丹参素体外促进胚胎干细胞向心肌分化作用研究
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摘要
目的探讨丹参素体外促进胚胎干细胞向心肌细胞分化的作用。方法通过形成拟胚体(EBs)的方法诱导小鼠胚胎干细胞向心肌细胞分化模型,其中,仅加入分化培养基的EBs为自然分化组,向培养基中加入丹参注射液(10 mg/ml)、丹参素(20μg/ml)的EBs分别为丹参诱导分化组、丹参素诱导分化组。检测各组EBs的心肌分化率及八聚体结合转录因子4(OCT4)、α-肌球蛋白重链(α-MHC)蛋白表达水平。结果 EBs接种后4 d,可观察到EBs周边部出现自发的节律性收缩细胞的合胞体。接种后4～12 d,EBs的不同部位可观察到1个或多个有自发节律收缩的合胞体,且形态、收缩节律不相同。接种后第12天心肌细胞分化率,自然分化组为55.4%,丹参诱导分化组为78.4%,丹参素诱导分化组为70.7%。丹参诱导分化组、丹参素诱导分化组的心肌细胞分化率均显著高于自然分化组,差异有统计学意义(P<0.05)。分化培养第12天,丹参诱导分化组、丹参素诱导分化组干细胞标志蛋白OCT4表达水平明显低于自发分化组,心肌细胞标志蛋白α-MHC表达水平明显高于自发分化组,差异有统计学意义(P<0.05)。结论丹参的活性成份丹参素具有促进干细胞向心肌细胞分化的作用,考虑是丹参的心肌保护作用机制之一。
Objective To investigate the effect of Danshensu(Dss) on promoting the differentiation of stem cells into cardiomyocytes in vitro.Methods Mouse embryonic stem cells were induced to differentiate into cardiomyocytes by forming embryo-like body,in which the embryo-like body added with differentiation medium was the natural differentiation group,and the embryo-like body added with salvia miltiorrhiza injection(10 mg/ml) and danshen injection(20 μg/ml) were the induced differentiation group and Dss injection group,respectively.The myocardial differentiation rate,OCT-bound transcription factor 4(OCT4) and α-myosin heavy chain(α-MHC) protein expression levels in each group were detected.Results Spontaneous rhythmic contractile syncytes in the periphery of EBs were observed 4 days after EBs inoculation.Four to twelve days after inoculation,one or more syncytes with spontaneous rhythmic contraction could be observed at different sites of EBs,with different morphology and contraction rhythm.On the 12 th day after inoculation,the rate of myocardial cell differentiation was 55.4% in the natural differentiation group,78.4% in the Dss induced differentiation group,and 70.7% in the Dss induced differentiation group.The differentiation rate of cardiomyocytes in the induced differentiation group and the induced differentiation group of danshen root was significantly higher than that in the natural differentiation group,with statistically significant difference(P<0.05).On the 12 th day of differentiation culture,the expression levels of stem cell marker OCT4 in the Dss induced differentiation group and Dss induced differentiation group were significantly lower than those in the spontaneous differentiation group,and the expression levels of myocardial cell marker α-MHC were significantly higher than those in the spontaneous differentiation group(P<0.05).Conclusion Danshensu,the active ingredient of Salvia miltiorrhiza Bunge,can promote the differentiation of stem cells into cardiac muscle cells,which is considered to be one of the protective mechanisms of cardiac muscle of Salvia miltiorrhiza Bunge.
Objective To investigate the effect of Danshensu(Dss) on promoting the differentiation of stem cells into cardiomyocytes in vitro.Methods Mouse embryonic stem cells were induced to differentiate into cardiomyocytes by forming embryo-like body,in which the embryo-like body added with differentiation medium was the natural differentiation group,and the embryo-like body added with salvia miltiorrhiza injection(10 mg/ml) and danshen injection(20 μg/ml) were the induced differentiation group and Dss injection group,respectively.The myocardial differentiation rate,OCT-bound transcription factor 4(OCT4) and α-myosin heavy chain(α-MHC) protein expression levels in each group were detected.Results Spontaneous rhythmic contractile syncytes in the periphery of EBs were observed 4 days after EBs inoculation.Four to twelve days after inoculation,one or more syncytes with spontaneous rhythmic contraction could be observed at different sites of EBs,with different morphology and contraction rhythm.On the 12 th day after inoculation,the rate of myocardial cell differentiation was 55.4% in the natural differentiation group,78.4% in the Dss induced differentiation group,and 70.7% in the Dss induced differentiation group.The differentiation rate of cardiomyocytes in the induced differentiation group and the induced differentiation group of danshen root was significantly higher than that in the natural differentiation group,with statistically significant difference(P<0.05).On the 12 th day of differentiation culture,the expression levels of stem cell marker OCT4 in the Dss induced differentiation group and Dss induced differentiation group were significantly lower than those in the spontaneous differentiation group,and the expression levels of myocardial cell marker α-MHC were significantly higher than those in the spontaneous differentiation group(P<0.05).Conclusion Danshensu,the active ingredient of Salvia miltiorrhiza Bunge,can promote the differentiation of stem cells into cardiac muscle cells,which is considered to be one of the protective mechanisms of cardiac muscle of Salvia miltiorrhiza Bunge.
引文
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[2] Wang YQ,Zhuang G,Li SJ.Multiple on-line screening and identification methods for hydroxyl radical scavengers in Yudanshen[J].J Pharm Biomed Anal,2018,156:278-283.
[3] Li H,Xie YH,Yang Q,et al.Cardioprotective effect of paeonol and danshensu combination on isoproterenol-induced myocardial injury in rats[J].PLoS One,2012,7(11):e48872.
[4] Bao XY,Zheng Q,Tong Q,et al.Danshensu for myocardial ischemic injury:preclinical evidence and novel methodology of quality assessment tool[J].Front Pharmacol,2018,9:1445.
[5] Song Q,Zhang Y,Han X,et al.Potential mechanisms underlying the protective effects of salvianic acid A against atherosclerosis in vivo and vitro[J].Biomed Pharmacother,2019,109:945-956.
[6] Klug MG,Soonpaa MH,Koh GY,et al.Genetically selected cardiomyocytes from differentiating embryonic stem cells from stable intracardiac grafts[J].J Clin Invest,1996,98(1):216-224.
[7] Kehat I,Kenyagin-Karsenti D,Snir M,et al.Human embryonic stem cell can differentiation into myocytes with structural and functional properties of cardiomyocytes[J].J Clin Invest,2001,108(3):407-414.
[8] Lu CY,Lu PC,Chen PC.Utilization trends in traditional Chinese medicine for acute myocardialinfarction[J].J Ethnopharmacol,2019,241:112010.
[9] Chen W,Chen G.Danshen(salvia miltiorrhiza bunge):a prospective healing sage for cardiovascular diseases[J].Curr Pharm Des,2017,23(34):5125-5135.
[10] Wang L,Li Y,Deng W,et al.Cardio-protection of ultrafine granular powder for Salvia miltiorrhiza Bunge against myocardialinfarction[J].J Ethnopharmacol,2018,222:99-106.
[11] Wang D,Zhang S,Tang H,et al.Development of sustained-release pellets to modulate the in vivo processes of the main active components of Danshen:a pharmacokinetic and pharmacodynamic evaluation[J].Phytomedicine,2019,58:152793.
[12] Li K,Song J,Zhao Q,et al.Effective component of Salvia miltiorrhiza in promoting cardiomyogenic differentiation of human placenta derived mesenchymal stem cells[J].Int J Mol Med,2018,41(2):962-968.
[13] Asaka MN,Uranishi K,Suzuki A,et al.Link between embryonic stem cell pluripotency and homologous allelic pairing of OCT4 loci[J].Dev Growth Differ,2017,59(8):639-647.
[14] Wang Z,Huang J.Apela Promotes cardiomyocyte differentiation from transgenic human embryonic stem cell lines[J].Appl Biochem Biotechnol,2019.
[15] Jyoti S,Tandon S.Disruption of mitochondrial membrane potential coupled with alterations in cardiac biomarker expression as early cardiotoxic signatures in human ES cell-derived cardiac cells[J].Hum Exp Toxicol,2019.