Saponins from Sanguisorba officinalis Improve Hematopoiesis by Promoting Survival through FAK and Erk1/2 Activation and Modulating Cytokine Production in Bone Marrow

Abstract

Radix Sanguisorbae, the root of Sanguisorba officinalis L. is used as traditional Chinese medicine. In recent decades, it has been reported to be clinically effective against myelosuppression induced by chemotherapy and/ or radiotherapy. However, the underlining mechanism has not been well studied. In this work, we evaluated the hematopoietic effect of total saponins from S. officinalis L. on myelosuppressive mice induced by cyclophosphamide and by⁶⁰Co-γ-irradiation and confirmed the therapeutic effect. Then, we found total saponins and their characteristic constituents Ziyuglycoside I and Ziyuglycoside II can inhibit apoptosis of TF-1 cells caused by cytokine deprivation, and promote survival of mouse bone marrow nuclear cells through focal adhesion kinase (FAK) and extracellular signal-regulated kinase 1/2 (Erk1/2) activation in vitro. In addition, they can down-regulate macrophage inflammatory protein 2 (MIP-2), platelet factor 4 (PF4) and P-selectin secretion, which are reported to be suppressive to hematopoiesis, both in vitro and in vivo. These results suggest that promotion of survival through FAK and Erk1/2 activation and inhibition of suppressive cytokines in the bone marrow is likely to be the pharmacological mechanism underlying the hematopoietic effect of saponins from S. officinalis L.

Keywords: Sanguisorba officinalis L.; Ziyuglycoside I; Ziyuglycoside II; apoptosis; cytokine; hematopoiesis; myelosuppression; saponin.

FIGURE 1

Total saponins enhanced hematopoietic recovery in myelosuppression mouse model. (A) Ziyuglycoside I and ziyuglycoside II. (B) Peripheral blood WBC analysis on Day 7 after the injection of 100 mg/kg cyclophosphamide. ^###p < 0.001 compared to vehicle control, ^∗∗∗p < 0.001 compared to cyclophosphamide. (C) Peripheral blood WBC analysis on Day 13 after the 3.5Gy ⁶⁰Co-γ irradiation. ^###p < 0.001 compared to vehicle control, ^∗p < 0.05 ^∗∗∗p < 0.001 compared to ⁶⁰Co-γ. Data were presented as mean ± SD (n = 13).

FIGURE 2

Saponins from Sanguisorba officinalis L. promoted survival of bone marrow cells. (A) Mouse BMN cells were maintained in medium free of cytokine or complete medium (containing 50 ng/mL rmSCF, 10 ng/mL rmIL-3, 10 ng/mL rmGM-CSF, 3 U/mL Epo). Mouse BMNCs were seeded at a density of 7 × 10⁵ cells in 96-well plates in the presence or absence of saponins for 6 days. (B) TF-1 cells were cultured in medium free of GM-CSF 2 days before saponins treatment or complete medium (containing 2 ng/mL rhGM-CSF). TF-1 cells were seeded at a density of 1.5 × 10⁴ cells in 96-well plates and stimulated with saponins for 48 h. All experiments were repeated at least three times. Data were presented as mean ± SD.

FIGURE 3

Saponins from S. officinalis L. exerted anti-apoptotic effects on TF-1 Cells. (A–B) The activity of caspase3/7 in TF-1 cells. Assays were done in triplicate. Mean values with standard deviations are shown. (C) Change of protein expression patterns in TF-1 cells. Cells were cultured in medium free of GM-CSF for 2 days. Then cells were treated with saponins or vehicle for 48 h. Representative results of three independent experiments are shown.

FIGURE 4

Saponins from S. officinalis L. induced activation of FAK and ERK1/2. (A) In vitro kinase assay for tyrosine phosphorylation-associated FAK kinase activity. The assay was performed in triplicate. Data were presented as mean ± SD. (B–C) Western blotting analysis for ERK1/2 phosphorylation. The time point was 16h in (C). Representative results of three independent experiments are shown.

FIGURE 5

The inhibitor of FAK and Erk1/2 suppressed the pro-survival effect of saponins on bone marrow cells. (A) Viability of mouse BMN cells treated with PF 573228 (inhibitor of FAK), either alone or in combination with saponins. (B) Viability of mouse BMN cells treated with FR 180204 (inhibitor of Erk1/2), either alone or in combination with saponins. The experiment was repeated at least three times. Data were presented as mean ± SD.

FIGURE 6

Saponins from S. officinalis L. inhibited suppressive cytokine production. (A) Mouse cytokine antibody array showing fold change in gene expression patterns of MIP-2, PF4, and P-selectin in bone marrow and conditioned medium of BMSCs. To collect sample of bone marrow, mice were treated with total saponins and cyclophosphamide as described in Section “Animal Experiment”, and mouse bone marrow supernatant fluid was collected as mentioned in Section “Cytokine Arrays”, and cyclophosphamide treatment alone was used as control (n = 8). The conditioned medium of BMSCs was collected as mentioned in Section “Cytokine Arrays”, and vehicle treatment was used as control. The fold change value is calculated by dividing the data from treatment with control. (B) ELISA analysis for MIP-2 and PF4 in cell culture supernatant of mouse BMSCs. (C) Viability of mouse BMN cells co-cultured with BMSCs pretreated with saponins. The experiment was repeated at least three times. Data were presented as mean ± SD.