| Objectives:Salvia miltiorrhiza is one of the Chinese tradition medicine, and its water-soluble portion exhibits significant activity against a ngina pectoris and myocardial ischemic infarction. It was reported that protocatechuic aldehyde, extracted from the water-soluble fraction of Salvia and the leaves of the holly leaf, has strong anti-anoxia, and expanded coronary artery in order to increase blood flow. In recent years, with the discovery and development of stem cells, Salvia itself, as well as the component extracted from it, was involved in stem cell research. However, it is no reported that proliferation of rat c-kit(pos) cardiac stem cells(CSCs) stimulated with protocatechuic aldehyde is associated with Pi3k/Akt mediated cell cycle regulation in vitro.This study was to select suitable methods and culture conditions, so as to obtain Pure, bioactive and stable CSCs, which isolated from rat heart, meanwhile, the effect of PCA, which facilitated the proliferation of CSCs, was refered to cell cycle processes regulated by Pi3k-Akt signal pathway. Methods:C-kit+ CSCs were isolated from neonatal Sprague–Dawley rats(within 72 hours of birth), and then were treated with PCA(0.16, 0.42, 1.26, 3.78 and 11.34 m M) for 24, 48 and 72 h, the influence of PCAon CSCs was detected by MTT assay and flow cytometry, After sorting by flow cytometry, the cell cycle of CSCs, stimulated by PCA, were investigated by flow cytometry, RT- q PCR and western blot analysis were used to detected the gene(Pi3k, Akt, C yclin D, CDK4, C-kit, Sca-1, K i-67 and PCNA)or protein(Pi3k, Akt and PCNA). Results:3 days later, fibroblast-like cells escaped from the digested myocardial tissue. After 1 week, it showed that small, round, bright cells appears on the fibroblast layer, which arounded the tissue. Subculture, cell morphology was observed under inverted microscope, CSCs was homogeneous shuttle type, and formed the "sun-like" colonies.MTT assay showed that 24 h later, the CSCs, cultureed with PC A(0.16 m M, 0.42 m M, 1.26 m M, 3.78 m M and 11.34 m M), rapidly proliferated. When compared with the control group, CSCs proliferation was statistically significant(P<0.05 or P<0.01). The result of 48 h was similar to 24 h. However, 72 later, with the time gone, the proliferation of CSCs declined, but the groups, concluding 0.42 m M, 1.26 m M and3.78 m M, was statistically significant(P<0.05 or P<0.01).The percent of c-kit + CSCs, which cultured with PCA(0.42 m M, 1.26 m M and3.78 m M)for 24 h, reached 19.6%,41.0% and 17.6%, it was a significant difference(P<0.05 or P<0.01) compared with control group(6.9%).CSCs, treated with PCA(0.42 m M, 1.26 m M and3.78 m M), in the S phase(10.27±2.61%,22.44±2.77% and 9.00±2.56%) was significantly higher than untreatment( 5.11±0.89%)(P<0.05 or P<0.01). Meanwhile, CSCs, treated with PCA(0.42 m M and3.78 m M), in the G2/M phase(8.72±2.57% and 7.34±2.87%) was significantly higher than untreatment(5.54±2.61%)(P <0.05), but CSCs cultured with 1.26 m M PCA decreased. CSCs, treated with PCA(0.42 m M, 1.26 m M and3.78 m M), in the G0/G1 phase(10.27±2.61%, 22.44±2.77% and 9.00±2.56%) was significantly lower than untreatment( 5.11±0.89%)(P<0.05 or P<0.01).Treated with 0.42 m M PCA, the m RNA expression levels of Akt(5.15±1.18), Cyclin D(1.90±0.27), CDK4(1.81±0.12), C-kit(2.61±1.00), K i-67(9.25±0.48) and PCNA(3.89±0.36) were significantly higher in CSCs(P<0.05 or P<0.01). Treated with 1.26 m M PCA, the m RNA expression levels of Pi3k(1.32±0.68), Akt(8.00±0.39), C yclin D(3.45±1.02), CDK4(2.03±0.02), C-kit(8.07±1.77), Sca-1(11.45±0.57), K i-67(22.26±0.66) and PCNA(4.09±0.32) were significantly higher in CSCs(P<0.05 or P<0.01). Treated with 3.78 m M PCA, the m RN A expression levels of Akt(2.26±0.73), C yclin D(1.56±0.18), CDK4(1.37±0.34), C-kit(1.83±0.34) and K i-67(4.14±0.78) were significantly higher in CSCs(P<0.05 or P<0.01).Treated with PCA(0.42 m M, 1.26 m M and3.78 m M), protein expressions of Pi3 k is 2.66±0.22, 1.86±0.19 and 1.57±0.18, which was significantly upregulated(P<0.05 or P<0.01). Similarly, protein expressions of Akt1 is 1.92±0.22, 1.57±0.20 and 0.75±0.11, which was significantly upregulated except the 3.78 m M group(P<0.05 or P<0.01). In addition, protein expressions of Akt1 is 2.01±0.27, 1.88±0.19 and 1.23±0.23, which was significantly upregulated except the 3.78 m M group(P<0.05 or P<0.01). Conclusions:These findings demonstrated that C-kit+ CSCs exist in neonatal Sprague–Dawley rat heart, and CSCs have the potential for selfrenewal and the ability to undergo a theoretically endless number of divisions. PCA can maintain the CSCs and stimulate their proliferation through the Pi3k-Akt pathway, and that could be successful in repairing the injured heart of patients in future. |
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