title: Danshen-Gegen decoction exerts proliferative effect on rat cardiac myoblasts H9c2 via MAPK and insulin pathways.
abstract: BACKGROUND
  Danshen (root of Salvia miltiorrhiza) and Gegen (roots of Pueraria lobata) are traditional Chinese medicines that have been used in combination for cardiovascular disease treatment.
  
  
  OBJECTIVE
  The present study was performed to investigate the effect of Danshen-Gegen decoction on rat myocardium cell line H9c2 and the possible molecular mechanisms.
  
  
  METHODS
  Rat heart myocardium H9c2 cells were treated with or without Danshen-Gegen decoction (DG) ranging from 10 to 1000μg/ml for 24h. Cell viability was measured by Alarma blue assay and cell proliferation assay was performed by BrdU Cell Proliferation ELISA kit. The activation of mitogen-activated protein kinase and insulin pathways was analyzed by Luminex technology and the growth factors and cytokine expression of H9c2 cells induced by DG was evaluated by protein array. Moreover, a rat functional specific cDNA microarray was constructed to study the gene expression profiles of H9c2 cells upon the DG treatment at 50μg/ml for 24h.
  
  
  RESULTS
  DG promoted H9c2 cell viability and cell proliferation at dose-dependent manner within the range between 0 and 250μg/ml. A Bio-Plex assay kit (Bio-Rad Bioscience) was used to detect the expression level of phosphoprotein as well as total proteins involved in the MAPK and insulin pathways. Significant phosphorylation of ERK, c-Jun, JNK, p38, AKT, IGF-IR, IRS-1and I kappa B were observed after DG treatment at 2h or 4h. A rat cytokine antibody array was used to detect and quantify 22 growth factors and cytokines in samples collected from the control and DG treated H9c2 cells. In the category of growth factors, GM-CSF, CNIF and b-NGF were stimulated by DG, while the expression of TIMP-1 was suppressed. For cytokine expression, it was found that DG stimulated three interleukin subclasses, IL-1α, 1X and 6, respectively. However, the expression of pro-inflammatory factors such as TNF-α and IFN-γ were down-regulated significantly. Moreover, the microarray analysis revealed that DG significantly up-regulated anti-apoptosis related genes such as Cdkn2c and Ppp3ca, and several cardiovascular disease suppressers and anti-inflammatory mediators; on the other hand, pro-apoptotic related genes including Caspase and Tnf-α were down-regulated by DG. Based on the results, a tentative scheme was proposed to show that the activation of the MAPK and insulin pathways are involved in the bioactive effect of Danshen-Gegen decoction on cardiomyocytes.
  
  
  CONCLUSIONS
  Our study suggested that Danshen-Gegen decoction has proliferative effect on myocardium cells via MAPK and insulin signaling pathways. The molecular mechanism of the action may include the up-regulation of IRS/AKT and JNK pathways as well as the inhibition of TNF and p38 pathways.
authors: Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong.
authors: Chi Chun
authors: CC
authors: Fong
authors: Fan
authors: F
authors: Wei
authors: Yao
authors: Y
authors: Chen
authors: Wai Kin
authors: WK
authors: Yu
authors: Chi Man
authors: CM
authors: Koon
authors: Ping Chung
authors: PC
authors: Leung
authors: Kwok Pui
authors: KP
authors: Fung
authors: Clara Bik San
authors: CB
authors: Lau
authors: Mengsu
authors: M
authors: Yang
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D002318:Cardiovascular Diseases / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D016207:Cytokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004365:Drugs, Chinese Herbal / Q000494:pharmacology* / Q000627:therapeutic use; D015870:Gene Expression / Q000187:drug effects; D018836:Inflammation Mediators / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D032386:Myoblasts, Cardiac / Q000187:drug effects* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation; D008517:Phytotherapy; D018517:Plant Roots; D029908:Pueraria; D051381:Rats; D027561:Salvia miltiorrhiza; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D016207:Cytokines; D004365:Drugs, Chinese Herbal; D018836:Inflammation Mediators; D007328:Insulin; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D020928:Mitogen-Activated Protein Kinases