Structure-activity Relationships And Related Mechanisms Of Icariin And Its Two Homologues In Cardiac Differentiation Of Mouse Embryonic Stem Cells

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 cellsTo 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 vitroThis section was aimed to analysis the role of nuclear factor-κB(NF-κB), activation protein-1(AP-1) and Ca²⁺ 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([Ca²⁺]_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.Summary1.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[Ca²⁺])_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.