Five Active Components Compatibility of Astragali Radix and Angelicae Sinensis Radix Protect Hematopoietic Function Against Cyclophosphamide-Induced Injury in Mice and t-BHP-Induced Injury in HSCs

Abstract

Although the compatibility of Astragali Radix (AR) and Angelicae Sinensis Radix (ASR) has favorable effect on promoting hematopoiesis in traditional Chinese medicine (TCM), the main active components and pharmacological mechanism are unknown. We investigated the five active components and its mechanisms in vitro and in vivo. Five active components of Astragalus glycosides (AST), Formononetin (FRM), Ferulic acid (FRA), Calycosin (CAL), and Calycosin-7-glucoside (CLG), which could be absorbed in intestinal tract, were detected in this study. The peripheral blood, hematopoietic growth factors (HGFs), and hematopoietic progenitor cells (HPCs) colony were observed to evaluate the effect of these five active components promoting hematopoiesis. Furthermore, hematopoietic stem cell (HSC) proliferation, aging, cycle, and related proteins were detected to explore the mechanism of these five components promoting HSC proliferation. i) The in vivo experiments showed that the combination of the five active components could remarkably increase the number of RBCs, WBCs, PLTs, and content of Hb in peripheral blood and the area of bone marrow hematopoietic tissue, as well as thrombopoietin (TPO), erythropoietin (EPO), granulocyte-macrophage colony stimulating factor (GM-CSF), and colony of CFU-GM, CFU-MK, CFU-E, and BFU-E in serum. Each of these five components promoted the recovery of RBCs and Hb, and increased TPO, CFU-MK, and CFU-E. All components except for AST increased the CFU-GM. FRA increased the number of WBCs, the area of bone marrow hematopoietic tissue, and BFU-E. FRA and AST promoted PLT recovery. FRA and CAL improved the content of GM-CSF. FRA, CAL, and CLG improved the content of EPO. ii) The in vitro experiments showed that FRA, FRM, and AST significantly promoted cell proliferation, reduced the positive rate and G0/G1 cells, and increased G2/M + S cells and the expression of cyclin D1 and CDK4 proteins in aging HSCs. Furthermore, the combination of five components had the best effect. Taken together, the five active components of AST, FRM, FRA, CAL, and CLG were the main pharmacodynamic substances of the AR-ASR compatibility, which promoted hematopoiesis. The combination of them had a synergistic effect. The mechanism of promoting hematopoiesis may be relevant to regulating cyclin-related proteins, promoting cell cycle transformation, and promoting HSC proliferation.

Keywords: angelicae sinensis radix; astragali radix; cyclin; hematopoietic stem sell; hemopoiesis.

Figure 1

Staining identification of progenitor cells (magnification: X 400) (A) BFU-E, red cells were positive cells; (B) CFU-GM, bright red cells were positive cells; (C) CFU-E, red cells were positive cells; (D) CFU-MK, brown cells were positive cells. Scale bars, 50 µm.

Figure 2

The positive rate of sca-1+ HSCs was detected by flow cytometry. (A) Percentage of sca-1+ HSCs before sorting in 3.6% (B) Percentage of sca-1+ HSCs after sorting is 92.39%.

Figure 3

UPLC-MS assay of the 5 main active components. (I) UPLC fingerprints of the five components in each reference substance (left). The peak time was evaluated using UPLC assay, and peak of each component is indicated by an arrow (A) The peak time of FRA was 12 min, (B) The peak time of FRM was 21 min, (C) The peak time of AST was 21 min, (D) The peak time of CAL was 17 min, (E) The peak time of FRA was CLG 11 min. (II) UPLC fingerprints of five components of aqueous extract (right). The peak time was evaluated using UPLC assay, and peak of each component is indicated by an arrow. (A) The peak time of FRA was 12 min, (B) The peak time of FRM was 21 min, (C) The peak time of AST was 22 min, (D) The peak time CAL was 17 min, (E) The peak time of FRA was CLG 11 min. a, FRA; b, FRM; c, AST; d, CAL; e, CLG.

Figure 4

ACC improved the peripheral hemogram and added the area of bone marrow hematopoietic tissue. (a–d) A total of 500 uL of blood by heart puncture was put into the tube contatining EDTA, RBC, WBC, PLT, and Hb were measured with an automated hematology analyzer, respectively. (e) Bone marrow hematopoietic tissue was observed with a microscope, and the percentage of hematopoietic tissue area to marrow cavity was compared with Image-Pro Plus. The data are presented as mean ± SD. N = 12. ^∇∇ P < 0.01 vs BLK, ^# P < 0.5, ^## P < 0.01 vs. MDL, *P <0.05, **P < 0.01 vs. ACC. (f) The area of bone marrow hematopoietic tissue in each group (H&E staining, magnification: 100 X). Three visual fields were obtained in each slide by microscope. The percentage of the bone marrow hematopoietic tissue area in the bone marrow cavity area was calculated using Image-Pro Plus software and the mean value as the bone marrow hematopoietic tissue area. The data are presented as the means ± SD. N = 12. Scale bards, 500 µm. A, CTL; B, MDL; C, FRA;D, AST; E, FROM; F, CAL; G, CLG; H, ACC; I, AA.

Figure 5

ACC increases the level of HGFs in serum. (A) Content of GM-CSF in the serum in each group. (B) Content of TPO in serum in each group. (C) Content of EPO in serum in each group. The data are presented as mean ± SD.N = 12. ^∇ P < 0.05 vs. CTL; ^∇∇ P < 0.01 vs. CTL, ^# P < 0.05, ^## P < 0.01 vs. MDL, *P < 0.05, **P < 0.01 vs. ACC. A, CTL; B, MDL; C, FRA; D.AST, E. FMlF, CAL; G, CLG; H, ACC; I, AA.

Figure 6

ACC increases hematopoietic progenitor cells colony. (A–B) The mice were gavaged with saline (25 ml/kg) in the control group (CTL) and model group (MDL) for 7days. (C–I) The mice were gavaged with drugs, respectively, for 7 days. To count the number of colonies under the microscope, we countedmore than eight cells as a colony in CFU-E, more than 50 cells as a colony in BFU-E and CFU-GM, more than three cells as a colony in CFU-MK, and the colony number of HPCs is the total colonies of every well under microscopes. (A) The comparison of CFU-GM count under microscopes in each group. (B) The comparison of CFU-MK count under microscopes in each group. (C) The comparison of CFU-E count under microscopes in each group. (D) The comparison of BFU-GM count under microscopes in each group. The data are presented as mean ± SD.N = 12. ^∇ P<0.05 vs. CTL; ^∇∇ P<0.01 vs. CTL, ^# P<0.05, ^## P<0.01 vs. model, *P<0.05, **P<0.01 vs. ACC. A, CTL; B, MDL; C, FRA; D, AST; E, FRM; F, CAL; G, CLG; H, ACC; I, AA.

Figure 7

Effect of five active components on aging cell proliferation. HSCs of 4–6 generations were inoculated into 96-well plates, and the cells were synchronized G0 phase cultured with serum-free culture medium for 12 h, then treated with t-BHP(100 µmol/L) and joined different concentrations of the five active components in each drug group, respectively, for 24 h. Next, the cells’ proliferative activity was detected by assaying the OD value of a 450-nm laser using CCK-8. The rate of HSCs proliferation was calculated by the following equation: proliferation rate (%) = [(OD of drug-treated–OD of CTL)-1] x 100%. The HSCs were pretreated with t-BHP in the MDL and drug groups, and nontreated HSCs were used as CTL (Figure A–E). (A) The t-BHP-treated HSCs proliferation. (C) The effect of AST (0, 2, 4, 8, 16, 32 µg/ml) on t-BHP treated HSCs proliferation. (D) The effect of CAL (0, 2.5, 5, 10, 20, 40 µ g/mL) on t-BHP-treated HSCs proliferation. (E) The effect of CLG (0,1.25, 2.5, 5, 10, 20 µg/ml) on t-BHP-treated HSCs proliferation. The data are presented as the mean ± SD. N = 4.

Figure 8

(I) Aging HSCs induced by t-BHP were improved bt drug treatment. Aging cells were assayed by SA-β-galactose glucoside enzyme staining method. The positive cells are indicaed by blue (arrow). Magnification: 400x. Scale bars. 50 µm. (II) The influence of each drug on HSC aging and proliferation. After determining the effective synergistic compatibility of the main components (FAR, AST, FRM) and minor components (CAL, CLG) on HSCs proliferation, HSCs of 4-6 generations were inoculated into 96-well plates and synchronized G₀ phase cultured with serum-free culture medium for 12 h, then cultured with complete medium containing t-BHP for 24 h. Meanwhile, we joined the different active ingredients or drug containing plasma into medium. There were 10 groups: (A) Blank group (BLK), (B) model group (MDL), (C) blank plasma group (BP), (D) FRA group (9 µg/ml), (E) AST group (8 µg/ml), (F) FRM group (6 µg/ml), (G) CAL group (10 µg/ml), (H) CLG group (10 µg/ml), (I) ACC group (FAR 9 µg/ml + AST 8 µg/ml + FRM 6 µg/ml + CAL 10 µg/ml + CLG 10 µg/ml), (J) drig containing plasma (DCP) group (AR 19 g/kg + ASR 19 g/kg). Groups A, B, and C joined the blank plasma, and groups D, E, F, G, H, and I joined different efficient compositions, respectively. Group J joined the drug containing plasma. Afterward, to assay the proliferation rate with CCK-* as previously mentioned, 400 cells was tha aging rate. The data are presented as the means ± SD. N = 4 ^∇∇ P<0.01 vs. BLK, ^## P < 0.001 vs. model, *P <0.05, **P<0.001 vs. ACC, ^•• P<0.01 vs. BP. A, CTL; B, MDL; C, BP; D, FRA; E, AST; F, FRM; G, CAL; H, CLG; O, ACC; J, DCP.

Figure 9

The influence of each drug on HSC cycle. Cell cycle was determined by flow cytometry, 1 x 10⁶. HSCs were collected after centrifugation, washed once with 1 ml PBS, fixed with 70% ice ethanol overnight, and incubated at 37ºC for 30 min with 100 µl bovine pancreas DNasel (Bp DNasel, 1g/l). Afterward, cells were stained with 1 ml propidium iodide (PI) away from light at 4ºC for 30 min, and the cell number was not less than 2 x 10⁵. The percentage of cells (G0 phase, G1 phase, G2 phase, S phase, M phase) was counted, respectively, and the cells at the G0, G1 phase were quiescent cells and the cells at the G2, S, and M phases were proliferating phase cells. The percentage of cells in G0, G1, G2, S and M phases was counted, and the cells in the G0/G1 phase were quiescent cells, whereas the other cells in the G2, S, and M phases were proliferating cells. The data are presented as the means ± SD.N = 4. ^∇∇ P< . 01 vs. CTL, ^## P < 0.01 vs. model, *P<0.05, **P<0.01 vs. ACC, •P<0.05 vs. BP. A, CTL; B, MDL; C, BPl; D, FRA; E, ASTL; F, FRM; G, CAL; H, CLGL; I, ACC; J, DCP.

Figure 10

(I) The influence of each drug on HSC cyclin D1 and CDK4. The protein level of cyclin D1 and CDK 4 was assessed using Western blot. The integrated optical density of target bands. The data are presented as the means ± SD.N = 4 ^∇ P<0.05 vs. CTL; ^∇∇ P<0.01 vs, CTL, ^## P<0.01 vs. model, *P<0.05, **P<0.01 vs. SAI, ^• P<0.05 vs. BP. A, CTL; B, MDL; C, BP; D, FRA; E, AST; F, FRM; G, CAL; H, CLG; I, ACC; J, DCP. (II) The expression profiles of cyclin D1 and CDK 4 proteins in each group were detected by Western blot. A, CTL; B, MDL; C, BP; D. ACC; E, DCP; F, FRA; G, AST; H, FRM; I, CAL; J, CLG.