MI in vivo, and with cellular experiments in vitro. Immunohistochemistry revealed the increased protein expression of CXCR4 in MI groups compared to control groups and the increase was more significant with RGE than NS in chronic stage after MI. Western blots analysis revealed the same findings for CXCR4 protein expression in ischemic myocardial tissue. As for SDF-1a, the expression increased with RGE but not with NS at week 1 compared to day 3. And there was no SB-705498 web statistical difference between the two treatments in chronic stage after MI. RT-PCR showed the changes in mRNA level of SDF-1a/CXCR4 cascade. The fluctuation of mRNA expression was just like what it showed in protein expression by Western blot. These suggested that RGE might activate SDF-1a/CXCR4 cascade mainly by upregulating transcription and translation of CXCR4. To further certify the effect of RGE on SDF-1a/CXCR4 cascade, EPCs were stimulated with RGE-PBS solutions in vitro. The proliferation of EPCs decreased with 500 and 1000 mg/ml RGE solution, so we chose other 4 kinds of solutions in subsequent experiments. Through Western blot and RT-PCR, we chose the optimal RGE-PBS solution concentration and duration for SDF-1a/CXCR4 cascade. For CXCR4, the optimal concentration is 25 mg/ml and the optimal duration is 48 h. For SDF-1a, the optimal concentration is 50 mg/ml and the optimal duration is 24 h. The optimal concentration and duration were used to stimulate EPCs and the tube-formation capacity of EPCs was tested. The inhibitor group, blocking the SDF-1a/CXCR4 cascade with AMD3100, showed poor functional EPCs that barely participated in capillary-like tube Group NS-b prior after NS-m prior after NS-MI prior after RGE-b prior after RGE-m prior after RGE-MIprior after LV left-ventricular end-diastolic volume; LV-s, left-ventricular end-systolic volume; LV-mass, relative mass of left ventricle; LVEF, the 
left-ventricular ejection fraction; LVFS, the Left-ventricular fractional shortening; E/A, the radio of peak E velocity and peak A velocity at mitral valve protocol; NS-b, NS blank; NS-m, NS mock; RGE-b, RGE blank; RGE-m, RGE mock; prior, before MI; after, after MI and before being killed. Data are mean 6 SD. e P,0.05, f P,0.01 vs. the same animal before MI; g P,0.05, h P,0.01 vs. blank group. doi:10.1371/journal.pone.0054303.t005 formation, CXCR4 showed poor expression and its ligand SDF-1a showed over-expression. The CXCR4 optimal concentration and duration most significantly increased EPCs participating in capillary-like tube formation as compared with the other groups. The optimal concentration and duration for SDF-1a had a similar but not as obvious an effect as for CXCR4. These revealed that RGE was able to increase EPCs participating in capillary-like tube formation by 14530216 up-regulating SDF-1a/CXCR4 cascade expression in vitro. And this effect of RGE could be reversed by CXCR4 specific inhibitor AMD3100. Discussion The traditional Chinese herb Rehmannia glutinosa can promote bone-marrow proliferation and protect the ischemic myocardium without mechanism studied. And the EPCs are attractive targets for repair of the ischemic myocardium. Therefore, we investigated the effect 23838678 of RGE on EPCs in a rat model of MI. In preliminary experiments, 3 different oral doses of RGE given to normal rats could increase the number of EPCs in peripheral blood and bone marrow at 8th to 16th weeks. Among these, the high dose had the most significant effect at 8th to 12th weeks. The preliminary e