淫羊藿苷及其同系物诱导小鼠胚胎干细胞体外定向分化心肌细胞构效关系及相关信号调控研究
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摘要
心脏疾病一直是严重威胁人类健康的重大疾病之一,通常认为,心肌细胞在出生后不能再生,损伤死亡后,健存心肌通过肥大加以代偿,梗死心肌被瘢痕组织替代,发生心室重构,最终导致心力衰竭。现有治疗手段诸如药物、介入、外科手术等并不能促使心肌再生,治疗效果十分有限。心脏移植由于供体心脏来源有限、费用昂贵及移植排斥反应等问题也限制了其推广应用。而细胞移植为心脏坏死区细胞重建及衰竭心脏功能恢复提供了一种崭新方法。
胚胎干细胞(Embryonic stem cells,ES cells)能在体外长期传代培养并保持高度未分化,给予合适培养条件,可在体外分化为外、中、内胚层任何类型细胞。研究证明,ES细胞体外向心肌细胞分化特异基因表达图式重演了体内心肌发育基因表达,分化得到心肌细胞具有体内正常心肌或原代培养心肌特征和功能。因此,ES细胞体外定向分化为心肌细胞已逐渐成为公认的体内心肌分化替代模型用于各种目的研究,如心脏发育生理学、心脏病理学及心脏疾病治疗学等。
在药物研究领域,通过在谱系特异启动子控制下基因敲入表达报告基因(通常为胎蛋白或荧光酶),ES细胞可提供高通量筛选平台,确定能诱导ES细胞定向分化为初级前体细胞的先导化合物。对先导进行药效基团优化,可获得低分子量化合物控制初级前体细胞分化。按照此模式可筛选使特定后期前体细胞分化为终端成熟细胞的化合物,而终端成熟细胞(如心肌细胞,特定神经细胞亚型)对特异性作用药物发现非常有价值。
尽管人们对ES细胞及其心肌细胞定向分化的认识和利用已有了长足进步,但仍存在一系列悬而未决的问题或发展中的“路障”。目前对于ES细胞诱导分化为心肌细胞准确调控机制尚不清楚,培养与诱导条件没有统一标准。人们还不能完全控制ES细胞定向分化为心肌细胞,还不知道ES细胞定向分化时是否有基因突变发生。只有准确掌握ES细胞分化调控机制,才有可能在分子水平上精确诱导ES细胞定向分化成心肌细胞,并消除一些不利因素。
具有诱导ES体外定向分化心肌细胞作用的小分子化合物的发现,为ES细胞基础研究及临床应用提供了强有力的研究工具。这些小分子化合物作用机制的阐明不仅有助于控制ES细胞命运,同时还可以提高心肌细胞诱导分化率,这不仅是ES细胞临床治疗必需,而且有助于心脏发育早期分子机制研究,也是进行新药创新重要手段。ES细胞是目前细胞工程研究最活跃领域,随着基础、应用研究的进一步深化,利用化学小分子化合物诱导ES细胞定向分化技术会在相当大程度上引发医学领域的重大变革,成为21世纪生命科学领域热点。
本课题前期研究发现,淫羊藿苷(ICA)及其同系物淫羊藿素(ICT)和去甲淫羊藿素(DICT)均能有效促进小鼠ES细胞体外定向分化为心肌细胞,作用强度依次为ICA＞ICT＞DICT,并且细胞内活性氧(Reactive Oxygen Species,ROS)在ICA诱导分化过程中扮演了重要角色。相反,对三者体外抗氧化活性研究显示,DICT体外抗氧化活性最强,ICT次之,ICA在体外几乎不表现抗氧化活性。ICA,ICT和DICT均为异戊烯基黄酮类化合物,研究显示,当基本结构相同时,黄酮类化合物的抗氧化活性往往与结构有关。ICA,ICT和DICT结构差异导致的体外抗氧化活性差异是否是其促分化效果不同的原因,尚未被证实。因此,本研究拟通过考察ICA,ICT和DICT对ROS信号通路的调控作用,初步对其促分化作用构效关系进行研究,以期揭示此类异戊烯基黄酮化合物促分化作用物质基础,为新的促心肌定向分化先导化合物发现提供依据。进一步我们对其相关信号调控进行了研究,为揭示ICA,ICT和DICT促心肌定向分化深层次机制,阐明其诱导分化药理作用提供实验依据,也为发展其新应用提供理论支持。
1 ICA及其同系物诱导ES细胞定向分化心肌细胞构效关系研究
为探索ICA、ICT和DICT诱导ES细胞体外定向分化心肌细胞构效关系,本研究考察了ICA和ICT启动心肌定向分化过程中对ROS信号通路调控作用,采用荧光分光光度计检测了ICA和ICT对拟胚体(embryoid bodies,EBs)内ROS水平影响。共孵育抗氧化剂vitamin E或pyrrolidine dithiocarbamate(PDTC),还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide-adenine dinucleotide phosphate,NADPH)氧化酶抑制剂apocynin,评价ROS在ICA促心肌定向分化中作用及其与NADPH氧化酶关系。Western blot和免疫荧光法检测ICA和ICT作用后胞外信号调节激酶(extracellular signal-regulated kinase,ERK)、c-Jun氮末端蛋白激酶(c-junN-terminal kinase,JNK)和丝裂素活化蛋白激酶p38(mitogen-activated proteinkinase p38,p38MAPK)磷酸化水平变化,并观察选择性抑制p38MAPK及ERK后对ICA诱导心肌细胞定向分化的影响。同时考察ICA和ICT介入后,NOX4NADPH氧化酶基因及蛋白表达。
实验结果显示,ICA作用EBs 2h后能显著提高细胞内ROS水平,当体系中共孵育vitamin E,PDTC或apocynin时,ICA促ROS产生作用消失。选择性清除细胞内ROS显著抑制ICA促心肌细胞定向分化作用,表现为心肌分化率降低,肌细胞增强因子2C(myocyte enhancer factor 2C,MEF2C)基因表达减少。p38MAPK和ERK1,2磷酸化水平分别在ICA介入培养体系30 min或2 h后上升至最高,而JNK磷酸化水平在观察的0～4 h内无明显变化。上述ICA诱导的丝裂素活化蛋白激酶(MAPKs)激活作用均可被vitamin E拮抗。p38MAPK抑制剂SB203580或ERK1,2抑制剂U0126对信号通路进行选择性抑制后,能有效拮抗ICA促分化作用,表现为心肌细胞分化率显著降低,心肌MEF2C基因表达显著减少。
进一步研究显示,ICA和ICT介入分化体系30min后能显著增加Nox4 NADPH氧化酶基因表达,作用2 h后能明显上调其蛋白表达,但ICA和ICT对其诱导效应无显著差异。ICT作用后EBs内活性氧升高水平,p38MAPK和ERK1,2磷酸化程度均较ICA组少。提示ICA和ICT具有同等效应激活细胞内ROS作用(对NOX4 NADPH氧化酶诱导作用相似),ICT本身具备的抗氧化活性在激活NOX4同时清除了部分ROS,从而导致其下游p38MAPK和ERK1,2激活效应削弱,最终导致其促心肌细胞定向分化作用相对较弱。
本实验结果提示ICA、ICT和DICT促心肌细胞体外定向分化作用物质基础是其母核本身,但母核上羟基位置与数目差异导致的抗氧化活性差异影响了其诱导小鼠ES细胞体外定向分化心肌细胞效应。
2 ROS信号通路在ICA启动心肌细胞定向分化中作用研究
第一部分研究从化合物构效关系层面进一步明确了细胞内ROS水平在ES细胞体外定向分化心肌细胞中的作用,基于上述研究结果,为进一步明确ROS信号通路在ICA促心肌细胞定向分化过程中的角色,本部分着重对核因子-κB(nuclearfactor-κB,NF-κB),活性蛋白(activation protein-1,AP-1),及Ca~(2+)信号通路进行了考察。采用Western blot法检测ICA介入体系后IκBα和p-IκBα表达,胞质和胞核内NF-κB p65表达,及c-fos和c-jun表达。荧光分光光度计法检测ICA和ICT介入后对EBs内Ca~(2+)水平影响,共孵育Ca~(2+)螯合剂EGTA或胞内Ca~(2+)清除剂BAPTA/AM后,评价EBs内Ca~(2+)水平对ICA促心肌细胞定向分化作用影响。Westernblot法检测ICA介入后EBs内钙调神经磷酸酶(calcineurin,CaN)及钙调蛋白激酶Ⅱ(Ca~(2+)/calmodulin-dependent kinaseⅡ,CaMKⅡ)表达,并观察选择性抑制CaN及CaMKⅡ后对ICA诱导ES细胞体外定向分化为心肌细胞影响。进一步考察ICA介入后EBs内组蛋白去乙酰基转移酶4(histone deacetylases 4,HDAC4)表达及其与CaMKⅡ关系。最后,共孵育vitamin E、SB203580或U0126,评价ICA诱导心肌细胞体外定向分化过程中ROS-MAPKs信号通路与NF-κB、AP-1及Ca~(2+)信号通路关系。
结果显示,ICA作用30 min后,EBs内p-IκBα表达显著增加,IκBα表达显著减少。ICA与EBs共孵育2 h后,胞核内NF-κB p65表达显著增加,胞浆中表达相应减少。c-fos和c-jun基因或蛋白表达在ICA作用30min或2 h后显著上调。
ICA介入5 min后细胞内Ca~(2+)浓度([Ca~(2+)]_i)即显著上升,共孵育EGTA或BAPTA/AM后,发现其能有效拮抗ICA促心肌细胞体外定向分化作用,表现为心肌分化率降低,心肌MEF2C核转录减少,肌小节蛋白α-辅肌动蛋白(α-actinin)和肌钙蛋白T(Troponin-T)表达下调。ICA作用48 h后CaN及CaMKⅡ表达显著上升,此作用可被EGTA或BAPTA/AM拮抗。选择性抑制CaMKⅡ显著拮抗ICA促分化作用,但CaN抑制后对ICA促分化作用并无显著影响。ICA作用48 h后HDAC4表达显著降低,选择性抑制CaMKⅡ后能显著抑制ICA降低HDAC4表达作用。
共孵育vitamin E后,发现其能有效拮抗ICA诱导的NF-κB,Ap-1激活和[Ca~(2+)]_i增加。SB203580或U0126对信号通路进行选择性抑制后,ICA诱导NF-κB和AP-1激活作用明显降低,但对[Ca~(2+)]_i无显著影响。
结果提示,ICA促进ESN胞体外定向分化为心肌细胞过程中,一方面,激活的ROS通过p38MAPK和ERK1,2传递信号,使NF-κB抑制因子IκBα发生自身磷酸化并迅速降解,NF-κB和IκBα解离,转位进入细胞核,同时,活化的p38MAPK和ERK1,2上调c-fos和c-jun表达;另一方面,ROS通过激活细胞内Ca~(2+),活化CaMKⅡ,导致下游HDAC4降解,其对MEF2C的转录抑制作用降低,MEF2C转录活性增加,从而促进心肌分化。
以上实验表明:
1.ICA、ICT和DICT促ES细胞体外定向分化心肌细胞作用与其母核本身密切相关,但母核上羟基位置与数目差异导致的抗氧化活性差异,可影响该类化合物诱导心肌细胞定向分化效应的强弱。
2.ICA促进EBs向心肌细胞分化过程中,内源性ROS水平迅速升高,一方面,激活的ROS通过p38MAPK和ERK1,2传递信号,使NF-κB抑制因子IκBα发生自身磷酸化并迅速降解,NF-κB和IκBα解离,转位进入细胞核,同时,活化的p38MAPK和ERK1,2上调c-fos和c-jun表达;另一方面,ROS通过激活细胞内Ca~(2+),活化CaMKⅡ,导致下游HDAC4降解,其对MEF2c的转录抑制作用降低,MEF2C转录活性增加,从而促进心肌分化。
Heart disease is one of the greatest diseases which seriously threaten human health.Generally,cardiomyocytes are terminally differentiated and have only limited regenerative capacity after injuries such as myocardial infarctions and heart failure. Therapeutic efficacy of drugs,intervention or surgery is extremely limited since they can not promote the regenerative capacity of cardiomyocytes.Moreover,heart transplantation is limited by its source,cost and graft rejective reaction.Thus,the transplantation of functional cardiomyocytes into the damaged myocardium would have therapeutic potential.
Embryonic stem(ES) cells can be cultured and maintained in undifferentiated state in vitro.Unlimited differentiation potential is one of the marked characters of ES cells,in other words,ES cells can differentiate into cell types of all three primary germ layers if cultured under the correct conditions.Accumulated evidence suggested that cardiomyocyte differentiation of ES cells in vitro faithfully recapitulated cardiomyocyte differentiation in vivo,and cardiomyocytes derived from ES cells display properties similar to those observed in vivo or in primary cultures. Thus cardiomyocyte differentiation of ES cells is considered as an efficiency model in vitro for cardiac developmental biology,cardiac pathology and cardiac disease therapy.
As to pharmacology,"knock-in" technology in mouse ES cells is well-established and given the wide potential for its application in drug discovery.ES cells can be engineered to express reportor genes(usually GFP or luciferase) under control of a lineage-specific promoter,which offers the opportunity for high-throughput screening to identify leads,which induce differentiation towards the precutors of cardiomyocytes.Optimisation of the pharmacophore around the lead would generate low molecular weight compounds for control of differentiation to an early precursor cell.One can envisage later stages which would specify later precursor cells and ultimately fully differentiated cells.Therefore,the application of ES cells in drug discovery will have drawn more attention.
There is still some "barricade" in ES cell application in spite of the tremendous development in cell biology.We now know little about accurate regulatory mechanism of cardiomyocyte differentiation.No standard protocol for ES cell cultivation or induction is available.Cardiomyocytes specific differentiation of ES cells can not faithfully fulfill,whether there is gene mutation during differentiation is largely unknown.Only the accurate regulatory mechanism uncovered,cardiomyocyte specific differentiation of ES cells can realize.Many problem needs solving, especially how to direct cardiomyocyte differentiation more efficiently and selectively. Cell-based phenotypic and pathway specific screens of natural products and synthetic compounds have recently provided a number of small molecules that can be used to selectively control stem cell proliferation and differentiation.Such molecules will likely provide new insights into stem cell biology,and may ultimately contribute to effective medicines for tissue repair and regeneration.ES cells are one of the greatest leading fields in cell engineering.Along with the progress of research,it will likely initiate innovation in medical domain,and will become hot spot in life science of 21 st century.
Our previous work has demonstrated that icariin(ICA) and its two homologues icaritin(ICT) and desmethylicaritin(DICT) could significantly stimulated cardiac differentiation of mouse ES cells in vitro.The most effective differentiation was promoted by ICA,followed by ICT,while DICT displayed the weakest but still significant inducible effect,intracellular reactive oxygen species(ROS) signaling pathway play a vital role in ICA induced cardiomyogenesis.Contrarily,DICT demonstrated the strongest anti-oxidative activity while ICA displayed only little in vitro,which was well matched with the hydroxyl(OH) numbers and the positions in the molecular structures.It has been implicated that flavonoids exhibit an anti-oxidative effect,which may play an important role in preventing or delaying disease process,such as cardiovascular disease,the anti-oxidative activity of flavonoids depend on the OH numbers and the positions in the structure.In the present studies,in order to reveal the underlying mechanism of prenylflavonoids in cardiomyocyte differentiation,the relationship between structures and inducing effects was firstly assessed,and then its related mechanisms was investigated.
1 Structure-activity relationships of ICA and its two homologues in cardiac differentiation of ES cells
To investigate the structure-activity relationships that involved in ICA and its two homologues in cardiac differentiation of mouse ES cells.ICA and ICT were employed to explore the mechanisms involved in the ROS signaling pathways. Intracellular ROS after ICA and ICT administration was assessed by fluorescence spectrophotometry.Free radical scavenger vitamin E or pyrrolidine dithiocarbamate (PDTC),the nicotinamide-adenine dinucleotide phosphate(NADPH) oxidase inhibitor apocynin was added to evaluate the role of ROS in ICA induced cardiomyogenesis.The expression of phosphorylated mitogen-activated protein kinase p38(p38MAPK),extracellular signal-regulated kinase(ERK) or c-jun N-terminal kinase(JNK) was assessed by western blot and immunofluorescence, ERK inhibitor U0126 or p38MAPK antagonist SB203580 was added to investigate the role of ERK1,2 and p38MAPK in ICA induced cardiomyogenesis.NADPH oxidase subunit Nox4 gene and protein expression after ICA and ICT treatment was evaluated synchronously.
The results demonstrated that ICA,ICT,and DICT could significantly stimulated cardiac differentiation of ES cells in vitro,the most effective differentiation was promoted by ICA,followed by ICT,while DICT displayed the weakest but still significant inducible effect.Using a cell-free assay,DICT demonstrated the strongest anti-oxidative activity while ICA displayed only little.
Treatment with ICA 2 h rapidly elevated intracellular ROS in embryoid bodies (EBs),free radical scavengers vitamin E or pyrrolidine dithiocarbamate(PDTC),the NADPH-oxidase inhibitor apocynin inhibited ICA induced ROS generation.ROS elimination significantly inhibited ICA induced cardiomyogenesis.ERK1,2 and p38MAPK activation was observed at 2 h or 30 min after ICA administration, however,phosphorylated JNK was not observed during 0-4 h after ICA treatment. The observed effects were abolished by Vitamin E,indicating the role of ROS in MAPKs activation.The cardiomyogenesis was significantly inhibited in the presence of ERK1,2 or p38MAPK inhibitor U0126 or SB203580,pointing towards that NADPH-ROS-MAPKs signaling cascades play a vital role in ICA induced cardiomyogenesis.
Compared to ICA,ICT generated less ROS,and followed by less activation of ERK1,2 and p38MAPK,but its effect on up-regulation of NADPH oxidase subunit NOX4 was the same as that of ICA.The results point toward that there is no difference of ICA and ICT on the activation of intracellular ROS,for their effects on the activation of NOX4 were similar;the ROS eliminated by ICT on account of its anti-oxidative activity resulted in its weaker effects on the activation of ERK1,2,and p38MAPK,and leading to lower enhancing effect on cardiomyogenesis consequently.
It is concluded that the promoting effects of ICA and its two homologues ICT, and DICT on cardiomyocyte differentiation of mouse ES cells lie on prenylflavonoids themselves,but anti-oxidative activity determined by the OH numbers and the positions in the structure do influence the effects.
2 Involvement of ROS signaling cascades in ICA induced cardiomyocyte differentiation of mouse ES cells in vitro
This section was aimed to analysis the role of nuclear factor-κB(NF-κB), activation protein-1(AP-1) and Ca~(2+) signaling cascades in ICA induced cardiomyogenesis. First,the expression of IκB,p-IκB,..NF-κB p65 in cytoplasm or nucleus,c-fos and c-jun was assessed by western-blot.Intracellular calcium concentration([Ca~(2+)]_i) was further evaluated by fluorescence spectrophotometry,Extracellular or cytoplasmic activated CaMKII followed by HDAC4 degradation,resulting in dissociation of MEF2C from HDAC4 and the stimulation MEF2C transcriptional activity,which leads to the stimulation of cardiac differentiation from mouse ES cells in vitro.
Summary
1.The promoting effects of ICA and its two homologues ICT,and DICT on cardiomyocyte differentiation of mouse ES cells lie on prenylflavonoids themselves, but anti-oxidative activity determined by the OH numbers and the positions in the structure do influence the effects.
2.ROS signaling cascades is essential for the inducible function of ICA on cardiomyocyte differentiation of mouse ES cells in vitro.On one hand,ROS activate ERK1,2 and p38MAPK,which subsequently phosphorylated IκB followed by IκB degradation,resulting in dissociation of the NF-κB:IκB complex and thereafter translocation of the released NF-κB into the nucleus.What is more,the activated p38MAPK and ERK1,2 up-regulated c-fos and c-jun.On the other hand,ROS increases[Ca~(2+)])_i,subsequently activated CaMKII followed by HDAC4 degradation, resulting in dissociation of MEF2C from HDAC4 and the stimulation MEF2C transcriptional activity,which leads to the stimulation of cardiac differentiation from mouse ES cells in vitro.
胚胎干细胞(Embryonic stem cells,ES cells)能在体外长期传代培养并保持高度未分化,给予合适培养条件,可在体外分化为外、中、内胚层任何类型细胞。研究证明,ES细胞体外向心肌细胞分化特异基因表达图式重演了体内心肌发育基因表达,分化得到心肌细胞具有体内正常心肌或原代培养心肌特征和功能。因此,ES细胞体外定向分化为心肌细胞已逐渐成为公认的体内心肌分化替代模型用于各种目的研究,如心脏发育生理学、心脏病理学及心脏疾病治疗学等。
在药物研究领域,通过在谱系特异启动子控制下基因敲入表达报告基因(通常为胎蛋白或荧光酶),ES细胞可提供高通量筛选平台,确定能诱导ES细胞定向分化为初级前体细胞的先导化合物。对先导进行药效基团优化,可获得低分子量化合物控制初级前体细胞分化。按照此模式可筛选使特定后期前体细胞分化为终端成熟细胞的化合物,而终端成熟细胞(如心肌细胞,特定神经细胞亚型)对特异性作用药物发现非常有价值。
尽管人们对ES细胞及其心肌细胞定向分化的认识和利用已有了长足进步,但仍存在一系列悬而未决的问题或发展中的“路障”。目前对于ES细胞诱导分化为心肌细胞准确调控机制尚不清楚,培养与诱导条件没有统一标准。人们还不能完全控制ES细胞定向分化为心肌细胞,还不知道ES细胞定向分化时是否有基因突变发生。只有准确掌握ES细胞分化调控机制,才有可能在分子水平上精确诱导ES细胞定向分化成心肌细胞,并消除一些不利因素。
具有诱导ES体外定向分化心肌细胞作用的小分子化合物的发现,为ES细胞基础研究及临床应用提供了强有力的研究工具。这些小分子化合物作用机制的阐明不仅有助于控制ES细胞命运,同时还可以提高心肌细胞诱导分化率,这不仅是ES细胞临床治疗必需,而且有助于心脏发育早期分子机制研究,也是进行新药创新重要手段。ES细胞是目前细胞工程研究最活跃领域,随着基础、应用研究的进一步深化,利用化学小分子化合物诱导ES细胞定向分化技术会在相当大程度上引发医学领域的重大变革,成为21世纪生命科学领域热点。
本课题前期研究发现,淫羊藿苷(ICA)及其同系物淫羊藿素(ICT)和去甲淫羊藿素(DICT)均能有效促进小鼠ES细胞体外定向分化为心肌细胞,作用强度依次为ICA＞ICT＞DICT,并且细胞内活性氧(Reactive Oxygen Species,ROS)在ICA诱导分化过程中扮演了重要角色。相反,对三者体外抗氧化活性研究显示,DICT体外抗氧化活性最强,ICT次之,ICA在体外几乎不表现抗氧化活性。ICA,ICT和DICT均为异戊烯基黄酮类化合物,研究显示,当基本结构相同时,黄酮类化合物的抗氧化活性往往与结构有关。ICA,ICT和DICT结构差异导致的体外抗氧化活性差异是否是其促分化效果不同的原因,尚未被证实。因此,本研究拟通过考察ICA,ICT和DICT对ROS信号通路的调控作用,初步对其促分化作用构效关系进行研究,以期揭示此类异戊烯基黄酮化合物促分化作用物质基础,为新的促心肌定向分化先导化合物发现提供依据。进一步我们对其相关信号调控进行了研究,为揭示ICA,ICT和DICT促心肌定向分化深层次机制,阐明其诱导分化药理作用提供实验依据,也为发展其新应用提供理论支持。
1 ICA及其同系物诱导ES细胞定向分化心肌细胞构效关系研究
为探索ICA、ICT和DICT诱导ES细胞体外定向分化心肌细胞构效关系,本研究考察了ICA和ICT启动心肌定向分化过程中对ROS信号通路调控作用,采用荧光分光光度计检测了ICA和ICT对拟胚体(embryoid bodies,EBs)内ROS水平影响。共孵育抗氧化剂vitamin E或pyrrolidine dithiocarbamate(PDTC),还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide-adenine dinucleotide phosphate,NADPH)氧化酶抑制剂apocynin,评价ROS在ICA促心肌定向分化中作用及其与NADPH氧化酶关系。Western blot和免疫荧光法检测ICA和ICT作用后胞外信号调节激酶(extracellular signal-regulated kinase,ERK)、c-Jun氮末端蛋白激酶(c-junN-terminal kinase,JNK)和丝裂素活化蛋白激酶p38(mitogen-activated proteinkinase p38,p38MAPK)磷酸化水平变化,并观察选择性抑制p38MAPK及ERK后对ICA诱导心肌细胞定向分化的影响。同时考察ICA和ICT介入后,NOX4NADPH氧化酶基因及蛋白表达。
实验结果显示,ICA作用EBs 2h后能显著提高细胞内ROS水平,当体系中共孵育vitamin E,PDTC或apocynin时,ICA促ROS产生作用消失。选择性清除细胞内ROS显著抑制ICA促心肌细胞定向分化作用,表现为心肌分化率降低,肌细胞增强因子2C(myocyte enhancer factor 2C,MEF2C)基因表达减少。p38MAPK和ERK1,2磷酸化水平分别在ICA介入培养体系30 min或2 h后上升至最高,而JNK磷酸化水平在观察的0～4 h内无明显变化。上述ICA诱导的丝裂素活化蛋白激酶(MAPKs)激活作用均可被vitamin E拮抗。p38MAPK抑制剂SB203580或ERK1,2抑制剂U0126对信号通路进行选择性抑制后,能有效拮抗ICA促分化作用,表现为心肌细胞分化率显著降低,心肌MEF2C基因表达显著减少。
进一步研究显示,ICA和ICT介入分化体系30min后能显著增加Nox4 NADPH氧化酶基因表达,作用2 h后能明显上调其蛋白表达,但ICA和ICT对其诱导效应无显著差异。ICT作用后EBs内活性氧升高水平,p38MAPK和ERK1,2磷酸化程度均较ICA组少。提示ICA和ICT具有同等效应激活细胞内ROS作用(对NOX4 NADPH氧化酶诱导作用相似),ICT本身具备的抗氧化活性在激活NOX4同时清除了部分ROS,从而导致其下游p38MAPK和ERK1,2激活效应削弱,最终导致其促心肌细胞定向分化作用相对较弱。
本实验结果提示ICA、ICT和DICT促心肌细胞体外定向分化作用物质基础是其母核本身,但母核上羟基位置与数目差异导致的抗氧化活性差异影响了其诱导小鼠ES细胞体外定向分化心肌细胞效应。
2 ROS信号通路在ICA启动心肌细胞定向分化中作用研究
第一部分研究从化合物构效关系层面进一步明确了细胞内ROS水平在ES细胞体外定向分化心肌细胞中的作用,基于上述研究结果,为进一步明确ROS信号通路在ICA促心肌细胞定向分化过程中的角色,本部分着重对核因子-κB(nuclearfactor-κB,NF-κB),活性蛋白(activation protein-1,AP-1),及Ca~(2+)信号通路进行了考察。采用Western blot法检测ICA介入体系后IκBα和p-IκBα表达,胞质和胞核内NF-κB p65表达,及c-fos和c-jun表达。荧光分光光度计法检测ICA和ICT介入后对EBs内Ca~(2+)水平影响,共孵育Ca~(2+)螯合剂EGTA或胞内Ca~(2+)清除剂BAPTA/AM后,评价EBs内Ca~(2+)水平对ICA促心肌细胞定向分化作用影响。Westernblot法检测ICA介入后EBs内钙调神经磷酸酶(calcineurin,CaN)及钙调蛋白激酶Ⅱ(Ca~(2+)/calmodulin-dependent kinaseⅡ,CaMKⅡ)表达,并观察选择性抑制CaN及CaMKⅡ后对ICA诱导ES细胞体外定向分化为心肌细胞影响。进一步考察ICA介入后EBs内组蛋白去乙酰基转移酶4(histone deacetylases 4,HDAC4)表达及其与CaMKⅡ关系。最后,共孵育vitamin E、SB203580或U0126,评价ICA诱导心肌细胞体外定向分化过程中ROS-MAPKs信号通路与NF-κB、AP-1及Ca~(2+)信号通路关系。
结果显示,ICA作用30 min后,EBs内p-IκBα表达显著增加,IκBα表达显著减少。ICA与EBs共孵育2 h后,胞核内NF-κB p65表达显著增加,胞浆中表达相应减少。c-fos和c-jun基因或蛋白表达在ICA作用30min或2 h后显著上调。
ICA介入5 min后细胞内Ca~(2+)浓度([Ca~(2+)]_i)即显著上升,共孵育EGTA或BAPTA/AM后,发现其能有效拮抗ICA促心肌细胞体外定向分化作用,表现为心肌分化率降低,心肌MEF2C核转录减少,肌小节蛋白α-辅肌动蛋白(α-actinin)和肌钙蛋白T(Troponin-T)表达下调。ICA作用48 h后CaN及CaMKⅡ表达显著上升,此作用可被EGTA或BAPTA/AM拮抗。选择性抑制CaMKⅡ显著拮抗ICA促分化作用,但CaN抑制后对ICA促分化作用并无显著影响。ICA作用48 h后HDAC4表达显著降低,选择性抑制CaMKⅡ后能显著抑制ICA降低HDAC4表达作用。
共孵育vitamin E后,发现其能有效拮抗ICA诱导的NF-κB,Ap-1激活和[Ca~(2+)]_i增加。SB203580或U0126对信号通路进行选择性抑制后,ICA诱导NF-κB和AP-1激活作用明显降低,但对[Ca~(2+)]_i无显著影响。
结果提示,ICA促进ESN胞体外定向分化为心肌细胞过程中,一方面,激活的ROS通过p38MAPK和ERK1,2传递信号,使NF-κB抑制因子IκBα发生自身磷酸化并迅速降解,NF-κB和IκBα解离,转位进入细胞核,同时,活化的p38MAPK和ERK1,2上调c-fos和c-jun表达;另一方面,ROS通过激活细胞内Ca~(2+),活化CaMKⅡ,导致下游HDAC4降解,其对MEF2C的转录抑制作用降低,MEF2C转录活性增加,从而促进心肌分化。
以上实验表明:
1.ICA、ICT和DICT促ES细胞体外定向分化心肌细胞作用与其母核本身密切相关,但母核上羟基位置与数目差异导致的抗氧化活性差异,可影响该类化合物诱导心肌细胞定向分化效应的强弱。
2.ICA促进EBs向心肌细胞分化过程中,内源性ROS水平迅速升高,一方面,激活的ROS通过p38MAPK和ERK1,2传递信号,使NF-κB抑制因子IκBα发生自身磷酸化并迅速降解,NF-κB和IκBα解离,转位进入细胞核,同时,活化的p38MAPK和ERK1,2上调c-fos和c-jun表达;另一方面,ROS通过激活细胞内Ca~(2+),活化CaMKⅡ,导致下游HDAC4降解,其对MEF2c的转录抑制作用降低,MEF2C转录活性增加,从而促进心肌分化。
Heart disease is one of the greatest diseases which seriously threaten human health.Generally,cardiomyocytes are terminally differentiated and have only limited regenerative capacity after injuries such as myocardial infarctions and heart failure. Therapeutic efficacy of drugs,intervention or surgery is extremely limited since they can not promote the regenerative capacity of cardiomyocytes.Moreover,heart transplantation is limited by its source,cost and graft rejective reaction.Thus,the transplantation of functional cardiomyocytes into the damaged myocardium would have therapeutic potential.
Embryonic stem(ES) cells can be cultured and maintained in undifferentiated state in vitro.Unlimited differentiation potential is one of the marked characters of ES cells,in other words,ES cells can differentiate into cell types of all three primary germ layers if cultured under the correct conditions.Accumulated evidence suggested that cardiomyocyte differentiation of ES cells in vitro faithfully recapitulated cardiomyocyte differentiation in vivo,and cardiomyocytes derived from ES cells display properties similar to those observed in vivo or in primary cultures. Thus cardiomyocyte differentiation of ES cells is considered as an efficiency model in vitro for cardiac developmental biology,cardiac pathology and cardiac disease therapy.
As to pharmacology,"knock-in" technology in mouse ES cells is well-established and given the wide potential for its application in drug discovery.ES cells can be engineered to express reportor genes(usually GFP or luciferase) under control of a lineage-specific promoter,which offers the opportunity for high-throughput screening to identify leads,which induce differentiation towards the precutors of cardiomyocytes.Optimisation of the pharmacophore around the lead would generate low molecular weight compounds for control of differentiation to an early precursor cell.One can envisage later stages which would specify later precursor cells and ultimately fully differentiated cells.Therefore,the application of ES cells in drug discovery will have drawn more attention.
There is still some "barricade" in ES cell application in spite of the tremendous development in cell biology.We now know little about accurate regulatory mechanism of cardiomyocyte differentiation.No standard protocol for ES cell cultivation or induction is available.Cardiomyocytes specific differentiation of ES cells can not faithfully fulfill,whether there is gene mutation during differentiation is largely unknown.Only the accurate regulatory mechanism uncovered,cardiomyocyte specific differentiation of ES cells can realize.Many problem needs solving, especially how to direct cardiomyocyte differentiation more efficiently and selectively. Cell-based phenotypic and pathway specific screens of natural products and synthetic compounds have recently provided a number of small molecules that can be used to selectively control stem cell proliferation and differentiation.Such molecules will likely provide new insights into stem cell biology,and may ultimately contribute to effective medicines for tissue repair and regeneration.ES cells are one of the greatest leading fields in cell engineering.Along with the progress of research,it will likely initiate innovation in medical domain,and will become hot spot in life science of 21 st century.
Our previous work has demonstrated that icariin(ICA) and its two homologues icaritin(ICT) and desmethylicaritin(DICT) could significantly stimulated cardiac differentiation of mouse ES cells in vitro.The most effective differentiation was promoted by ICA,followed by ICT,while DICT displayed the weakest but still significant inducible effect,intracellular reactive oxygen species(ROS) signaling pathway play a vital role in ICA induced cardiomyogenesis.Contrarily,DICT demonstrated the strongest anti-oxidative activity while ICA displayed only little in vitro,which was well matched with the hydroxyl(OH) numbers and the positions in the molecular structures.It has been implicated that flavonoids exhibit an anti-oxidative effect,which may play an important role in preventing or delaying disease process,such as cardiovascular disease,the anti-oxidative activity of flavonoids depend on the OH numbers and the positions in the structure.In the present studies,in order to reveal the underlying mechanism of prenylflavonoids in cardiomyocyte differentiation,the relationship between structures and inducing effects was firstly assessed,and then its related mechanisms was investigated.
1 Structure-activity relationships of ICA and its two homologues in cardiac differentiation of ES cells
To investigate the structure-activity relationships that involved in ICA and its two homologues in cardiac differentiation of mouse ES cells.ICA and ICT were employed to explore the mechanisms involved in the ROS signaling pathways. Intracellular ROS after ICA and ICT administration was assessed by fluorescence spectrophotometry.Free radical scavenger vitamin E or pyrrolidine dithiocarbamate (PDTC),the nicotinamide-adenine dinucleotide phosphate(NADPH) oxidase inhibitor apocynin was added to evaluate the role of ROS in ICA induced cardiomyogenesis.The expression of phosphorylated mitogen-activated protein kinase p38(p38MAPK),extracellular signal-regulated kinase(ERK) or c-jun N-terminal kinase(JNK) was assessed by western blot and immunofluorescence, ERK inhibitor U0126 or p38MAPK antagonist SB203580 was added to investigate the role of ERK1,2 and p38MAPK in ICA induced cardiomyogenesis.NADPH oxidase subunit Nox4 gene and protein expression after ICA and ICT treatment was evaluated synchronously.
The results demonstrated that ICA,ICT,and DICT could significantly stimulated cardiac differentiation of ES cells in vitro,the most effective differentiation was promoted by ICA,followed by ICT,while DICT displayed the weakest but still significant inducible effect.Using a cell-free assay,DICT demonstrated the strongest anti-oxidative activity while ICA displayed only little.
Treatment with ICA 2 h rapidly elevated intracellular ROS in embryoid bodies (EBs),free radical scavengers vitamin E or pyrrolidine dithiocarbamate(PDTC),the NADPH-oxidase inhibitor apocynin inhibited ICA induced ROS generation.ROS elimination significantly inhibited ICA induced cardiomyogenesis.ERK1,2 and p38MAPK activation was observed at 2 h or 30 min after ICA administration, however,phosphorylated JNK was not observed during 0-4 h after ICA treatment. The observed effects were abolished by Vitamin E,indicating the role of ROS in MAPKs activation.The cardiomyogenesis was significantly inhibited in the presence of ERK1,2 or p38MAPK inhibitor U0126 or SB203580,pointing towards that NADPH-ROS-MAPKs signaling cascades play a vital role in ICA induced cardiomyogenesis.
Compared to ICA,ICT generated less ROS,and followed by less activation of ERK1,2 and p38MAPK,but its effect on up-regulation of NADPH oxidase subunit NOX4 was the same as that of ICA.The results point toward that there is no difference of ICA and ICT on the activation of intracellular ROS,for their effects on the activation of NOX4 were similar;the ROS eliminated by ICT on account of its anti-oxidative activity resulted in its weaker effects on the activation of ERK1,2,and p38MAPK,and leading to lower enhancing effect on cardiomyogenesis consequently.
It is concluded that the promoting effects of ICA and its two homologues ICT, and DICT on cardiomyocyte differentiation of mouse ES cells lie on prenylflavonoids themselves,but anti-oxidative activity determined by the OH numbers and the positions in the structure do influence the effects.
2 Involvement of ROS signaling cascades in ICA induced cardiomyocyte differentiation of mouse ES cells in vitro
This section was aimed to analysis the role of nuclear factor-κB(NF-κB), activation protein-1(AP-1) and Ca~(2+) signaling cascades in ICA induced cardiomyogenesis. First,the expression of IκB,p-IκB,..NF-κB p65 in cytoplasm or nucleus,c-fos and c-jun was assessed by western-blot.Intracellular calcium concentration([Ca~(2+)]_i) was further evaluated by fluorescence spectrophotometry,Extracellular or cytoplasmic activated CaMKII followed by HDAC4 degradation,resulting in dissociation of MEF2C from HDAC4 and the stimulation MEF2C transcriptional activity,which leads to the stimulation of cardiac differentiation from mouse ES cells in vitro.
Summary
1.The promoting effects of ICA and its two homologues ICT,and DICT on cardiomyocyte differentiation of mouse ES cells lie on prenylflavonoids themselves, but anti-oxidative activity determined by the OH numbers and the positions in the structure do influence the effects.
2.ROS signaling cascades is essential for the inducible function of ICA on cardiomyocyte differentiation of mouse ES cells in vitro.On one hand,ROS activate ERK1,2 and p38MAPK,which subsequently phosphorylated IκB followed by IκB degradation,resulting in dissociation of the NF-κB:IκB complex and thereafter translocation of the released NF-κB into the nucleus.What is more,the activated p38MAPK and ERK1,2 up-regulated c-fos and c-jun.On the other hand,ROS increases[Ca~(2+)])_i,subsequently activated CaMKII followed by HDAC4 degradation, resulting in dissociation of MEF2C from HDAC4 and the stimulation MEF2C transcriptional activity,which leads to the stimulation of cardiac differentiation from mouse ES cells in vitro.
引文
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