IL-1β及ox-LDL对心室重构的影响及他汀药物的干预作用
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摘要
急性心肌梗死(AMI)后的心室重构是心室扩张、心功能不全甚至死亡的主要原因,它是一个复杂的动态的而且是时间依赖性的过程。心室重构不仅影响心肌细胞,使心肌细胞凋亡、坏死、肥大、延长,还表现心肌纤维化,即细胞外基质(ECM)的合成和降解的动态平衡被打破,心肌间质和心肌内小血管周围胶原过度增多、沉积,并且出现炎性细胞浸润。导致心肌纤维化形成的病理生理机制与心脏压力负荷过重以及炎症、肾素-血管紧张素-醛固酮系统(RAS)等因素有关。上述因素作用于细胞外基质,使细胞外基质胶原的合成减少、分解增多以及成分改变,促使心室的扩张,影响心室的结构和功能,最终导致心脏功能的减弱甚至心室破裂。
心脏中75%的细胞是非心肌细胞,其中产生细胞外物质的成纤维细胞占90%-95%,由此可见成纤维细胞功能的改变是影响心室重构的重要因素。心肌成纤维细胞产生大部分基质大分子包括胶原和结构蛋白,主要是Ⅰ和Ⅲ型胶原,形成胶原网络结构,维持心脏的几何形态,并使心脏成为一个具有高度协调的舒缩器官。而有序的细胞外基质的降解是心脏生长、重构、修复的关键,其主要是由基质金属蛋白酶(MMPs)和其抑制剂(TIMPs)之间的相互作用来完成的。MMPs是一组以锌离子为辅助因子的蛋白酶家族,心脏的MMPs主要由成纤维细胞、内皮细胞、炎症细胞等细胞产生。MMPs在心脏基质的重构中发挥重要作用,一种MMPs可以直接降解某种或几种细胞外基质,也可以通过激活其它类型的MMPs而产生作用,从而形成瀑布效应;在心脏中,MMPs不仅对心脏基质成分起降解作用,而且调节胶原蛋白的合成,使正常胶原蛋白被升高的MMPs降解并被缺乏连接结构的纤维性间质所取代,使心肌纤维化增加。MMP-2、MMP-9是其中重要的组成成分,起降解变性的胶原纤维、其他类型的胶原和弹力蛋白。一旦MMPs结合到胶原纤维并开始作用时,它们能持续的作用直至所有的胶原都降解完毕或者被TIMPs抑制。AMI时MMPs/TIMPs的失衡,使得MMPs增加或者TIMPs的减少,或者MMPs/TIMPs比率增高,促使不良重构的发生。
动脉粥样硬化(AS)是导致AMI发生的根本原因。目前认为其发生发展是一个动态过程,涉及感染、炎症、免疫多种机制。而氧化低密度脂蛋白(ox-LDL)始终贯穿其中,它被巨噬细胞吞噬,转化成泡沫细胞,形成脂质斑块。不稳定的脂质斑块破裂导致血栓形成,发生AMI。发生AMI后,血液中的IL-1、IL-6、血小板源生长因子(PDGF)、肿瘤坏死因子α(TNF-α)等促炎因子的浓度大幅度升高,可促进心肌细胞的肥大和凋亡,同时它们还可能通过影响了细胞外基质,从两方面同时影响了心室的不良重构。IL-1β是AMI时升高的众多的促炎因子之一,它具有免疫调节活性作用,对不同来源的细胞有不同的生物学作用。IL-1β能通过细胞表面的白介素受体(IL-1R)减少胶原的合成及MAPK途径来刺激心肌成纤维细胞的迁移,从而参与心室的不良重构。然而对于AMI发生后,ox-LDL是否同时促进心室的重构,两者之间是否协同促进心室重构的发生,目前研究不多。已有研究表明ox-LDL可以使高同型半胱氨酸血症的患者外周血单核细胞的MMP-9的表达增高,而正常人MMP-9表达不增高。ox-LDL还可以通过羰基蛋白形成和激活表皮生长因子受体信号途径来促使MMP-2生成增多,降解胶原。
由此可见,AMI后的心室重构是个复杂的过程,在此过程中,ox-LDL、IL-1β等多种因素介导的细胞外基质降解和合成的动态平衡得以打破,使得正常结构的胶原组织降解,合成的无序的胶原纤维,使得心室重构的发生,最终导致心室功能不良。AMI后的抗重构治疗的目的是防止、限制甚至逆转不良的重构,阻止心室扩张、功能失调、无能和死亡。其中一个重要的方面就是在重构过程中保护细胞外基质。由于AMI后心室重构从心肌梗死的愈合、修复直至数月甚至数年,在此过程中,机械变形和增加的室壁压力持续作用于梗死区和非梗死区,促进心室的扩张、纤维化,故治疗的起始点和持续时间是关键所在,需要早期和长期的抗重构治疗。而一旦已经发生心衰,尽管心室重构可以停止,但心衰改善不明显。目前抗心肌重构治疗的药物主要有ACEI、ARB、醛固酮拮抗剂、β-受体拮抗剂等等。新近发现他汀类药物除调脂外,尚有抗炎、抗氧化、减少炎性递质水平、抑制凝血因子激活、抑制自身抗体形成、抑制内皮细胞粘附分子和组织因子表达、降低斑块中T细胞和巨噬细胞活性以及维持斑块稳定等作用。为此应用他汀类药物早期干预,是否可以延缓或者改善心室的重构,从而改善MI后患者的死亡率呢?我们用IL-1β联合ox-LDL刺激心肌成纤维细胞,观察MMPs的改变及胶原合成的变化,并用辛伐他汀(Sim)进行干预,为临床进行抗心室重构治疗奠定一定的基础。
本实验分为三个部分,首先通过~3H-脯氨酸掺入法、明胶酶谱法、蛋白质免疫印迹法和逆转录聚合酶链反应等方法,检测IL-1β对体外培养的心肌成纤维细胞胶原合成及促进胶原降解的基质金属蛋白酶MMP-2、MMP-9表达的影响。接着于体外提取并氧化低密度脂蛋白,并采用上述方法检测ox-LDL对心肌成纤维细胞胶原合成及MMP-2、MMP-9表达的影响。最后联合IL-1β和ox-LDL作用于成纤维细胞,并应用Sim干预上述因子,探讨Sim对上述因子作用下的心肌成纤维细胞增殖、胶原的合成和降解的影响。结果分述如下:
第一部分:IL-1β对心肌成纤维细胞合成和降解胶原的影响
方法:
体外培养乳鼠心肌成纤维细胞。加入IL-1β(0.01 ng/ml、0.1 ng/ml、1 ng/ml、10 ng/ml、100ng/ml)干预24h后,观察细胞形态,MTT法观察细胞增殖情况,~3H-胸腺嘧啶核苷(~3H-TdR)掺入法观察细胞DNA合成,~3H-脯氨酸(~3H-Pro)掺入法观察胶原的合成。明教酶谱法观察MMPs活性,Western-blot观察MMP-2和MMP-9的蛋白表达,RT-PCR观察MMP-2和MMP-9的mRNA表达。
结果:
1.IL-1β作用于心肌成纤维细胞24h后,0.01 ng/ml IL-1β对细胞增殖、DNA的合成没有明显影响。0.1ng/ml IL-1β可显著性抑制细胞活性及减少~3H-TdR掺入量(P＜0.05)。随着浓度增高,上述抑制作用逐渐增强,1～100 ng/ml较0.1ng/ml组抑制作用明显增强(P＜0.05)。
2.0.01ng/ml IL-1β干预24h后对CFs合成胶原无明显影响。随着浓度的升高,0.1ng/ml IL-1β即可显著性抑制细胞胶原的合成(P＜0.05)。1～100 ng/ml IL-1β抑制细胞胶原合成的作用更强(P＜0.01),但10～100 ng/ml之间作用无显著性差异。
3.小剂量的IL-1β干预后,MMP-2的活性即升高,并呈浓度依赖性增高趋势。同时MMP-9的活性也显著性增高,与MMP-2不同,0.01～1ng/ml IL-1β作用后MMP-9的升高程度无显著性差异。
4.与空白对照组比较,IL-1β干预后可显著性升高MMP-2蛋白表达水平,按0.01～0.1ng/ml分别升高1.35倍和1.56倍(P＜0.05),1～100ng/ml分别升高2.15倍、2.34倍、2.41倍(P＜0.01)。MMP-9蛋白表达也显著性升高,按0.01ng/ml即可显著性升高MMP-9蛋白表达1.38倍(P＜0.05),0.1～100ng/ml分别2.37倍、2.56倍、2.60倍、2.67倍(P＜0.01)。
5.IL-1β作用于CFs 24小时后能浓度依赖性增加MMPs mRNA表达。与空白对照组比较,MMP-2 mRNA表达能显著性增加,按0.01ng/ml～100ng/ml不同浓度分别升高1.81倍(P＜0.05)、2.17倍、2.18倍、2.27倍、2.31倍(P＜0.01)。MMP-9 mRNA表达也能显著性升高,分别1.36倍(P＜0.05)、1.49倍、1.50倍、1.51倍、1.52倍(P＜0.01)。
结论:
一定浓度的IL-1β抑制CFs的增殖及DNA的合成,并可能通过该作用降低胶原的合成,同时IL-1β还能增加MMPs的活性,起到降解胶原的作用。上述作用呈浓度依赖性,与心肌梗死面积越大,炎症因子浓度越高,心室重构越严重相符合,提示IL-1β在心室重构过程中起一定的作用。
第二部分:氧化低密度脂蛋白(ox-LDL)对心肌成纤维细胞合成和降解胶原的影响
方法:分离并氧化低密度脂蛋白。体外培养乳鼠心肌成纤维细胞,加入ox-LDL(10、20、50、100μg/ml)干预24 h,观察细胞形态,~3H-TdR掺入法观察细胞DNA合成,~3H-Pro掺入法观察细胞胶原的合成,明胶酶谱法测定基质金属蛋白酶MMP-2、MMP-9活性,Western-blot检测MMP-2和MMP-9蛋白表达水平,RT-PCR检测MMP-2、MMP-9的mRNA表达。
结果:
1.ox-LDL对细胞~3H-TdR掺入率的影响与其浓度密切相关。用ox-LDL干预24h后可见10μg╱ml和20μg╱ml即显著性增加细胞~3H-TdR掺入率(P＜0.05),随着浓度的增高(50～100μg/ml),掺入率也随之增高(P＜0.01),100μg/ml的增强作用最强。
2.ox-LDL对细胞~3H-Pro掺入率的影响也与其浓度密切相关。用ox-LDL干预24h后可见10μg/ml即显著性减少细胞~3H-Pro掺入率(P＜0.05),而随着浓度的增高(20～100μg/ml),掺入率也迅速降低(P＜0.01),100μg╱ml的抑制作用最强。
3.ox-LDL干预24小时后能增加条件培养基中MMP-2和MMP-9的活性。与空白对照组相比,ox-LDL干预后可显著性增加细胞MMP-2活性(P＜0.05),但各剂量组之间无显著性差异。10μg/ml ox-LDL作用于CFs后,MMP-9的活性轻微增高,但无显著性差异;随浓度的升高,MMP-9的活性也显著性增高(P＜0.05)。
4.与空白对照组比较,10μg/ml的ox-LDL即能显著性升高MMP-2蛋白表达1.17倍(P＜0.05),而20～100μg╱ml作用更强并呈浓度依赖性,分别升高1.32倍、1.42倍、1.53倍(P＜0.01)。10μg/mL ox-LDL不影响MMP-9蛋白的表达;20μg/ml ox-LDL能使MMP-9蛋白表达显著性升高1.17倍(P＜0.05),而50～100μg/ml ox-LDL作用更强,分别使MMP-9蛋白升高1.39倍和1.52倍(P＜0.01)。
5.10～20μg/ml ox-LDL使MMP-2 mRNA表达分别升高1.01倍、1.07倍。而50～100μg/ml ox-LDL则使MMP-2 mRNA表达分别升高1.21倍、1.33倍,与前两组和对照组比较,呈明显上升趋势。与对照组比较,10～20μg/ml ox-LDL干预细胞24h后,对细胞MMP-9的mRNA表达无显著性影响,而50μg/ml和100μg/ml ox-LDL则显著性升高MMP-9的mRNA表达1.18倍和1.22倍(P＜0.05)。
结论:
高剂量的ox-LDL作用于心肌成纤维细胞后,能促进细胞DNA合成,但减少细胞合成胶原并增加MMP-2和MMP-9的活性和mRNA表达。但低剂量的ox-LDL对mRNA的影响不大。提示ox-LDL可能使CFs变性,导致胶原合成减少。高剂量时能通过转录前和转录后水平影响MMPs分泌,而低剂量时只能通过转录后水平促进MMPs分泌,从而增加胶原的分解,促进心室重构的发生。
第三部分:辛伐他汀(Sim)干预IL-1和ox-LDL对心肌成纤维细胞合成和降解胶原的影响
方法:分离并氧化低密度脂蛋白(ox-LDL)。体外培养乳鼠心肌成纤维细胞,设立空白对照组、IL-1β组(10ng/ml)、Sim(10μmol/L)+IL-1β组(10ng/ml)、ox-LDL组(50μg/ml)、Sim(10μmol/L)+ox-LDL组(50μg/ml)、ox-LDL(50μg/ml)+IL-1β(10ng/ml)组、Sim(10μmol/L)+ox-LDL(50μg/ml)+IL-1β(10ng/ml)组。干预24 h,观察细胞形态,~3H-TdR掺入法观察细胞DNA合成,~3H-Pro掺入法观察胶原的合成,明胶酶谱法测定基质金属蛋白酶MMP-2、MMP-9活性,Western-blot检测MMP-2和MMP-9蛋白表达水平,RT-PCR检测MMP-2、MMP-9的mRNA表达。
结果:
1.与对照组比较,IL-1β能使CFs的~3H-TdR掺入率明显降低(P＜0.05),而ox-LDL使CFs ~3H-TdR掺入率显著性升高(P＜0.05);IL-1β+ox-LDL组也明显降低~3H-TdR掺入率(P＜0.05),且与ox-LDL单独作用相比较,有显著性差异(P＜0.01)。应用Sim干预后,能改善IL-1β或ox-LDL对细胞DNA的影响,与对照组比较无明显区别。而IL-1β+ox-LDL+Sim组与IL-1β+ox-LDL组相比,明显增加细胞DNA合成,与对照组比较无明显差异。
2.与对照组相比,IL-1β和ox-LDL单独作用时均能使CFs ~3H-Pro掺入率显著性降低(P＜0.01);IL-1β+ox-LDL协同作用能使胶原的合成进一步降低(P＜0.01),而且其与IL-1β组相比也有显著性差异(P＜0.05)。用Sim干预后,各组细胞胶原合成能力得到明显改善(P＜0.05),但均未达到对照组水平。
3.IL-1β和ox-LDL单独作用细胞后均能使MMP-2活性明显升高(P＜0.01)。两者联合应用后,使得MMP-2活性更进一步升高(P＜0.01)。用Sim干预后,能显著性降低IL-1β和ox-LDL单独作用对MMP-2的影响(P＜0.05),且与对照组比较无显著性差异(P＞0.05);还可显著性抑制IL-1β和ox-LDL协同产生的作用(P＜0.01),但未能到达对照组水平(P＜0.05)。与对照组相比,IL-1β和ox-LDL单独作用细胞后均能使MMP-9活性明显升高(P＜0.05)。两者联合应用后,发挥协同作用,使得MMP-9活性进一步升高(P＜0.01),但与单因素作用相比,无显著性差异。用Sim干预后,能显著性降低上述因素对MMP-9的影响(P＜0.05),其中IL-1β+Sim组和ox-LDL+Sim组与对照组比较无显著性差异,IL-1β+ox-LDL+Sim组则部分降低两者作用,但未能到达对照组水平。
4.与空白对照组比较,IL-1β和ox-LDL单独干预细胞24h后,即能显著性升高MMP-2蛋白的表达,分别升高1.59倍和1.23倍(P＜0.01),两者协同作用后,可使MMP-2水平进一步升高到1.74倍,且与单因素作用组相比有显著性差异(P＜0.05)。用Sim干预后,均能显著性降低上述因素对MMP-2的影响。1L-1β+Sim组升高1.23倍,与IL-1β组相比,显著降低MMP-2蛋白水平(P＜0.05);ox-LDL+Sim组升高1.23倍,与ox-LDL组相比,也显著降低MMP-2蛋白水平(P＜0.05);IL-1β+ox-LDL+Sim组升高1.37倍,与IL-1β+ox-LDL组相比,显著降低MMP-2蛋白水平(P＜0.01)。但均未能恢复对照组水平(P＜0.05)。与空白对照组比较,IL-1β和ox-LDL也能显著性升高条件培养基中MMP-9蛋白的表达(分别升高1.71倍和1.48倍,P＜0.01),两者协同作用后,MMP-9水平进一步升高为1.72倍(P＜0.01),且与ox-LDL单独作用相比,有显著差异(P＜0.05),但与IL-1β单独作用相比,无明显差异。用Sim干预后,能显著性降低上述因素对MMP-9的影响。IL-1β+Sim组(升高1.18倍)与IL-1β组相比,ox-LDL+Sim组升高(1.20倍)与ox-LDL组相比,IL-1β+ox-LDL+Sim组(升高1.43倍)与IL-1β+ox-LDL组相比,均显著降低MMP-9蛋白水平,但与对照组比较,未能恢复正常水平。
5.与空白对照组比较,IL-1β和ox-LDL单独干预细胞后,均能显著性升高条件培养基中MMP-2 mRNA的表达(分别升高1.57倍和1.37倍),且IL-1β组升高程度较高(P＜0.01)。两者协同作用后,MMP-2 mRNA水平进一步升高至1.68倍(P＜0.01),与ox-LDL单独作用相比,有显著性差异,但与IL-1β单独作用相比,无明显差异。用Sim干预后,能显著性降低上述因素对MMP-2 mRNA的影响。IL-1β+Sim组(升高1.24倍)与IL-1β组相比,ox-LDL+Sim组(升高1.08倍)与ox-LDL组相比,IL-1β+ox-LDL+Sim组(升高1.34)倍与IL-1β+ox-LDL组相比,显著降低MMP-2 mRNA水平(P＜0.05)。除ox-LDL+Sim组外,其余两组与对照组比较,均未能恢复对照组水平。IL-1β和ox-LDL单独干预细胞24h后,均能显著性升高条件培养基中MMP-9 mRNA的表达,且IL-1β升高程度较高(IL-1β组升高1.50倍,P＜0.01;ox-LDL组升高1.27倍,P＜0.05);两者协同作用后,MMP-9 mRNA水平进一步升高(1.64倍,P＜0.01)。与ox-LDL组相比较,作用有显著性差异(P＜0.05),但与IL-1β组相比,无明显差异。用Sim干预后,能显著性降低上述因素对MMP-9 mRNA的影响。ox-LDL+Sim组升高1.10倍,与ox-LDL组相比,显著降低MMP-9 mRNA水平(P＜0.05),且恢复至对照组水平;IL-1β+Sim组干预后升高1.19倍,与IL-1β组相比,显著降低MMP-9 mRNA水平(P＜0.05),但未能恢复至对照组水平;IL-1β+ox-LDL+Sim组升高1.31倍,与IL-1β+ox-LDL组相比,同样显著降低MMP-9 mRNA水平(P＜0.05),也未能恢复对照组水平(P＜0.05)。
结论:
IL-1β和ox-LDL联合作用后,可抑制CFs DNA的合成,降低胶原的合成,并通过转录前和转录后水平调节MMPs,增加MMP-2和MMP-9 mRNA、蛋白的表达及生物活性,起到降解胶原的作用。Sim不仅能改善IL-1β和ox-LDL单独作用时对细胞胶原合成和降解的影响,也可改善两者联合应用后的影响。提示MI后的心室重构是多因素作用的结果,早期应用他汀类药物能改善心室不良重构。
Ventricular remodeling in the process of post- myocardial infarction is the major mechanism of heart failure.It contributes to ventricular dilation and dysfunction, patients' disability and death.Ventricular remodeling is a complex dynamic process including cardiomyocyte hypertrophy,apoptosis,necrosis and extracellular matrix (ECM) remodeling,especially dynamic equilibrium of synthesis and decomposition of collagen,the major composition of ECM.Pressure overload,inflammations and rennin- angiotensin-aldosterone systems(RAS) and some other factors are involoved in the process and cause collagen synthesis decreasing,degradation and denaturation. Those changes promote the ventricular dilation and rupture.
The 75%cells of heart are non-cardiocyte and cardiac fibroblasts(CFs) are the major composition.So we can see that CFs play important role in the process of post-MI remodeling.Most collagen and skeleton protein were synthesized by CFs, especially the typeⅠand typeⅢcollagen,which induced the collagen network structure formation and to maintain the fuction of heart.The ordered degradation of ECM is the key point of heart grow,remodeling and repair.Collagen,the major componant of ECM,whose degradation is regulated by the balance between MMPs and its inhibitor called TIMPs in ECM.MMPs are a class of zinc-dependent enzymes that secreted in a proenzyme form and require proteolytic cleavage for activation and have a high specificity for components of the ECM.It participated in ECM turnover and vascular wall remodeling,angiogenesis,and atherosclerosis.MMPs selectively degrade extracellular proteins such as the fibrillar collagens.MMP-2 and MMP-9 are the important composition of MMPs family which can degrades the denatured collagen fibrosis and elastin.The inbalance of MMPs/TIMPs after AMI caused dynamic equilibrium of synthesis and decomposition of collagen and promoted the adverse ventricular remodeling.
Atherosclerosis(AS) is presently one of the most harmful human diseases with highest morbidity.It has been proved that AS has complex factors at multiple levels and infection,inflammation,and immunity were involoved in the process of initiation and progress.Oxidized low density lipoprotein(ox-LDL),one of the initiations which could be englobed by macrophage,formed foam cells and plaque.AMI was happened after the unstable plaque was ruputure.Interleukin-1β(IL-1β),Interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) are pro-inflammatory cytokines that have been found to be elevated in the serum of patients who underwent MI.This initiates pro-inflammatory cytokines which may play important role in the process of post-MI remodeling.Previous study showed that IL-1β,TNF-αdecreased collagen synthesis without change on cell number or total protein synthesis,its also increased total MMPs activity,causing specific increases in the bands corresponding to MMP-13, MMP-2,and MMP-9 and the expression of proMMP-2 and proMMP-3 mRNA.But the effects of ox-LDL on the post-MI ventricular remodeling and the interaction with the elevating of pro-inflammatory cytokines are not very clear.
From the above,we can see the post-MI ventrivular is a complicated process and ox-LDL,IL-1β,IL-6 and TNF-αis all involoved in.They caused imbalance between the synthesis and degradation of collagen,damaged the integrity of ECM,initiated the ventricular remodeling and dysfuction.The aim of anti-remodeling is to prevent, restrict and reverse the adverse remodeling in order to protect the ruction of cardiac ventricle.One aspect is to recorve the integrity of ECM.The onset and duration of theraphy is the key point in the treatment of ventricular remodeling because it was originated in early time after AMI and maintained for several months to years.ACEI, ARB,aldosterone antagonist and beta blosks were used to anti-remodeling theraphy. Statin is best known for its antilipidemic action and use in cardiovascular disease due to its inhibition of 3-hydroxy-3-methylglutaryl CoenzymeA(HMG CoA) reductase,a key enzyme in the cholesterol synthesis pathway.Recently,Statins were founded can reduce adverse ventricular remodeling independently of their cholesterol-lowering ability.So we used IL-1β,one of the pro-inflammation cytokines,combined with ox-LDL to work on the cardiac fibroblast of neonatal SD rat and then interfere with Simvastatin,observed the effects on collagen synthesis and the activity of matrix metalloproteinases.
In this study,we firstly used ~3H-thymidine(~3H-TdR) incorporation,~3H-proline (~3H-Pro) incorporation,gelatinase zymography,fluorescence microscope, Western-blot and reverse transcription PCR(RT-PCR) to measure the effect of IL-1βon the collagen synthesis,decomposion and the expression of Matrix Metalloproteinases(MMPs) in cardiac fibroblasts respectively.Then LDL was separated from human serum and oxidized in vitro and CFs was treated with different concentration of it.The proliferation of CFs,the collagen synthesisi and expression of MMP-2 and MMP-9 were observed as before.At last,we combined IL-1βwith ox-LDL to work on the CFs and pretreated with Simvastatin respectively.The effect of Simvastatin on the proliferation of CFs,the collagen synthesisi and decomposition were studied.We got the result as follows:
PartⅠ:Interleukin-1βinhibits collagen synthesis and promotes it decomposition in cultured cardiac fibroblast.
Methods:Cardiac fibroblast of neonatal Sprague-Dawley rat were cultured in vitro and treated with IL-1β(0.01ng/mL,0.1ng/mL,1ng/mL,10ng/mL 100ng/mL) for 24h.Cell proliferation was measured by MTT assay.Cell DNA synthesis was measured by ~3H-TdR incorporation and collagen synthesis was measured by ~3H-Pro incorporation.MMPs activity was measured by gelatinase zymography.MMP-2, MMP-9 protein levels were measured by Western-blot.The mRNA expression of MMP-2 and MMP-9 was detected by RT-PCR.
Results:
1.Compared with the control group,the cell proliferation and the incorporation of ~3H-TdR and ~3H-Pro was decreased in high dose IL-1β(≥0.1ng/ml) but not in low dose(0.01ng/ml).
2.0.01ng/ml IL-1βhad no effects on the collagen synthesis of CFs. 0.1～100ng/ml IL-1βmarked inhibites ~3H-Pro incorporation in concengtration dependent way.
3.IL-1βcould significantly increased MMP-2 activities in concentration dependent way and MMP-9 activities were increased also.But it was different with MMP-2 levels,the extent of elevation between 0.01ng/ml to 1ng/ml had no significant difference.
4.IL-1βalso dose dependently increased the protein levels of MMP-2(fold change,0.01ng/mL,1.35;0.1ng/mL,1.56;1ng/mL,2.15;10ng/mL,2.34;100ng/mL, 2.41) and MMP-9(fold change,0.01ng/mL,1.38;0.1ng/mL,2.37;1ng/mL,2.56; 10ng/mL,2.60;100ng/mL,2.67) respectively.
5.Similar results were observed in the mRNA expression of MMP-2(fold change,0.01ng/mL,1.81,0.1ng/mL,2.17;1ng/mL,2.18;10ng/mL,2.27;100ng/mL, 2.31) and MMP-9(fold change,0.01ng/mL,1.36;0.1ng/mL,1.49;1ng/mL,1.50; 10ng/mL,1.51;100ng/mL,1.52).
Conclusion:These results suggest that IL-1βdecreases collagen synthesis and activates MMPs through transcriptional and posttranslational processes that degrade collagen in dose dependent way.Elevation of IL-1βafter infarction myocardial is possibly involved in the process of post-myocardial infarction ventricle remodeling, and that the concentration of IL-1βmay the major factor which affects the degree of ventricle remodeling.
PartⅡ:ox-LDL inhibits collagen synthesis and promotes it decomposition in cultured cardiac fibroblast.
Methods:ox-LDL was seprated and oxidated in vitro.Cardiac fibroblast of neonatal SD rat were cultured and treated with ox-LDL(10,20,50,100μg/ml) for 24h.Cell DNA synthesis was measured by ~3H-TdR incorporation,~3H-Pro incorporation,MMPs activity,protein levels of MMP-2 and MMP-9 were measured as partⅠ.The mRNA expression of MMP-2 and MMP-9 was detected by RT-PCR also.
Results:
1.Compared with the control group,the cell proliferation and the incorporation of ~3H-TdR were increased in ox-LDL treated group in concentration way.
2.The effect of ox-LDL on ~3H-Pro incorporation were associated with its concentration.10μg/ml ox-LDL could decrease the ~3H-Pro incorporation and 100μg/ml ox-LDL had the most power.
3.ox-LDL could significantly increased MMP-2 activities in concentration dependent way,MMP-9 activities were increased also in high dose of ox-LDL. 10μg/ml ox-LDL could slightly promate the MMP-9 activities,but had no significant difference with control group.
4.ox-LDL also dose dependently increased the protein levels of MMP-2 (foldchange,1.17,1.32,1.42,1.53 respectively).10μg/ml ox-LDL have no effect on the protein level of MMP-9.20μg/ml～100μg/ml could significantly enhance the protein expression of MMP-9(fold change,1.17,1.39,1.52,respectively).
5.10μg/ml and 20μg/ml ox-LDL had no effect on the mRNA expression of MMP-2 and MMP-9.50μg/ml and 100μg/ml ox-LDL could significantly increase the mRNA expression of MMP-2(foldchange 1.21 and 1.33)and MMP-9(foldchange 1.18 and 1.22).
Conclusion:ox-LDL decreases collagen synthesis with the cell proliferation in the same time.It activates MMPs through transcriptional and posttranslational processes that degrade collagen in high concentration.But in low dose,it only could through posttranslational processes to increase the MMPs only.These results suggest that hyperlipemia may not only initiate the coronay artery disease,hut involvded in the whole course from onset to ventricle remodeling.
PartⅢ:Simvastatin prevent the effect of IL-1βand ox-LDL on the collagen synthesis and decomposition in cultured cardiac fibroblast.
Methods:ox-LDL was seprated and oxidated as partⅡ.Cardiac fibroblast of neonatal SD rat were cultured and treated with IL-1β(10 ng/ml),Simvastatin (10μmol/L)+IL-1β(10 ng/ml),ox-LDL(50μg/ml),Simvastatin+ox-LDL(10μmol/L and 50μg/ml),IL-1β+ox-LDL(10 ng/ml and 50μg/ml),Simvastatin+IL-1β+ox-LDL (10μmol/L,10 ng/ml and 50μg/ml) respectively for 24h.In all study,Simvastatin were pretreated for 30min.Cell DNA synthesis was measured by ~3H-TdR incorporation,~3H-Pro incorporation,MMPs activity,protein levels of MMP-2 and MMP-9 were measured as partⅠ.The mRNA expression of MMP-2 and MMP-9 was detected by RT-PCR also.
Results:
1.The same effects on the DNA synthesis of IL-1βand ox-LDL were observed as before when treated solo and pretreated with Simvastatin could prevent those effects.The synthesis of DNA was decreased when cotreated with IL-1βand ox-LDL. Simvastatin could inhibite those effects.
2.The same effects on the collagen synthesis of IL-1βand ox-LDL were observed as before when treated single and pretreated with Simvastatin could reverse those effects.After cotreated with IL-1βand ox-LDL,the collagen synthesis was highly decreased than every single treatment.Interfere with Simvastatin could promote collagen synthesis,but couldn't restore to the nomal.
3.Pretreated with Simvastatin could significantly inhibit the effects of IL-1βand ox-LDL single on the MMPs activity to the normal levels.After cotreated with IL-1βand ox-LDL,the MMPs activity was slightly increased,but had no significant difference with each single factor.Interfere with Simvastatin could promote collagen synthesis,but couldn't restore to the nomal.
4.Comparing with control group,treated with IL-1βor ox-LDL could marked increased the protein content of MMP-2 and MMP-9.After cotreated with IL-1βand ox-LDL,the protein levels of MMP-2 and MMP-9 were significantly higher than each single factor.Interfere with Simvastatin could reverse those effects of each single factor to the normal levels.Although it could significantly decreased those protein levels in the combined group,it still was higher than control group.
5.Comparing with control group,treated with IL-1βor ox-LDL could marked increased the mRNA expression of MMP-2 and MMP-9,especially in the treating of IL-1β.And those effects were significantly higher after cotreated with IL-1βand ox-LDL than each single factor.Interfere with Simvastatin could reverse those effects of each single factor to the normal levels,but not in the combined group as protein leves.
Conclusion:Combine IL-1βwith ox-LDL could decreases DNA synthesis and collagen synthesis in the same time.They could modulate the activity of MMPs through transcriptional and posttranslational processes together which promote the decomposion of collagen.Simvastatin could prevent those effects through transcriptional and posttranslational processes also.These results suggest that hyperlipemia and inflammation were all involove in the post-MI ventricular remoding. And interfere with Simvastatin early may inhibit those adverse effects.
心脏中75%的细胞是非心肌细胞,其中产生细胞外物质的成纤维细胞占90%-95%,由此可见成纤维细胞功能的改变是影响心室重构的重要因素。心肌成纤维细胞产生大部分基质大分子包括胶原和结构蛋白,主要是Ⅰ和Ⅲ型胶原,形成胶原网络结构,维持心脏的几何形态,并使心脏成为一个具有高度协调的舒缩器官。而有序的细胞外基质的降解是心脏生长、重构、修复的关键,其主要是由基质金属蛋白酶(MMPs)和其抑制剂(TIMPs)之间的相互作用来完成的。MMPs是一组以锌离子为辅助因子的蛋白酶家族,心脏的MMPs主要由成纤维细胞、内皮细胞、炎症细胞等细胞产生。MMPs在心脏基质的重构中发挥重要作用,一种MMPs可以直接降解某种或几种细胞外基质,也可以通过激活其它类型的MMPs而产生作用,从而形成瀑布效应;在心脏中,MMPs不仅对心脏基质成分起降解作用,而且调节胶原蛋白的合成,使正常胶原蛋白被升高的MMPs降解并被缺乏连接结构的纤维性间质所取代,使心肌纤维化增加。MMP-2、MMP-9是其中重要的组成成分,起降解变性的胶原纤维、其他类型的胶原和弹力蛋白。一旦MMPs结合到胶原纤维并开始作用时,它们能持续的作用直至所有的胶原都降解完毕或者被TIMPs抑制。AMI时MMPs/TIMPs的失衡,使得MMPs增加或者TIMPs的减少,或者MMPs/TIMPs比率增高,促使不良重构的发生。
动脉粥样硬化(AS)是导致AMI发生的根本原因。目前认为其发生发展是一个动态过程,涉及感染、炎症、免疫多种机制。而氧化低密度脂蛋白(ox-LDL)始终贯穿其中,它被巨噬细胞吞噬,转化成泡沫细胞,形成脂质斑块。不稳定的脂质斑块破裂导致血栓形成,发生AMI。发生AMI后,血液中的IL-1、IL-6、血小板源生长因子(PDGF)、肿瘤坏死因子α(TNF-α)等促炎因子的浓度大幅度升高,可促进心肌细胞的肥大和凋亡,同时它们还可能通过影响了细胞外基质,从两方面同时影响了心室的不良重构。IL-1β是AMI时升高的众多的促炎因子之一,它具有免疫调节活性作用,对不同来源的细胞有不同的生物学作用。IL-1β能通过细胞表面的白介素受体(IL-1R)减少胶原的合成及MAPK途径来刺激心肌成纤维细胞的迁移,从而参与心室的不良重构。然而对于AMI发生后,ox-LDL是否同时促进心室的重构,两者之间是否协同促进心室重构的发生,目前研究不多。已有研究表明ox-LDL可以使高同型半胱氨酸血症的患者外周血单核细胞的MMP-9的表达增高,而正常人MMP-9表达不增高。ox-LDL还可以通过羰基蛋白形成和激活表皮生长因子受体信号途径来促使MMP-2生成增多,降解胶原。
由此可见,AMI后的心室重构是个复杂的过程,在此过程中,ox-LDL、IL-1β等多种因素介导的细胞外基质降解和合成的动态平衡得以打破,使得正常结构的胶原组织降解,合成的无序的胶原纤维,使得心室重构的发生,最终导致心室功能不良。AMI后的抗重构治疗的目的是防止、限制甚至逆转不良的重构,阻止心室扩张、功能失调、无能和死亡。其中一个重要的方面就是在重构过程中保护细胞外基质。由于AMI后心室重构从心肌梗死的愈合、修复直至数月甚至数年,在此过程中,机械变形和增加的室壁压力持续作用于梗死区和非梗死区,促进心室的扩张、纤维化,故治疗的起始点和持续时间是关键所在,需要早期和长期的抗重构治疗。而一旦已经发生心衰,尽管心室重构可以停止,但心衰改善不明显。目前抗心肌重构治疗的药物主要有ACEI、ARB、醛固酮拮抗剂、β-受体拮抗剂等等。新近发现他汀类药物除调脂外,尚有抗炎、抗氧化、减少炎性递质水平、抑制凝血因子激活、抑制自身抗体形成、抑制内皮细胞粘附分子和组织因子表达、降低斑块中T细胞和巨噬细胞活性以及维持斑块稳定等作用。为此应用他汀类药物早期干预,是否可以延缓或者改善心室的重构,从而改善MI后患者的死亡率呢?我们用IL-1β联合ox-LDL刺激心肌成纤维细胞,观察MMPs的改变及胶原合成的变化,并用辛伐他汀(Sim)进行干预,为临床进行抗心室重构治疗奠定一定的基础。
本实验分为三个部分,首先通过~3H-脯氨酸掺入法、明胶酶谱法、蛋白质免疫印迹法和逆转录聚合酶链反应等方法,检测IL-1β对体外培养的心肌成纤维细胞胶原合成及促进胶原降解的基质金属蛋白酶MMP-2、MMP-9表达的影响。接着于体外提取并氧化低密度脂蛋白,并采用上述方法检测ox-LDL对心肌成纤维细胞胶原合成及MMP-2、MMP-9表达的影响。最后联合IL-1β和ox-LDL作用于成纤维细胞,并应用Sim干预上述因子,探讨Sim对上述因子作用下的心肌成纤维细胞增殖、胶原的合成和降解的影响。结果分述如下:
第一部分:IL-1β对心肌成纤维细胞合成和降解胶原的影响
方法:
体外培养乳鼠心肌成纤维细胞。加入IL-1β(0.01 ng/ml、0.1 ng/ml、1 ng/ml、10 ng/ml、100ng/ml)干预24h后,观察细胞形态,MTT法观察细胞增殖情况,~3H-胸腺嘧啶核苷(~3H-TdR)掺入法观察细胞DNA合成,~3H-脯氨酸(~3H-Pro)掺入法观察胶原的合成。明教酶谱法观察MMPs活性,Western-blot观察MMP-2和MMP-9的蛋白表达,RT-PCR观察MMP-2和MMP-9的mRNA表达。
结果:
1.IL-1β作用于心肌成纤维细胞24h后,0.01 ng/ml IL-1β对细胞增殖、DNA的合成没有明显影响。0.1ng/ml IL-1β可显著性抑制细胞活性及减少~3H-TdR掺入量(P＜0.05)。随着浓度增高,上述抑制作用逐渐增强,1～100 ng/ml较0.1ng/ml组抑制作用明显增强(P＜0.05)。
2.0.01ng/ml IL-1β干预24h后对CFs合成胶原无明显影响。随着浓度的升高,0.1ng/ml IL-1β即可显著性抑制细胞胶原的合成(P＜0.05)。1～100 ng/ml IL-1β抑制细胞胶原合成的作用更强(P＜0.01),但10～100 ng/ml之间作用无显著性差异。
3.小剂量的IL-1β干预后,MMP-2的活性即升高,并呈浓度依赖性增高趋势。同时MMP-9的活性也显著性增高,与MMP-2不同,0.01～1ng/ml IL-1β作用后MMP-9的升高程度无显著性差异。
4.与空白对照组比较,IL-1β干预后可显著性升高MMP-2蛋白表达水平,按0.01～0.1ng/ml分别升高1.35倍和1.56倍(P＜0.05),1～100ng/ml分别升高2.15倍、2.34倍、2.41倍(P＜0.01)。MMP-9蛋白表达也显著性升高,按0.01ng/ml即可显著性升高MMP-9蛋白表达1.38倍(P＜0.05),0.1～100ng/ml分别2.37倍、2.56倍、2.60倍、2.67倍(P＜0.01)。
5.IL-1β作用于CFs 24小时后能浓度依赖性增加MMPs mRNA表达。与空白对照组比较,MMP-2 mRNA表达能显著性增加,按0.01ng/ml～100ng/ml不同浓度分别升高1.81倍(P＜0.05)、2.17倍、2.18倍、2.27倍、2.31倍(P＜0.01)。MMP-9 mRNA表达也能显著性升高,分别1.36倍(P＜0.05)、1.49倍、1.50倍、1.51倍、1.52倍(P＜0.01)。
结论:
一定浓度的IL-1β抑制CFs的增殖及DNA的合成,并可能通过该作用降低胶原的合成,同时IL-1β还能增加MMPs的活性,起到降解胶原的作用。上述作用呈浓度依赖性,与心肌梗死面积越大,炎症因子浓度越高,心室重构越严重相符合,提示IL-1β在心室重构过程中起一定的作用。
第二部分:氧化低密度脂蛋白(ox-LDL)对心肌成纤维细胞合成和降解胶原的影响
方法:分离并氧化低密度脂蛋白。体外培养乳鼠心肌成纤维细胞,加入ox-LDL(10、20、50、100μg/ml)干预24 h,观察细胞形态,~3H-TdR掺入法观察细胞DNA合成,~3H-Pro掺入法观察细胞胶原的合成,明胶酶谱法测定基质金属蛋白酶MMP-2、MMP-9活性,Western-blot检测MMP-2和MMP-9蛋白表达水平,RT-PCR检测MMP-2、MMP-9的mRNA表达。
结果:
1.ox-LDL对细胞~3H-TdR掺入率的影响与其浓度密切相关。用ox-LDL干预24h后可见10μg╱ml和20μg╱ml即显著性增加细胞~3H-TdR掺入率(P＜0.05),随着浓度的增高(50～100μg/ml),掺入率也随之增高(P＜0.01),100μg/ml的增强作用最强。
2.ox-LDL对细胞~3H-Pro掺入率的影响也与其浓度密切相关。用ox-LDL干预24h后可见10μg/ml即显著性减少细胞~3H-Pro掺入率(P＜0.05),而随着浓度的增高(20～100μg/ml),掺入率也迅速降低(P＜0.01),100μg╱ml的抑制作用最强。
3.ox-LDL干预24小时后能增加条件培养基中MMP-2和MMP-9的活性。与空白对照组相比,ox-LDL干预后可显著性增加细胞MMP-2活性(P＜0.05),但各剂量组之间无显著性差异。10μg/ml ox-LDL作用于CFs后,MMP-9的活性轻微增高,但无显著性差异;随浓度的升高,MMP-9的活性也显著性增高(P＜0.05)。
4.与空白对照组比较,10μg/ml的ox-LDL即能显著性升高MMP-2蛋白表达1.17倍(P＜0.05),而20～100μg╱ml作用更强并呈浓度依赖性,分别升高1.32倍、1.42倍、1.53倍(P＜0.01)。10μg/mL ox-LDL不影响MMP-9蛋白的表达;20μg/ml ox-LDL能使MMP-9蛋白表达显著性升高1.17倍(P＜0.05),而50～100μg/ml ox-LDL作用更强,分别使MMP-9蛋白升高1.39倍和1.52倍(P＜0.01)。
5.10～20μg/ml ox-LDL使MMP-2 mRNA表达分别升高1.01倍、1.07倍。而50～100μg/ml ox-LDL则使MMP-2 mRNA表达分别升高1.21倍、1.33倍,与前两组和对照组比较,呈明显上升趋势。与对照组比较,10～20μg/ml ox-LDL干预细胞24h后,对细胞MMP-9的mRNA表达无显著性影响,而50μg/ml和100μg/ml ox-LDL则显著性升高MMP-9的mRNA表达1.18倍和1.22倍(P＜0.05)。
结论:
高剂量的ox-LDL作用于心肌成纤维细胞后,能促进细胞DNA合成,但减少细胞合成胶原并增加MMP-2和MMP-9的活性和mRNA表达。但低剂量的ox-LDL对mRNA的影响不大。提示ox-LDL可能使CFs变性,导致胶原合成减少。高剂量时能通过转录前和转录后水平影响MMPs分泌,而低剂量时只能通过转录后水平促进MMPs分泌,从而增加胶原的分解,促进心室重构的发生。
第三部分:辛伐他汀(Sim)干预IL-1和ox-LDL对心肌成纤维细胞合成和降解胶原的影响
方法:分离并氧化低密度脂蛋白(ox-LDL)。体外培养乳鼠心肌成纤维细胞,设立空白对照组、IL-1β组(10ng/ml)、Sim(10μmol/L)+IL-1β组(10ng/ml)、ox-LDL组(50μg/ml)、Sim(10μmol/L)+ox-LDL组(50μg/ml)、ox-LDL(50μg/ml)+IL-1β(10ng/ml)组、Sim(10μmol/L)+ox-LDL(50μg/ml)+IL-1β(10ng/ml)组。干预24 h,观察细胞形态,~3H-TdR掺入法观察细胞DNA合成,~3H-Pro掺入法观察胶原的合成,明胶酶谱法测定基质金属蛋白酶MMP-2、MMP-9活性,Western-blot检测MMP-2和MMP-9蛋白表达水平,RT-PCR检测MMP-2、MMP-9的mRNA表达。
结果:
1.与对照组比较,IL-1β能使CFs的~3H-TdR掺入率明显降低(P＜0.05),而ox-LDL使CFs ~3H-TdR掺入率显著性升高(P＜0.05);IL-1β+ox-LDL组也明显降低~3H-TdR掺入率(P＜0.05),且与ox-LDL单独作用相比较,有显著性差异(P＜0.01)。应用Sim干预后,能改善IL-1β或ox-LDL对细胞DNA的影响,与对照组比较无明显区别。而IL-1β+ox-LDL+Sim组与IL-1β+ox-LDL组相比,明显增加细胞DNA合成,与对照组比较无明显差异。
2.与对照组相比,IL-1β和ox-LDL单独作用时均能使CFs ~3H-Pro掺入率显著性降低(P＜0.01);IL-1β+ox-LDL协同作用能使胶原的合成进一步降低(P＜0.01),而且其与IL-1β组相比也有显著性差异(P＜0.05)。用Sim干预后,各组细胞胶原合成能力得到明显改善(P＜0.05),但均未达到对照组水平。
3.IL-1β和ox-LDL单独作用细胞后均能使MMP-2活性明显升高(P＜0.01)。两者联合应用后,使得MMP-2活性更进一步升高(P＜0.01)。用Sim干预后,能显著性降低IL-1β和ox-LDL单独作用对MMP-2的影响(P＜0.05),且与对照组比较无显著性差异(P＞0.05);还可显著性抑制IL-1β和ox-LDL协同产生的作用(P＜0.01),但未能到达对照组水平(P＜0.05)。与对照组相比,IL-1β和ox-LDL单独作用细胞后均能使MMP-9活性明显升高(P＜0.05)。两者联合应用后,发挥协同作用,使得MMP-9活性进一步升高(P＜0.01),但与单因素作用相比,无显著性差异。用Sim干预后,能显著性降低上述因素对MMP-9的影响(P＜0.05),其中IL-1β+Sim组和ox-LDL+Sim组与对照组比较无显著性差异,IL-1β+ox-LDL+Sim组则部分降低两者作用,但未能到达对照组水平。
4.与空白对照组比较,IL-1β和ox-LDL单独干预细胞24h后,即能显著性升高MMP-2蛋白的表达,分别升高1.59倍和1.23倍(P＜0.01),两者协同作用后,可使MMP-2水平进一步升高到1.74倍,且与单因素作用组相比有显著性差异(P＜0.05)。用Sim干预后,均能显著性降低上述因素对MMP-2的影响。1L-1β+Sim组升高1.23倍,与IL-1β组相比,显著降低MMP-2蛋白水平(P＜0.05);ox-LDL+Sim组升高1.23倍,与ox-LDL组相比,也显著降低MMP-2蛋白水平(P＜0.05);IL-1β+ox-LDL+Sim组升高1.37倍,与IL-1β+ox-LDL组相比,显著降低MMP-2蛋白水平(P＜0.01)。但均未能恢复对照组水平(P＜0.05)。与空白对照组比较,IL-1β和ox-LDL也能显著性升高条件培养基中MMP-9蛋白的表达(分别升高1.71倍和1.48倍,P＜0.01),两者协同作用后,MMP-9水平进一步升高为1.72倍(P＜0.01),且与ox-LDL单独作用相比,有显著差异(P＜0.05),但与IL-1β单独作用相比,无明显差异。用Sim干预后,能显著性降低上述因素对MMP-9的影响。IL-1β+Sim组(升高1.18倍)与IL-1β组相比,ox-LDL+Sim组升高(1.20倍)与ox-LDL组相比,IL-1β+ox-LDL+Sim组(升高1.43倍)与IL-1β+ox-LDL组相比,均显著降低MMP-9蛋白水平,但与对照组比较,未能恢复正常水平。
5.与空白对照组比较,IL-1β和ox-LDL单独干预细胞后,均能显著性升高条件培养基中MMP-2 mRNA的表达(分别升高1.57倍和1.37倍),且IL-1β组升高程度较高(P＜0.01)。两者协同作用后,MMP-2 mRNA水平进一步升高至1.68倍(P＜0.01),与ox-LDL单独作用相比,有显著性差异,但与IL-1β单独作用相比,无明显差异。用Sim干预后,能显著性降低上述因素对MMP-2 mRNA的影响。IL-1β+Sim组(升高1.24倍)与IL-1β组相比,ox-LDL+Sim组(升高1.08倍)与ox-LDL组相比,IL-1β+ox-LDL+Sim组(升高1.34)倍与IL-1β+ox-LDL组相比,显著降低MMP-2 mRNA水平(P＜0.05)。除ox-LDL+Sim组外,其余两组与对照组比较,均未能恢复对照组水平。IL-1β和ox-LDL单独干预细胞24h后,均能显著性升高条件培养基中MMP-9 mRNA的表达,且IL-1β升高程度较高(IL-1β组升高1.50倍,P＜0.01;ox-LDL组升高1.27倍,P＜0.05);两者协同作用后,MMP-9 mRNA水平进一步升高(1.64倍,P＜0.01)。与ox-LDL组相比较,作用有显著性差异(P＜0.05),但与IL-1β组相比,无明显差异。用Sim干预后,能显著性降低上述因素对MMP-9 mRNA的影响。ox-LDL+Sim组升高1.10倍,与ox-LDL组相比,显著降低MMP-9 mRNA水平(P＜0.05),且恢复至对照组水平;IL-1β+Sim组干预后升高1.19倍,与IL-1β组相比,显著降低MMP-9 mRNA水平(P＜0.05),但未能恢复至对照组水平;IL-1β+ox-LDL+Sim组升高1.31倍,与IL-1β+ox-LDL组相比,同样显著降低MMP-9 mRNA水平(P＜0.05),也未能恢复对照组水平(P＜0.05)。
结论:
IL-1β和ox-LDL联合作用后,可抑制CFs DNA的合成,降低胶原的合成,并通过转录前和转录后水平调节MMPs,增加MMP-2和MMP-9 mRNA、蛋白的表达及生物活性,起到降解胶原的作用。Sim不仅能改善IL-1β和ox-LDL单独作用时对细胞胶原合成和降解的影响,也可改善两者联合应用后的影响。提示MI后的心室重构是多因素作用的结果,早期应用他汀类药物能改善心室不良重构。
Ventricular remodeling in the process of post- myocardial infarction is the major mechanism of heart failure.It contributes to ventricular dilation and dysfunction, patients' disability and death.Ventricular remodeling is a complex dynamic process including cardiomyocyte hypertrophy,apoptosis,necrosis and extracellular matrix (ECM) remodeling,especially dynamic equilibrium of synthesis and decomposition of collagen,the major composition of ECM.Pressure overload,inflammations and rennin- angiotensin-aldosterone systems(RAS) and some other factors are involoved in the process and cause collagen synthesis decreasing,degradation and denaturation. Those changes promote the ventricular dilation and rupture.
The 75%cells of heart are non-cardiocyte and cardiac fibroblasts(CFs) are the major composition.So we can see that CFs play important role in the process of post-MI remodeling.Most collagen and skeleton protein were synthesized by CFs, especially the typeⅠand typeⅢcollagen,which induced the collagen network structure formation and to maintain the fuction of heart.The ordered degradation of ECM is the key point of heart grow,remodeling and repair.Collagen,the major componant of ECM,whose degradation is regulated by the balance between MMPs and its inhibitor called TIMPs in ECM.MMPs are a class of zinc-dependent enzymes that secreted in a proenzyme form and require proteolytic cleavage for activation and have a high specificity for components of the ECM.It participated in ECM turnover and vascular wall remodeling,angiogenesis,and atherosclerosis.MMPs selectively degrade extracellular proteins such as the fibrillar collagens.MMP-2 and MMP-9 are the important composition of MMPs family which can degrades the denatured collagen fibrosis and elastin.The inbalance of MMPs/TIMPs after AMI caused dynamic equilibrium of synthesis and decomposition of collagen and promoted the adverse ventricular remodeling.
Atherosclerosis(AS) is presently one of the most harmful human diseases with highest morbidity.It has been proved that AS has complex factors at multiple levels and infection,inflammation,and immunity were involoved in the process of initiation and progress.Oxidized low density lipoprotein(ox-LDL),one of the initiations which could be englobed by macrophage,formed foam cells and plaque.AMI was happened after the unstable plaque was ruputure.Interleukin-1β(IL-1β),Interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) are pro-inflammatory cytokines that have been found to be elevated in the serum of patients who underwent MI.This initiates pro-inflammatory cytokines which may play important role in the process of post-MI remodeling.Previous study showed that IL-1β,TNF-αdecreased collagen synthesis without change on cell number or total protein synthesis,its also increased total MMPs activity,causing specific increases in the bands corresponding to MMP-13, MMP-2,and MMP-9 and the expression of proMMP-2 and proMMP-3 mRNA.But the effects of ox-LDL on the post-MI ventricular remodeling and the interaction with the elevating of pro-inflammatory cytokines are not very clear.
From the above,we can see the post-MI ventrivular is a complicated process and ox-LDL,IL-1β,IL-6 and TNF-αis all involoved in.They caused imbalance between the synthesis and degradation of collagen,damaged the integrity of ECM,initiated the ventricular remodeling and dysfuction.The aim of anti-remodeling is to prevent, restrict and reverse the adverse remodeling in order to protect the ruction of cardiac ventricle.One aspect is to recorve the integrity of ECM.The onset and duration of theraphy is the key point in the treatment of ventricular remodeling because it was originated in early time after AMI and maintained for several months to years.ACEI, ARB,aldosterone antagonist and beta blosks were used to anti-remodeling theraphy. Statin is best known for its antilipidemic action and use in cardiovascular disease due to its inhibition of 3-hydroxy-3-methylglutaryl CoenzymeA(HMG CoA) reductase,a key enzyme in the cholesterol synthesis pathway.Recently,Statins were founded can reduce adverse ventricular remodeling independently of their cholesterol-lowering ability.So we used IL-1β,one of the pro-inflammation cytokines,combined with ox-LDL to work on the cardiac fibroblast of neonatal SD rat and then interfere with Simvastatin,observed the effects on collagen synthesis and the activity of matrix metalloproteinases.
In this study,we firstly used ~3H-thymidine(~3H-TdR) incorporation,~3H-proline (~3H-Pro) incorporation,gelatinase zymography,fluorescence microscope, Western-blot and reverse transcription PCR(RT-PCR) to measure the effect of IL-1βon the collagen synthesis,decomposion and the expression of Matrix Metalloproteinases(MMPs) in cardiac fibroblasts respectively.Then LDL was separated from human serum and oxidized in vitro and CFs was treated with different concentration of it.The proliferation of CFs,the collagen synthesisi and expression of MMP-2 and MMP-9 were observed as before.At last,we combined IL-1βwith ox-LDL to work on the CFs and pretreated with Simvastatin respectively.The effect of Simvastatin on the proliferation of CFs,the collagen synthesisi and decomposition were studied.We got the result as follows:
PartⅠ:Interleukin-1βinhibits collagen synthesis and promotes it decomposition in cultured cardiac fibroblast.
Methods:Cardiac fibroblast of neonatal Sprague-Dawley rat were cultured in vitro and treated with IL-1β(0.01ng/mL,0.1ng/mL,1ng/mL,10ng/mL 100ng/mL) for 24h.Cell proliferation was measured by MTT assay.Cell DNA synthesis was measured by ~3H-TdR incorporation and collagen synthesis was measured by ~3H-Pro incorporation.MMPs activity was measured by gelatinase zymography.MMP-2, MMP-9 protein levels were measured by Western-blot.The mRNA expression of MMP-2 and MMP-9 was detected by RT-PCR.
Results:
1.Compared with the control group,the cell proliferation and the incorporation of ~3H-TdR and ~3H-Pro was decreased in high dose IL-1β(≥0.1ng/ml) but not in low dose(0.01ng/ml).
2.0.01ng/ml IL-1βhad no effects on the collagen synthesis of CFs. 0.1～100ng/ml IL-1βmarked inhibites ~3H-Pro incorporation in concengtration dependent way.
3.IL-1βcould significantly increased MMP-2 activities in concentration dependent way and MMP-9 activities were increased also.But it was different with MMP-2 levels,the extent of elevation between 0.01ng/ml to 1ng/ml had no significant difference.
4.IL-1βalso dose dependently increased the protein levels of MMP-2(fold change,0.01ng/mL,1.35;0.1ng/mL,1.56;1ng/mL,2.15;10ng/mL,2.34;100ng/mL, 2.41) and MMP-9(fold change,0.01ng/mL,1.38;0.1ng/mL,2.37;1ng/mL,2.56; 10ng/mL,2.60;100ng/mL,2.67) respectively.
5.Similar results were observed in the mRNA expression of MMP-2(fold change,0.01ng/mL,1.81,0.1ng/mL,2.17;1ng/mL,2.18;10ng/mL,2.27;100ng/mL, 2.31) and MMP-9(fold change,0.01ng/mL,1.36;0.1ng/mL,1.49;1ng/mL,1.50; 10ng/mL,1.51;100ng/mL,1.52).
Conclusion:These results suggest that IL-1βdecreases collagen synthesis and activates MMPs through transcriptional and posttranslational processes that degrade collagen in dose dependent way.Elevation of IL-1βafter infarction myocardial is possibly involved in the process of post-myocardial infarction ventricle remodeling, and that the concentration of IL-1βmay the major factor which affects the degree of ventricle remodeling.
PartⅡ:ox-LDL inhibits collagen synthesis and promotes it decomposition in cultured cardiac fibroblast.
Methods:ox-LDL was seprated and oxidated in vitro.Cardiac fibroblast of neonatal SD rat were cultured and treated with ox-LDL(10,20,50,100μg/ml) for 24h.Cell DNA synthesis was measured by ~3H-TdR incorporation,~3H-Pro incorporation,MMPs activity,protein levels of MMP-2 and MMP-9 were measured as partⅠ.The mRNA expression of MMP-2 and MMP-9 was detected by RT-PCR also.
Results:
1.Compared with the control group,the cell proliferation and the incorporation of ~3H-TdR were increased in ox-LDL treated group in concentration way.
2.The effect of ox-LDL on ~3H-Pro incorporation were associated with its concentration.10μg/ml ox-LDL could decrease the ~3H-Pro incorporation and 100μg/ml ox-LDL had the most power.
3.ox-LDL could significantly increased MMP-2 activities in concentration dependent way,MMP-9 activities were increased also in high dose of ox-LDL. 10μg/ml ox-LDL could slightly promate the MMP-9 activities,but had no significant difference with control group.
4.ox-LDL also dose dependently increased the protein levels of MMP-2 (foldchange,1.17,1.32,1.42,1.53 respectively).10μg/ml ox-LDL have no effect on the protein level of MMP-9.20μg/ml～100μg/ml could significantly enhance the protein expression of MMP-9(fold change,1.17,1.39,1.52,respectively).
5.10μg/ml and 20μg/ml ox-LDL had no effect on the mRNA expression of MMP-2 and MMP-9.50μg/ml and 100μg/ml ox-LDL could significantly increase the mRNA expression of MMP-2(foldchange 1.21 and 1.33)and MMP-9(foldchange 1.18 and 1.22).
Conclusion:ox-LDL decreases collagen synthesis with the cell proliferation in the same time.It activates MMPs through transcriptional and posttranslational processes that degrade collagen in high concentration.But in low dose,it only could through posttranslational processes to increase the MMPs only.These results suggest that hyperlipemia may not only initiate the coronay artery disease,hut involvded in the whole course from onset to ventricle remodeling.
PartⅢ:Simvastatin prevent the effect of IL-1βand ox-LDL on the collagen synthesis and decomposition in cultured cardiac fibroblast.
Methods:ox-LDL was seprated and oxidated as partⅡ.Cardiac fibroblast of neonatal SD rat were cultured and treated with IL-1β(10 ng/ml),Simvastatin (10μmol/L)+IL-1β(10 ng/ml),ox-LDL(50μg/ml),Simvastatin+ox-LDL(10μmol/L and 50μg/ml),IL-1β+ox-LDL(10 ng/ml and 50μg/ml),Simvastatin+IL-1β+ox-LDL (10μmol/L,10 ng/ml and 50μg/ml) respectively for 24h.In all study,Simvastatin were pretreated for 30min.Cell DNA synthesis was measured by ~3H-TdR incorporation,~3H-Pro incorporation,MMPs activity,protein levels of MMP-2 and MMP-9 were measured as partⅠ.The mRNA expression of MMP-2 and MMP-9 was detected by RT-PCR also.
Results:
1.The same effects on the DNA synthesis of IL-1βand ox-LDL were observed as before when treated solo and pretreated with Simvastatin could prevent those effects.The synthesis of DNA was decreased when cotreated with IL-1βand ox-LDL. Simvastatin could inhibite those effects.
2.The same effects on the collagen synthesis of IL-1βand ox-LDL were observed as before when treated single and pretreated with Simvastatin could reverse those effects.After cotreated with IL-1βand ox-LDL,the collagen synthesis was highly decreased than every single treatment.Interfere with Simvastatin could promote collagen synthesis,but couldn't restore to the nomal.
3.Pretreated with Simvastatin could significantly inhibit the effects of IL-1βand ox-LDL single on the MMPs activity to the normal levels.After cotreated with IL-1βand ox-LDL,the MMPs activity was slightly increased,but had no significant difference with each single factor.Interfere with Simvastatin could promote collagen synthesis,but couldn't restore to the nomal.
4.Comparing with control group,treated with IL-1βor ox-LDL could marked increased the protein content of MMP-2 and MMP-9.After cotreated with IL-1βand ox-LDL,the protein levels of MMP-2 and MMP-9 were significantly higher than each single factor.Interfere with Simvastatin could reverse those effects of each single factor to the normal levels.Although it could significantly decreased those protein levels in the combined group,it still was higher than control group.
5.Comparing with control group,treated with IL-1βor ox-LDL could marked increased the mRNA expression of MMP-2 and MMP-9,especially in the treating of IL-1β.And those effects were significantly higher after cotreated with IL-1βand ox-LDL than each single factor.Interfere with Simvastatin could reverse those effects of each single factor to the normal levels,but not in the combined group as protein leves.
Conclusion:Combine IL-1βwith ox-LDL could decreases DNA synthesis and collagen synthesis in the same time.They could modulate the activity of MMPs through transcriptional and posttranslational processes together which promote the decomposion of collagen.Simvastatin could prevent those effects through transcriptional and posttranslational processes also.These results suggest that hyperlipemia and inflammation were all involove in the post-MI ventricular remoding. And interfere with Simvastatin early may inhibit those adverse effects.
引文
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5 Sun Y,Zhang JQ,Zhang J,et al.Cardiac remodeling by fibrous tissue after infarction in rats[J].J Lab Clin Med,2000,135(4):316-323.
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8 Yang F,Liu YH,Yang XP,et al.Myocardial infarction and cardiac remodelling in mice[J].Exp Physiol,2002,87(5):547-555.
9 Weber KT.Monitoring tissue repair and fibrosis from a distance[J].Circulation,1997,96(8):2488-2492.
10 Haas TL,Davis SJ,Madri JA.Three-dimensional type Ⅰ collagen lattices induce coordinate expression of matrix metalloproteinases MT1-MMP and MMP-2 in microvascular endothelial cells[J].J Biol Chem,1998,273(6):3604-3610.
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13 Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry[J]. Circ Res,2003,92(8):827-839.
14 Skiles JW, Gonnella NC, Jeng AY. The design, structure, and clinical update of small molecular weight matrix metalloproteinase inhibitors[J]. Curr Med Chem,2004,11(22):2911-2977.
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