【作者】 赵宇光；

【导师】 李薇；

【作者基本信息】 吉林大学 ， 内科学， 2011， 博士

【摘要】 背景:糖尿病能够引起机体多种组织器官损伤甚至功能衰竭。随着人们生活水平的提高,肥胖人群逐年增加,2型糖尿病的发病率逐年上升,其并发症糖尿病心肌病(DCM)日益受到重视。高脂能够诱导心肌细胞发生凋亡,而心肌细胞凋亡在DCM的发病机制中占有重要地位,然而,有关高脂诱导心肌细胞凋亡的具体机制目前尚未彻底阐明。有关基质细胞衍生因子-1(stromal cell derived factor-1,SDF-1)的很多研究证明,SDF-1参与调节许多重要的生物过程,包括心脏和神经元发育、干细胞动员、血管新生、肿瘤发生及转移等。SDF-1调节这些迥异的生物进程是通过其经典的细胞表面特异性趋化因子受体4(CXCR4)实现的。但近年来研究发现,CXCR4并不是SDF-1在细胞表面的唯一受体,除CXCR4外还存在CXCR7,但CXCR7并不能广泛表达,其与CXCR4相比具有不同的生物学功能。关于SDF-1对心脏系统的影响,既往研究主要集中于其动员干细胞至心脏损伤的部位,并通过血管新生修复心脏损伤,但SDF-1对脂毒性所致的心脏细胞损伤是否具有直接保护作用目前尚未有相关报道。目的:本研究在体外用饱和脂肪酸棕榈酸酯(palmitate,Pal)模拟2型糖尿病时机体内的高脂环境,来处理大鼠胚胎心肌细胞(H9c2),一方面拟阐明脂毒性诱导心肌细胞发生凋亡的机制;另一方面,探讨SDF-1β对高脂诱导的H9c2细胞凋亡是否具有保护作用,若具有保护作用,拟揭示其分子机制。方法:1. Pal诱导H9c2细胞凋亡及机制研究(1)首先用Pal处理H9c2细胞,做剂量效应及时间效应实验,分别用western-blot检测cleaved-caspase3和细胞死亡ELISA试剂盒检测DNA链断裂来观察Pal诱导细胞的凋亡效应,从而选择出Pal诱导H9c2细胞凋亡的最适剂量及最佳处理时间。(2)用western-blot的方法检测Pal诱导细胞凋亡不同信号通路上的关键蛋白(p-p53、p53、Bax、Bcl-2、AIF、GRP78、caspase12、CHOP and caspase8)进而确定Pal诱导H9c2心肌细胞凋亡的具体途径。(3)用western-blot的方法检测3-NT,观察Pal能否引起H9c2细胞的硝化损伤。(4)用western-blot的方法检测不同的阻滞剂或清除剂(apocynin、MnTMPyP、L-NAME、urate、4-PBA)对Pal诱导细胞硝化损伤、内质网应激(endoplasmic reticulum stress,ERS)及凋亡的影响,并明确硝化损伤、ERS及凋亡这三种现象之间是否有内在联系。2. SDF-1β预防Pal诱导的心肌细胞凋亡及机制研究(1)用western-blot检测cleaved-caspase3和细胞死亡ELISA试剂盒检测DNA链断裂,拟观察不同剂量的SDF-1β对Pal诱导的细胞凋亡是否具有预防保护作用,从中选择出具有保护作用的最适剂量进而做机制研究。(2)用western-blot的方法观察不同的阻滞剂(PI3K阻滞剂:LY294002;ERK阻滞剂:U0126;p38 MAPK阻滞剂:SB203580;AMPK阻滞剂:compound C;SDF-1/CXCR4拮抗剂:AMD3100)和siRNA(p38βMAPK siRNA、CXCR7 siRNA、IL-6 siRNA)能否阻滞SDF-1β对心肌细胞的预防保护作用;再用激动剂(AMPK激动剂:AICAR)或重组IL-6观察是否同样能够起到预防心肌细胞凋亡的作用。进而分析具体哪条信号转导通路参与SDF-1β保护Pal诱导心肌细胞凋亡的过程。结果:1. Pal诱导H9c2细胞凋亡的最佳剂量和时间点分别是62.5μM和15 h。2.经Pal处理后,H9c2细胞的GRP78、caspase12、CHOP表达升高,而Bax/Bcl-2比值、p-p53/p53比值、AIF和caspase8表达没有发生变化,说明Pal不通过线粒体途径和死亡受体途径、而通过ERS途径引起细胞凋亡。3. Pal能够使3-NT表达升高,说明Pal能够引起心肌细胞硝化损伤。4.用不同阻滞剂或清除剂分别阻断硝化损伤或ERS这部分实验证实Pal诱导的H9c2细胞硝化损伤和ERS均发生于凋亡之前,且ERS的发生是硝化损伤途径依赖性的。5. SDF-1β的剂量效应实验证明其在100 nM时能够显著抑制Pal诱导的心肌细胞凋亡。6. SDF-1β预防Pal诱导心肌细胞凋亡的作用是通过激活p38 MAPK实现的,用p38阻滞剂SB203580能够完全阻断SDF-1β的心肌保护作用。p38βsiRNA同样能够完全抑制SDF-1β的心肌保护作用,进而说明SDF-1β预防心肌细胞凋亡的作用是通过激活p38β亚型实现的。7. SDF-1β的心肌保护作用是通过激活AMPK实现的,SDF-1β激活p38 MAPK的作用是AMPK依赖性的,AMPK阻滞剂compound C能够阻断SDF-1β的保护作用,而AMPK激动剂AICAR同SDF-1β一样,能够预防Pal引起的心肌细胞凋亡。8. SDF-1β能够促进H9c2细胞分泌IL-6进而保护Pal诱导的细胞凋亡,这种效应是通过激活AMPK及p38 MAPK实现的。9. IL-6 siRNA能够阻断SDF-1β的心肌保护作用,而重组IL-6能够预防Pal诱导的H9c2细胞凋亡。10. H9c2细胞表面存在CXCR7受体,SDF-1β保护Pal诱导心肌细胞凋亡的作用不是通过其细胞表面受体CXCR4、而是通过CXCR7实现的。这种效应能够被CXCR7 siRNA所阻断,同时,CXCR7 siRNA还能够阻断SDF-1β对AMPK及p38 MAPK的激活作用。结论:1. Pal引起H9c2细胞凋亡是通过诱导细胞硝化损伤和ERS实现的;凋亡的发生是ERS依赖性的,而ERS的发生是硝化损伤依赖性的。2. SDF-1β能够预防Pal引起的心肌细胞硝化损伤、ERS和凋亡。3. SDF-1β的心肌保护作用是通过与细胞表面受体CXCR7结合,进而激活AMPK和p38 MAPK,促进IL-6生成来实现的。创新点:1.阐明Pal诱导心肌细胞凋亡的分子机制,即Pal通过诱导细胞硝化损伤和ERS进而引起心肌细胞凋亡。2.证明SDF-1β对Pal诱导的心肌细胞凋亡具有保护作用。3.证明H9c2细胞表面有CXCR7表达。4.阐明SDF-1β预防Pal诱导心肌细胞凋亡的分子机制,即通过与CXCR7结合,进而激活AMPK和p38 MAPK,促进IL-6生成来实现的。更多还原

【Abstract】 Background:Diabetes can cause damages or even failures on many organs and tissues. With the improvement of living standard, more people suffered obesity, and morbidity of type 2 diabetes increase gradually by year, diabetic cardiomyopathy (DCM), as one of its major complications has been received more attentions than ever before. Lipotoxicity can cause cardiac cell death which plays a critical role in the pathogenesis of DCM, but its exact mechanisms remain largely unknown.The chemokine stromal cell derived factor (SDF-1) regulates many essential biological processes, including cardiac and neuronal development, stem cell mobilization, neovascularization, angiogenesis, tumorigenesis and metastasis. It is generally believed that SDF-1 mediates these many disparate processes via its classic specific cell surface receptor known as chemokine receptor 4 (CXCR4). However, increasing evidences indicate the existence of an alternate receptor, CXCR7, which does not widely express and has different biological functions compared to CXCR4.Previous studies regarding the effect of SDF-1 on cardiac system have mainly been focused on the homing of stem cells into the damaged heart and its role in recovering the damaged heart via its angiogenesis action. However, whether SDF-1 has direct protection of the cardiac cells from lipotoxic effect has not been approached.Objectives:By using palmitate (Pal), as one of saturated free fatty acid, to mimic the environment of hyperlipidemia under the condition of type 2 diabetes, we have several objectives for the present study to be determined: First, to elucidate the possible mechanisms of Pal-induced cardiac cell death. Second, to observe whether SDF-1βcould protect the cardiac cells from Pal-induced apoptosis, and if so, to explore the possible mechanisms. To these ends, in vitro cultured cardiac H9c2 cells, in combination of pharmacological inhibitors and siRNA approaches were used.Methods:1. Pal-induced H9c2 cell death and its mechanisms(1) H9c2 cells were exposed to Pal for dose and time course studies. Cell death ELISA assay and western-blot assay were used to detect DNA fragmentation and cleaved-caspase3 separately.(2) To determine which apoptosis pathway involved in Pal-induced cardiac cell death, we examined the critical proteins of relevant apoptosis pathway (p-p53, p53, Bax, Bcl-2, AIF, GRP78, caspase12, CHOP and caspase8) by western-blot assay.(3) 3-NT accumulation was determined by western-blot assay to observe whether Pal induced nitrosative damage or not.(4) Different inhibitors or scavengers (apocynin, MnTMPyP, L-NAME, urate and 4-PBA) were used to determine 3-NT formation and clarify the relationship of nitrosative damage, ER stress and apoptosis.2. The preventive effect of SDF-1βon Pal-induced cardiac cell death and possible mechanisms(1) Dose effect of SDF-1βon Pal-induced cell death was determined by DNA fragmentation via cell death ELISA assay and cleaved-caspase3 via western-blot assay.(2) Different pharmacological inhibitors or siRNAs (PI3K inhibitor: LY294002; ERK inhibitor: U0126; p38MAPK inhibitor: SB203580; AMPK inhibitor: Compound C; SDF-1/CXCR4 inhibitor: AMD3100; IL-6 siRNA; p38βMAPK siRNA; CXCR7 siRNA) were used to observe whether they can block the protective effect of SDF-1βor not. Furthermore, specific activator (AMPK activator: AICAR) or stimulant (rIL-6) were also used to observe whether they had same protective effect as SDF-1βon Pal-induced apoptosis.Results:1. The best dosage and time point for Pal-induced H9c2 cell apoptosis is Pal at 62.5μM for 15 h.2. The expression of GRP78, CHOP and caspase12 were up-regulated after Pal treatment; Bax/Bcl-2 ratio, p-p53/p53 ratio, AIF and caspase8 were no changes after Pal treatment. These results suggest ER stress pathway, but not mitochondrial pathway or death receptor pathway was involved in Pal-induced cardiac cell death.3. Pal can induce cardiac cell nitrosative damage due to the increase of 3-NT accumulation.4. Pal-induced cardiac cell apoptosis was through nitrosative damage-mediated ER stress production.5. SDF-1βat 100nM can significantly inhibit Pal-induced cell apoptosis.6. The preventive effect of SDF-1βon Pal-induced cardiac cell death was through p38 MAPK activation. P38 inhibitor (SB203580) can totally block the protective effect of SDF-1β, and specific p38βsiRNA had same effect as SB203580.7. The preventive effect of SDF-1βon Pal-induced cardiac cell death was through AMPK activation, and p38 MAPK activation was AMPK dependent. AMPK inhibitor (compound C) can abolish SDF-1βprotective effect, whereas AMPK activator (AICAR) has same protective effect as SDF-1β.8. SDF-1βcan stimulate IL-6 production, and this effect was AMPK and p38 MAPK dependent.9. Specific IL-6 siRNA can abolish protective effect of SDF-1β, whereas recombinant IL-6 has same protective effect as SDF-1β.10. CXCR7 exists on the surface of H9c2 cardiac cells. The preventive effect of SDF-1βon Pal-induced cell death is mediated via CXCR7 but not CXCR4, specific CXCR7 siRNA can totally abolish SDF-1βprotective effect through inhibiting AMPK-mediated p38 MAPK activation. Conclusions:1. Exposure of H9c2 cells to Pal causes nitrosative damage and ER stress related apoptotic cell death.2. SDF-1βcan protect cardiac cells from Pal-induced nitrosative damage, ER stress and cell death.3. The cardiac protective effect of SDF-1βis mediated through its interaction with CXCR7, and activation of AMPK and p38 MAPK-mediated IL-6 production.Innovations:1. We elucidated the mechanisms of Pal-induced H9c2 cardiac cell death, i.e. Pal causes nitrosative damage and ER stress related apoptotic cell death.2. We demonstrated the protective effect of SDF-1βon Pal-induced cardiac cell death.3. We demonstrated that CXCR7 exists on the surface of H9c2 cells.4. We elucidated the mechanisms of SDF-1β’s preventive effect on Pal-induced cardiac cell apoptosis through its interaction with CXCR7, and activation of AMPK and p38βMAPK-mediated IL-6 production.更多还原