Klotho蛋白改善应激诱导的心肌损伤的机制研究
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摘要
背景：
心室重构(Cardiac remodeling)是心脏对外界各种应激性刺激作出的共同病理反应，主要表现为心脏结构、大小及功能的改变。心室重构最终会导致心脏舒缩功能下降，是心力衰竭发病的根本原因。中国老龄化程度的不断加剧，以及高血压、冠心病、瓣膜病等心血管病发病率的逐年升高，使得心衰的发病率及病死率逐年增加，成为影响老年人生活质量及生存率的主要疾病之一。在众多引起心室重构的疾病中，心肌梗死后心衰重构是最主要的病因之一，尽管随着临床医疗水平的进步，如冠脉造影及支架植入术以及冠脉搭桥手术的开展，更大程度的挽救了濒死的心肌，使梗死后心室重构的发生及发展显著改善，但是这些措施仍不能完全逆转心室重构的发生以及心衰临床症状的恶化。研究表明，神经体液因子在心室重构的病理过程中发挥着重要作用。早期阶段，β肾上腺素能受体的激活可以诱导心肌细胞收缩力增强，代偿前后负荷增高对机体的影响。但是，长期的β肾上腺素能受体的激活将导致病理性的心肌损伤，引起心室重构。异丙肾上腺素(Isoproterenol，ISO)作为β肾上腺素能受体激动剂，是常用的诱导实验动物心肌损伤的诱导剂。心肌损伤主要包括心肌细胞丢失、肥大以及间质胶原含量的改变。寻求逆转上述病理改变的关键分子对于降低心衰的发病率及猝死率具有重要意义。
Klotho蛋白是新近发现的具有抗衰老作用的蛋白，通过膜结合型和分泌型两种形式发挥生物学效应。基于文献及前期的研究工作，我们推测Klotho蛋白可能是具有心血管保护作用的激素样蛋白分子。首先Klotho蛋白具有抗氧化应激特性，而氧化应激广泛参与了许多疾病的发生发展，包括心室重构；其次Klotho蛋白可通过增加NO生物活性或调控血管内皮细胞上的钙离子通道维持血管内皮完整性及内皮功能；再次Klotho蛋白具有降压作用，而压力负荷增高是导致心衰发病的主要原因；另外Klotho蛋白还可抑制内皮细胞凋亡及炎症反应。临床研究表明血浆中低水平的Klotho蛋白可能是慢性肾脏疾病相关的心血管疾病的高危因素，而我们的前期工作也发现Klotho基因的单核苷酸多态性与动脉硬化等心血管疾病密切相关，因此我们有足够的证据认为Klotho蛋白可能对心室重构具有抑制作用。而且最近的研究发现，ISO可导致小鼠心脏收缩功能显著下降，而Klotho基因敲除可进一步加重这一下降效果，提示Klotho蛋白对心脏收缩功能具有保护作用。
心肌细胞丢失是心脏收缩功能下降的主要原因，其中心肌细胞的丢失主要源于心肌细胞的凋亡及坏死，而心肌细胞凋亡是慢性心室重构过程中心肌细胞丢失的主要原因，是导致心脏功能失调甚至猝死的关键因素。内质网(Endoplasmic reticulum，ER)是除了线粒体外又一个参与细胞凋亡的重要器官，其主要功能包括调节钙离子平衡、参与蛋白质的折叠及成熟等。当内质网的平衡被破坏时(如钙离子平衡被破坏、蛋白质生成过多或蛋白质折叠错误)，内质网将诱导相关伴侣蛋白如糖调节蛋白78(GRP78)、热休克蛋白47(HSP47)的表达，此过程称为内质网应激。内质网伴侣分子的增加不仅是内质网应激发生的标志，同时也是内质网应激严重程度的重要体现。早期阶段的内质网应激是机体对外界刺激作出的适应性反应，主要通过促进钙离子平衡、抑制蛋白质运输到内质网、增强蛋白质正确折叠及运输以维持内质网稳态，但长期或严重的内质网应激将导致凋亡信号通路的激活，其中促凋亡转录因子CHOP的表达增加是重要的内质网介导凋亡的效应分子。近年来的研究表明，内质网应激在心肌细胞凋亡中同样发挥重要的作用。研究发现，抑制内质网应激可有效缓解心肌细胞的损伤。同样，ISO可以诱导心肌细胞株发生凋亡，其中伴随内质网伴侣蛋白及促凋亡转录因子CHOP的表达明显升高，说明ISO通过激活内质网应激介导的凋亡通路诱导细胞凋亡。而Klotho蛋白维持心脏收缩功能的作用是否与抑制ER应激介导的CHOP凋亡信号有关还未见报道。
细胞主要通过调节细胞内的信号通路对外界刺激作出反应。 MAPK(Mitogen-activated protein kinases)是普遍存在于细胞内的信号转导通路，由胞外信号调节蛋白激酶(ERK1/2), p38和c-Jun氨基末端激酶(JNK)组成，调节细胞的增殖、分化，在ER应激引起的凋亡中也发挥重要的调节作用。而分泌型Klotho蛋白作为循环中的蛋白分子，能否通过该信号通路参与ER应激介导的凋亡还未完全阐明，需要进一步研究明确。
实验目的:
本实验以Klotho蛋白的抗凋亡作用作为切入点，在构建ISO诱导小鼠心室重构模型的基础上，研究Klotho蛋白对心室重构特别是心肌细胞凋亡的影响。并进一步明确Klotho蛋白的抗凋亡机制是否与ER应激介导的CHOP凋亡信号通路密切相关。最后通过信号通路研究，明确Klotho蛋白调节MAPK信号通路的具体情况，以及其调节MAPK信号通路与ER应激介导的凋亡之间的关系。
主要实验方法及实验结果：
1.取6-8周龄的Balb/C品系，重量约20g的雄性小鼠，随机分为对照组（CON）、ISO组。其中CON组每天背部皮下注射100ul生理盐水，共注射9天；ISO组每天以5mg/kg/d剂量背部皮下注射ISO（溶解于100ul生理盐水中），共注射9天。在注射后的第9天在电子天平上称小鼠的重量，处死小鼠，并轻柔的切除小鼠的心脏组织称心脏的重量，计算HW/BW的比值。每个离体的心脏平均切割成6块，其中3块心肌组织用锡箔纸包好，放入冻存管内，液氮罐中保存，用于后续的Realtime-PCR实验。剩下的心肌组织浸泡在4%多聚甲醛溶液中，固定24小时后，石蜡包埋制作石蜡块。用切片机切割4um厚度的石蜡切片，对连续的4um心肌石蜡切片行HE染色观察心肌的形态改变。同时对心肌石蜡切片进行Masson染色，对其胶原容积分数进行定量分析，然后应用Tunel染色技术对石蜡切片进行凋亡检测，分别计数检测心肌细胞的凋亡率。研究结果表明持续小剂量皮下注射ISO可诱导小鼠心肌细胞肥大凋亡、肌纤维紊乱及心肌间质成纤维细胞增生及胶原蛋白沉积。通过测定HW/BW的比值，我们发现ISO可诱导小鼠心心脏体重指数增加，且具有统计学差异(P<0.05)，这种作用与心肌细胞的肥大增生及胶原纤维增生密切相关。HE染色结果发现持续皮下注射ISO(9天)可引起心肌细胞的肥大及肌纤维排列的紊乱。除了心肌细胞的病变外，我们还发现ISO可诱导心肌间质中成纤维细胞增殖显著增加，说明皮下注射ISO也诱导了心肌间质的纤维化反应。同时我们应用Realtime-PCR技术观察心肌间质胶原蛋白的mRNA水平，结果表明ISO可显著增加collegen mRNA的表达水平。Masson染色观察心肌间质胶原纤维的含量发现，在ISO组中心肌间质的胶原容积比例显著高于对照组，由此说明ISO可成功诱导心肌间质纤维化；最后我们应用Tunel染色技术检测心肌细胞的凋亡率，结果表明皮下注射ISO可增加心肌细胞的凋亡率，且具有统计学意义(P<0.05)。由此说明ISO作为β肾上腺素能受体激动剂，可成功用于构建小鼠心室重构模型。
2.取6-8周龄的Balb/C品系，重量约20g的雄性小鼠，随机分为以下各组：CON组：背部皮下注射生理盐水(100ul，qd，共9天)，同时腹腔注射生理盐水(100ul，qod，共4天)；ISO组：背部皮下注射ISO(5mg/kg/d，qd，共9天)，同时腹腔注射生理盐水(0.1ml，qod，共4天)；ISO+KL组：背部皮下注射ISO(5mg/kg/d，gd，共9天)，同时腹腔注射Klotho蛋白(0.01mg/kg，qod，共4天)。按照上述的方法对各组进行操作，分别在ISO注射的第2，5，9天处死小鼠。首先，我们应用ELISA技术检测腹腔注射重组Klotho蛋白对小鼠血循环中Klotho蛋白水平的影响。并分别应用HE染色、Masson染色、TUNEL染色检测心肌组织的结构改变及凋亡情况，ROS检测技术测定组织中ROS的含量，最后应用免疫组织化学染色、Real-time PCR技术、Western blot技术测定心脏组织中ER应激介导的凋亡信号分子GRP78、HSP47及促凋亡转录因子CHOP的mRNA水平及蛋白水平。同时通过体外培养大鼠胚胎来源的H9c2心肌细胞，进一步观察Klotho蛋白对ISO诱导H9c2心肌细胞肥大、凋亡及ER应激介导的凋亡信号通路的影响。结果表明腹腔注射Klotho蛋白后，血清中Klotho蛋白的水平显著增高(P<0.05)。HE染色结果表明，重组Klotho蛋白抑制ISO诱导的小鼠心脏组织的病理变化。在CON组，心肌纤维走形正常；而ISO注射之后小鼠心脏组织的HE染色图片表现为心肌纤维走形紊乱、间质成纤维细胞显著增生，表现为细胞核密度的增加以及单核细胞局限性浸润，暗示了ISO组中严重的心肌损伤；而重组Klotho蛋白可以缓解上述改变，提示Klotho蛋白具有心肌保护作用。Masson染色结果表明，从ISO注射后的第5天开始，心肌间质及血管周的胶原纤维含量明显高于对照组，而Klotho蛋白可抑制胶原纤维的增加。TUNEL染色结果表明ISO注射的第9天，心肌细胞的凋亡率显著增加，而Klotho蛋白可抑制ISO诱导的小鼠心肌细胞的凋亡。ROS是公认的细胞凋亡诱导因素，和上述研究结果一致，ISO组中ROS的含量显著增加，而在ISO+KL组ROS含量被抑制。由此说明了Klotho蛋白的心肌保护作用及抗凋亡作用。进而我们应用免疫组化及Western blot技术检测了ER应激相关蛋白分子的表达情况，结果证实ISO可诱导内质网应激相关伴侣分子GRP78、HSP47及促凋亡转录因子CHOP的表达，而Klotho蛋白对其有显著的抑制效果。而Realtime-PCR结果也表明，Klotho蛋白可抑制ISO诱导的GRP78、HSP47及促凋亡转录因子CHOP mRNA水平的增加。体外研究表明10uM ISO可诱导心肌细胞凋亡，而重组Klotho蛋白显著减少ISO诱导的H9c2心肌细胞的凋亡。研究同样发现ISO促进ROS含量的生成，而重组Klotho蛋白可抑制ROS含量的增加。而且1ug/ml的重组Klotho蛋白可显著抑制ISO诱导的H9c2心肌细胞中GRP78、HSP47及CHOP的显著增加。
3.培养H9c2心肌细胞，待细胞达到80~90%融合时接种到6孔板内。为了明确MAPK信号通路在Klotho蛋白抵抗内质网应激介导的凋亡中的作用，分别加入不同的MAPK抑制剂刺激H9c2细胞，随机分为：ISO组(加入2mlDMEM培养液)、ISO+KL组(加入2ml含有0.1ug/ml Klotho蛋白的DMEM培养液)，ISO+SB203580(加入2ulSB203580+2mlDMEM培养液)，ISO+PD98059(加入10ul PD98058+2mlDMEM培养液)，ISO+SP600125(加入10ul SP600125+2ml DMEM培养液)，培养箱孵育1h后，再加入10uM ISO刺激细胞，继续培养箱内培养细胞。应用流式细胞技术及Western blot技术检测细胞的凋亡率及ER应激相关蛋白的表达。同时，为了明确Klotho蛋白对MAPK信号通路的直接影响，分别用ISO和/或不同浓度的Klotho蛋白(0.01ug/ml、0.1ug/ml、1ug/ml、10ug/ml)刺激H9c2心肌细胞，然后应用Western blot技术观察P38、JNK以及ERK1/2的磷酸化情况。结果表明Klotho蛋白可显著抑制ISO诱导的心肌细胞凋亡，而加入SB203580或SP600125后Klotho蛋白的抗凋亡作用依然明显，但是加入PD98059后心肌细胞的凋亡率未见明显改变，而进一步应用MAPK抑制剂检测内质网应激相关蛋白的表达水平表明，SB203580、SP600125和Klotho蛋白均可显著降低GRP78、HSP47和CHOP蛋白的表达，但PD98059对GRP78、HSP47和CHOP蛋白表达水平的影响未见显著差异，推测Klotho蛋白可能通过P38和JNK信号通路调节内质网应激介导的细胞凋亡。进一步观察不同浓度的Klotho蛋白对MAPK信号通路的影响发现，不同浓度的Klotho蛋白(0.01-10ug/ml)不能明显影响ERK1/2的活化，但是Klotho蛋白可以剂量依赖性的抑制ISO诱导的P38和JNK的磷酸化，提示Klotho蛋白的抗凋亡作用与抑制P38及JNK的活化有关。
结论：
1.持续小剂量皮下注射ISO9天后可诱导Balb/C小鼠心肌细胞肥大、凋亡、肌纤维紊乱及心肌间质纤维化，说明ISO皮下注射可成功诱导小鼠心室重构，能有效模拟慢性心衰的病理过程；
2. Klotho蛋白可抑制ISO诱导Balb/C小鼠心室重构的发生发展，更重要的是研究证实Klotho蛋白可抑制ISO诱导的小鼠心肌细胞及H9c2细胞株的凋亡，减少内质网应激相关蛋白GRP78、HSP47蛋白以及促凋亡转录因子CHOP的表达，表明Klotho蛋白的抗凋亡作用与抑制心肌细胞中ER应激介导凋亡信号通路有关；
3. P38抑制剂SB203580和JNK抑制剂SP600125与Klotho蛋白对H9c2心肌细胞的抗凋亡作用具有相似的效果，但ERK1/2抑制剂PD98059则不能显著影响细胞的凋亡率，Klotho蛋白呈剂量依赖性抑制P38和JNK的磷酸化，而不能明显影响ERK1/2的磷酸化，提示Klotho蛋白抗ER应激介导凋亡的作用部分依赖于对P38和JNK信号通路的抑制。
Background：
Cardiac remodeling is the common pathological alteration in heart responsing tostimuli, which is charactered by the change of the structure, volume and function of heart.Chronic cardiac remodeling finally induced the decline of cardiac diastolic and systolicfunction. Since the global aging population increasing, the incidence and mortality of heartfailure increase year by year, which is a major disease affecting the life quality and survivalof the older. Cardiac remodeling after myocardial infarction is one of the most main cause.Although the development of clinical medical level has saved the dying myocardium at agreater degree, such measures still can’t completely reverse the development of ventricularremodeling and heart failure. Researchers show that neurohumoral factors play animportant role in the pathological process of ventricular remodeling. At early stage, theactivation of β-adrenergic receptor can enhance contraction of myocardial responsing to thechange of blood flow. However, long-term activation of β-adrenergic receptor inducespathological myocardial hypertrophy, apoptosis and interstitial fibrosis, and finally resultsin cardiac dysfunction. Isoproterenol, as β-adrenergic agonists, is commonly used as anideal inducer for cardiac injury, which includes myocyte hypertrophy, apoptosis andinterstitial fibrosis. To explore the key factor reversing the above pathological changes is ofgreat significance in the reduction of heart failure and sudden death.
Klotho protein is a newly discovered anti-aging factor, which plays biological effectsthrough the membrane-bound type and secreted type. Based on the literature and previousresearches, we speculated that Klotho protein can play a cardiovascular protection role.Firstly, Klotho protein can resist to oxidation stress, which is widely involved in thedevelopment of many disease, including ventricular remodeling. Secondly, Klotho proteincan increase the biological activity of nitric oxide (NO), or regulate the calcium ionchannels on vascular endothelial cells to maintain endothelial integrity and endothelialfunction. Thirdly, Klotho protein control hypertensive, and the increased pressure load is the main cause of chronic hear failure. In addition, Klotho protein inhibits endothelial cellapoptosis and inflammation. Clinical studies show that low level of plasma Klotho proteinmay be related to cardiovascular disease induced by chronic kidney disease, our previouswork also found the single nucleotide polymorphisms in Klotho gene are closely associatedwith cardiovascular diseases such as arteriosclerosis, so it is reasonable to speculate thatKlotho protein can inhibit ventricular remodeling. Recent study found that ISO can result ina significant reduction of the cardiac systolic function in mice, and the effect can beenhancedin Klotho deficient mice, suggesting that Klotho protein plays a protective role oncardiac systolic function.
Myocardial cell apoptosis is an important pathological change of ventricularremodeling, which is the key factors that lead to cardiac dysfunction and even sudden death.In addition to mitochondria, endoplasmic reticulum is another important organ involved inapoptosis, whose main functions include regulating calcium level, protein folding andmature. When the balance of the endoplasmic reticulum was disrupted (such as damagedcalcium level, excessive generation of protein or protein misfolding), it would induce theexpression of endoplasmic reticulum chaperone such as glucose regulatory protein78andheat shock protein47，and this process is known as endoplasmic reticulum stress. At theinitial stage, endoplasmic reticulum stress is the body's adaptive response to externalstimuli, which will help to maintain the balance of internal environment. Chronicendoplasmic reticulum stress will lead to the activation of apoptosis signaling pathways,including the induction of pro-apoptosis transcription factors CHOP. CHOP signalingpathway is one of the key apoptotic signaling pathways mediated by endoplasmic reticulumstress. Endoplasmic reticulum stress mediated apoptosis is the hot studying field. A largenumber of experiments showed that excessive or chronic ER stress can lead to the apoptosisof myocardial cells in vivo and in vitro. Studies reported that ISO could induce apoptosis ofcultured myocardial cell accompanied by the increased expression of the endoplasmicreticulum chaperone and pro-apoptosis transcription factors CHOP. And the inhibition ofendoplasmic reticulum stress can prevent cardiomyocytes apoptosis. And whether theprotection of cardiac systolic function by Klotho protein is related to CHOP-mediatedapoptosis signal induced by ER stress has not been reported.
Cells respond to external stimuli mainly through intracellular signaling pathways. Mitogen-activated protein kinases is an ubiquitous intracellular signaling pathways,including extracellular signal regulated kinase1/2, p38and c-Jun NH2-terminal kinase.Mitogen-activated protein kinases play an important role in cells’ proliferation,differentiation, and it also involves in ER stress-mediated apoptosis. Whether Klothoprotein regulates ER stress mediated apoptosis through these signal pathways has not beenfully elucidated and need further research.
Objective：
This experiment made anti-apoptosis effect of Klotho protein as the breakthroughpoint to explorer the effect of Klotho protein on ventricular remodeling especiallymyocardial cell apoptosis based on the construction of ventricular remodeling mouse model.And further study was performed to define whether the anti-apoptotic mechanism of Klothoprotein was related with ER stress mediated apoptosis signal. Finally, it intended todetermine whether Klotho inhibited ER stress mediated apoptosis through regulation ofMAPK signal pathway through signal study.
The main experimental methods and results:
1. PART ONE:6-8weeks of Balb/C male mice weighing about20g were randomlydivided into control group and ISO group. CON group: subcutaneous injection of100ulsaline solution, total9days; ISO group: subcutaneous injection of5mg/kg ISO, dissolved in100ul saline solution, total9days. At the9thof ISO injection, mice were weight on theelectronic balance, and then execute them and remove their hearts gently. Finally wecalculate the ratio of HW/BW. Cut each cardiac tissue into6pieces,3piece of whichpacked with tinfoil, and then put them into the frozen storage tube, preserved in liquidnitrogen tank to performe quantitative PCR test. The rest of the myocardial tissue soak in4%paraformaldehyde solution for24hours, and then embed with paraffin.4um paraffinsection was cut on slicer. Serial4um paraffin sections were used to performe HE staining,Masson staining, Tunel staining for morphological analysis, quantitative analysis ofcollagen and apoptosis. Results indicated that subcutaneous injection of ISO could causecardiomyocytes hypertrophy and the disorders of muscle fibers. By measuring the ratio ofHW/BW, we also found that ISO could increase the ratio of HW/BW statistically, whichwas closely related to cardiomyocytes hypertrophy. HE staining technology found thatcontinuous subcutaneous injection ISO daily (9days) could cause myocardial cell hypertrophy and make muscle fibers arranged in disorder. In addition to the pathologicalchanges of myocardial cells, we also found that fibroblast infiltration and proliferationsignificantly increased in ISO group, suggesting the subcutaneous injection of ISO inducedmyocardial interstitial fibrosis. And Masson staining found that the collagen volume ofmyocardial interstitium in ISO group wss significantly higher than the control group, whichindicated that ISO could induced myocardial fibrosis successfully. Real time PCR wasperformed to detect college mRNA levels in the control group and the ISO group. Resultfound that ISO could increase the college mRNA level. Subcutaneous injection of ISO canincrease the rate of myocardial cell apoptosis. Tunel staining found that the apoptosis rateof myocardial cells in ISO group was higher than in the CON group. Therefore ISO, as abeta-adrenergic agonist, can be used to construct the ventricular remodeling mouse modelsuccessfully.
2. PART TWO:6-8weeks of Balb/C male mice weighing about20g were randomlydivided into the following groups. CON group: subcutaneous injection of saline solution(100ul, qd, a total of9days), and intraperitoneal injection of saline solution (100ul, qod, atotal of4days). ISO group: subcutaneous injection of ISO (at the concentration of5mg/kg/d, qd a total of9days), and intraperitoneal injection of saline solution100ul, qod, atotal of4days). ISO+KL group: subcutaneous injection of ISO (at the concentration of5mg/kg, a total of9days), and intraperitoneal injections of Klotho protein (at theconcentration of0.01mg/kg, qod, a total of4days). Mice were decapitated at2,5,9day afterISO injection. We determine the serum Klotho protein levels in mice after intraperitonealinjection of Klotho protein by ELISA technology. And HE staining, Massonn staining andTUNEL staining were performed to determine the pathological changes of heart tissue.ROS detection technology was performed to detect the ROS production in heart.Immunohistochemical staining, Western blot and Real-time PCR was used to determine theprotein level and mRNA level of ER stress related protein GRP78, HSP47andpro-apoptosis transcription factors CHOP. At the same time, H9c2cells were cultured toobserve the effect of Klotho protein on ISO induced cardiacmyocytes injury. Resultsshowed that serum Klotho protein level was increased significantly after the injection ofKlotho protein (P <0.05). We observed that Klotho protein inhibit the pathologic changesin heart tissue induced by ISO. In the control group, myocardial fibers were normally arranged, however cardiac tissue showed irregular myocardial fibrosis and fibroblastshyperplasia after ISO injection, charactered by increased nucleus density and mononuclearcell infiltrates. These suggested serious pathological changes in the ISO group. However,Klotho can relieve the above change, suggesting cardiac protective effect of Klotho protein.Masson stain experiments showed the collagen of interstitial and perivascular issignificantly higher from5days after the injection of ISO in ISO group than in CON group.However Klotho can reduce myocardial fibrosis, showed that Klotho proteins inhibit ISOcaused cardiac structural changes. TUNEL staining experiments showed that Klotho proteincould reduce ISO-induce apoptosis of myocardial cells. At9th day after ISO stimulation,ISO significantly increases the percentage of TUNEL positive cells. The Klotho proteinscan significantly reduced the percentage of TUNEL positive cells, with statistical difference(P <0.05). In accordance with the result, the generation of ROS in ISO group increasedsignificantly, and it was significantly inhibitte in ISO+KL group. Further research showedthat anti-apoptotic effect of Klotho protein was related to the inhibition of ER stress-relatedapoptotic signals in heart tissue. Immunohistochemistry, Western blot and Real-time PCRtechniques had confirmed that Klotho proteins could significantly reduce the protein leveland mRNA level of ER chaperonin GRP78, HSP47and CHOP. In vitro study had shownthat10um ISO induced a significant increase in myocardial cell apoptosis, and Klothoprotein significantly inhibit H9c2cell apoptosis induced by ISO. The study also found thatISO promote the generation of ROS, and Klotho inhibited the generation of ROS. And1ug/ml Klotho proteins can significantly suppress the ER stress protein markers GRP78HSP47and CHOP expression induced by the ISO statistically. So Klotho can improve thepathological change of heart through inhibiting ER stress-mediated apoptosis.
3. PART THREE：Cultured H9c2cells were passaged into6-well plates when cells reachedto80-90%confluence. In order to determine the role of MAPK signaling pathways in theinhibition of endoplasmic reticulum stress mediated apoptosis by Klotho, different MAPKinhibitors was used to stimulate H9c2cells. Then they were randomly divided into differentgroups: ISO group (2ml DMEM), ISO+KL group (2ml DMEM containing0.1ug/mlKlotho protein), ISO+SB20380(2ul SB203580+2ml DMEM), ISO+PD98059(10ulPD98058+2ml DMEM), ISO+SP600125(10ul SP600125+2ml DMEM). They wereincubated in an incubator for1h, and then10uM ISO were added to each group. Flow cytometry and Western blot technique were performed to detect cell apoptosis and theexpression of ER stress-related proteins. At the same time, in order to detect the directimpact of Klotho protein of MAPK signal pathway, the cells were exposed to ISO and/ordifferent concentrations of Klotho protein (0.01ug/ml,0.1ug/ml,1ug/ml,10ug/ml).Western blot technique was applied to detect the phosphorylation of P38MAPK, JNK andERK1/2. The results showed that SB203580and SP600125reduced H9c2cell apoptosislike Klotho protein, but PD98059did not affect the apoptosis significantly. In addition, theexpression of GRP78, HSP47and CHOP can be inhibited by Klotho, SB203580orSP600125statistically, but not significantly inhibited by PD98059.These results indicatedthat Klotho may inhibit cell apoptosis through P38MAPK and JNK.10uM ISO can activatethe MAPK signaling pathway in H9c2cells, and different concentrations of Klotho(0.01-10ug/ml) does not significantly affect the level of phosphorylated ERK1/2, but0.1ug/ml and1ug/ml of Klotho significantly stimulated the phosphorylation of JNK andP38MAPK. The results showed that the anti-apoptosis effect of Klotho was related to thedownregulation of P38and JNK pathway.
Conclusion:
1. Subcutaneous injection of ISO can induce myocardial hypertrophy, apoptosis,muscle fiber disorder and interstitial myocardial fibrosis, indicating that subcutaneousinjection of ISO can successfully construct ventricular remodeling mouse model whichcan effectively simulate the pathological process of chronic heart failure；
2. Klotho protein inhibited the development of chronic heart failure in ISO-inducedBalb/C mouse model. Importantly this study confirmed that Klotho protein can reduce theISO-induced apoptosis and the expression of endoplasmic reticulum stress-related proteinGRP78, HSP47and pro-apoptosis transcription factor CHOP in heart tissue and culturedcardiomyocytes, indicating the anti-apoptosis role of Klotho protein was related with theinhibition of ER stress-related apoptosis signal pathway;
3. P38inhibitor, SB203580and JNK inhibitor, SP600125have the same anti-apoptosiseffect with Klotho protein in ISO-stimulated H9c2cells, however ERK1/2inhibitor,PD98059did not affect the apoptosis significantly. Klotho protein (0.01-10ug/ml)significantly inhibited the phosphorylation of JNK and P38, but did not significantly affectthe phosphorylation of ERK1/2, suggesting that anti-apoptosis effect of Klotho protein was partially dependent on the inhibition of JNK and P38MAPK signaling pathway.
心室重构(Cardiac remodeling)是心脏对外界各种应激性刺激作出的共同病理反应，主要表现为心脏结构、大小及功能的改变。心室重构最终会导致心脏舒缩功能下降，是心力衰竭发病的根本原因。中国老龄化程度的不断加剧，以及高血压、冠心病、瓣膜病等心血管病发病率的逐年升高，使得心衰的发病率及病死率逐年增加，成为影响老年人生活质量及生存率的主要疾病之一。在众多引起心室重构的疾病中，心肌梗死后心衰重构是最主要的病因之一，尽管随着临床医疗水平的进步，如冠脉造影及支架植入术以及冠脉搭桥手术的开展，更大程度的挽救了濒死的心肌，使梗死后心室重构的发生及发展显著改善，但是这些措施仍不能完全逆转心室重构的发生以及心衰临床症状的恶化。研究表明，神经体液因子在心室重构的病理过程中发挥着重要作用。早期阶段，β肾上腺素能受体的激活可以诱导心肌细胞收缩力增强，代偿前后负荷增高对机体的影响。但是，长期的β肾上腺素能受体的激活将导致病理性的心肌损伤，引起心室重构。异丙肾上腺素(Isoproterenol，ISO)作为β肾上腺素能受体激动剂，是常用的诱导实验动物心肌损伤的诱导剂。心肌损伤主要包括心肌细胞丢失、肥大以及间质胶原含量的改变。寻求逆转上述病理改变的关键分子对于降低心衰的发病率及猝死率具有重要意义。
Klotho蛋白是新近发现的具有抗衰老作用的蛋白，通过膜结合型和分泌型两种形式发挥生物学效应。基于文献及前期的研究工作，我们推测Klotho蛋白可能是具有心血管保护作用的激素样蛋白分子。首先Klotho蛋白具有抗氧化应激特性，而氧化应激广泛参与了许多疾病的发生发展，包括心室重构；其次Klotho蛋白可通过增加NO生物活性或调控血管内皮细胞上的钙离子通道维持血管内皮完整性及内皮功能；再次Klotho蛋白具有降压作用，而压力负荷增高是导致心衰发病的主要原因；另外Klotho蛋白还可抑制内皮细胞凋亡及炎症反应。临床研究表明血浆中低水平的Klotho蛋白可能是慢性肾脏疾病相关的心血管疾病的高危因素，而我们的前期工作也发现Klotho基因的单核苷酸多态性与动脉硬化等心血管疾病密切相关，因此我们有足够的证据认为Klotho蛋白可能对心室重构具有抑制作用。而且最近的研究发现，ISO可导致小鼠心脏收缩功能显著下降，而Klotho基因敲除可进一步加重这一下降效果，提示Klotho蛋白对心脏收缩功能具有保护作用。
心肌细胞丢失是心脏收缩功能下降的主要原因，其中心肌细胞的丢失主要源于心肌细胞的凋亡及坏死，而心肌细胞凋亡是慢性心室重构过程中心肌细胞丢失的主要原因，是导致心脏功能失调甚至猝死的关键因素。内质网(Endoplasmic reticulum，ER)是除了线粒体外又一个参与细胞凋亡的重要器官，其主要功能包括调节钙离子平衡、参与蛋白质的折叠及成熟等。当内质网的平衡被破坏时(如钙离子平衡被破坏、蛋白质生成过多或蛋白质折叠错误)，内质网将诱导相关伴侣蛋白如糖调节蛋白78(GRP78)、热休克蛋白47(HSP47)的表达，此过程称为内质网应激。内质网伴侣分子的增加不仅是内质网应激发生的标志，同时也是内质网应激严重程度的重要体现。早期阶段的内质网应激是机体对外界刺激作出的适应性反应，主要通过促进钙离子平衡、抑制蛋白质运输到内质网、增强蛋白质正确折叠及运输以维持内质网稳态，但长期或严重的内质网应激将导致凋亡信号通路的激活，其中促凋亡转录因子CHOP的表达增加是重要的内质网介导凋亡的效应分子。近年来的研究表明，内质网应激在心肌细胞凋亡中同样发挥重要的作用。研究发现，抑制内质网应激可有效缓解心肌细胞的损伤。同样，ISO可以诱导心肌细胞株发生凋亡，其中伴随内质网伴侣蛋白及促凋亡转录因子CHOP的表达明显升高，说明ISO通过激活内质网应激介导的凋亡通路诱导细胞凋亡。而Klotho蛋白维持心脏收缩功能的作用是否与抑制ER应激介导的CHOP凋亡信号有关还未见报道。
细胞主要通过调节细胞内的信号通路对外界刺激作出反应。 MAPK(Mitogen-activated protein kinases)是普遍存在于细胞内的信号转导通路，由胞外信号调节蛋白激酶(ERK1/2), p38和c-Jun氨基末端激酶(JNK)组成，调节细胞的增殖、分化，在ER应激引起的凋亡中也发挥重要的调节作用。而分泌型Klotho蛋白作为循环中的蛋白分子，能否通过该信号通路参与ER应激介导的凋亡还未完全阐明，需要进一步研究明确。
实验目的:
本实验以Klotho蛋白的抗凋亡作用作为切入点，在构建ISO诱导小鼠心室重构模型的基础上，研究Klotho蛋白对心室重构特别是心肌细胞凋亡的影响。并进一步明确Klotho蛋白的抗凋亡机制是否与ER应激介导的CHOP凋亡信号通路密切相关。最后通过信号通路研究，明确Klotho蛋白调节MAPK信号通路的具体情况，以及其调节MAPK信号通路与ER应激介导的凋亡之间的关系。
主要实验方法及实验结果：
1.取6-8周龄的Balb/C品系，重量约20g的雄性小鼠，随机分为对照组（CON）、ISO组。其中CON组每天背部皮下注射100ul生理盐水，共注射9天；ISO组每天以5mg/kg/d剂量背部皮下注射ISO（溶解于100ul生理盐水中），共注射9天。在注射后的第9天在电子天平上称小鼠的重量，处死小鼠，并轻柔的切除小鼠的心脏组织称心脏的重量，计算HW/BW的比值。每个离体的心脏平均切割成6块，其中3块心肌组织用锡箔纸包好，放入冻存管内，液氮罐中保存，用于后续的Realtime-PCR实验。剩下的心肌组织浸泡在4%多聚甲醛溶液中，固定24小时后，石蜡包埋制作石蜡块。用切片机切割4um厚度的石蜡切片，对连续的4um心肌石蜡切片行HE染色观察心肌的形态改变。同时对心肌石蜡切片进行Masson染色，对其胶原容积分数进行定量分析，然后应用Tunel染色技术对石蜡切片进行凋亡检测，分别计数检测心肌细胞的凋亡率。研究结果表明持续小剂量皮下注射ISO可诱导小鼠心肌细胞肥大凋亡、肌纤维紊乱及心肌间质成纤维细胞增生及胶原蛋白沉积。通过测定HW/BW的比值，我们发现ISO可诱导小鼠心心脏体重指数增加，且具有统计学差异(P<0.05)，这种作用与心肌细胞的肥大增生及胶原纤维增生密切相关。HE染色结果发现持续皮下注射ISO(9天)可引起心肌细胞的肥大及肌纤维排列的紊乱。除了心肌细胞的病变外，我们还发现ISO可诱导心肌间质中成纤维细胞增殖显著增加，说明皮下注射ISO也诱导了心肌间质的纤维化反应。同时我们应用Realtime-PCR技术观察心肌间质胶原蛋白的mRNA水平，结果表明ISO可显著增加collegen mRNA的表达水平。Masson染色观察心肌间质胶原纤维的含量发现，在ISO组中心肌间质的胶原容积比例显著高于对照组，由此说明ISO可成功诱导心肌间质纤维化；最后我们应用Tunel染色技术检测心肌细胞的凋亡率，结果表明皮下注射ISO可增加心肌细胞的凋亡率，且具有统计学意义(P<0.05)。由此说明ISO作为β肾上腺素能受体激动剂，可成功用于构建小鼠心室重构模型。
2.取6-8周龄的Balb/C品系，重量约20g的雄性小鼠，随机分为以下各组：CON组：背部皮下注射生理盐水(100ul，qd，共9天)，同时腹腔注射生理盐水(100ul，qod，共4天)；ISO组：背部皮下注射ISO(5mg/kg/d，qd，共9天)，同时腹腔注射生理盐水(0.1ml，qod，共4天)；ISO+KL组：背部皮下注射ISO(5mg/kg/d，gd，共9天)，同时腹腔注射Klotho蛋白(0.01mg/kg，qod，共4天)。按照上述的方法对各组进行操作，分别在ISO注射的第2，5，9天处死小鼠。首先，我们应用ELISA技术检测腹腔注射重组Klotho蛋白对小鼠血循环中Klotho蛋白水平的影响。并分别应用HE染色、Masson染色、TUNEL染色检测心肌组织的结构改变及凋亡情况，ROS检测技术测定组织中ROS的含量，最后应用免疫组织化学染色、Real-time PCR技术、Western blot技术测定心脏组织中ER应激介导的凋亡信号分子GRP78、HSP47及促凋亡转录因子CHOP的mRNA水平及蛋白水平。同时通过体外培养大鼠胚胎来源的H9c2心肌细胞，进一步观察Klotho蛋白对ISO诱导H9c2心肌细胞肥大、凋亡及ER应激介导的凋亡信号通路的影响。结果表明腹腔注射Klotho蛋白后，血清中Klotho蛋白的水平显著增高(P<0.05)。HE染色结果表明，重组Klotho蛋白抑制ISO诱导的小鼠心脏组织的病理变化。在CON组，心肌纤维走形正常；而ISO注射之后小鼠心脏组织的HE染色图片表现为心肌纤维走形紊乱、间质成纤维细胞显著增生，表现为细胞核密度的增加以及单核细胞局限性浸润，暗示了ISO组中严重的心肌损伤；而重组Klotho蛋白可以缓解上述改变，提示Klotho蛋白具有心肌保护作用。Masson染色结果表明，从ISO注射后的第5天开始，心肌间质及血管周的胶原纤维含量明显高于对照组，而Klotho蛋白可抑制胶原纤维的增加。TUNEL染色结果表明ISO注射的第9天，心肌细胞的凋亡率显著增加，而Klotho蛋白可抑制ISO诱导的小鼠心肌细胞的凋亡。ROS是公认的细胞凋亡诱导因素，和上述研究结果一致，ISO组中ROS的含量显著增加，而在ISO+KL组ROS含量被抑制。由此说明了Klotho蛋白的心肌保护作用及抗凋亡作用。进而我们应用免疫组化及Western blot技术检测了ER应激相关蛋白分子的表达情况，结果证实ISO可诱导内质网应激相关伴侣分子GRP78、HSP47及促凋亡转录因子CHOP的表达，而Klotho蛋白对其有显著的抑制效果。而Realtime-PCR结果也表明，Klotho蛋白可抑制ISO诱导的GRP78、HSP47及促凋亡转录因子CHOP mRNA水平的增加。体外研究表明10uM ISO可诱导心肌细胞凋亡，而重组Klotho蛋白显著减少ISO诱导的H9c2心肌细胞的凋亡。研究同样发现ISO促进ROS含量的生成，而重组Klotho蛋白可抑制ROS含量的增加。而且1ug/ml的重组Klotho蛋白可显著抑制ISO诱导的H9c2心肌细胞中GRP78、HSP47及CHOP的显著增加。
3.培养H9c2心肌细胞，待细胞达到80~90%融合时接种到6孔板内。为了明确MAPK信号通路在Klotho蛋白抵抗内质网应激介导的凋亡中的作用，分别加入不同的MAPK抑制剂刺激H9c2细胞，随机分为：ISO组(加入2mlDMEM培养液)、ISO+KL组(加入2ml含有0.1ug/ml Klotho蛋白的DMEM培养液)，ISO+SB203580(加入2ulSB203580+2mlDMEM培养液)，ISO+PD98059(加入10ul PD98058+2mlDMEM培养液)，ISO+SP600125(加入10ul SP600125+2ml DMEM培养液)，培养箱孵育1h后，再加入10uM ISO刺激细胞，继续培养箱内培养细胞。应用流式细胞技术及Western blot技术检测细胞的凋亡率及ER应激相关蛋白的表达。同时，为了明确Klotho蛋白对MAPK信号通路的直接影响，分别用ISO和/或不同浓度的Klotho蛋白(0.01ug/ml、0.1ug/ml、1ug/ml、10ug/ml)刺激H9c2心肌细胞，然后应用Western blot技术观察P38、JNK以及ERK1/2的磷酸化情况。结果表明Klotho蛋白可显著抑制ISO诱导的心肌细胞凋亡，而加入SB203580或SP600125后Klotho蛋白的抗凋亡作用依然明显，但是加入PD98059后心肌细胞的凋亡率未见明显改变，而进一步应用MAPK抑制剂检测内质网应激相关蛋白的表达水平表明，SB203580、SP600125和Klotho蛋白均可显著降低GRP78、HSP47和CHOP蛋白的表达，但PD98059对GRP78、HSP47和CHOP蛋白表达水平的影响未见显著差异，推测Klotho蛋白可能通过P38和JNK信号通路调节内质网应激介导的细胞凋亡。进一步观察不同浓度的Klotho蛋白对MAPK信号通路的影响发现，不同浓度的Klotho蛋白(0.01-10ug/ml)不能明显影响ERK1/2的活化，但是Klotho蛋白可以剂量依赖性的抑制ISO诱导的P38和JNK的磷酸化，提示Klotho蛋白的抗凋亡作用与抑制P38及JNK的活化有关。
结论：
1.持续小剂量皮下注射ISO9天后可诱导Balb/C小鼠心肌细胞肥大、凋亡、肌纤维紊乱及心肌间质纤维化，说明ISO皮下注射可成功诱导小鼠心室重构，能有效模拟慢性心衰的病理过程；
2. Klotho蛋白可抑制ISO诱导Balb/C小鼠心室重构的发生发展，更重要的是研究证实Klotho蛋白可抑制ISO诱导的小鼠心肌细胞及H9c2细胞株的凋亡，减少内质网应激相关蛋白GRP78、HSP47蛋白以及促凋亡转录因子CHOP的表达，表明Klotho蛋白的抗凋亡作用与抑制心肌细胞中ER应激介导凋亡信号通路有关；
3. P38抑制剂SB203580和JNK抑制剂SP600125与Klotho蛋白对H9c2心肌细胞的抗凋亡作用具有相似的效果，但ERK1/2抑制剂PD98059则不能显著影响细胞的凋亡率，Klotho蛋白呈剂量依赖性抑制P38和JNK的磷酸化，而不能明显影响ERK1/2的磷酸化，提示Klotho蛋白抗ER应激介导凋亡的作用部分依赖于对P38和JNK信号通路的抑制。
Background：
Cardiac remodeling is the common pathological alteration in heart responsing tostimuli, which is charactered by the change of the structure, volume and function of heart.Chronic cardiac remodeling finally induced the decline of cardiac diastolic and systolicfunction. Since the global aging population increasing, the incidence and mortality of heartfailure increase year by year, which is a major disease affecting the life quality and survivalof the older. Cardiac remodeling after myocardial infarction is one of the most main cause.Although the development of clinical medical level has saved the dying myocardium at agreater degree, such measures still can’t completely reverse the development of ventricularremodeling and heart failure. Researchers show that neurohumoral factors play animportant role in the pathological process of ventricular remodeling. At early stage, theactivation of β-adrenergic receptor can enhance contraction of myocardial responsing to thechange of blood flow. However, long-term activation of β-adrenergic receptor inducespathological myocardial hypertrophy, apoptosis and interstitial fibrosis, and finally resultsin cardiac dysfunction. Isoproterenol, as β-adrenergic agonists, is commonly used as anideal inducer for cardiac injury, which includes myocyte hypertrophy, apoptosis andinterstitial fibrosis. To explore the key factor reversing the above pathological changes is ofgreat significance in the reduction of heart failure and sudden death.
Klotho protein is a newly discovered anti-aging factor, which plays biological effectsthrough the membrane-bound type and secreted type. Based on the literature and previousresearches, we speculated that Klotho protein can play a cardiovascular protection role.Firstly, Klotho protein can resist to oxidation stress, which is widely involved in thedevelopment of many disease, including ventricular remodeling. Secondly, Klotho proteincan increase the biological activity of nitric oxide (NO), or regulate the calcium ionchannels on vascular endothelial cells to maintain endothelial integrity and endothelialfunction. Thirdly, Klotho protein control hypertensive, and the increased pressure load is the main cause of chronic hear failure. In addition, Klotho protein inhibits endothelial cellapoptosis and inflammation. Clinical studies show that low level of plasma Klotho proteinmay be related to cardiovascular disease induced by chronic kidney disease, our previouswork also found the single nucleotide polymorphisms in Klotho gene are closely associatedwith cardiovascular diseases such as arteriosclerosis, so it is reasonable to speculate thatKlotho protein can inhibit ventricular remodeling. Recent study found that ISO can result ina significant reduction of the cardiac systolic function in mice, and the effect can beenhancedin Klotho deficient mice, suggesting that Klotho protein plays a protective role oncardiac systolic function.
Myocardial cell apoptosis is an important pathological change of ventricularremodeling, which is the key factors that lead to cardiac dysfunction and even sudden death.In addition to mitochondria, endoplasmic reticulum is another important organ involved inapoptosis, whose main functions include regulating calcium level, protein folding andmature. When the balance of the endoplasmic reticulum was disrupted (such as damagedcalcium level, excessive generation of protein or protein misfolding), it would induce theexpression of endoplasmic reticulum chaperone such as glucose regulatory protein78andheat shock protein47，and this process is known as endoplasmic reticulum stress. At theinitial stage, endoplasmic reticulum stress is the body's adaptive response to externalstimuli, which will help to maintain the balance of internal environment. Chronicendoplasmic reticulum stress will lead to the activation of apoptosis signaling pathways,including the induction of pro-apoptosis transcription factors CHOP. CHOP signalingpathway is one of the key apoptotic signaling pathways mediated by endoplasmic reticulumstress. Endoplasmic reticulum stress mediated apoptosis is the hot studying field. A largenumber of experiments showed that excessive or chronic ER stress can lead to the apoptosisof myocardial cells in vivo and in vitro. Studies reported that ISO could induce apoptosis ofcultured myocardial cell accompanied by the increased expression of the endoplasmicreticulum chaperone and pro-apoptosis transcription factors CHOP. And the inhibition ofendoplasmic reticulum stress can prevent cardiomyocytes apoptosis. And whether theprotection of cardiac systolic function by Klotho protein is related to CHOP-mediatedapoptosis signal induced by ER stress has not been reported.
Cells respond to external stimuli mainly through intracellular signaling pathways. Mitogen-activated protein kinases is an ubiquitous intracellular signaling pathways,including extracellular signal regulated kinase1/2, p38and c-Jun NH2-terminal kinase.Mitogen-activated protein kinases play an important role in cells’ proliferation,differentiation, and it also involves in ER stress-mediated apoptosis. Whether Klothoprotein regulates ER stress mediated apoptosis through these signal pathways has not beenfully elucidated and need further research.
Objective：
This experiment made anti-apoptosis effect of Klotho protein as the breakthroughpoint to explorer the effect of Klotho protein on ventricular remodeling especiallymyocardial cell apoptosis based on the construction of ventricular remodeling mouse model.And further study was performed to define whether the anti-apoptotic mechanism of Klothoprotein was related with ER stress mediated apoptosis signal. Finally, it intended todetermine whether Klotho inhibited ER stress mediated apoptosis through regulation ofMAPK signal pathway through signal study.
The main experimental methods and results:
1. PART ONE:6-8weeks of Balb/C male mice weighing about20g were randomlydivided into control group and ISO group. CON group: subcutaneous injection of100ulsaline solution, total9days; ISO group: subcutaneous injection of5mg/kg ISO, dissolved in100ul saline solution, total9days. At the9thof ISO injection, mice were weight on theelectronic balance, and then execute them and remove their hearts gently. Finally wecalculate the ratio of HW/BW. Cut each cardiac tissue into6pieces,3piece of whichpacked with tinfoil, and then put them into the frozen storage tube, preserved in liquidnitrogen tank to performe quantitative PCR test. The rest of the myocardial tissue soak in4%paraformaldehyde solution for24hours, and then embed with paraffin.4um paraffinsection was cut on slicer. Serial4um paraffin sections were used to performe HE staining,Masson staining, Tunel staining for morphological analysis, quantitative analysis ofcollagen and apoptosis. Results indicated that subcutaneous injection of ISO could causecardiomyocytes hypertrophy and the disorders of muscle fibers. By measuring the ratio ofHW/BW, we also found that ISO could increase the ratio of HW/BW statistically, whichwas closely related to cardiomyocytes hypertrophy. HE staining technology found thatcontinuous subcutaneous injection ISO daily (9days) could cause myocardial cell hypertrophy and make muscle fibers arranged in disorder. In addition to the pathologicalchanges of myocardial cells, we also found that fibroblast infiltration and proliferationsignificantly increased in ISO group, suggesting the subcutaneous injection of ISO inducedmyocardial interstitial fibrosis. And Masson staining found that the collagen volume ofmyocardial interstitium in ISO group wss significantly higher than the control group, whichindicated that ISO could induced myocardial fibrosis successfully. Real time PCR wasperformed to detect college mRNA levels in the control group and the ISO group. Resultfound that ISO could increase the college mRNA level. Subcutaneous injection of ISO canincrease the rate of myocardial cell apoptosis. Tunel staining found that the apoptosis rateof myocardial cells in ISO group was higher than in the CON group. Therefore ISO, as abeta-adrenergic agonist, can be used to construct the ventricular remodeling mouse modelsuccessfully.
2. PART TWO:6-8weeks of Balb/C male mice weighing about20g were randomlydivided into the following groups. CON group: subcutaneous injection of saline solution(100ul, qd, a total of9days), and intraperitoneal injection of saline solution (100ul, qod, atotal of4days). ISO group: subcutaneous injection of ISO (at the concentration of5mg/kg/d, qd a total of9days), and intraperitoneal injection of saline solution100ul, qod, atotal of4days). ISO+KL group: subcutaneous injection of ISO (at the concentration of5mg/kg, a total of9days), and intraperitoneal injections of Klotho protein (at theconcentration of0.01mg/kg, qod, a total of4days). Mice were decapitated at2,5,9day afterISO injection. We determine the serum Klotho protein levels in mice after intraperitonealinjection of Klotho protein by ELISA technology. And HE staining, Massonn staining andTUNEL staining were performed to determine the pathological changes of heart tissue.ROS detection technology was performed to detect the ROS production in heart.Immunohistochemical staining, Western blot and Real-time PCR was used to determine theprotein level and mRNA level of ER stress related protein GRP78, HSP47andpro-apoptosis transcription factors CHOP. At the same time, H9c2cells were cultured toobserve the effect of Klotho protein on ISO induced cardiacmyocytes injury. Resultsshowed that serum Klotho protein level was increased significantly after the injection ofKlotho protein (P <0.05). We observed that Klotho protein inhibit the pathologic changesin heart tissue induced by ISO. In the control group, myocardial fibers were normally arranged, however cardiac tissue showed irregular myocardial fibrosis and fibroblastshyperplasia after ISO injection, charactered by increased nucleus density and mononuclearcell infiltrates. These suggested serious pathological changes in the ISO group. However,Klotho can relieve the above change, suggesting cardiac protective effect of Klotho protein.Masson stain experiments showed the collagen of interstitial and perivascular issignificantly higher from5days after the injection of ISO in ISO group than in CON group.However Klotho can reduce myocardial fibrosis, showed that Klotho proteins inhibit ISOcaused cardiac structural changes. TUNEL staining experiments showed that Klotho proteincould reduce ISO-induce apoptosis of myocardial cells. At9th day after ISO stimulation,ISO significantly increases the percentage of TUNEL positive cells. The Klotho proteinscan significantly reduced the percentage of TUNEL positive cells, with statistical difference(P <0.05). In accordance with the result, the generation of ROS in ISO group increasedsignificantly, and it was significantly inhibitte in ISO+KL group. Further research showedthat anti-apoptotic effect of Klotho protein was related to the inhibition of ER stress-relatedapoptotic signals in heart tissue. Immunohistochemistry, Western blot and Real-time PCRtechniques had confirmed that Klotho proteins could significantly reduce the protein leveland mRNA level of ER chaperonin GRP78, HSP47and CHOP. In vitro study had shownthat10um ISO induced a significant increase in myocardial cell apoptosis, and Klothoprotein significantly inhibit H9c2cell apoptosis induced by ISO. The study also found thatISO promote the generation of ROS, and Klotho inhibited the generation of ROS. And1ug/ml Klotho proteins can significantly suppress the ER stress protein markers GRP78HSP47and CHOP expression induced by the ISO statistically. So Klotho can improve thepathological change of heart through inhibiting ER stress-mediated apoptosis.
3. PART THREE：Cultured H9c2cells were passaged into6-well plates when cells reachedto80-90%confluence. In order to determine the role of MAPK signaling pathways in theinhibition of endoplasmic reticulum stress mediated apoptosis by Klotho, different MAPKinhibitors was used to stimulate H9c2cells. Then they were randomly divided into differentgroups: ISO group (2ml DMEM), ISO+KL group (2ml DMEM containing0.1ug/mlKlotho protein), ISO+SB20380(2ul SB203580+2ml DMEM), ISO+PD98059(10ulPD98058+2ml DMEM), ISO+SP600125(10ul SP600125+2ml DMEM). They wereincubated in an incubator for1h, and then10uM ISO were added to each group. Flow cytometry and Western blot technique were performed to detect cell apoptosis and theexpression of ER stress-related proteins. At the same time, in order to detect the directimpact of Klotho protein of MAPK signal pathway, the cells were exposed to ISO and/ordifferent concentrations of Klotho protein (0.01ug/ml,0.1ug/ml,1ug/ml,10ug/ml).Western blot technique was applied to detect the phosphorylation of P38MAPK, JNK andERK1/2. The results showed that SB203580and SP600125reduced H9c2cell apoptosislike Klotho protein, but PD98059did not affect the apoptosis significantly. In addition, theexpression of GRP78, HSP47and CHOP can be inhibited by Klotho, SB203580orSP600125statistically, but not significantly inhibited by PD98059.These results indicatedthat Klotho may inhibit cell apoptosis through P38MAPK and JNK.10uM ISO can activatethe MAPK signaling pathway in H9c2cells, and different concentrations of Klotho(0.01-10ug/ml) does not significantly affect the level of phosphorylated ERK1/2, but0.1ug/ml and1ug/ml of Klotho significantly stimulated the phosphorylation of JNK andP38MAPK. The results showed that the anti-apoptosis effect of Klotho was related to thedownregulation of P38and JNK pathway.
Conclusion:
1. Subcutaneous injection of ISO can induce myocardial hypertrophy, apoptosis,muscle fiber disorder and interstitial myocardial fibrosis, indicating that subcutaneousinjection of ISO can successfully construct ventricular remodeling mouse model whichcan effectively simulate the pathological process of chronic heart failure；
2. Klotho protein inhibited the development of chronic heart failure in ISO-inducedBalb/C mouse model. Importantly this study confirmed that Klotho protein can reduce theISO-induced apoptosis and the expression of endoplasmic reticulum stress-related proteinGRP78, HSP47and pro-apoptosis transcription factor CHOP in heart tissue and culturedcardiomyocytes, indicating the anti-apoptosis role of Klotho protein was related with theinhibition of ER stress-related apoptosis signal pathway;
3. P38inhibitor, SB203580and JNK inhibitor, SP600125have the same anti-apoptosiseffect with Klotho protein in ISO-stimulated H9c2cells, however ERK1/2inhibitor,PD98059did not affect the apoptosis significantly. Klotho protein (0.01-10ug/ml)significantly inhibited the phosphorylation of JNK and P38, but did not significantly affectthe phosphorylation of ERK1/2, suggesting that anti-apoptosis effect of Klotho protein was partially dependent on the inhibition of JNK and P38MAPK signaling pathway.
引文
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