Fig 2: RORα1 mediates BavaC-stimulated EPO expression(A) RORα binding sites in the human, mouse, and rat EPO promoters. (B) Transient transfected EA.hy926 cells containing RORα (3×RORE) reporter luciferase plasmids were stimulated with the indicated doses of BavaC for 16 h, and RORα1 luciferase activity was measured (n = 3). Data are expressed as mean ± SD. **P < 0.01 vs. vehicle control. (C and D) Transient transfected EA.hy926 cells cells containing human EPO promoter reporter luciferase plasmids were stimulated with the indicated doses of the RORα activator CGP52608, or with 2 μM BavaC or BavaC plus the RORα antagonist VPR66 for 16 h, and EPO luciferase activity was measured (each n = 3). Data are expressed as the mean ± SD. *P < 0.05 vs. vehicle control, **P < 0.01 vs. vehicle control; ##P < 0.01 vs. BavaC alone. (E and F) Rat bone marrow stromal cells with or without 2 μM BavaC stimulation and/or with or without VPR66 treatment for 7 days. Immunofluorescence staining involved labeling with anti-vWF (green) and anti-CD34 (red) antibodies. Data are presented as mean ± SD, n = 3, *P < 0.05 vs. control group; #P < 0.05 vs. sham-operation group. Bar = 20 μm. (G and H) The cells were or were not incubated with 2 μM BavaC, and/or were incubated with VPR66 in the EBM-2 basal medium for 7 days. The cells were labeled with anti-vWF (green) or anti-CD34 (red) antibodies, and the ratios of CD34, vWF and vWF/CD34 cells were measured using flow cytometry. Data are presented as mean ± SD, n = 3, *P < 0.05, total number of EPCs in the second and fourth zones vs. the control.

Fig 3: Identification of ADSCs by multi-lineage differentiation and cell surface markers.(a) Passage 3 ADSCs show fibroblast-like morphology (original magnification 100×). (b) Oil red stain shows lipid droplets 1 week after the induction of adipogenic differentiation. (c) ADSCs pellets were stained positively with toluidine blue after induction of chondrogenic differentiation at 14 days. (d) Alkaline phosphatase was detected in the cytoplasm 14 days after induction of osteogenic differentiation. The scale bar is 100 μm. (e) Specific cell surface markers were detected by flow cytometry. The ADSCs were negative for the hematopoietic lineage markers CD34 and CD45. The fractions of CD44-, CD90-, and CD105-positive cells were 67.63%, 68.13% and 92.43%, respectively, indicating their mesenchymal origin. The experiments were performed three times, and representative images are shown. Values are the mean ± SEM.

Fig 4: Flow cytometry revealed double positive expression of CD34 and PDGFRα (A) before and (B) after cell sorting. (C) Quantification of CD34/PDGFRα double positive cells. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05. CD34, cluster of differentiation 34; PDGFRα, platelet derived growth factor receptor α.

Fig 5: BavaC increases the circulating EPC ratio(A and B) Wistar rats in the BavaC-treated group were given 3 mg/kg BavaC for 7 days by intragastric administration. The rats in the control group were given the adjuvant (CMC-Na) only. Non-erythrocytes of rats were labeled with anti-CD31 (green) or anti-CD34 (red) antibodies, respectively, and analyzed by flow cytometry. Data are presented as mean ± SD, n = 3, *P < 0.05 vs. control group; ** P < 0.01 vs. control group. (C and D) Wistar rats were treated or not treated with 3 mg/kg BavaC for 1, 3, 7, and 14 days by intragastric administration. Non-erythrocytes were labeled with anti-vWF (green) or anti-CD34 (red) antibodies, respectively, and analyzed by flow cytometry. Data are presented as mean ± SD, n = 4, *P < 0.05 vs. model group.