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. 2012;7(6):e39327.
doi: 10.1371/journal.pone.0039327. Epub 2012 Jun 25.

Ginger stimulates hematopoiesis via Bmp pathway in zebrafish

Karine F Ferri-Lagneau  1 , Karni S Moshal, Matthew Grimes, Braden Zahora, Lishuang Lv, Shengmin Sang, Tinchung Leung
Affiliations

Ginger stimulates hematopoiesis via Bmp pathway in zebrafish

Karine F Ferri-Lagneau et al. PLoS One. 2012.

Abstract

Background: Anemia is a hematologic disorder with decreased number of erythrocytes. Erythropoiesis, the process by which red blood cells differentiate, are conserved in humans, mice and zebrafish. The only known agents available to treat pathological anemia are erythropoietin and its biologic derivatives. However, erythropoietin therapy elicits unwanted side-effects, high cost and intravenous or subcutaneous injection, warranting the development of a more cost effective and non-peptide alternative. Ginger (Zingiber officinale) has been widely used in traditional medicine; however, to date there is no scientific research documenting the potential of ginger to stimulate hematopoiesis.

Methodology/principal findings: Here, we utilized gata1:dsRed transgenic zebrafish embryos to investigate the effect of ginger extract on hematopoiesis in vivo and we identified its bioactive component, 10-gingerol. We confirmed that ginger and 10-gingerol promote the expression of gata1 in erythroid cells and increase the expression of hematopoietic progenitor markers cmyb and scl. We also demonstrated that ginger and 10-gingerol can promote the hematopoietic recovery from acute hemolytic anemia in zebrafish, by quantifying the number of circulating erythroid cells in the dorsal aorta using video microscopy. We found that ginger and 10-gingerol treatment during gastrulation results in an increase of bmp2b and bmp7a expression, and their downstream effectors, gata2 and eve1. At later stages ginger and 10-gingerol can induce bmp2b/7a, cmyb, scl and lmo2 expression in the caudal hematopoietic tissue area. We further confirmed that Bmp/Smad pathway mediates this hematopoiesis promoting effect of ginger by using the Bmp-activated Bmp type I receptor kinase inhibitors dorsomorphin, LND193189 and DMH1.

Conclusions/significance: Our study provides a strong foundation to further evaluate the molecular mechanism of ginger and its bioactive components during hematopoiesis and to investigate their effects in adults. Our results will provide the basis for future research into the effect of ginger during mammalian hematopoiesis to develop novel erythropoiesis promoting agents.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Ginger extract and its purified phenolic compounds promote Tg(gata1:dsRed) fluorescence and gata1 mRNA expression.
(A) Bright field (top left) and Tg(gata1:dsRed) fluorescence of zebrafish embryos at about 22 hpf, before the onset of circulation (anterior to the left). Exposure to ginger extract or its compounds 8-gingerol (8-G), 10-gingerol (10-G), 8-shogaol (8-S) and 10-shogaol (10-S) promoted Tg(gata1:dsRed) fluorescent erythroid cell development in the ICM and PBI (arrows), as compared to control embryos. N = 35 embryos per group. In this panel, we show an embryo treated with a lower concentration of 6-S (2 µg/ml) as this compound was toxic at higher doses. Scale bar = 400 µm. (B) Whole-mount in situ hybridization of ginger or 10-G treated embryos (8 hpf to 21 hpf exposure) revealed increased expression of gata1 transcript at 22 hpf. N = 50 embryos per group. Scale bar = 350 µm. (C) At 48 hpf, control embryos at the top; ginger or 10-G treated embryos at the bottom. Scale bar = 500 µm. Fluorescent erythrocytes circulating in the axial vasculature (arrows) and in the pericardial space (arrow heads).
Figure 2
Figure 2. Ginger/10-G treatment increases hematopoietic progenitor markers expression.
Zebrafish embryos were treated with ginger or 10-G from 9 to 21 hpf. (A) Tg(gata1:dsRed) for erythrocyte and Tg(flk1:GFP) for blood vessels, double-fluorescent overlay pictures of embryos at 22 hpf after exposure to ginger or 10-G. Hypertrophy of the PBI vascular plexus in Tg(flk1:GFP) after ginger or 10-G treatment, with Tg(gata1:dsRed) red fluorescent erythrocytes accumulated inside the honeycomb-like vasculature (arrows). Scale bars = 500 µm. Whole-mount in situ hybridization of c-myb (B) and scl (C) in zebrafish embryos at 22 hpf. Both hematopoietic progenitor markers were up-regulated in primitive hematopoietic tissues (ICM+PBI) upon ginger (arrow head) or 10-G (arrow) exposure. Scale bar = 350 µm.
Figure 3
Figure 3. Ginger/10-G treatment during gastrulation promotes bmp2b/7a and Bmp target gene expression in zebrafish embryos.
(A) Treatment of late gastrulae with ginger at 15 or 20 µg/ml induces the mercedes mutant-like phenotype (partial duplication of the tail fin) at 1 dpf in 8% or 10% of the treated embryos, respectively. Thus, the zebrafish embryos exposed to ginger extract mimic the phenotype of the ogon mutant, which has a mutation in sizzled, a bmp suppressor gene, at 1 dpf. (B) bmp7a expression was strongly increased and extended to the entire blastoderm at 60% epiboly, following short-term exposure to ginger (5 µg/ml) or 10-G (1 µg/ml) from sphere (4 hpf) to 60% epiboly (7 hpf) stages. (C) Up-regulation and extension of the expression domain were observed for bmp2b at 60% epiboly. (D–E) Accordingly, BMP target genes were up-regulated after ginger/10G treatment from the sphere stage (4 hpf) to 7 hpf, as illustrated by enhanced eve1 extended towards the dorsal side (arrow heads), a ventral mesoderm marker (D), and gata2, a non-neural ectoderm marker (E), in zebrafish embryos at 60% epiboly. Pictures on left panels show gastrulae, dorsal side to the right (B–E) and statistics tables (right panels) are representative of three independent experiments. N = number of embryos per group. Scale bars = 250 µm.
Figure 4
Figure 4. Ginger/10-G treatment after gastrulation promotes bmp2b/7a in the developing caudal hematopoietic tissue.
(A–B) Zebrafish embryos were treated with ginger (5 µg/ml) or 10-G (2 µg/ml) from 10 to 48 hpf, followed by whole-mount in situ hybridization of bmp2b (A) and bmp7a (B). Both bmp2b and bmp7a were up-regulated locally in the CHT (and underlying fin) upon ginger or 10-G exposure (whereas they are not expressed in the CHT of control embryos at 48 hpf). Scale bars = 700 µm.
Figure 5
Figure 5. Ginger/10-G treatment increases circulating erythrocytes in anemic zebrafish.
(A) A schematic representing the timing and experimental protocol of the anemic procedure, which is required for accurate measurements of the number of circulating erythroid cells within the caudal dorsal aorta. (B–C) Quantitation of Tg(gata1:dsRed) fluorescent erythroid cells, within the caudal artery, in anemic zebrafish embryos with ginger treatment from 2 to 5 dpf (B) or 10-G treatment from 2 to 6 dpf (C), respectively (See Figure 6 A and B for detailed quantitative analyses). (D) A cartoon illustrating the tail region filmed for quantitative analyses is shown. Note the morphological difference between erythroblasts (round progenitors, light blue-colored) and mature erythrocytes (elongated cells, dark blue-colored). The green box represents the filmed area for quantitative measurements of circulating erythrocytes, with dimensions: 327 µm x 246 µm. Data are represented as mean ± SEM. p values were determined by using the Student′s t-test. *, # represent statistically significant values of p = 1.5×10−10 and 9.4×10−6, respectively.
Figure 6
Figure 6. Ginger or 10-G exposure promotes erythrocyte recovery from anemia via a Bmp/Smad signal-dependent mechanism.
Bmp/Smad inhibition abolishes the hematopoiesis promoting effect of ginger and 10-G. (A–B) The effect of ginger on hematopoiesis was quantitated in zebrafish embryos after phenylhydrazine (PHZ) induced acute hemolytic anemia, followed by extensive washes and treatments with ginger extract (A) or 10-G (B) with or without dorsomorphin (DMP; 0.1 µM). Ginger and 10-G promote hematopoietic recovery in PHZ treated embryos. Videos of circulating erythrocytes were analyzed and erythrocyte numbers for “PHZ+ginger” and “PHZ+ginger+DMP” assays were calculated and normalized with blood flow (velocity) using the PHZ control value as a reference. Tables summarize the results of one representative experiment. Experiments were repeated 3 times. n = number of embryos analyzed per group. p values were determined by using the Student’s t-test. (C) Regions of erythropoiesis promoted by ginger and 10-G are indicated on cartoons of zebrafish embryos at 22 hpf (primitive wave; before circulation), and at 5–6 dpf during the definitive wave of hematopoiesis. DMP-mediated inhibition of Bmp/Smad signal refers to Figures S5, S6, 6 and S7 data. DMH1-mediated inhibition of Bmp/Smad signaling refers to Figures S5, S6 and S8 data. During the primitive wave of hematopoiesis, expression of gata1 and Tg(gata1:dsRed) were increased in the ICM and PBI, as shown in Figure 1, and the hematopoietic progenitor markers cmyb and scl were up-regulated in the same hematopoietic tissues, as shown in Figure 2. During the definitive wave, Tg(gata1:dsRed) circulating cells were promoted at 5/6 dpf upon ginger/10-G exposure (Figures 5 and 6), and the hematopoietic progenitor markers cmyb, scl and lmo2 were up-regulated in the CHT/hemogenic endothelium at 6 dpf (cmyb, Figure 7) or in the CHT only at 5 dpf (scl and lmo2, Figure S9).
Figure 7
Figure 7. Effect of ginger and 10-G treatments on c-myb expression in zebrafish embryos at 6 dpf.
(A–C) Ginger (B) or 10-G (C) treatment from 2 dpf to 6 dpf promotes cmyb expression in the CHT (black arrows) and the hemogenic endothelium (white arrows) along the ventral wall of the dorsal aorta (AGM equivalent) in the trunk and tail regions of normal embryos. (D–F) In phenylhydrazine-induced anemic embryos, ginger (E) or 10-G (F) treatment similarly promotes cmyb expression both in the CHT (black arrows) and the hemogenic endothelium (white arrows). (G–H) Graphical representation of the percentage of embryos showing cmyb expression in the CHT (G) and in the hemogenic endothelium (H). CTRL: control; PHZ: phenylhydrazine; n = number of embryos. Scale bar = 500 µm.
Figure 8
Figure 8. Over-expression of bmp2b specifically localized in the CHT area at 79 hpf upon ginger or 10-G exposure in normal and in anemic zebrafish embryos.
Whole-mount in situ hybridization of bmp2b. (A–C, left) Normal non-anemic control embryos or embryos treated with ginger/10-G. (D–F, right) Anemic control embryos or anemic embryos treated with ginger/10-G. Anemic groups were treated with 0.5 µM PHZ from 33 to 48 hpf. Embryos express bmp2b in the CHT region (arrows) following exposure to ginger (B, E) or 10-G (C, F). (G) A table shows the percentage of embryos with bmp2b expression in the CHT area at 79 hpf. Scale bars = 420 µm.
Figure 9
Figure 9. Over-expression of bmp7a specifically localized in the CHT region at 79 hpf upon ginger or 10-G exposure in normal and in anemic zebrafish embryos.
Whole-mount in situ hybridization of bmp7a. (A–C, left) Normal non-anemic control embryos or embryos treated with ginger/10-G. (D–F, right) Anemic control embryos or anemic embryos treated with ginger/10-G. Anemic group were treated with 0.5 µM PHZ from 33 to 48 hpf. Embryos express bmp7a in the CHT area (arrows) following exposure to ginger (B, E) or 10-G (C, F). (G) A table shows the percentage of embryos with bmp7a expression in the CHT region at 79 hpf. Scale bars = 420 µm.
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