pathogenesis of DCM. We also hypothesized that NF-κB and TXNIP might be links between ROS and NLRP3activation.
Objective
(1) We established a type2diabetes rat model and explored the expression level of NLRP3inflammasome in different stages of IDCM;
(2) We induced the cardiac NLRP3silencing by lenti-NLRP3-miRNA, and explore the role of NLRP3in DCM.
(3) We further explored the mechanism of the regulation of NLRP3activation in high glucose-treated H9c2cells.
Methods
Induction of type2diabetes in rats
We randomized120Sprague-Dawley rats into4groups (n=30per group):control, diabetes mellitus (DM), DM+vehicle, DM+NLRP3-miRNA. All rats were housed at22℃with12h light-dark cycles. The control group was fed the basal diet, and the other groups a high fat diet (HF diet,16%fat and0.25%cholesterol).4weeks later, the intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed to determine insulin resistance. In the groups of DM, DM+vehicle and DM+NLRP3-miRNA rats with reduced insulin sensitivity will receive a single intraperitoneal injection of streptozotocin (STZ;35mg/kg). After one week of STZ injection, blood glucose and insulin levels were measured after rats fasted overnight. Insulin sensitivity index (ISI) was calculated by ISI=In[(INS×BG)-1] method. Only rats with blood glucose level>11.1mmol/L and reduced insulin sensitivity were used as type2diabetes rats in the study.
Lenti-NLRP3-miRNA
According to RNAi principle, we designed4pieces of miRNA against rat NLRP3and constructed pcDNA6.2-GW/EmGFP-NLRP3-miRNA plasmid. Then we selected the most effective NLRP3-miRNA plasmid and then synthesized the shuttle pDONR221-EmGFP-NLRP3-miRNA plasmid. Finally, the lentiviral vector of pLenti6.3—EmGFP-NLRP3-miRNA was constructed.
Gene silencing therapy
According to previous studies, diabetic rats showed onset of cardiac dysfunction after8weeks of STZ injection. Our gene silencing treatment occurred after8weeks of STZ injection. The groups of DM+vehicle and DM+NLRP3-miRNA rats in our study received a jugular-vein injection of a total lentivector dose of1×108TU/rat vehicle (empty virus) or NLRP3-miRNA. After8weeks of lentivector injection, rats of DM+vehicle and DM+NLRP3-miRNA were killed. The heart was excised and immediately frozen to determine the transfection efficiency under fluorescence microscope. The age matched rats of control and DM were killed at the same time point (n=10per group).
Cardiac expression pattern of NLRP3inflammasome
For the analysis of NLRP3inflammasome and IL-1β expression, cardiac samples of DM rats were collected at0,4,8,12,16and20weeks (n=3) after STZ injection, and control rats (n=3) were collected at20weeks after diabetic rats received STZ.
Serum measurements
At the end point of gene silencing therapy, serum blood glucose, TG and TC analysis were tested in rats of the4groups.
Enzyme Linked Immunosorbent Assay (ELISA)
At the end point of gene silencing therapy, serum insulin was tested in rats of the4groups.
Echocardiography
Transthoracic echocardiography analysis was performed after the rats anesthetized. And we collected the parameters of left ventricular end diastolic diameter (LVEDd), left ventricular ejection fraction (LVEF), fractional shortening (FS), peak E to peak A ratio (E/A), and early to late diastolic velocity ratio (E'/A').
Histological examination
The hearts were arrested with10%KC1, and heart weight was measured immediately. The images of whole heart were obtained by camera. Paraffin sections from the midpoint of heart were stained with hematoxylin and eosin, and the images were obtained by camera.
Measurement of myocardial fibrosis
Masson's trichrome and Sirius red staining were used for detecting collagen. The fibrotic area fraction was analyzed by automated image analysis.
Transmission electron microscopy (TEM)
About1mm3tissue was obtained from the left ventricle of rats and fixed in buffer. The following preparation of TEM was performed according to the guidance.
Immunohistochemistry
Immunohistochemistry was performed in the paraffin section of the left ventricular tissue of different groups of rats. We used antibodies against caspase-1and IL-1β. Data were analyzed by Image-Pro Plus6.0.
Real-time RT-PCR
Real-time RT-PCR was used to explore the relative expression of NLRP3, ASC, caspase-1and IL-1β. The primer sequences were shown in table1. The relative expression of genes was calculated by the2-ΔΔCT method.
Western blot
Western blot was used to explore the protein levels of NF-κB, TXNIP, NLRP3, ASC, caspase-1, IL-1β, collagen I, collagen III, and β-actin. The protein bands were developed by the use of chemiluminescence, and quantified by densitometric analysis.
TUNEL Assay
Detection of fragmentation of DNA was performed using an ApopTag in situ apoptosis detection kit. Deparaffinized sections were treated with3%hydrogen peroxide in methanol for10min. The sections were washed in PBS twice for5min each. After adding the equilibration buffer, sections were treated with TdT-enzyme at37℃for1h. The sections were then rinsed with PBS and incubated with digoxigenin-conjugated antibodies at37℃for30min. Then sections were colorized with DAB. Finally, the stained sections were examined under microscope.
Cell culture and treatment
H9c2cardiomyocytes were cultured as described previously. During the treatment period, H9c2cells were cultured in normal glucose medium with minimal essential medium for12h, followed by the exposure of control glucose (Ctrl,5.5mM), medium glucose (MG,25mM), high glucose (HG,33.3mM), and osomotic control (OC,27.5mM mannose) for24,36, or48h. In ROS inhibition experiment, cells were treated with HG for36h in the presence of10mmol/L N-acetylcysteine or the relevant vehicle control (PBS).
Lentivirus transfection
H9c2cardiomyocytes plated at a density of1×105/cm2were infected with vehicle (empty lentivirus) or lentivrius-NLRP3-miRNA at10multiplicity of infection (MOI) in serum-free media for2h, followed by incubation with DMEM containing10%FBS for an additional48h before processing. The transfection efficiency was observed by fluorescence microscope. In addition, NLRP3knockdown was assessed in transfected cells, and cells were used only if NLRP3mRNA was decreased by70%compared with vehicle.
Immunofluorescence
Immunofluorescence was used to detect the localization of caspase-1in H9c2cells. We used antibodies against caspase-1. The fluorescence was visualized by confocal microscopy and analyzed by Image-Pro Plus6.0.
Caspase-1activity assay
Caspase-1activity was measured by using colorimetric assay. This assay was designed in consideration of the ability of caspase-1to change acetyl-Tyr-Val-Ala-Asp p-nitroaniline (Ac-YVAD-pNA) into the yellow formazan product p-nitroaniline (pNA).
ROS levels
Intracellular generation of ROS was tested by peroxide-sensitive fluorescent probe2',7'-dichlorofluorescein diacetate (DCFH-DA).
Cell death assay
Cell death was assessed by TUNEL assay and EthD-Ⅲ/calcein AM staining. The virtually nonfluorescent cell-permeant calcein AM is enzymically converted to the brightly fluorescent calcein, producing a bright green fluorescence in live cell. EthD-III enters dead cells, thereby producing an intense red fluorescence in dead cells.
Statistical analysis
Data are expressed as mean±SEM. Differences among experimental groups were analyzed by ANOVA, followed by the Tukey-Kramer post hoc test and independent samples t test. Analysis involved use of SPSS v18.0. Significance was defined as p<0.05.
Result
HF induced insulin resistance
After4weeks of HF diet, IPGTT and IPITT were performed in all groups. In IPGTT, the blood glucose in HF group was significantly higher than that in control group at all of the time points tested except at15min (p<0.05-p<0.01). The AUC for glucose level was higher in HF group than that in control (p<0.01). Similarly, the result observed by IPITT revealed impaired insulin sensitivity in rats with HF diet (p<0.05-p<0.01).
NLRP3inflammasome and IL-1β were activated in DCM
When compared with the control rats, diabetic rats showed elevated mRNA levels of NLRP3, ASC, caspase-1and IL-1β after4or8weeks of diabetes (all p<0.01). Moreover, NLRP3inflammasome components and IL-1β mRNA levels reached highest value after8weeks of diabetes, and stayed at relatively high value until20weeks of diabetes (all p<0.01). The protein levels of NLRP3, ASC, pro-caspase-1, activated caspasep-1(caspase-1p20) and mature IL-1β (IL-1β p17) were higher in DM than control group (all p<0.01). The protein expression of NLRP3, ASC and caspase-1achieved the highest levels after8weeks of diabetes and kept at relatively high levels until20weeks of diabetes (all p＜0.01). The activated caspase-1and IL-1β exhibited the similar protein expression pattern (both p＜0.01).
NLRP3gene silencing improved metabolism abnormalities
The metabolic characteristic of the experimental animals analyzed in this study are shown in Table2. In the DM group, blood glucose, TC, TG and INS were remarkably higher than the control (all p＜0.01). ISI in DM group was lower than control group (p＜0.01). NLRP3gene silencing was insufficient to improve the systemic metabolic disturbance.
Cardiac NLRP3expression was suppressed by gene silencing
After10weeks of NLRP3silencing treatment, transfection efficiency was checked in all groups. As compared with vehicle treatment, NLRP3-miRNA treatment decreased the mRNA and protein levels of cardiac NLRP3(both p<0.01). In addition, the protein levels of activated caspaspe-1and mature IL-1β were lower in NLRP3-miRNA treated diabetic rats than the vehicle treated rats (both p<0.01). Immunohistochemistry revealed that increased caspase-1predominantly localized in perinuclear area, while elevated IL-1β showed diffused distribution pattern in DCM (both p<0.01).
NLRP3gene silencing alleviated left ventricular disfunction in DCM
The results of echocardiography showed that LVEDd of DM rat was larger than control (p<0.01). LVEF, FS, E/A and E'/A'were lower in DM than control p<0.01). When compared with vehicle, increased LVEF, FS, E/A, E'/A'and decreased LVEDd were observed in NLRP3-miRNA group (p<0.05-p<0.01).
NLRP3gene silencing reversed myocardial remodeling
The DM group showed the phenotype of eccentric ventricular hypertrophy, which was characterized with larger chamber size and thicker ventricular wall. The ratio of heart weight to body weight was larger for DM than the control group (p<0.01). TUNEL result showed that the percentage of dead cell was obviously higher in DM group than control (p<0.01). The ultrastructure of cardiomyocyte in control rats showed typical symmetric myofibrils, well-organized Z lines with sarcomeres, and packed mictochontria beside the fibers. In contrast, DM rats showed severe damage of the left ventricular ultrastructure, including destruction of myofibrils, swollen mitochondria with disorganized cristae, excess glycogen lysis, and accumulated lipids. Moreover, the diabetic group showed increased interstitial cardiac fibrosis as compared with the control (p＜0.01). Coincident with cardiac fibrosis, the protein levels of collagen Ⅰ and collagen Ⅲ, and the collagen I-to-III ratio were significantly higher in DM group than the control (all p＜0.01).
With NLRP3gene silencing, heart weight to body weight ratio and cell death were significantly decreased in the NLRP3-miRNA group versus the vehicle group (both p<0.01). NLRP3-miRNA treatment normalized alterations in myofilaments and mitochondria, along with reduced glycogen lysis and lipid accumulation in diabetic rats. Moreover, cardiac fibrosis area, collagen I, collagen III, and the collagen I to III ratio were lower in the DM+NLRP3-miRNA group than vehicle group (all p<0.01).
The increased expression of NLRP3inflammasome and IL-1β were induced by high glucose
Different levels of glucose caused a concentration-dependent increase of NLRP3, ASC, caspase-1and IL-1β in H9c2cells in24to48h (all p<0.01). Except for caspase-1, the mRNA levels of NLRP3inflammasome and IL-1β were increased in a time-dependent pattern with high glucose (both p<0.05-p<0.01). Likewise, the protein levels of all NLRP3inflammasome components and mature IL-1β were increased significantly at high glucose as compared with control and medium glucose in36and48h (all p<0.01). Moreover, the increase of NLRP3, ASC and mature IL-1β protein showed the highest level at36h, and persisted for48h with high glucose incubation (all p<0.05). Thus, we chose high glucose as the stimulation, and chose36h as the stimulated time in subsequent experiments. The expression of NLRP3inflammasome and mature IL-1β in H9c2cells with control glucose or isotonic mannose had no significant change at the same time point tested.
High glucose induced caspase-1activation and cell death in H9c2cells
High glucose significantly increased the level of activated caspase-1as compared with control and medium glucose (p<0.05-p<0.01). Immunofluorescence result showed the accumulation of caspase-1in the cytoplasm of H9c2cells incubated with medium and high glucose. Coincident with caspase-1activation in high glucose, TUNEL result revealed increased cell death with nucleus DNA damage (p<0.01), and EthD-Ⅲ/calceim AM staining showed elevated cell death with damaged cell membrane as compared with control and medium glucose (p<0.01).
NLRP3was involved in cell death induced by high glucose
To explore the role of NLRP3in high glucose-induced cell death of H9c2, we inhibited the expression of NLRP3by lentivirial NLRP3-miRNA. The transfection efficacy of lentivirial NLRP3-miRNA in H9c2cardiomyocytes reached80%. Both the mRNA and protein levels of NLRP3in cells transfected with NLRP3-miRNA were significantly lower than the vehicle (both p<0.01). After inhibiting the expression of NLRP3, the protein levels of activated caspase-1and mature IL-1β induced by high glucose decreased significantly as compared with vehicle (p<0.05-p<0.01). In keeping with these observations, the dead cell rate detected by TUNEL was obviously lower in HG+NLRP3-miRNA than HG+vehicle (p<0.01). To avoid the interference of lentivector fluorescence, we did not use calcein AM in the detection of live cells. The high glucose induced-cell-death rate detected by EthD-Ⅲ were significantly decreased in NLRP3-miRNA treated cells as compared with vehicle (p<0.01).
NF-κB and TXNIP were associated with the ROS-induced NLRP3inflammasome activation
Glucose treatment promoted ROS generation in H9c2. The increase of ROS was dose dependent (all p<0.01). Coincidence with ROS production, the phosphorylation of NF-kB p65was increased in medium and high glucose treated cells. And the expression of TXNIP also exhibited a dose-dependent manner (all p<0.01). Pretreatment of cells with NAC inhibited high glucose-induced increase in intracellular ROS activity (p<0.01). Intriguingly, the levels of NF-kB phosphorylation and TXNIP were also lower in the NAC treated group than the PBS treated group (all p<0.01). In keeping with the decreased expression of NF-kB and TXNIP, the expression of all component of NLRP3, ASC, pro-caspaspe-1, activated caspase-1and mature IL-1β were down-regulated with pretreatment of NAC as compared with PBS (all p<0.01).
Conclusion
(1) High glucose-induced ROS accelerated the expression of NLRP3inflammasome. NF-kB and TXNIP might be involved in the inhibition of RSV on NLRP3inflammasome.
(2) We found the increased expression of NLRP3inflammasome in cardiac tissue at the early stage of diabetes. NLRP3inflammasome accelerated the process of DCM by inducing cardiac inflammation, cardiomyocyte dysfunction and interstitial fibrosis.
(3) The cardiac NLRP3gene silencing was effective by using the lenti-NLRP3-miRNA in vivo.
(4) The NLRP3gene silencing therapy ameliorated the structural and functional disorder in DCM.
Background
Diabetic cardiomyopathy (DCM), a significant contributor to morbidity and mortality in diabetes, is characterized by early-onset diastolic and late-onset systolic dysfunction without coronary artery disease, hypertension, or valvular heart disease. Accumulating evidence indicates that inflammation is an important pathogenic factor in DCM. Therefore, investigation of the inflammatory mechanism is the keystone in the management of DCM.
Previous studies showed that interleukin-1β (IL-1β) is an important proinflammatory cytokine in the development of DCM. IL-1β activation is mainly mediated the protein platform "the inflammasome". Inflammasome is a multiprotein complex which exists in the cytoplasm. Different inflammasomes have been identified, including nucleotide-binding oligomerization domain-like receptors (NLRs) and absent in melanoma2(AIM2). NOD like receptor3(NLRP3) is a member of NLRs. NLRP3inflammasome consists of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1. Recently, several studies suggested that NLRP3inflammasome plays a critical role in the inflammatory process of diabetes and diabetic complications, such as diabetic nephropathy and diabetic retinopathy. Recent observations indicate that thioredoxin interacting/inhibiting protein (TXNIP) can bind NLRP3directly and lead to NLRP3inflammasome assembly under oxidative stress. And our previous research indicated that NLRP3inflammasome could contribute to the process of DCM.
In DCM, mitogen-activated protein kinases (MAPKs) are the most important pathways induced by high glucose levels and are involved in inflammation. Also, activation of MAPKs can contribute to the development of cardiac fibrosis.
Rosuvastatin (RSV), a member of3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, has a number of pleiotropic properties, such as anti-inflammation, antioxidation and cardiac remodeling attenuation. The cardioprotective effect of RSV has been demonstrated in animal models of hypertrophy, myocardial infarction and experimental autoimmune myocarditis. However, the mechanisms by which RSV protects against DCM are incompletely understood.
The main purpose of the present study was to determine whether NLRP3and MAPKs were important targets for RSV in exerting its protective effect on DCM. And we tried to explore the potential value of NLRP3inflammasome in the treatment of DCM.
Objective
(1) We established a type2diabetes rat model and evaluated the effect of RSV on the process of DCM.
(2) We also evaluated whether NLRP3was important target for RSV in exerting its protective effect on DCM.
(3) We tried to explore the mechanism by which RSV inhibites NLRP3inflammasome in the process of DCM.
Methods
Induction of type2diabetes in rats
We randomized105Sprague-Dawley rats (100-120g) into7groups (n=15per group):control, high fat (HF), diabetes mellitus (DM), HF+RSV10mg/kg (10mg/kg, body weight, daily), HF+RSV15mg/kg, DM+RSV10mg/kg and DM+RSV15mg/kg. The control group was fed the basal diet, and the other groups a high fat diet (HF diet,16%fat and0.25%cholesterol).4weeks later, the intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed to determine insulin resistance. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ;35mg/kg) to rats in the groups of DM, DM+RSV10mg/kg and DM+RSV15mg/kg. One week later, blood glucose levels were measured after rats fasted overnight. Only rats with blood glucose level≥11.1mmol/L were used as diabetes in the study. The oral administration of RSV was started in the ninth week after STZ injection, and was continued for8weeks. Rats were weighted before they were killed after8weeks of RSV treatment. The experiments complied with the Animal Management Rule of Chinese Ministry of Health (documentation no.55,2001), and experimental protocols were approved by the Shandong University Animal Care Committee.
Lenti-NLRP3-miRNA
According to RNAi principle, we design4pieces of miRNA against rat NLRP3and constructed pcDNA6.2-GW/EmGFP-NLRP3-miRNA plasmid. Then we selected the most effective NLRP3-miRNA plasmid and then synthesized the shuttle pDONR221-EmGFP-NLRP3-miRNA plasmid. Finally, the lentiviral vector of pLenti6.3—EmGFP-NLRP3-miRNA was constructed.
Gene silencing therapy
Another90Sprague-Dawley rats (100-120g) were randomized into6groups (n=15per group):vehicle+control, vehicle+DM, vehicle+DM+RSV15mg/kg (the dose of15mg/kg was the most effective in our drug experiment), NLRP3microRNA (NLRP3-miRNA)+control, NLRP3-miRNA+DM, and NLRP3-miRNA+RSV15mg/kg. After8weeks of STZ injection, diabetic rats showed onset of cardiac dysfunction. Then all6groups of rats received a jugular-vein injection of a total lentivector dose of1×108TU/rat NLRP3-miRNA or vehicle. RSV supplementation for the2groups was performed at the same time for8weeks. After8weeks of lentivector injection, rats were killed. The heart was excised and immediately frozen to determine the transfection efficiency under fluorescence microscope.
Serum measurements
After rats fasted overnight, we collected jugular blood samples. Serum triglycerides (TG), total cholesterol (TC), insulin (INS) were determined. The insulin sensitivity index (ISI) was calculated by ISI=In[(FINS×FBG)-1] method.
Echocardiography
Transthoracic echocardiography was performed in rats anesthetized with10%chloral hydrate. The derived echocardiography parameters included left ventricular ejection fraction (LVEF), fractional shortening (FS), peak E to peak A ratio (E/A), and early (E') to late (A') diastolic velocity ratio (E'/A').
Measurement of myocardial fibrosis
Masson's trichrome and Sirius red staining were used for detecting collagen. The fibrotic area fraction was analyzed by automated image analysis.
Transmission electron microscopy (TEM)
About1mm3tissue was obtained from the left ventricle of rats and fixed in buffer. The following preparation of TEM was performed according to the guidance.
Real-time RT-PCR
Real-time RT-PCR was used to explore the relative expression of NLRP3, ASC, caspase-1, IL-1β, collagen I and collagen III. The relative expression of genes was calculated by the2-ΔΔCT method.
Western blot
Western blot was used to explore the relative protein expression of NLRP3, ASC, pro-caspase-1, caspase-1, IL-1β, and the phosphorylation of ERK1/2, P38, and JNK. The protein bands were developed by the use of chemiluminescence, and quantified by densitometric analysis.
Statistical analysis
Analysis involved use of SPSS v18.0. Data are expressed as mean±SEM. Differences among experimental groups were analyzed by ANOVA, followed by the Tukey-Kramer post hoc test and independent samples t test. Significance was defined as p<0.05.
Results
HF induced insulin resistance
After4weeks of HF diet, insulin tolerance was determined by IPGTT and IPITT. In IPGTT, the blood glucose in HF group was significantly higher than that in control group at all of the time points tested except at15min (p<0.05-p<0.01). The AUC for glucose level was higher in HF group than that in control one (p<0.01). Similarly, the result observed by IPITT revealed blood glucose in HF group was significantly higher than that in control group at all of the time points tested except at15min (p<0.05-p<0.01). The AUC for glucose level was higher in HF group than that in control one (p<0.01).
RSV had limited effect on systemic metabolic abnormities
DM rats showed more severe hyperglycemia, hyperlipidemia and impaired insulin sensitivity than control and HF rats (both p<0.01). The metabolic state was worse for HF group than control group (p<0.05-p<0.01).
RSV affected TC regulation. TC level was lower in DM rats receiving10and15mg/kg RSV than no treatment (p<0.05). TC level was lower with HF+RSV15mg/kg group than HF group (p<0.05). Neither10nor15mg/kg RSV improved glucose dysregulation, TG level disturbance, insulin imbalanee, or impaired insulin sensitivity in DM and HF rats.
RSV alleviated DM-induced left-ventricular dysfunction
LVEF, FS, E/A and E'/A were lower with DM than control. E/A and E'/A'was lower with HF group than control (p<0.05-p<0.01).
After treatment, LVEF was improved with DM+RSV15mg/kg as compared with DM alone, with no significant improvement with DM+RSV10mg/kg, or HF+RSV10or15mg/kg over DM and HF, respectively (p<0.05). RSV treatment did not significantly prevent FS in DM or HF rats. For diastolic function, DM+RSV15mg/kg increased both E/A and E'/A'as compared with DM alone (both p<0.05). DM+RSV10mg/kg also improved E'/A'(p<0.05).
RSV alleviated myocardial remodeling
The ratio of heart weight to body weight was larger for DM and HF than the control group (p<0.05-p<0.01). Diabetes induced interstitial fibrosis, increased collagen mRNA expression, and increased the collagen I to III ratio (p<0.05-p<0.01). Similarly, HF rats showed increased fibrosis and collagen content as compared with control rats (p<0.05-p<0.01). TEM revealed typical symmetric myofibrils, well-organized Z-lines with sarcomeres, and packed mitochondria beside fibers in control rats. In contrast, DM rats showed severe damage of the left ventricular ultrastructure, including destruction of myofibrils, degenerated Z-lines, swollen mitochondria with vacuoles and disorganized cristae, coalescence of irregular mitochondria, and accumulated lipids. The HF rats showed moderate destruction of the left ventricular ultrastructure.
RSV15mg/kg treatment decreased heart weight to body weight ratio, fibrosis area and collagen disorders in DM rats (all p<0.01), but had little effect on HF rats. RSV10mg/kg restored only the disordered fibrosis and collagen I content in DM rats (p<0.05-p<0.01). After treatment with RSV, the diabetic and HF rats showed improved cardiac ultrastructure.
RSV inhibited NLRP3inflammasome activation
We investigated the mRNA and protein levels of TXNIP, NLRP3, ASC, pro-caspase-1, activated caspase-1and activated IL-1β in left ventricular tissue of rats. The mRNA expression of TXNIP, NLRP3, ASC, caspase-1and IL-1β was significantly higher in DM than control rats (all p<0.01). The protein levels of TXNIP, NLRP3, ASC, pro-caspase-1, activated caspase-1and maturation of IL-1β were higher in DM than control rats (p<0.05-p<0.01). Similarly, the mRNA and protein levels of TXNIP, NLRP3inflammasome and IL-1β were higher in HF than control rats (p<0.05-p<0.01).
RSV15mg/kg suppressed the increased mRNA levels of TXNIP, NLRP3inflammasome and IL-1β induced by diabetes (p<0.05-p<0.01). The mRNA levels of TXNIP and IL-1β were lower with DM+RSV10mg/kg than DM alone (both p<0.05). Except for caspase-1mRNA, similar reduced levels were observed in HF with RSV treatment than HF alone and control group (p<0.05-p<0.01). Protein levels of TXNIP, NLPR3inflammasome and mature IL-1β were lower with DM+RSV15mg/kg than DM alone (p<0.05-p<0.01). DM+RSV10mg/kg only decreased pro-caspase-1protein level as compared with DM alone (p<0.05). In addition, DM+RSV15mg/kg decreased protein levels of TXNIP and ASC, total and activated caspase-1, and activated IL-1β as compared with DM+RSV10mg/kg (all p<0.01). Similar results were observed in RSV-treated HF groups (p<0.05-p<0.01).
RSV inhibited the phosphorylation of MAPK signaling pathways
We further investigated the activation of MAPK signaling pathways. DM rats showed the greatest activation of ERK1/2, p38and JNK (p<0.05-p<0.01). The HF group showed increased phosphorylation of MAPKs as compared with control group (p<0.05-p<0.01).
The phosphorylation of MAPKs was suppressed in RSV-treated DM rats as compared with DM alone (p<0.05-p<0.01), and RSV15mg/kg was better than10mg/kg in inhibiting ERK1/2phosphorylation (p<0.01). Similar results were found in RSV-treated HF rats (p<0.05-p<0.01).
Detection of cardiac NLRP3expression with gene silencing
After8weeks of NLRP3silencing treatment, transfection efficiency was checked in all groups. As compared with vehicle treatment, NLRP3-miRNA treatment decreased the mRNA and protein levels of cardiac NLRP3and mature IL-1β (p<0.05-p<0.01).
RSV alleviated diabetes-induced cardiac dysfunction by inhibiting NLRP3expression
After8weeks of NLRP3-miRNA treatment, LVEF, FS, E/A and E'/A'were improved in the DM rats as compared with vehicle treatment (all p<0.05). NLRP3miRNA treatment increased E/A and E'/A'in DM+RSV15mg/kg rats as compared with vehicle treatment (both p<0.05).
In vehicle-treated rats, diabetes-induced severe systolic and diastolic dysfunction was ameliorated with RSV supplementation (all p<0.05). The cardioprotective effect of RSV in diabetes was abrogated more in NLRP3-miRNA-treated rats than vehicle-treated rats.
RSV reversed diabetes-induced myocardial disorder with inhibited NLRP3
In DM rats, NLRP3-mi
糖尿病性心肌病(diabetic cardiomyopathy, DCM)是由糖尿病引起的,并以心肌重构和心功能下降为特征,最终导致心力衰竭的心肌病变。糖尿病状态下,高糖刺激活性氧(reactive oxygen species, ROS)生成增多被认为是DCM发生和发展的重要原因。过多的ROS刺激多种炎症因子表达升高,如核因子kB(nuclear factor kappa-light-chain-enhancer of activated B cells, NF-kB)、硫氧还蛋白相互作用蛋白(thioredoxin interacting/inhibiting protein, TXNIP)及炎症小体。既往研究表明,炎症小体与胰岛素抵抗、2型糖尿病及糖尿病并发症关系密切。但目前炎症小体在DCM中的作用及其调控机制尚未见报道。
炎症小体是一类分布于细胞胞浆中的蛋白复合体,它可以感受细胞内多种与免疫及死亡相关的信号刺激,参与细胞功能调控。目前已发现多个炎症小体家族成员,主要包括：NLRs (nucleotide binding oligomerization domain-like receptors)家族及AIM2(absent in melanoma2)家族。NLRP3(NOD-like receptor protein3)是NLRs家族中的一员。当接收到相关信号刺激后,NLRP3将与含CARD结构域的凋亡相关颗粒样蛋白(apoptosis-associated speck-like protein containing CARD, ASC)及含半胱氨酸的天冬氨酸蛋白水解酶1(cysteinyl aspartate specific proteinase-1, caspase-1)组成炎症复合体,促进caspase-1前体(pro-caspase-1)的自我剪切,形成具有活性的caspase-1p20/p10复合物,最终促进白介素1β (interleukin1β, IL-1β)前体蛋白pro-IL-1β水解成具有活性的IL-1p。既往研究表明,IL-1p在心肌细胞凋亡中具有重要作用。
NLRP3炎症小体的异常激活通常表现在两个方面：一方面,NLRP3表达上调；另一方面,NLRP3炎症小体形成复合物,促进caspase-1及IL-1p等效应分子的产生。在巨噬细胞中,NF-kB可促进NLRP3表达上调。在HEK293T细胞中,TXNIP可促进NLRP3炎症复合物的形成,最终促进caspase-1及IL-1β等效应分子的产生。但是在高糖刺激的心肌细胞中,NF-kB及TXNIP是否与NLRP3炎症小体的激活相关,目前尚未见报道。
近期研究显示,caspase-1除了可以促进IL-1β成熟外,还可以促进细胞程序性死亡,这一过程被称为“细胞焦亡(pyroptosis)"。细胞焦亡是一种与炎症相关,依赖于caspase-1的程序性死亡方式。焦亡的细胞同时具备细胞凋亡及细胞坏死的部分形态特征。这主要表现在：细胞焦亡存在DNA片段化损伤,并在脱氧核糖核苷酸末端转移酶介导的粘性末端标记检测(terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, TUNEL)中呈现阳性；细胞焦亡时胞膜完整性消失,胞内炎症物质会通过膜上的孔隙释放到细胞间质,胞外物质会从孔隙渗入胞浆,促进细胞器及细胞浆肿胀,最终导致细胞破裂。因此,不能通过完整细胞膜而只能通过形成孔隙的胞膜的染料可将活细胞与焦亡细胞区分开,如EthD-Ⅲ染料,它可将焦亡细胞染成阳性,但不能将正常细胞染色。焦亡现象最初是在巨噬细胞及树突细胞等髓系细胞受到病原体刺激后观察到的。近期研究显示,一些非病原体也可刺激非髓系细胞出现焦亡现象。但是,高糖刺激的心肌细胞是否发生焦亡,目前未见报道。
在糖尿病小鼠及大鼠模型中,心肌组织的电镜结果显示大量心肌细胞呈现与焦亡细胞类似的形态特征,如线粒体肿胀空泡化、肌纤维紊乱溶解、细胞肿胀、及细胞膜模糊不清等。此外,我们在预实验中发现细胞焦亡关键因子caspase-1在DCM大鼠模型心肌组织中表达升高。据此,我们提出假设：糖尿病状态下,心肌细胞ROS生成增多。ROS通过NF-kB及TXNIP促进NLRP3炎症小体过度激活。NLRP3炎症小体的异常激活通过促进心肌间质炎症反应、心肌细胞焦亡及心肌纤维化等途径导致心脏结构和功能异常。
研究目的
(1)建立2型DCM模型,探究NLRP3炎症小体在DCM病程不同阶段的表达水平；
(2)利用NLRP3-miRNA'慢病毒对DCM大鼠进行体内干预,探究NLRP3炎症小体在DCM中的作用；
(3)探究NLRP3炎症小体影响DCM发生发展的相关机制。
研究方法
2型糖尿病大鼠模型
将120只5周龄雄性Sprague-Dawley (SD)大鼠随机分为4组(每组30只)：对照组(control组)、糖尿病组(DM组)、糖尿病+空载体慢病毒组(DM+vehicle组)及糖尿病+NLRP3-miRNA慢病毒组(DM+NLRP3-miRNA组)。Control组给予基础饮食,主要成分为：5%脂质,不添加胆固醇。其余3组给予高脂饮食喂养,主要成分为：16%脂质及0.25%胆固醇。4周后4组大鼠均行腹腔葡萄糖耐量实验(Intraperitoneal glucose tolerance test, IPGTT)及腹腔胰岛素耐量实验(Intraperitoneal insulin tolerance test, IPITT)。DM组、DM+vehicle组及DM+NLRP3-miRNA组出现胰岛素抵抗的大鼠接受单次腹腔STZ注射,剂量为35mg/kgo1周后,检测4组大鼠的空腹血糖(BG)及胰岛素(insulin, INS),计算胰岛素敏感指数(ISI), ISI=In[(INS×BG)"1]。DM组、DM+vehicle组及DM+NLRP3-miRNA组大鼠连续两次空腹血糖≥11.1mmol/L,且存在胰岛素抵抗则认为2型糖尿病造模成功。
糖尿病性心肌病模型
糖尿病大鼠造模成功后,继续给予高脂喂养。至STZ注射后8周,检测大鼠心功能,糖尿病大鼠出现左室舒张功能障碍则提示开始进展为DCM。至STZ注射后16周,检测大鼠心脏结构及功能,糖尿病大鼠心脏组织呈现心肌间质纤维化及左室肥厚的,并出现左室收缩及舒张功能障碍,则提示DCM模型已经成功建立。
基因沉默技术
根据RNAi干扰技术的原则,设计4对针对大鼠NLRP3基因的miRNA片段,并分别构建4对pcDNA6.2-GW/EmGFP-NLRP3-miRNA质粒,经筛选后,选择沉默效率最高的质粒,随后构建pDONR221-EmGFP-NLRP3-miRNA重组穿梭质粒,最终构建pLenti6.3—EmGFP-NLRP3-miRNA慢病毒载体。
慢病毒干预
在STZ腹腔注射8周后,分别给予DM+vehicle组及DM+NLRP3-miRNA组慢病毒空载体(vehicle)或NLRP3-miRNA慢病毒(NLRP3-miRNA)干预8周。病毒干预8周后,对DM+vehicle组及DM+NLRP3-miRNA组全部大鼠实施安乐死,随机抽取部分control及DM组大鼠(每组10只)实施安乐死。立即心脏取材,行冰冻切片检测心肌病毒转染效率。
NLRP3炎症小体表达趋势
Control组及DM组大鼠,分别在STZ注射后的第0、4、8、12、16及20周时取材,每组取3只,然后进行相关指标的检测。
血清学检测
待实验结束时,大鼠饥饿过夜,抽取外周静脉血,分别检测血清中血糖(blood glucose)、甘油三酯(triglyceride, TG)及总胆固醇(total cholesterol,TC)含量。
酶联免疫吸附试验(Enzyme Linked Immunosorbent Assay, ELISA)
利用ELISA测定大鼠外周血血清中胰岛素的水平。
心脏彩超
大鼠经10%水合氯醛麻醉后,行经胸心脏彩超,收集LVEDd、LVEF、F/S、 E/A及E’/A’等数据评价大鼠左室功能。
组织学检测
大鼠实施安乐死后,立即心脏取材,随后称重、测量心脏大小并留取图像。于乳头肌水平横切心脏,制成石蜡切片,于乳头肌平面连续切片,行苏木精-伊红(hematoxylin-eosinstaining, H&E)染色后留取图像。
心肌纤维化
对左室心肌组织行Masson染色、天狼猩红染色,使用图像分析软件对染色结果进行分析,评价左室心肌纤维化程度。
透谢电镜
大鼠实施安乐死后,立即取出心脏,于左室壁取1mm3小组织块,立即放入戊二醛固定液中,为透射电镜下观察左室心肌超微结构做准备。
免疫组织化学
利用免疫组织化学的方法,观察caspase-1及IL-1β在组织中的分布。
实时定量RT-PCR
取新鲜左室心肌组织,利用实时定量RT-PCR的方法检测大鼠左室心肌中NLRP3, ASC, caspase-1及IL-1βmRNA表达水平。
Western blot
取新鲜左室心肌组织,利用Western blot方法检测大鼠左室心肌中TXNIP、 NF-kB、NLRP3、ASC、pro-caspase-1、 caspase-1及IL-1β的表达。
TUNEL检测
利用TUNEL检测大鼠左室组织中,心肌细胞核出现DNA损伤的情况。
细胞培养及干预
培养大鼠H9c2心肌细胞系,分为3组：基础糖培养组(control组)、中等浓度糖刺激组(median glucose, MG组)及高浓度糖刺激组(high glucose, HG组)。在进行干预前,利用无血清基础糖培养基进行饥饿处理。随后给予含不同浓度葡萄糖的培养基进行不同时间梯度的干预。在ROS抑制实验中,我们选择N-乙酰半胱氨酸(N-acetylcysteine, NAC)作为抑制剂对心肌细胞进行预处理。
心肌细胞病毒转染
利用vehicle或NLRP3-miRNA慢病毒对心肌细胞进行干预,以探究NLRP3在心肌细胞中的作用及调控机制。
免疫荧光
利用免疫荧光技术观察caspase-1在心肌细胞中的分布。
Caspase-1活性检测
利用caspase-1活性检测试剂盒检测高糖刺激下caspase-1的活性。
ROS检测
利用DCFH-DA检测高糖刺激下心肌细胞ROS的产生。细胞焦亡检测
利用TUNEL检测心肌细胞核DNA损伤,利用EthD-III染色监测心肌细胞胞膜损伤,利用calcein-AM检测细胞活性。
数据分析
利用SPSS v18.0处理数据,连续变量以均数±标准误来表示。利用单因素方差分析(one-way ANOVA)和Tukey-Kramer post hoc检验比较多组间的连续变量。利用独立样本t检验比较两组间的连续变量。当p<0.05时认为有统计学意义。
研究结果高脂饮食4周可诱导大鼠出现胰岛素抵抗
IPGTT结果显示,与基础饮食相比,高脂喂养大鼠4周后,在IPGTT的Omin、30min、60min、120min时血糖值均明显升高(p<0.05～p<0.01)；血糖曲线下面积(AUC)明显增大(p<0.01)。IPITT实验也呈现类似结果(p<0.05～p<0.01)。
NLRP3炎症小体及IL-1β在糖尿病性心肌病中表达增高
与对照组相比,糖尿病大鼠在STZ注射后第4或第8周起即出现NLRP3、 ASC、caspase-1及IL-1β表达升高(p<0.01)。此外,NLRP3炎症小体各组分及IL-1β的mRNA水平多在STZ注射后第8周时达到顶峰,并持续高表达至第20周(p<0.01)。与control组相比,NLRP3、ASC、pro-caspase-1、活化的caspase-1(caspase-1p20)及活化的IL-1β (IL-1β p17)在DM组中表达升高,且多在STZ注射后第8周达到顶峰,并持续高表达至第20周(p<0.01)。
NLRP3基因沉默不能改善糖尿病引起的代谢异常
在DM组大鼠中,血糖、血脂及胰岛素水平均显著高于control组(p<0.01),胰岛素敏感指数显著低于control组(p<0.01)。利用NLRP3-miRNA进行体内干预后,并不能改善糖尿病引起的代谢异常。基因干预能有效抑制心肌组织NLRP3表达
与vehicle干预组相比,NLRP3-miRNA干预后,心肌组织中NLRP3的mRNA及蛋白水平明显降低(p<0.01)。此外,心肌组织中caspase-1和IL-1β的表达也明显降低(p<0.01)。免疫组化结果显示,在糖尿病大鼠的心肌组织中,caspase-1主要分布在细胞核周围,IL-1β主要呈弥散分布(p<0.01)。NLRP3基因沉默可改善DCM左室功能异常
与control组比较,DM组的LVEF、FS、E/A及E’/A’均降低(p<0.01),LVEDd增大(p<0.01)。将NLRP3基因沉默后,糖尿病引起的LVEF、FS、E/A及E’/A’降低均明显增加(p<0.05-p<0.01), LVED明显降低(p<0.01)。
NLRP3基因沉默改善DCM心肌重构
与control组比较,DM组心肌重构的特点是：心肌细胞DNA损伤增多,心肌细胞超微结构受损,1型及3型胶原表达增加,1型及3型胶原比例增高,间质纤维化增多,心脏向心性肥厚,心身比增大等。
与vehicle组相比,NLRP3基因沉默可明显改善DCM心肌重构,主要表现为：心肌细胞DNA损伤减少,1型及3型胶原表达降低,1型及3型胶原比例降低,间质纤维化减少,身心比降低等。
高糖刺激心肌细胞NLRP3炎症小体及L-1β表达增高
与control组相比,MG或HG刺激24至48小时,可引起H9c2心肌细胞NLRP3, ASC, caspase-1及IL-1β的mRNA表达升高,且此升高呈现糖浓度依赖性(p<0.01)。MG或HG刺激心肌细胞24至48小时,NLRP3炎症小体各组分及IL-1βp17的表达较control组均升高(p<0.01)。HG刺激时,NLRP3、ASC及IL-1β p17在36小时表达量最高,并持续到48小时(p<0.05)。据此,在后续的实验中,我们选择HG为刺激条件,36小时为刺激时间。与基础培养基相比,与MG、HG渗透压匹配的甘露醇并不能引起NLRP3炎症小体及IL-1p表达升高。
高糖刺激诱导H9c2细胞caspase-1表达升高及细胞焦亡
与control及MG组比较,HG组中的caspase-1p20表达明显增高(p<0.05～p<0.01)。免疫荧光结果显示,MG及HG可促进caspase-1在心肌细胞胞质中聚集,同时我们还发现,与control组相比,心肌细胞核DNA的损伤及心肌细胞膜的破坏随着caspase-1表达升高而加重(p<0.01)。
NLRP3在高糖引起的细胞焦亡中发挥重要的作用
我们利用NLRP3-miRNA抑制心肌细胞内NLRP3的表达,探究NLRP3在心肌焦亡中的作用。在确保病毒转染效率达80%以上的前提下,NLRP3-miRNA能够有效抑制靶基因mRNA及蛋白的表达(p<0.01)。随着NLRP3蛋白的抑制,高糖刺激下caspase-1及IL-1β的活性也明显受到抑制(p<0.05-p<0.01)。与此同时,高糖刺激下心肌细胞核DNA损伤及细胞膜受损较之前有明显的改善(p<0.05)。
NF-kB及TXNIP在ROS刺激NLRP3激活中发挥重要作用
与control组相比,MG及HG组H9c2细胞ROS生成增多(p<0.01)。同时,MG及HG组NF-kB p65磷酸化及TXNIP表达较control组增多(p<0.01)。利用NAC预处理细胞能够明显减少细胞内ROS产生(p<0.01)。随着ROS的生成减少, NF-kB p65磷酸化及TXNIP表达减少,同时NLRP3、ASC、pro-caspase-1、caspase p20及IL-1βp17的表达降低(p<0.01)。研究结论
(1)糖尿病状态下,心肌细胞焦亡及心肌纤维化促进心脏结构和功能异常；
(2)在糖尿病早期,心肌组织NLRP3炎症小体即表达升高；
(3)利用NLRP3-miRNA慢病毒实施体内干预后,心肌组织NLRP3的表达被有效抑制；
(4)抑制心肌组织NLRP3表达后,糖尿病引起的心肌结构和功能的异常明显改善；
(5)高糖刺激可诱导心肌细胞ROS生成增多,ROS可促进NLRP3炎症小体表达升高,增高的caspase-1可促进心肌细胞出现细胞焦亡的特征；
(6) NF-kB及TXNIP可能介导了ROS促进NLRP3炎症小体表达增多。
研究背景
糖尿病性心肌病(diabetic cardiomyopathy, DCM)是以舒张功能显著受损和收缩功能轻度下降为特征的心肌病变,是糖尿病病人的主要死亡原因之一。既往研究表明,炎症反应在DCM中发挥着重要作用。因此,对DCM中炎症反应的相关调控机制进行深入研究并寻找相关治疗策略具有重要意义。
促炎因子白介素1β (interleukin-1β, IL-1β)在DCM的发病过程中起到非常重要的作用。既往研究表明,IL-1β的激活主要受炎症小体(inflammasome)调控。炎症小体是位于细胞内的一种多聚蛋白复合体,其家族成员包括：NLRs (nucleotide binding oligomerization domain-like receptors)家族及AIM2(absent in melanoma2)家族。NLRP3(NOD like receptor protein3, NLRP3)是NLRs亚组中的一员。NLRP3炎症小体由NLRP3,含CARD结构域的凋亡相关颗粒样蛋白(apoptosis-associated speck-like protein containing CARD, ASC)和含半胱氨酸的天冬氨酸蛋白水解酶(?)(cysteinyl aspartate specific proteinase-1, caspase-1)组成。近来有研究表明,NLRP3炎症小体在糖尿病、糖尿病肾病及糖尿病视网膜病变等疾病的炎症反应中起到非常重要的作用。此外,在高糖诱导的氧化应激反应中,NLRP3炎症小体通过与硫氧还蛋白相互作用蛋白(thioredoxin interacting/inhibiting protein, TXNIP)直接结合而被激活,继而促发下游的炎症反应。我们前期的实验结果表明,NLRP3炎症小体通过促进炎症反应、心肌损伤及心肌纤维化而加速DCM的发生和发展。既往研究表明丝裂原活化蛋白激酶(mitogen-activatedproteinkinases, MAPKs)在DCM的炎症反应中发挥着重要作用。
瑞舒伐他汀是一种HMG-CoA还原酶抑制剂。研究显示,瑞舒伐他汀除了具有调脂作用外,还具有抗炎、抗氧化及逆转心肌重构的作用。在心肌肥厚、心肌梗死及实验性自身免疫性心肌炎的动物模型中,瑞舒伐他汀的心脏保护作用已经得到证实。但是,具有抗炎作用的瑞舒伐他汀能否影响DCM中异常表达的NLRP3炎症小体及MAPKs通路,目前尚未见报道。瑞舒伐他汀能否通过NLRP3炎症小体及MAPKs通路改善心肌重构,目前尚未见报道。
据此,本课题拟构建DCM大鼠模型,并研究瑞舒伐他汀在DCM发生、发展中的作用,探讨瑞舒伐他汀是否通过调控NLRP3炎症小体及MAPKs通路发挥心肌保护作用,进一步研究NLRP3炎症小体在DCM中潜在临床价值。
研究目的
(1)建立DCM大鼠模型,探讨RSV在DCM进展中的作用；
(2)探讨NLRP3炎症小体及MAPKs通路在RSV对DCM大鼠心肌保护过程中的作用；
(3)探讨RSV抑制NLRP3炎症小体的可能机制。
研究方法
2型糖尿病大鼠模型
选择105只健康雄性Sprague-Dawley (SD)大鼠(山东省中医药大学实验动物中心),平均体重为100-120g。将大鼠随机分为7组：对照组(control组),高脂饮食组(HF组),糖尿病组(DM组),高脂饮食+瑞舒伐他汀10mg/kg组(HF+RSV10mg/kg组),高脂饮食+瑞舒伐他汀15mg/kg组(HF+RSV15mg/kg组),糖尿病+瑞舒伐他汀10mg/kg组(DM+RSV10mg/kg组),糖尿病+瑞舒伐他汀15mg/kg组(DM+RSV15mg/kg组)。Control组给予基础饲料喂养,主要成分为：5%脂质,不添加胆固醇。其余6组给予高脂饮食喂养,主要成分为：16%脂质,0.25%胆固醇。4周后7组大鼠均行腹腔葡萄糖耐量实验(IPGTT)及腹腔胰岛素耐量实验(IPITT)。DM组、DM+RSV10mg/kg组及DM+RSV15mg/kg组出现胰岛素抵抗的大鼠接受单次腹腔STZ注射,剂量为35mg/kg。1周后,检测DM组、DM+RSV lOmg/kg组及DM+RSV15mg/kg组大鼠的空腹血糖(BG)及胰岛素(INS),计算胰岛素敏感指数(ISI), ISI=In[INS×BG)-1]连续两次空腹血糖≥11.1mmol/L,且存在胰岛素抵抗则认为2型糖尿病造模成功。在行STZ腹腔注射8周后,HF+RSV10mg/kg组、HF+RSV15mg/kg、DM+RSV10mg/kg组及DM+RSV15mg/kg分别给予瑞舒伐他汀干预8周。瑞舒伐他汀干预8周后,7组大鼠均实施安乐死。
糖尿病性心肌病模型
糖尿病大鼠造模成功后,继续给予高脂喂养。至STZ注射后8周,检测大鼠心功能,糖尿病大鼠出现左室舒张功能障碍则提示开始进展为DCM。至STZ注射后16周,检测大鼠心脏结构及功能,糖尿病大鼠心脏组织呈现心肌间质纤维化及左室肥厚的,并出现左室收缩及舒张功能障碍,则提示DCM模型已经成功建立。基因沉默技术
根据RNAi干扰技术的原则,设计4对针对大鼠NLRP3基因的miRNA片段,并分别构建4对pcDNA6.2-GW/EmGFP-NLRP3-miRNA质粒,经筛选后,选择沉默效率最高的质粒,随后构建pDONR221-EmGFP-NLRP3-miRNA重组穿梭质粒,最终构建pLenti6.3-EmGFP-NLRP3-miRNA慢病毒载体。
大鼠体内转染慢病毒载体
选择90只健康雄性SD大鼠(山东省中医药大学实验动物中心),平均体重为100-120g。将大鼠随机分为6组：对照组+空载体组(control+vehicle组),糖尿病组+空载体组(DM+vehicle组),糖尿病+瑞舒伐他汀15mg/kg+空载体组(DM+RSV15mg/kg+vehicle组),对照组+NLRP3-miRNA慢病毒干扰组(control+NLRP3-miRNA组),糖尿病组+NLRP3-miRNA慢病毒干扰组(DM+NLRP3-miRNA组),糖尿病+瑞舒伐他汀15mg/kg+NLRP3-miRNA慢病毒干扰组(DM+RSV15mg/kg+NLRP3-miRNA组)。于STZ注射8周后,分别给予6组大鼠vehicle或NLRP3-miRNA慢病毒行体内干预,同时还需给予DM+RSV15mg/kg+vehicle组及DM+RSV15mg/kg+NLRP3-miRNA组大鼠瑞舒伐他汀干预,慢病毒及瑞舒伐他汀的干预时间均为8周,慢病毒注射剂量为1×108TU/只。干预8周后给予大鼠安乐死,心脏取材后行冰冻切片,检测心肌内病毒转染效率。
血清学指标检测
于实验结束时,行血清学检测。大鼠禁食禁水12小时后,取外周静脉血。检测血糖(blood glucose, BG)、血清甘油三酯(triglyceride, TG)、总胆固醇(total cholesterol, TC)、胰岛素(INS),并计算胰岛素敏感指数(ISI)。
心脏彩超
大鼠经10%水合氯醛麻醉后,行经胸心脏彩超,收集LVEF、F/S、E/A以及E’/A’等数据评价大鼠左室功能。
心肌纤维化
对左室心肌组织行Masson染色、天狼猩红染色,使用图像分析软件对染色结果进行分析,评价左室心肌纤维化程度。
透谢电镜
大鼠行安乐死后,立即心脏取材,于左室壁上取1mm3小组织块,立即放入戊二醛固定液中,为透射电镜下观察左室心肌超微结构做准备。
实时定量PCR
取新鲜左室心肌组织,通过实时定量荧光PCR技术检测TXNIP、NLRP3、 ASC、caspase-1、IL-1β、collagen I及collagen III的mRNA相对表达量。Western blot检测
取新鲜左室心肌组织,提取蛋白,利用Western blot检测心肌组织内TXNIIP、 NLRP3、ASC、活化与非活化的caspase-1、活化的IL-1β及磷酸化和非磷酸化的ERK1/2、p38. JNK的蛋白表达量。
统计学分析
利用SPSS v18.0处理数据,连续变量以均数±标准误来表示。利用单因素方差分析和Tukey-Kramer post hoc检验比较多组间的连续型变量。利用独立样本t检验比较两组间的连续变量。当p<0.05时认为有统计学意义。
研究结果
高脂饮食4周可诱导大鼠出现胰岛素抵抗
IPGTT结果显示,与基础饮食相比,高脂喂养大鼠4周后,在IPGTT的0min、30min、60min、120min的血糖值均明显升高(p<0.05～p<0.01)；血糖曲线下面积(AUC)明显增大(p<0.01)。IPITT的结果显示,与基础饮食相比,高脂喂养大鼠4周后,在IPITT的Omin、30min、60min、120min的血糖值均明显升高(p<0.05～p<0.01)；血糖曲线下面积(AUC)明显增大(p<0.01)。
瑞舒伐他汀对糖尿病大鼠血糖及胰岛素水平无明显影响
与Control及HF组大鼠相比,DM组大鼠表现出高血糖、高血脂、低胰岛素水平及胰岛素敏感性指数下降等特点(p<0.01)。与Control组比较,HF也存在代谢异常的情况(p<0.05～p<0.01)。
与未治疗组相比,10mg/kg及15mg/kg瑞舒伐他汀均能降低DM及HF组的胆固醇水平(p<0.05),且15mg/kg瑞舒伐他汀降脂效果优于10mg/kg (p<0.05)。与未用药组相比,10mg/kg或15mg/kg的瑞舒伐他汀均未能逆转DM组及HF组出现的血糖、甘油三酯及胰岛素异常。
瑞舒伐他汀改善糖尿病引起的左室功能异常
DM组的LVEF、FS、E/A及E’/A’均低于Control组,HF组的E/A及E’/A’均低于Control组(p<0.05～p<0.01)。
与未治疗组相比,15mg/kg瑞舒伐他汀可以提高DM组的LVEF、E/A及E’/A’(p<0.05)。10mg/kg瑞舒伐他汀可提高DM组的E'/A'(p<0.05)。
瑞舒伐他汀改善糖尿病引起的心肌重构
与Control组相比,DM组及HF组的心身比(heart weight to body weight, HW/BW)均增加(p<0.05～p<0.01)。糖尿病诱导心肌间质纤维化,促进胶原I及胶原Ⅲ的(?)nRNA表达增加,促进胶原Ⅰ/Ⅲ的增大。与Control组相比,HF组也呈现心肌纤维化增多、胶原(?)nRNA表达增加的现象(p<0.05-p<0.01)。Control组大鼠左室心肌组织透射电镜结果显示：心肌纤维成对称性分布,Z线有序排列在肌节周围,线粒体规则分布在肌纤维附近。DM组电镜结果显示：心肌超微结构混乱无序,部分区域心肌纤维溶解,Z线退化,线粒体肿胀并出现空泡化,线粒体聚集、融合、内部线粒体嵴混乱。HF组电镜结果显示左室心肌组织超微结构亦出现破坏,但破坏程度较DM组轻。
与未治疗组相比,15mg/kg瑞舒伐他汀可降低DM组的HW/BW、左室纤维化面积、胶原Ⅰ/Ⅲ的表达量及比例的异常(p<0.01)。10mg/kg瑞舒伐他汀仅能改善心肌纤维化及胶原Ⅰ的表达异常(p<0.05～p<0.01)。但与未治疗组相比,瑞舒伐他汀未能有效改善HF组HW/BW及胶原Ⅰ及Ⅲ的表达量及比例异常。电镜结果显示,经瑞舒伐他汀干预后,DM组及HF组的左心室心肌组织超微结构较未治疗组有明显改善。
瑞舒伐他汀抑制炎症小体激活
我们检测不同组别大鼠左室心肌组织中TXNIP、NLRP3、ASC、未活化及活化的caspase-1(pro-caspase-1, caspase-1p20)及IL-1β (IL-1β, IL-1β p17)的mRNA及蛋白的表达水平。DM组中TXNIP、NLRP3、ASC、caspase-1及IL-1p的mRNA表达水平均高于control组(p<0.01)。DM组TXNIP、NLRP3、ASC、pro-caspase-1、 caspase-1p20及IL-1β p17蛋白表达水平均高于control组(p<0.05～p<0.01)。与control组相比,HF组的TXNIP、NLRP3炎症小体及IL-1β的mRNA及蛋白表达量明显增高(p<0.05～p<0.01)。
与未治疗组比较,15mg/kg瑞舒伐他汀可降低糖尿病引起的TXNIP、NLRP3炎症小体及IL-1β的mRNA表达(p<0.05-p<0.01)。10mg/kg瑞舒伐他汀也可明显降低TXNIP及IL-1β的mRNA表达水平。与未治疗组比较,瑞舒伐他汀降低了HF组TXNIP、NLRP3、ASC及IL-1p的mRNA表达水平(p<0.05～p<0.01)。与未治疗组相比,15mg/kg瑞舒伐他汀降低糖尿病引起的TXNIP.NLRP3炎症小体、ⅠL-1βp17蛋白表达升高(p<0.05-p<0.01).10mg/kg瑞舒伐他汀只降低糖尿病引起的pro-caspase-1蛋白表达升高(p<0.05)。此外,15mg/kg瑞舒伐他汀的对TXNIP、 ASC、pro-caspase-1、caspase-1p20及IL-1β p17蛋白表达的抑制效果优于10mg/kg(p<0.01)。对HF组进行相同干预,观察到类似结果。
瑞舒伐他汀抑制MAPK信号通路
我们检测不同组别大鼠左室心肌组织中磷酸化及未磷酸化的ERK1/2, p38及JNK的表达水平。与对照组相比,DM及HF组中磷酸化的ERK1/2, p38及JNK表达升高(p<0.05-p<0.01)。与未治疗组比较,10mg/kg及15mg/kg均能降低DM组磷酸化的ERK1/2,p38及JNK(p<0.05-p<0.01)。此外,15mg/kg瑞舒伐他汀抑制磷酸化的ERK1/2的作用优于10mg/kg瑞舒伐他汀。在瑞舒伐他汀干预的HF组中也发现了类似结果(p<0.05～p<0.01)。
检测体内传染后NLRP3沉默效率
行NLRP3-miRNA慢病毒体内转染8周后,检测心肌组织转染效率。与转染空载体组相比,转染NLRP3-miRNA'慢病毒组可明显抑制大鼠心肌组织中NLRP3的mRNA及蛋白水平的表达。此外,随着NLRP3表达的降低,IL-1β p17的表达也降低(p<0.05～p<0.01)。
瑞舒伐他汀通过抑制NLRP3改善糖尿病引起的心功能损害
进行8周的病毒干预后,与空载体组相比,NLRP3-miRNA干预组能显著改善糖尿病引起的LVEF、FS、E/A及E’/A’降低(p<0.05)。此外,与空载体组相比,NLRP3-miRNA干预组能显著提高DM+RSV15mg/kg组的E/A及E'/A'(p<0.05)。
在空载体转染的糖尿病大鼠中,给予瑞舒伐他汀干预能明显改善心脏的收缩及舒张功能(p<0.05)。但与空载体组相比,NLRP3-miRNA干预组中瑞舒伐他汀的这种心脏保护作用并不明显。
瑞舒伐他汀通过抑制NLRP3减轻心肌损伤
与空载体组相比,NLRP3-miRNA可以降低DM组的心身比、间质纤维化、胶原Ⅰ及Ⅲ的(?)nRNA表达及比例(p<0.05-p<0.01)。NLRP3-miRNA还能显著改善心肌超微结构的损伤,包括逆转降解的Z线、紊乱的心肌纤维、肿胀的线粒体及积聚的脂肪微粒。与空载体联合15mg/kg瑞舒伐他汀相比,NLRP3-miRNA联合15mg/kg瑞舒伐他汀能更显著地改善糖尿病引起的心脏结构和功能的改变(p<0.05～p<0.01)。
瑞舒伐他汀的心肌保护作用在空载体干预组中非常明显,但这种保护作用在NLRP3-miRNA干预组中并不明显。
研究结论
(1) NLRP3炎症小体介导了瑞舒伐他汀对DCM的心脏保护作用；MAPKs通路可能参与了瑞舒伐他汀对DCM的心脏保护作用；
(2)长期给予瑞舒伐他汀干预可剂量依赖性地改善糖尿病性心肌病左室重构及收缩和舒张功能障碍；
(3)瑞舒伐他汀的心肌保护作用包括：抑制心肌炎症反应；减轻心肌细胞的肌纤维紊乱、Z线降解、线粒体肿胀融合及脂质沉积等超微结构的破坏；抑制胶原纤维的产生及沉积；
(4) TXNIP可能介导了瑞舒伐他汀抑制NLRP3炎症小体的作用。
研究背景
原发性扩张型心肌病(idiopathic dilated cardiomyopathy, IDCM)是一种病因不明,以心肌受累为基础,以心室扩张及收缩功能受损为特点的疾病。IDCM是继冠状动脉疾病及高血压疾病之后,引起心功能衰竭的第三大常见疾病。临床资料显示, IDCM患者通常需要反复住院治疗,部分患者预后不良。流行病学资料显示,IDCM的治疗已经成为公共卫生医疗系统的负担。因此,寻找与IDCM预后相关、并能提供干预预警信号及临床治疗靶点的因子,已经成为目前研究的热点。
虽然IDCM的病因不明,但研究显示病毒感染、炎症、自身免疫异常及基因突变均能促进IDCM的发生及发展。其中,炎症反应在IDCM的病理生理过程中发挥重要作用。白介素1β (interleukin-1β, IL-1β)是一种重要的促炎因子,其在多种心血管疾病中均表达上调。研究显示,IL-1β的成熟主要受炎症小体调控。炎症小体是一种蛋白复合物,主要位于细胞质内,其家族成员主要包括NLRs (nucleotide binding oligomerization domain-like receptors)家族及AIM2(absent in melanoma2)家族。NLRP3(NOD like receptor protein3)是NLRs亚组中的一员。NLRP3炎症小体由NLRP3、含CARD结构域的凋亡相关颗粒样蛋白(apoptosis-associated speck-like protein containing CARD, ASC)及含半胱氨酸的天冬氨酸蛋白水解酶1(cysteinyl aspartate specific proteinase-1, caspase-1)组成。当NLRP3炎症小体在相关信号的刺激下形成复合物之后,pro-caspase-1将会自我剪切,最终具有活性的caspase-1p10/p20蛋白,活化的caspase-1又可促进IL-1β从非活性的前体pro-IL-1β转化成具有活性的IL-1β。
外周血单核淋巴细胞(peripheral blood mononuclear cells, PBMCs)是血液循环中NLRP3炎症小体的主要来源。在系统性红斑狼疮、风湿性关节炎及系统性硬化等疾病中,PBMCs中NLRP3炎症小体的异常表达在疾病病程中起到重要的作用。近期的研究显示,NLRP3炎症小体在心肌缺血再灌注、急性心肌梗死及心衰等动物模型中表达上调,并促进心肌炎症损伤及纤维化,最终导致心功能异常。我们的前期结果表明,NLRP3炎症小体在糖尿病性心肌病中发挥重要作用。这些研究提NLRP3炎症小体的异常激活与心血管疾病密切相关,PBMCs中NLRP3炎症小体的异常激活在疾病的发展过程中具有重大意义。但目前,IDCM患者PBMCs中NLRP3炎症小体的表达尚未见报道。
据此,本实验将检测IDCM患者PBMCs中NLRP3炎症小体的表达水平,并探究IDCM患者体内NLRP3炎症小体表达水平与患者预后的相关性。
研究目的
(1)探究NLRP3炎症小体在IDCM患者PBMCs中的表达水平；
(2)探究NLRP3炎症小体与IDCM患者临床指标的相关性；
(3)探究NLRP3炎症小体在IDCM患者预后评估中的价值。
研究方法
研究对象
连续收录就诊于山东大学齐鲁医院的IDCM患者54人(IDCM组),收录健康志愿者20人(Control组)。IDCM患者同时患有以下疾病者不能纳入实验：活动期的心肌炎、冠脉疾病、心脏瓣膜病、高血压、急性或慢性炎症性疾病、糖尿病、甲状腺功能异常、痛风及类风湿性关节炎。在患者入院及健康志愿者入组时开始收集临床资料,主要包括：年龄、性别、血压、12导联心电图(12-lead electrocardiography, ECG)、心脏彩超检查结果、纽约心功能分级(New York Heart Association, NYHA)、血常规检查结果、血清N末端B型脑钠肽(N-terminal pro-brain natriuretic peptide, NT-pro BNP)检查结果等。IDCM患者住院期间均得到规范治疗。本实验设计已通过山东大学齐鲁医院伦理委员会审核。
PBMCs分离和纯化
收集研究对象的外周血,并分离出血清及PBMCs,分别存于-80℃备用。实时定量PCR
利用实时定量方法检测研究对象PBMCs中NLRP3、ASC、caspase-1及IL-1β的mRNA表达水平。
流式细胞术
分别利用固定剂和打孔剂处理PBMCs,随后分别使用NLRP3特异性一抗及带有FITC的二抗孵育PBMCs,处理完毕后利用流式细胞术检测PBMCs胞浆中NLRP3的蛋白表达水平。
Western blot
从研究对象PBMCs中提取蛋白,利用western blot方法检测ASC、pro-caspase-1及活化的caspase-1的蛋白表达。
酶联免疫反应(Enzyme-linked immunosobent assay, ELISA)
利用ELISA检测研究对象外周血血清中IL-1β的含量。随访
IDCM患者从山东大学齐鲁医院出院后,将患者因心功能恶化而再入院定为终点事件,对患者进行为期6个月的随访,并记录IDCM患者的再入院的时间。
统计分析
SPSS18.0用于统计分析PASS用于效能计算。连续变量以均数±标准差的形式表示,分类变量以百分数形式表示。t检验用于两组连续变量间统计分析。方差分析用于多组连续变量间统计分析。卡方检验用于分类变量统计分析。偏相关分析用于目标因子与临床指标相关性的统计分析。Cox回归分析用于IDCM患者6个月内再入院的影响因素的统计分析。Kaplan-Meier法用于2组IDCM患者6个月再入院率的统计分析,并绘制时间-效应曲线Log-rank检验用于比较2组IDCM患者6个月再入院率的比较。双侧p<0.05时认为有统计学意义。
研究结果
研究对象基本信息
IDCM组及Control组的临床资料如表1所示。在所研究的参数中,仅左室射血分数(left ventricular ejection farction, LVEF)(p<0.001)、NT-proBNP(p<0.001)及外周血单核细胞计数(p=0.008)在IDCM组及control组中存在统计学差异。
NLRP3炎症小体及IL-1β在IDCM患者PBMCs中表达上调
入院24小时内,在未进行治疗干预前,IDCM组PBMCs中NLRP3炎症小体及IL-1β mRNA表达水平较control组明显升高(3.50±2.02vs.0.90±0.27,3.54±1.90vs.0.80±0.22,3.82±2.79vs.0.88±0.11,3.96士2.21vs.0.73±0.25,p<0.001)。在IDCM组中,出院时NLRP3.ASC.caspase-1及IL-1β的mRNA水平较入院时降低(1.74±1.20vs.3.50±2.02,1.81±1.17vs.3.54±1.90,2.19±1.53vs.3.82±2.79,2.20±1.23vs.3.96±2.21,p<0.001),但仍高于control组(1.74±1.20vs.0.90±0.27,p<0.05;1.81±1.17vs.0.80±0.22,p<0.01；2.19±1.53vs.0.88±0.11,p<0.01;2.20±1.23vs.0.73±0.25,p<0.05).
入院24小时内,在未进行治疗干预前,IDCM组PBMCs中NLRP3.ASC. pro-caspase-1.caspase-1蛋白表达水平及血清中IL-1p蛋白表达水平较control组明显升高(p<0.01)。在IDCM组中,出院时NLRP3.ASC.caspase-1及IL-1p的蛋白水平较入院时均降低(p<0.01),但仍高于control组(p<0.01)。在IDCM组中,PBMCs中pro-caspase-1蛋白表达在入院时和出院时的表达水平未见明显差异。
IDCM患者NLRP3炎症小体的表达水平与临床参数相关
入院时,剔除了年龄、性别等影响因素后,IDCM组PBMCs中NLRP3的mRNA水平与LVEF(r=-0.520,p<0.05).单核细胞数(r=0.613,p<0.05)及NT-pro BNP水平(r==0.514,p<0.05)相关。IDCM组PBMCs中ASC的mRNA水平仅与LVEF(r=-0.334,p<0.05)及单核细胞数(r=0.592,p<0.05)相关。
NLRP3mRNA表达水平是IDCM患者6个月内再入院的独立危险因素
将IDCM患者出院时的LVEF及NLRP3.ASC.pro-caspase-1及IL-1β的mRNA表达水平纳入Cox多因素回归分析模型,结果显示：LVEF(RR:0.001,95%置信区间：0.000-0.128,p<0.001),NLRP3mRNA水平(RR:1.643,95%置信区间：1.193-2.264,p=0.002),IL-1p mRNA水平(RR：1.512,95%置信区间：1.131-2.031,p=0.032)是IDCM患者6个月内再入院的独立危险因素。
NLRP3mRNA表达水平与IDCM预后相关
在IDCM患者中,出院时NLRP3mRNA表达量低的患者再入院的风险低于NLRP3mRNA表达水平高的患者(p<0.05)。
研究结论
(1) NLRP3炎症小体在IDCM患者PBMCs中表达上调；
(2) IDCM患者NLRP3及ASC mRNA表达水平与心功能相关；
(3) IDCM患者中,NLRP3mRNA高表达可提示患者半年再入院风险增加。
Background
Diabetic cardiomyopathy (DCM), an important complication of diabetes, is characterized by consistent diastolic dysfunction and ventricular mass. In addition, DCM is a common cause of heart failure. In the state of diabetes, hyperglycemia-induced reactive oxygen species (ROS) generation is considered to be responsible for progression and development of DCM. The increased ROS could induce a number of cytokine and inflammatory factors, such as nuclear factor-kB (NF-kB), thioredoxin interacting/inhibiting protein (TXNIP), and inflammasome. Although inflammasome was shown to be involved in the pathogenic mechanisms of type2diabetes and its complications, the potential role and regulatory mechanism of inflammasome in DCM has remained largely unexplored.
Inflammasomes are multi-protein platforms that interact with various immune and cell death pathways. Different inflammasomes have been identified, including nucleotide-binding oligomerization domain-like receptors (NLRs) and absent in melanoma2(AIM2). NLRP3, the most extensively studied NLRs, forms a complexes comprised of the apoptosis associated speck like protein (ASC), and the serine protease caspase-1. Upon activation, NLRP3forms a complex with its adaptor ASC, which facilitates the autocatalytic activation of pro-caspase-1and the formation of an active caspase-1p10/20tetramer. The activated caspase-1can process pro-IL-1β into its mature form, which is important in cardiomyocyte apoptosis.
The activation of NLRP3inflammasome consist of two characteristics, including the the up-regulation of NLRP3and the production of caspase-1and IL-1β. Previous research showed that NF-κB could induce NLRP3expression. Thioredoxin interacting/inhibiting protein (TXNIP) can bind NLRP3directly and lead to NLRP3inflammasome assembly in HEK293T cells. However, little is known about whether NF-κB and TXNIP participate in the regulation of NLRP3in hyperglycemia-treated cardiomyocyte.
In addition to resulting in the maturation of IL-1β, activated caspase-1can induce a distinct form of programmed cell death called "pyroptosis". Pyroptosis, a highly inflammatory form of cell death, is dependent on caspase-1activity. The morphology of pyroptosis shares the unique characteristics with both apoptosis and necrosis. As in apoptotic cell, pyroptotic cells incur DNA damage and become positive in the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. As necrosis, pyroptosis results in pore formation in the cell membrane, release of pro-inflammatory cytosolic content, and cell lysis. Therefore, membrane impermeant dyes such as EthD-III stain pyroptotic cells by entering through the pores, but do not stain apoptotic cells. Pyroptosis is initially described in macrophages and dendritic cells infected with different pathogens. Recent studies showed that pyroptosis could also occur in non-myeloid cells induced by non-infectious stimuli. However, it is not clear whether pyroptosis participates in hyperglycemia-induced cardiomyocyte death.
Electron microscopy studies of myocardium in diabetic mice and rats showed that the majority of dying cells had swollen fibril and mitochondria, which are the characteristics of cell swelling and lysis in pyroptosis. Activated caspase-1, the executor caspase of pyroptosis, is found to be elevated in DCM in a rat model. Thus, we hypothesized that pyroptosis, regulated by NLRP3inflammsome, might participate in the pathogenesis of DCM. We also hypothesized that NF-κB and TXNIP might be links between ROS and NLRP3activation.
Objective
(1) We established a type2diabetes rat model and explored the expression level of NLRP3inflammasome in different stages of IDCM;
(2) We induced the cardiac NLRP3silencing by lenti-NLRP3-miRNA, and explore the role of NLRP3in DCM.
(3) We further explored the mechanism of the regulation of NLRP3activation in high glucose-treated H9c2cells.
Methods
Induction of type2diabetes in rats
We randomized120Sprague-Dawley rats into4groups (n=30per group):control, diabetes mellitus (DM), DM+vehicle, DM+NLRP3-miRNA. All rats were housed at22℃with12h light-dark cycles. The control group was fed the basal diet, and the other groups a high fat diet (HF diet,16%fat and0.25%cholesterol).4weeks later, the intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed to determine insulin resistance. In the groups of DM, DM+vehicle and DM+NLRP3-miRNA rats with reduced insulin sensitivity will receive a single intraperitoneal injection of streptozotocin (STZ;35mg/kg). After one week of STZ injection, blood glucose and insulin levels were measured after rats fasted overnight. Insulin sensitivity index (ISI) was calculated by ISI=In[(INS×BG)-1] method. Only rats with blood glucose level>11.1mmol/L and reduced insulin sensitivity were used as type2diabetes rats in the study.
Lenti-NLRP3-miRNA
According to RNAi principle, we designed4pieces of miRNA against rat NLRP3and constructed pcDNA6.2-GW/EmGFP-NLRP3-miRNA plasmid. Then we selected the most effective NLRP3-miRNA plasmid and then synthesized the shuttle pDONR221-EmGFP-NLRP3-miRNA plasmid. Finally, the lentiviral vector of pLenti6.3—EmGFP-NLRP3-miRNA was constructed.
Gene silencing therapy
According to previous studies, diabetic rats showed onset of cardiac dysfunction after8weeks of STZ injection. Our gene silencing treatment occurred after8weeks of STZ injection. The groups of DM+vehicle and DM+NLRP3-miRNA rats in our study received a jugular-vein injection of a total lentivector dose of1×108TU/rat vehicle (empty virus) or NLRP3-miRNA. After8weeks of lentivector injection, rats of DM+vehicle and DM+NLRP3-miRNA were killed. The heart was excised and immediately frozen to determine the transfection efficiency under fluorescence microscope. The age matched rats of control and DM were killed at the same time point (n=10per group).
Cardiac expression pattern of NLRP3inflammasome
For the analysis of NLRP3inflammasome and IL-1β expression, cardiac samples of DM rats were collected at0,4,8,12,16and20weeks (n=3) after STZ injection, and control rats (n=3) were collected at20weeks after diabetic rats received STZ.
Serum measurements
At the end point of gene silencing therapy, serum blood glucose, TG and TC analysis were tested in rats of the4groups.
Enzyme Linked Immunosorbent Assay (ELISA)
At the end point of gene silencing therapy, serum insulin was tested in rats of the4groups.
Echocardiography
Transthoracic echocardiography analysis was performed after the rats anesthetized. And we collected the parameters of left ventricular end diastolic diameter (LVEDd), left ventricular ejection fraction (LVEF), fractional shortening (FS), peak E to peak A ratio (E/A), and early to late diastolic velocity ratio (E'/A').
Histological examination
The hearts were arrested with10%KC1, and heart weight was measured immediately. The images of whole heart were obtained by camera. Paraffin sections from the midpoint of heart were stained with hematoxylin and eosin, and the images were obtained by camera.
Measurement of myocardial fibrosis
Masson's trichrome and Sirius red staining were used for detecting collagen. The fibrotic area fraction was analyzed by automated image analysis.
Transmission electron microscopy (TEM)
About1mm3tissue was obtained from the left ventricle of rats and fixed in buffer. The following preparation of TEM was performed according to the guidance.
Immunohistochemistry
Immunohistochemistry was performed in the paraffin section of the left ventricular tissue of different groups of rats. We used antibodies against caspase-1and IL-1β. Data were analyzed by Image-Pro Plus6.0.
Real-time RT-PCR
Real-time RT-PCR was used to explore the relative expression of NLRP3, ASC, caspase-1and IL-1β. The primer sequences were shown in table1. The relative expression of genes was calculated by the2-ΔΔCT method.
Western blot
Western blot was used to explore the protein levels of NF-κB, TXNIP, NLRP3, ASC, caspase-1, IL-1β, collagen I, collagen III, and β-actin. The protein bands were developed by the use of chemiluminescence, and quantified by densitometric analysis.
TUNEL Assay
Detection of fragmentation of DNA was performed using an ApopTag in situ apoptosis detection kit. Deparaffinized sections were treated with3%hydrogen peroxide in methanol for10min. The sections were washed in PBS twice for5min each. After adding the equilibration buffer, sections were treated with TdT-enzyme at37℃for1h. The sections were then rinsed with PBS and incubated with digoxigenin-conjugated antibodies at37℃for30min. Then sections were colorized with DAB. Finally, the stained sections were examined under microscope.
Cell culture and treatment
H9c2cardiomyocytes were cultured as described previously. During the treatment period, H9c2cells were cultured in normal glucose medium with minimal essential medium for12h, followed by the exposure of control glucose (Ctrl,5.5mM), medium glucose (MG,25mM), high glucose (HG,33.3mM), and osomotic control (OC,27.5mM mannose) for24,36, or48h. In ROS inhibition experiment, cells were treated with HG for36h in the presence of10mmol/L N-acetylcysteine or the relevant vehicle control (PBS).
Lentivirus transfection
H9c2cardiomyocytes plated at a density of1×105/cm2were infected with vehicle (empty lentivirus) or lentivrius-NLRP3-miRNA at10multiplicity of infection (MOI) in serum-free media for2h, followed by incubation with DMEM containing10%FBS for an additional48h before processing. The transfection efficiency was observed by fluorescence microscope. In addition, NLRP3knockdown was assessed in transfected cells, and cells were used only if NLRP3mRNA was decreased by70%compared with vehicle.
Immunofluorescence
Immunofluorescence was used to detect the localization of caspase-1in H9c2cells. We used antibodies against caspase-1. The fluorescence was visualized by confocal microscopy and analyzed by Image-Pro Plus6.0.
Caspase-1activity assay
Caspase-1activity was measured by using colorimetric assay. This assay was designed in consideration of the ability of caspase-1to change acetyl-Tyr-Val-Ala-Asp p-nitroaniline (Ac-YVAD-pNA) into the yellow formazan product p-nitroaniline (pNA).
ROS levels
Intracellular generation of ROS was tested by peroxide-sensitive fluorescent probe2',7'-dichlorofluorescein diacetate (DCFH-DA).
Cell death assay
Cell death was assessed by TUNEL assay and EthD-Ⅲ/calcein AM staining. The virtually nonfluorescent cell-permeant calcein AM is enzymically converted to the brightly fluorescent calcein, producing a bright green fluorescence in live cell. EthD-III enters dead cells, thereby producing an intense red fluorescence in dead cells.
Statistical analysis
Data are expressed as mean±SEM. Differences among experimental groups were analyzed by ANOVA, followed by the Tukey-Kramer post hoc test and independent samples t test. Analysis involved use of SPSS v18.0. Significance was defined as p<0.05.
Result
HF induced insulin resistance
After4weeks of HF diet, IPGTT and IPITT were performed in all groups. In IPGTT, the blood glucose in HF group was significantly higher than that in control group at all of the time points tested except at15min (p<0.05-p<0.01). The AUC for glucose level was higher in HF group than that in control (p<0.01). Similarly, the result observed by IPITT revealed impaired insulin sensitivity in rats with HF diet (p<0.05-p<0.01).
NLRP3inflammasome and IL-1β were activated in DCM
When compared with the control rats, diabetic rats showed elevated mRNA levels of NLRP3, ASC, caspase-1and IL-1β after4or8weeks of diabetes (all p<0.01). Moreover, NLRP3inflammasome components and IL-1β mRNA levels reached highest value after8weeks of diabetes, and stayed at relatively high value until20weeks of diabetes (all p<0.01). The protein levels of NLRP3, ASC, pro-caspase-1, activated caspasep-1(caspase-1p20) and mature IL-1β (IL-1β p17) were higher in DM than control group (all p<0.01). The protein expression of NLRP3, ASC and caspase-1achieved the highest levels after8weeks of diabetes and kept at relatively high levels until20weeks of diabetes (all p＜0.01). The activated caspase-1and IL-1β exhibited the similar protein expression pattern (both p＜0.01).
NLRP3gene silencing improved metabolism abnormalities
The metabolic characteristic of the experimental animals analyzed in this study are shown in Table2. In the DM group, blood glucose, TC, TG and INS were remarkably higher than the control (all p＜0.01). ISI in DM group was lower than control group (p＜0.01). NLRP3gene silencing was insufficient to improve the systemic metabolic disturbance.
Cardiac NLRP3expression was suppressed by gene silencing
After10weeks of NLRP3silencing treatment, transfection efficiency was checked in all groups. As compared with vehicle treatment, NLRP3-miRNA treatment decreased the mRNA and protein levels of cardiac NLRP3(both p<0.01). In addition, the protein levels of activated caspaspe-1and mature IL-1β were lower in NLRP3-miRNA treated diabetic rats than the vehicle treated rats (both p<0.01). Immunohistochemistry revealed that increased caspase-1predominantly localized in perinuclear area, while elevated IL-1β showed diffused distribution pattern in DCM (both p<0.01).
NLRP3gene silencing alleviated left ventricular disfunction in DCM
The results of echocardiography showed that LVEDd of DM rat was larger than control (p<0.01). LVEF, FS, E/A and E'/A'were lower in DM than control p<0.01). When compared with vehicle, increased LVEF, FS, E/A, E'/A'and decreased LVEDd were observed in NLRP3-miRNA group (p<0.05-p<0.01).
NLRP3gene silencing reversed myocardial remodeling
The DM group showed the phenotype of eccentric ventricular hypertrophy, which was characterized with larger chamber size and thicker ventricular wall. The ratio of heart weight to body weight was larger for DM than the control group (p<0.01). TUNEL result showed that the percentage of dead cell was obviously higher in DM group than control (p<0.01). The ultrastructure of cardiomyocyte in control rats showed typical symmetric myofibrils, well-organized Z lines with sarcomeres, and packed mictochontria beside the fibers. In contrast, DM rats showed severe damage of the left ventricular ultrastructure, including destruction of myofibrils, swollen mitochondria with disorganized cristae, excess glycogen lysis, and accumulated lipids. Moreover, the diabetic group showed increased interstitial cardiac fibrosis as compared with the control (p＜0.01). Coincident with cardiac fibrosis, the protein levels of collagen Ⅰ and collagen Ⅲ, and the collagen I-to-III ratio were significantly higher in DM group than the control (all p＜0.01).
With NLRP3gene silencing, heart weight to body weight ratio and cell death were significantly decreased in the NLRP3-miRNA group versus the vehicle group (both p<0.01). NLRP3-miRNA treatment normalized alterations in myofilaments and mitochondria, along with reduced glycogen lysis and lipid accumulation in diabetic rats. Moreover, cardiac fibrosis area, collagen I, collagen III, and the collagen I to III ratio were lower in the DM+NLRP3-miRNA group than vehicle group (all p<0.01).
The increased expression of NLRP3inflammasome and IL-1β were induced by high glucose
Different levels of glucose caused a concentration-dependent increase of NLRP3, ASC, caspase-1and IL-1β in H9c2cells in24to48h (all p<0.01). Except for caspase-1, the mRNA levels of NLRP3inflammasome and IL-1β were increased in a time-dependent pattern with high glucose (both p<0.05-p<0.01). Likewise, the protein levels of all NLRP3inflammasome components and mature IL-1β were increased significantly at high glucose as compared with control and medium glucose in36and48h (all p<0.01). Moreover, the increase of NLRP3, ASC and mature IL-1β protein showed the highest level at36h, and persisted for48h with high glucose incubation (all p<0.05). Thus, we chose high glucose as the stimulation, and chose36h as the stimulated time in subsequent experiments. The expression of NLRP3inflammasome and mature IL-1β in H9c2cells with control glucose or isotonic mannose had no significant change at the same time point tested.
High glucose induced caspase-1activation and cell death in H9c2cells
High glucose significantly increased the level of activated caspase-1as compared with control and medium glucose (p<0.05-p<0.01). Immunofluorescence result showed the accumulation of caspase-1in the cytoplasm of H9c2cells incubated with medium and high glucose. Coincident with caspase-1activation in high glucose, TUNEL result revealed increased cell death with nucleus DNA damage (p<0.01), and EthD-Ⅲ/calceim AM staining showed elevated cell death with damaged cell membrane as compared with control and medium glucose (p<0.01).
NLRP3was involved in cell death induced by high glucose
To explore the role of NLRP3in high glucose-induced cell death of H9c2, we inhibited the expression of NLRP3by lentivirial NLRP3-miRNA. The transfection efficacy of lentivirial NLRP3-miRNA in H9c2cardiomyocytes reached80%. Both the mRNA and protein levels of NLRP3in cells transfected with NLRP3-miRNA were significantly lower than the vehicle (both p<0.01). After inhibiting the expression of NLRP3, the protein levels of activated caspase-1and mature IL-1β induced by high glucose decreased significantly as compared with vehicle (p<0.05-p<0.01). In keeping with these observations, the dead cell rate detected by TUNEL was obviously lower in HG+NLRP3-miRNA than HG+vehicle (p<0.01). To avoid the interference of lentivector fluorescence, we did not use calcein AM in the detection of live cells. The high glucose induced-cell-death rate detected by EthD-Ⅲ were significantly decreased in NLRP3-miRNA treated cells as compared with vehicle (p<0.01).
NF-κB and TXNIP were associated with the ROS-induced NLRP3inflammasome activation
Glucose treatment promoted ROS generation in H9c2. The increase of ROS was dose dependent (all p<0.01). Coincidence with ROS production, the phosphorylation of NF-kB p65was increased in medium and high glucose treated cells. And the expression of TXNIP also exhibited a dose-dependent manner (all p<0.01). Pretreatment of cells with NAC inhibited high glucose-induced increase in intracellular ROS activity (p<0.01). Intriguingly, the levels of NF-kB phosphorylation and TXNIP were also lower in the NAC treated group than the PBS treated group (all p<0.01). In keeping with the decreased expression of NF-kB and TXNIP, the expression of all component of NLRP3, ASC, pro-caspaspe-1, activated caspase-1and mature IL-1β were down-regulated with pretreatment of NAC as compared with PBS (all p<0.01).
Conclusion
(1) High glucose-induced ROS accelerated the expression of NLRP3inflammasome. NF-kB and TXNIP might be involved in the inhibition of RSV on NLRP3inflammasome.
(2) We found the increased expression of NLRP3inflammasome in cardiac tissue at the early stage of diabetes. NLRP3inflammasome accelerated the process of DCM by inducing cardiac inflammation, cardiomyocyte dysfunction and interstitial fibrosis.
(3) The cardiac NLRP3gene silencing was effective by using the lenti-NLRP3-miRNA in vivo.
(4) The NLRP3gene silencing therapy ameliorated the structural and functional disorder in DCM.
Background
Diabetic cardiomyopathy (DCM), a significant contributor to morbidity and mortality in diabetes, is characterized by early-onset diastolic and late-onset systolic dysfunction without coronary artery disease, hypertension, or valvular heart disease. Accumulating evidence indicates that inflammation is an important pathogenic factor in DCM. Therefore, investigation of the inflammatory mechanism is the keystone in the management of DCM.
Previous studies showed that interleukin-1β (IL-1β) is an important proinflammatory cytokine in the development of DCM. IL-1β activation is mainly mediated the protein platform "the inflammasome". Inflammasome is a multiprotein complex which exists in the cytoplasm. Different inflammasomes have been identified, including nucleotide-binding oligomerization domain-like receptors (NLRs) and absent in melanoma2(AIM2). NOD like receptor3(NLRP3) is a member of NLRs. NLRP3inflammasome consists of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1. Recently, several studies suggested that NLRP3inflammasome plays a critical role in the inflammatory process of diabetes and diabetic complications, such as diabetic nephropathy and diabetic retinopathy. Recent observations indicate that thioredoxin interacting/inhibiting protein (TXNIP) can bind NLRP3directly and lead to NLRP3inflammasome assembly under oxidative stress. And our previous research indicated that NLRP3inflammasome could contribute to the process of DCM.
In DCM, mitogen-activated protein kinases (MAPKs) are the most important pathways induced by high glucose levels and are involved in inflammation. Also, activation of MAPKs can contribute to the development of cardiac fibrosis.
Rosuvastatin (RSV), a member of3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, has a number of pleiotropic properties, such as anti-inflammation, antioxidation and cardiac remodeling attenuation. The cardioprotective effect of RSV has been demonstrated in animal models of hypertrophy, myocardial infarction and experimental autoimmune myocarditis. However, the mechanisms by which RSV protects against DCM are incompletely understood.
The main purpose of the present study was to determine whether NLRP3and MAPKs were important targets for RSV in exerting its protective effect on DCM. And we tried to explore the potential value of NLRP3inflammasome in the treatment of DCM.
Objective
(1) We established a type2diabetes rat model and evaluated the effect of RSV on the process of DCM.
(2) We also evaluated whether NLRP3was important target for RSV in exerting its protective effect on DCM.
(3) We tried to explore the mechanism by which RSV inhibites NLRP3inflammasome in the process of DCM.
Methods
Induction of type2diabetes in rats
We randomized105Sprague-Dawley rats (100-120g) into7groups (n=15per group):control, high fat (HF), diabetes mellitus (DM), HF+RSV10mg/kg (10mg/kg, body weight, daily), HF+RSV15mg/kg, DM+RSV10mg/kg and DM+RSV15mg/kg. The control group was fed the basal diet, and the other groups a high fat diet (HF diet,16%fat and0.25%cholesterol).4weeks later, the intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed to determine insulin resistance. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ;35mg/kg) to rats in the groups of DM, DM+RSV10mg/kg and DM+RSV15mg/kg. One week later, blood glucose levels were measured after rats fasted overnight. Only rats with blood glucose level≥11.1mmol/L were used as diabetes in the study. The oral administration of RSV was started in the ninth week after STZ injection, and was continued for8weeks. Rats were weighted before they were killed after8weeks of RSV treatment. The experiments complied with the Animal Management Rule of Chinese Ministry of Health (documentation no.55,2001), and experimental protocols were approved by the Shandong University Animal Care Committee.
Lenti-NLRP3-miRNA
According to RNAi principle, we design4pieces of miRNA against rat NLRP3and constructed pcDNA6.2-GW/EmGFP-NLRP3-miRNA plasmid. Then we selected the most effective NLRP3-miRNA plasmid and then synthesized the shuttle pDONR221-EmGFP-NLRP3-miRNA plasmid. Finally, the lentiviral vector of pLenti6.3—EmGFP-NLRP3-miRNA was constructed.
Gene silencing therapy
Another90Sprague-Dawley rats (100-120g) were randomized into6groups (n=15per group):vehicle+control, vehicle+DM, vehicle+DM+RSV15mg/kg (the dose of15mg/kg was the most effective in our drug experiment), NLRP3microRNA (NLRP3-miRNA)+control, NLRP3-miRNA+DM, and NLRP3-miRNA+RSV15mg/kg. After8weeks of STZ injection, diabetic rats showed onset of cardiac dysfunction. Then all6groups of rats received a jugular-vein injection of a total lentivector dose of1×108TU/rat NLRP3-miRNA or vehicle. RSV supplementation for the2groups was performed at the same time for8weeks. After8weeks of lentivector injection, rats were killed. The heart was excised and immediately frozen to determine the transfection efficiency under fluorescence microscope.
Serum measurements
After rats fasted overnight, we collected jugular blood samples. Serum triglycerides (TG), total cholesterol (TC), insulin (INS) were determined. The insulin sensitivity index (ISI) was calculated by ISI=In[(FINS×FBG)-1] method.
Echocardiography
Transthoracic echocardiography was performed in rats anesthetized with10%chloral hydrate. The derived echocardiography parameters included left ventricular ejection fraction (LVEF), fractional shortening (FS), peak E to peak A ratio (E/A), and early (E') to late (A') diastolic velocity ratio (E'/A').
Measurement of myocardial fibrosis
Masson's trichrome and Sirius red staining were used for detecting collagen. The fibrotic area fraction was analyzed by automated image analysis.
Transmission electron microscopy (TEM)
About1mm3tissue was obtained from the left ventricle of rats and fixed in buffer. The following preparation of TEM was performed according to the guidance.
Real-time RT-PCR
Real-time RT-PCR was used to explore the relative expression of NLRP3, ASC, caspase-1, IL-1β, collagen I and collagen III. The relative expression of genes was calculated by the2-ΔΔCT method.
Western blot
Western blot was used to explore the relative protein expression of NLRP3, ASC, pro-caspase-1, caspase-1, IL-1β, and the phosphorylation of ERK1/2, P38, and JNK. The protein bands were developed by the use of chemiluminescence, and quantified by densitometric analysis.
Statistical analysis
Analysis involved use of SPSS v18.0. Data are expressed as mean±SEM. Differences among experimental groups were analyzed by ANOVA, followed by the Tukey-Kramer post hoc test and independent samples t test. Significance was defined as p<0.05.
Results
HF induced insulin resistance
After4weeks of HF diet, insulin tolerance was determined by IPGTT and IPITT. In IPGTT, the blood glucose in HF group was significantly higher than that in control group at all of the time points tested except at15min (p<0.05-p<0.01). The AUC for glucose level was higher in HF group than that in control one (p<0.01). Similarly, the result observed by IPITT revealed blood glucose in HF group was significantly higher than that in control group at all of the time points tested except at15min (p<0.05-p<0.01). The AUC for glucose level was higher in HF group than that in control one (p<0.01).
RSV had limited effect on systemic metabolic abnormities
DM rats showed more severe hyperglycemia, hyperlipidemia and impaired insulin sensitivity than control and HF rats (both p<0.01). The metabolic state was worse for HF group than control group (p<0.05-p<0.01).
RSV affected TC regulation. TC level was lower in DM rats receiving10and15mg/kg RSV than no treatment (p<0.05). TC level was lower with HF+RSV15mg/kg group than HF group (p<0.05). Neither10nor15mg/kg RSV improved glucose dysregulation, TG level disturbance, insulin imbalanee, or impaired insulin sensitivity in DM and HF rats.
RSV alleviated DM-induced left-ventricular dysfunction
LVEF, FS, E/A and E'/A were lower with DM than control. E/A and E'/A'was lower with HF group than control (p<0.05-p<0.01).
After treatment, LVEF was improved with DM+RSV15mg/kg as compared with DM alone, with no significant improvement with DM+RSV10mg/kg, or HF+RSV10or15mg/kg over DM and HF, respectively (p<0.05). RSV treatment did not significantly prevent FS in DM or HF rats. For diastolic function, DM+RSV15mg/kg increased both E/A and E'/A'as compared with DM alone (both p<0.05). DM+RSV10mg/kg also improved E'/A'(p<0.05).
RSV alleviated myocardial remodeling
The ratio of heart weight to body weight was larger for DM and HF than the control group (p<0.05-p<0.01). Diabetes induced interstitial fibrosis, increased collagen mRNA expression, and increased the collagen I to III ratio (p<0.05-p<0.01). Similarly, HF rats showed increased fibrosis and collagen content as compared with control rats (p<0.05-p<0.01). TEM revealed typical symmetric myofibrils, well-organized Z-lines with sarcomeres, and packed mitochondria beside fibers in control rats. In contrast, DM rats showed severe damage of the left ventricular ultrastructure, including destruction of myofibrils, degenerated Z-lines, swollen mitochondria with vacuoles and disorganized cristae, coalescence of irregular mitochondria, and accumulated lipids. The HF rats showed moderate destruction of the left ventricular ultrastructure.
RSV15mg/kg treatment decreased heart weight to body weight ratio, fibrosis area and collagen disorders in DM rats (all p<0.01), but had little effect on HF rats. RSV10mg/kg restored only the disordered fibrosis and collagen I content in DM rats (p<0.05-p<0.01). After treatment with RSV, the diabetic and HF rats showed improved cardiac ultrastructure.
RSV inhibited NLRP3inflammasome activation
We investigated the mRNA and protein levels of TXNIP, NLRP3, ASC, pro-caspase-1, activated caspase-1and activated IL-1β in left ventricular tissue of rats. The mRNA expression of TXNIP, NLRP3, ASC, caspase-1and IL-1β was significantly higher in DM than control rats (all p<0.01). The protein levels of TXNIP, NLRP3, ASC, pro-caspase-1, activated caspase-1and maturation of IL-1β were higher in DM than control rats (p<0.05-p<0.01). Similarly, the mRNA and protein levels of TXNIP, NLRP3inflammasome and IL-1β were higher in HF than control rats (p<0.05-p<0.01).
RSV15mg/kg suppressed the increased mRNA levels of TXNIP, NLRP3inflammasome and IL-1β induced by diabetes (p<0.05-p<0.01). The mRNA levels of TXNIP and IL-1β were lower with DM+RSV10mg/kg than DM alone (both p<0.05). Except for caspase-1mRNA, similar reduced levels were observed in HF with RSV treatment than HF alone and control group (p<0.05-p<0.01). Protein levels of TXNIP, NLPR3inflammasome and mature IL-1β were lower with DM+RSV15mg/kg than DM alone (p<0.05-p<0.01). DM+RSV10mg/kg only decreased pro-caspase-1protein level as compared with DM alone (p<0.05). In addition, DM+RSV15mg/kg decreased protein levels of TXNIP and ASC, total and activated caspase-1, and activated IL-1β as compared with DM+RSV10mg/kg (all p<0.01). Similar results were observed in RSV-treated HF groups (p<0.05-p<0.01).
RSV inhibited the phosphorylation of MAPK signaling pathways
We further investigated the activation of MAPK signaling pathways. DM rats showed the greatest activation of ERK1/2, p38and JNK (p<0.05-p<0.01). The HF group showed increased phosphorylation of MAPKs as compared with control group (p<0.05-p<0.01).
The phosphorylation of MAPKs was suppressed in RSV-treated DM rats as compared with DM alone (p<0.05-p<0.01), and RSV15mg/kg was better than10mg/kg in inhibiting ERK1/2phosphorylation (p<0.01). Similar results were found in RSV-treated HF rats (p<0.05-p<0.01).
Detection of cardiac NLRP3expression with gene silencing
After8weeks of NLRP3silencing treatment, transfection efficiency was checked in all groups. As compared with vehicle treatment, NLRP3-miRNA treatment decreased the mRNA and protein levels of cardiac NLRP3and mature IL-1β (p<0.05-p<0.01).
RSV alleviated diabetes-induced cardiac dysfunction by inhibiting NLRP3expression
After8weeks of NLRP3-miRNA treatment, LVEF, FS, E/A and E'/A'were improved in the DM rats as compared with vehicle treatment (all p<0.05). NLRP3miRNA treatment increased E/A and E'/A'in DM+RSV15mg/kg rats as compared with vehicle treatment (both p<0.05).
In vehicle-treated rats, diabetes-induced severe systolic and diastolic dysfunction was ameliorated with RSV supplementation (all p<0.05). The cardioprotective effect of RSV in diabetes was abrogated more in NLRP3-miRNA-treated rats than vehicle-treated rats.
RSV reversed diabetes-induced myocardial disorder with inhibited NLRP3
In DM rats, NLRP3-mi
引文
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