Enhancing Erythropoiesis by a Phytoestrogen Diarylheptanoid from Curcuma comosa

Erythropoietin (Epo) is widely used for the treatment of anemia; however, non-hematopoietic effects and cancer risk limit its clinical applications. Therefore, alternative molecules to improve erythropoiesis in anemia patients are urgently needed. Here, we investigated the potential effects of a phytoestrogen diarylheptanoid (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol, (ASPP 049) isolated from Curcuma comosa on promoting erythropoiesis. Treatment with C. comosa extract improved anemia symptoms demonstrated by increasing red blood cell numbers, hematocrit, and hemoglobin content in anemic mice. In addition, ASPP 049, the major compound isolated from C. comosa, enhanced the suboptimal Epo dosages to improve erythroid cell differentiation from hematopoietic stem cells, which was inhibited by the estrogen receptor (ER) antagonist, ICI 182,780. Moreover, the ASPP 049-activated Epo-Epo receptor (EpoR) complex subsequently induced phosphorylation of EpoR-mediated erythropoiesis pathways: STAT5, MAPK/ERK, and PI3K/AKT in Epo-sensitive UT-7 cells. Taken together, these results suggest that C. comosa extract and ASPP 049 increased erythropoiesis through ER- and EpoR-mediated signaling cascades. Our findings provide insight into the specific interaction between a phytoestrogen diarylheptanoid and Epo-EpoR in a hematopoietic system for the potential treatment of anemia.


C. Comosa Treatment of Anemic Mice
C57BL/6J mice were employed for induction of anemia. They were sub-lethally irradiated at 5 cGy using a cobalt-60 gamma source to deplete the bone marrow cells. Subsequently, the production of all types of blood cells and their related components including RBCs, hematocrit, hemoglobin, and reticulocytes were disrupted. Irradiated mice were randomly divided into three groups of nine animals each, then intraperitoneally injected with vehicle control or C. comosa extract (50 or 100 mg/kg of body weight (BW)) each day throughout the course of the experiment. Blood samples were collected from the retro-orbital plexus, and hematological parameters were determined with an MS9 analyzer (Melet Schloesing) at various time points.

In Vitro Erythroblast Culture
CD34 + HSCs isolated from human cord blood and leftover specimens (Miltenyi CD34 Progenitor Cell Isolation Kit, Miltenyi Biotech, Bergisch Gladbach, Germany) were cultured in the presence of interleukin-3 (10 ng/mL), stem cell factor (100 ng/mL) (all from Peprotech, Rocky Hill, NJ, USA), and Epo (0.1 or 1 U/mL, obtained from the Department of Hematology, Hôpital Necker, Paris, France) in Iscove's Modified Dulbecco's Medium (Gibco, USA) supplemented with 15% BIT 9500 (Stem Cell Technologies, Vancouver, BC, Canada) as previously described [22]. For human colony-forming cell (CFC) assays, CD34 + cells were cultured in MethoCult TM SF H4236 (Stem Cell Technologies, Canada) supplemented with interleukin-3 (10 ng/mL) and stem cell factor (50 ng/mL), as previously described [23], along with various concentrations of Epo, E2, and ASPP 049. Numbers of burst-forming unit-erythroid cells (BFU-Es) were scored after 14 days of culture according to the manufacturer's recommendations (Stem Cell Technologies, Canada). For the colony-forming cell (CFC) assay, the BFU-Es were recognized from the colonies with the characteristic of hemoglobinized cells, which are the red or brown colonies containing more than 200 erythroblasts in single or multiple clusters.
Murine HSCs were negatively sorted from the bone marrow of C57BL/6J mice using a magnetic lineage cell-depletion kit from Miltenyi Biotec. Sorted murine lineage-negative (Lin − ) cells were used for CFC assays employing MethoCult TM SF M3236 and liquid culture after 7 days of treatment with indicated concentrations of E2 and ASPP 049. Numbers of BFU-Es were analyzed according to the manufacturer's recommendations (Stem Cell Technologies, Canada) and cell viability was assessed by trypan blue exclusion assay.

Cytospin Preparations and Histological Staining
In vitro cultured erythroid cells were centrifuged onto slides for 3 min at 500 rpm using a Cytospin 4 (Thermo Scientific, USA), air dried, and fixed with methanol for 10 min. Next, cells were stained with Liu's stain according to the manufacturer's recommendations (BaSO Biotech, New Taipei City, Taiwan). Morphological characteristics of nuclei, enucleated RBCs, and mature RBCs were evaluated using light microscopy according to the manufacturer's recommendations.

Erythroid Cell Starvation, Stimulation, and Western Blot Analysis
Epo-sensitive UT-7 (UT7/Epo) cells were maintained in Minimum Essential Medium Alpha (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 1 U/mL Epo at 37 • C in a 5% CO 2 humidified atmosphere. To avoid exposure to growth factors, serum-starved cells were obtained by washing and culturing cells in a medium without fetal bovine serum for 16-18 h before experiments. Serum-starved UT-7/Epo cells were plated at a density of 2 × 10 6 cells/mL and treated with varying concentrations of Epo, ASPP 049, or Epo combined with ASPP 049. At various time points, treated cells were washed twice with cold phosphate-buffered saline and then lysed with modified radioimmunoprecipitation assay buffer (in mM: 50 Tris-HCl (pH 7.4), 150 NaCl, 1 EDTA, 1 NaF, 1 Na 3 VO 4 , 1 PMSF; plus 1% TritonX-100) containing a protease inhibitor cocktail and phosphatase inhibitor. After 20 min of incubation on ice, samples were centrifuged at 12,000 rpm for 20 min at 4 • C. An equal amount of protein was mixed with 4X Laemmli buffer and heated at 95 • C for 5 min. Samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequently transferred to a polyvinylidene fluoride membrane by electroblotting. Membranes were incubated overnight at 4 • C with primary antibodies. After washing five times with Tris-buffered saline containing Tween 20, membranes were incubated with HRP-conjugated secondary antibodies. Signals were detected with Immobilon ® Crescendo Western HRP Substrate (Millipore).

Molecular Docking and Binding Site Prediction
For molecular docking analysis, the crystal structure of the Epo-EpoR complex (PDB ID: 1EER) [24] was obtained from the Protein Data Bank. Briefly, after downloading the receptor in .pdb format, the unused portion was removed, and the polar hydrogen group was added and given charge using AutoDockTools 1.5.6. Next, the optimized ligand structure was evaluated at the B3LYP/6-31 + G(d,p) level of theory using the Gaussian09 program and changing the format from .log to .pdb with Open Babel 2.4.1. Subsequently, the receptor and ligand were saved in pdbqt format using AutoDockTools 1.5.6. Finally, the ligand was docked with the Epo-EpoR complex using Autodock Vina 1.1.2 software from The Scripps Research Institute [25]. Discovery Studio Visualizer 19.1.0 was used to visualize docking results.
The binding site of the Epo-EpoR complex was obtained by a GHECOM web server using the mathematical morphology of pockets on protein surface [26].

Statistical Analysis
Statistical significance was determined by one-way ANOVA followed by post hoc (Bonferroni) multiple comparisons between treatment groups using Prism version 8.0 for Windows (GraphPad Software). A two-tailed unpaired Student's t-test was used to compare the means of two groups. p < 0.05 was considered statistically significant.

C. comosa Extract Enhanced Recovery from Anemia
To investigate the potential of C. comosa to alleviate anemia symptoms, severe anemia was induced in C57BL/6J mice by exposure to sub-lethal total body irradiation to deplete bone marrow cells. In Figure 1, numbers of RBCs in control animals were 10.27 ± 0.15 × 10 6 /mm 3 before the induction of anemia and were progressively decreased with time, reaching the lowest level during days 14 to 18 (3.38 ± 0.25 × 10 6 /mm 3 and 3.34 ± 0.25 × 10 6 /mm 3 at day-14, and day-18, respectively). After day 18, RBC counts gradually increased with time. Treatment of the anemic mice with C. comosa extract at dosages of 50 and 100 mg/kg BW/day significantly increased RBC count ( Figure 1A), hematocrit ( Figure 1B), and Hb concentration ( Figure 1C) from day 18 onward, but reticulocyte levels were decreased from day 26 ( Figure 1D) of treatment compared with the vehicle control group. However, the extract slightly affected numbers of white blood cells and platelets ( Figure S1). These results suggest that C. comosa extract improved RBC count and Hb content, subsequently ameliorating anemia symptoms in a mouse model of anemia.  collected from the retro-orbital plexus, and hematological parameters were monitored with an MS9 analyzer. All data are expressed as mean ± SEM (n = 9). * p < 0.05 and ** p < 0.01, ASPP 049 compared with vehicle control treatments (ANOVA); # p < 0.05, ## p < 0.01, and ### p < 0.001, E2 compared with vehicle control treatment (ANOVA). BW, body weight; RBC, red blood cell. Black arrow indicates the induction of anemia.

ASPP 049-and E2-Enhanced Epo-Stimulated Murine Erythropoiesis
To explore the principle active component of C. comosa involved in enhancement of Epo-stimulated murine erythropoiesis, we investigated the effect of the major compound isolated from C. comosa (the diarylheptanoid, ASPP 049) on erythropoiesis using Lin − murine HSCs. These cells, which were isolated from the bone marrow of C57BL/6J mice, were treated with ASPP 049 or E2 in a methylcellulose medium for 7 days. We found that in combination with a suboptimal concentration of Epo (0.05 U/mL), 0.1, 1, and 10 μM ASPP 049 or 10 and 100 nM E2 significantly increased numbers of BFU-E colonies ( Figure 2B), whereas 50 μM ASPP 049 had no significant effect. In Figure 2C 7, 11, 14, 18, 21, and 26. Irradiated mice were intraperitoneally injected with vehicle control or C. comosa (50 or 100 mg/kg BW) each day. The blood sample was collected from the retro-orbital plexus, and hematological parameters were monitored with an MS9 analyzer. All data are expressed as mean ± SEM (n = 9). * p < 0.05 and ** p < 0.01, ASPP 049 compared with vehicle control treatments (ANOVA); # p < 0.05, ## p < 0.01, and ### p < 0.001, E2 compared with vehicle control treatment (ANOVA). BW, body weight; RBC, red blood cell. Black arrow indicates the induction of anemia.

ASPP 049 and E2 Enhanced the Effect of Epo to Promote Human Erythropoiesis
The effects of ASPP 049 on human erythropoietic cells derived from HSCs were next investigated using a CFC assay. As shown in Figure 3A, in combination with a suboptimal concentration of Epo (0.05 U/mL), 1 and 10 µM ASPP 049 significantly increased the number of BFU-E colonies formed by human CD34 + cells. In the absence of Epo, ASPP 049 (0.1, 1, and 10 µM) failed to induce the formation of BFU-E colonies ( Figure 3A). Consistent with the observed proliferative effects, a combination of 0.1 U/mL Epo with 1 µM ASPP 049 or 10 nM E2 significantly increased numbers of cumulative erythroid cells on day 11 ( Figure 3B). Therefore, 1 µM ASPP 049 and 0.1 U/mL Epo were chosen for subsequent experiments. The effect of ASPP 049 on erythropoiesis was further confirmed using cell surface markers of immature RBCs (CD71 and GPA). Combining Epo with 1 µM ASPP 049 or 10 nM E2 significantly increased expression of CD71 and GPA on days 10 and 14 of treatment ( Figure 3C). By day 14, ASPP 049 and E2 had increased expression of these cell surface markers from 62.6% to 73.4% and 69.2%, respectively ( Figure S2). Immunohistochemical staining of cytospin preparations of HSC-derived erythroblast cultures confirmed the effect of ASPP 049 in improving erythroid cell differentiation. On day 14 of culture, numbers of RBCs were greater in cultures treated with a combination of 0.1 U/mL Epo and 1 µM ASPP 049 compared with 0.1 U/mL Epo alone ( Figure S3). For all experiments, 1 U/mL Epo was used as a positive control. Collectively, these results suggest that ASPP 049 and E2 promoted Epo-stimulated erythropoiesis in human HSC-derived erythroblasts.  Figure S3). For all experiments, 1 U/mL Epo was used as a positive control. Collectively, these results suggest that ASPP 049 and E2 promoted Epo-stimulated erythropoiesis in human HSC-derived erythroblasts.

ASPP 049 Acted through Erα Activation to Induce Human Erythropoiesis
ERα has previously been implicated in HSC division and erythropoiesis [10,11]. To determine whether ERα participates in the regulation of ASPP 049-induced erythropoiesis, CD34 + HSCs were treated with ASPP 049 or E2 in the presence of the ER antagonist ICI 182,780 as indicated. As shown in Figure 4A, on day 11, both ASPP 049 and E2 significantly increased the cumulative erythroid cell number, but these effects were decreased in the presence of ICI 182,780. Moreover, the induction of erythroid precursor cells (BFU-E colonies) mediated by ASPP 049 and E2 was abolished by co-treatment with ICI 182,780 ( Figure 4B). However, with a higher dosage of Epo (1 U/mL), ICI 182,780 did not significantly reduce the enhancing effect of ASPP 049 or E2. The enhancing effect of ASPP 049 and E2 on the number of immature (CD71 + GPA + ) RBCs was slightly decreased by ICI 182,780 ( Figure 4C). Collectively, these results suggest that ERα is required for ASPP 049 or E2 to enhance Epo-induced erythropoiesis in human HSC-derived erythroblasts.

ASPP 049 Acted through Erα Activation to Induce Human Erythropoiesis
ERα has previously been implicated in HSC division and erythropoiesis [10,11]. To determine whether ERα participates in the regulation of ASPP 049-induced erythropoiesis, CD34 + HSCs were treated with ASPP 049 or E2 in the presence of the ER antagonist ICI 182,780 as indicated. As shown in Figure 4A, on day 11, both ASPP 049 and E2 significantly increased the cumulative erythroid cell number, but these effects were decreased in the presence of ICI 182,780. Moreover, the induction of erythroid precursor cells (BFU-E colonies) mediated by ASPP 049 and E2 was abolished by co-treatment with ICI 182,780 ( Figure 4B). However, with a higher dosage of Epo (1 U/mL), ICI 182,780 did not significantly reduce the enhancing effect of ASPP 049 or E2. The enhancing effect of ASPP 049 and E2 on the number of immature (CD71 + GPA + ) RBCs was slightly decreased by ICI 182,780 ( Figure 4C). Collectively, these results suggest that ERα is required for ASPP 049 or E2 to enhance Epo-induced erythropoiesis in human HSC-derived erythroblasts.  erythroid-derived HSCs was observed by trypan blue exclusion assay. (B) As indicated, CD34 + HSCs plated in MethoCult H4236 were treated with 1 µM ASPP 049 and 10 nM E2 in the presence of 0.05 U/mL Epo or 1 µM ICI 182,780; 1 U/mL Epo was used as a positive control. Numbers of BFU-Es were scored on day 14 according to the manufacturer's recommendations. (C) CD34 + HSCs were plated in human erythroid culture medium and treated with 1 µM ASPP 049 and 10 nM E2 in the presence of 0.1 U/mL of Epo or 1 µM ICI 182,780, as indicated. Expression of immature erythrocyte markers (GPA + CD71 + ) was detected by flow cytometry analysis. All data are expressed as mean ± SEM (n = 3). **** p < 0.0001, ASPP 049 compared with vehicle control treatments (ANOVA); #### p < 0.0001, E2 compared with vehicle treatments (ANOVA); $$$$ p < 0.001, 1 U/mL compared with vehicle control treatments (ANOVA). BFU-Es, burst-forming unit-erythroid cells; E2, 17β-estradiol; Epo, erythropoietin; HSCs, hematopoietic stem cells.

ASPP 049 Itself Is Unable to Induce Erythropoiesis of Erythroid Precursor Cells
Erythroid precursor (CD36 + ) cells are the targets to activate human RBC production by erythropoietic stimulating agents [27]. To assess whether ASPP 049 can induce erythropoiesis, the CD36 + cells were treated with 1 µM ASPP 049 or 10 nM E2 in the presence or absence of 0.1 or 1 U/mL Epo for 4 days. As shown in Figure 5A, ASPP 049 nor E2 could induce the proliferation of erythroid precursor cells. Consistent with immature RBC populations, ASPP 049, and E2 both failed to increase numbers of CD71 + GPA + cells in the presence of a suboptimal concentration of Epo (0.1 U/mL); 1 U/mL Epo was used as a positive control ( Figure 5B). Moreover, treatment with the ER-antagonist ICI 182,780 did not affect cumulative erythroid precursor cell number ( Figure S4). Together, these results indicate that erythroid precursor cells are not a target for ASPP 049-induced erythropoiesis.

ASPP 049 Potentiated the Effects of Epo Innactivating ERK, AKT, and STAT5 Phosphorylation
Epo-EpoR signaling is a major pathway in erythroid differentiation [4]. After Epo binds to EpoR, the cell surface form of EpoR is internalized and degraded [28,29], subsequently inducing signaling cascades including ERK, AKT, and STAT5 pathways [4]. Therefore, we investigated whether activation of erythropoiesis by ASPP 049 is mediated through the EpoR signaling pathway. UT7/Epo cells were used to determine the effect of ASPP 049 with Western blotting. We found that 10 µM ASPP 049 and 0.1 U/mL Epo markedly reduced protein expression of the cell surface form of EpoR (upper band). Moreover, ASPP 049 progressively increased phosphorylation of AKT(Ser473) from 10 to 120 min, as well as ERK(Thr202/Tyr204) at 30 min after treatment. However, treatment with a physiological dosage of Epo, AKT(Ser473), and ERK(Thr202/Tyr204) phosphorylation appeared to strongly affect the expression of AKT-ser473 and ERK-Thr202/Tyr204 phosphorylated protein, as clearly detected at early point (10 min) compared with ASPP 049. Interestingly, treatment with a combination of Epo and ASPP 049 effectively induced phosphorylation of AKT(Ser473) and ERK(Thr202/Tyr204), indicating an additive effect of these two compounds ( Figure 6A). As expected, phosphorylation of STAT5 was observed in ASPP 049and Epo-treated UT7/Epo cells and was delayed (observed at 60 min) in a co-treatment condition ( Figure 6B). These results were further confirmed to occur in a concentrationdependent treatment for 30 min, and we found that both ASPP 049 and Epo could reduce expression of the cell surface form of EpoR ( Figure 6C) and increase phosphorylation of AKT(Ser473) and ERK(Thr202/Tyr204), as shown in Figure 6D. This information suggests that ASPP 049 is, at least in part, involved in the EpoR signaling pathway.
induce the proliferation of erythroid precursor cells. Consistent with immature RBC pop-ulations, ASPP 049, and E2 both failed to increase numbers of CD71 + GPA + cells in the presence of a suboptimal concentration of Epo (0.1 U/mL); 1 U/mL Epo was used as a positive control ( Figure 5B). Moreover, treatment with the ER-antagonist ICI 182,780 did not affect cumulative erythroid precursor cell number ( Figure S4). Together, these results indicate that erythroid precursor cells are not a target for ASPP 049-induced erythropoiesis. Figure 5. ASPP 049 could not stimulate erythropoiesis of CD36 + erythroid precursor cells. CD36 + cells sorted from CD34 + HSCs cultured in erythroid medium were treated with 1 μM ASPP 049 and 10 nM E2 in the presence of 0.1 or 1 U/mL of Epo. Proliferation of CD36 + cells was observed by (A) trypan blue exclusion assay and expression of immature erythrocyte markers (GPA + CD71 + ) was detected by (B) flow cytometry analysis. All data are expressed as mean ± SEM (n = 3). E2, 17β-estradiol; Epo, erythropoietin; HSCs, hematopoietic stem cells; ns, non-significant. $$$ p < 0.001.

ASPP 049 Potentiated the Effects of Epo Innactivating ERK, AKT, and STAT5 Phosphorylation
Epo-EpoR signaling is a major pathway in erythroid differentiation [4]. After Epo binds to EpoR, the cell surface form of EpoR is internalized and degraded [28,29], subsequently inducing signaling cascades including ERK, AKT, and STAT5 pathways [4]. Therefore, we investigated whether activation of erythropoiesis by ASPP 049 is mediated through the EpoR signaling pathway. UT7/Epo cells were used to determine the effect of Figure 5. ASPP 049 could not stimulate erythropoiesis of CD36 + erythroid precursor cells. CD36 + cells sorted from CD34 + HSCs cultured in erythroid medium were treated with 1 µM ASPP 049 and 10 nM E2 in the presence of 0.1 or 1 U/mL of Epo. Proliferation of CD36 + cells was observed by (A) trypan blue exclusion assay and expression of immature erythrocyte markers (GPA + CD71 + ) was detected by (B) flow cytometry analysis. All data are expressed as mean ± SEM (n = 3). E2, 17β-estradiol; Epo, erythropoietin; HSCs, hematopoietic stem cells; ns, non-significant. $$$ p < 0.001.

ASPP 049-Mediated Epo-Epor Complex in Inducing ERK and AKT Phosphorylation
To evaluate whether the Epo-enhancing effect of ASPP 049 was associated with EpoRmediated signaling pathways, the binding affinity of ASPP 049 to EpoR was determined using molecular docking analysis. As ASPP 049 can modulate erythroid cell production only in the presence of Epo, a model of the Epo-EpoR complex was employed in our docking design. We found that ASPP 049 binds to this complex with a binding affinity equal to −6.2 kcal/mol ( Figure 7A and Figure S5A). This binding location was supported by GHECOM binding site prediction, as shown in the black circle in Figure S5B (colors represent possible areas for ligand binding sites). The two-dimensional interaction profile of ASPP 049 and the Epo-EpoR complex is shown in Figure 7B. ASPP 049 could form interactions with Arg21, Leu18, and Phe208. The aromatic ring was stabilized by Glu25, Leu27, and Phe208, while the remaining residues (shown in light green) contributed to ASPP 049 binding via van der Waals interactions. These results indicated that ASPP 049 can bind to the Epo-EpoR complex and initiate EpoR signaling. To further prove whether the EpoR is required for the Epo-enhancing effect of ASPP 049, we knocked down the EpoR in UT7/Epo cells using EpoR siRNA. The siRNA decreased EpoR protein levels in the UT7/Epo cells by~50% ( Figure 7C,D) compared to the control. We then investigated the effects of ASPP 049 and Epo on signaling cascade including AKT and ERK phosphorylation in the EpoR-knockdown UT7/Epo cells using Western blotting. We found that neither ASPP 049 nor suboptimal Epo induced the phosphorylation of AKT(Ser473) and ERK(Thr202/Tyr204) in the EpoR-knockdown cells ( Figure 7E,F). These effects remained intact in the siControl cells. Collectively, our data clearly suggest that ASPP 049 in cooperation with a suboptimal concentration of Epo activates the EpoR signaling cascades, leading to enhanced erythropoiesis. ological dosage of Epo, AKT(Ser473), and ERK(Thr202/Tyr204) phosphorylation appeared to strongly affect the expression of AKT-ser473 and ERK-Thr202/Tyr204 phosphorylated protein, as clearly detected at early point (10 min) compared with ASPP 049. Interestingly, treatment with a combination of Epo and ASPP 049 effectively induced phosphorylation of AKT(Ser473) and ERK(Thr202/Tyr204), indicating an additive effect of these two compounds ( Figure 6A). As expected, phosphorylation of STAT5 was observed in ASPP 049and Epo-treated UT7/Epo cells and was delayed (observed at 60 min) in a co-treatment condition ( Figure 6B). These results were further confirmed to occur in a concentrationdependent treatment for 30 min, and we found that both ASPP 049 and Epo could reduce expression of the cell surface form of EpoR ( Figure 6C) and increase phosphorylation of AKT(Ser473) and ERK(Thr202/Tyr204), as shown in Figure 6D. This information suggests that ASPP 049 is, at least in part, involved in the EpoR signaling pathway.

Discussion
The present study demonstrated a novel role of the phytoestrogen diarylheptanoid, ASPP 049, isolated from C. comosa, in enhancing Epo-induced erythropoiesis. In a mouse model of anemia, C. comosa extract improved Hb content and RBC counts and ameliorated anemic symptoms. ASPP 049, the major compound isolated from C. comosa, was found to and phospho-ERK (Thr202/Try204) normalized to total AKT and total ERK, respectively. All data are representative of three independent experiments. * p < 0.05 and ** p < 0.01, ASPP 049 or Epo compared with control treatments (Student's t-test).

Discussion
The present study demonstrated a novel role of the phytoestrogen diarylheptanoid, ASPP 049, isolated from C. comosa, in enhancing Epo-induced erythropoiesis. In a mouse model of anemia, C. comosa extract improved Hb content and RBC counts and ameliorated anemic symptoms. ASPP 049, the major compound isolated from C. comosa, was found to be the principal active component in exerting these effects. ASPP 049 enhanced Epoinduced erythropoiesis of murine Lin − and human HSCs; however, its effect required the presence of Epo. The molecular mechanism by which ASPP 049 stimulated erythropoiesis was mediated through ERα and EpoR signaling cascades, i.e., AKT, ERK, and STAT5 pathways. In addition, the effect of ASPP 049 was observed only during the early stage of erythropoiesis in erythroid progenitors, the effect was absent in precursor (CD36 + ) cells. This finding has a great impact on inducing the generation of erythroid cells, especially correcting an early defect in erythropoiesis. Moreover, its biological activity in combination with Epo may have therapeutic potential in anemia resulting from different causes.
ASPP 049 exhibits estrogenic activity [17] and induces osteoblast differentiation through Wnt/β-catenin and MAPK signaling pathways [18,19]. Indeed, these two pathways have been demonstrated to promote erythropoiesis both in vivo and ex vivo [30,31]. In the present study, the effects of a diarylheptanoid ASPP 049 and the underlying mechanism were evaluated on murine-and human-models of erythropoiesis. ASPP 049 and E2 could induce BFU-E colony formation and HSC proliferation of Lin − cells derived from mouse bone marrow. This result confirmed that the effect of C. comosa extract in accelerating recovery of anemic-induced mice involves the primary compound isolated from this extract, ASPP 049.
In human HSCs, both E2 and ASPP 049 synergistically worked with a suboptimal concentration of Epo to promote erythropoiesis, as observed by increased human BFU-E colony formation and erythroblast proliferation and differentiation. Although many factors are involved in control of processes for RBC formation, the Epo-EpoR signaling pathway is a major mediator [4]. Therefore, targeting this key pathway is commonly used for anemia treatment. Suboptimal production of erythropoietin in the kidney is one of the common causes of anemia in aging, in addition to bone marrow failure [32]. Currently, recombinant Epo is routinely used to treat patients with anemia caused by chronic kidney disease and myelosuppressive cancer chemotherapy [33]. However, treatment with high dosages of Epo is ineffective for some hematological diseases and may promote tumor progression [34,35]. Therefore, decreasing the concentration of Epo while maintaining effective treatment is ideal for medicating patients with anemia. Our findings indicate that Epo concentrations in our system were decreased to a level approximately 10 times lower than that of the normal condition (0.05-0.1 vs. 1 U/mL, respectively). However, the addition of 1 µM ASPP 049 together with 0.1 U/mL Epo clearly increased the immature erythrocyte population. Our results suggest an alternative treatment using a low concentration of Epo while maintaining therapeutic effectiveness, which would limit any adverse effect associated with conventional Epo-related therapy. As there are several causes contribute to anemia in aging, ASPP 049, which induces proliferation of HSCs and possesses antiinflammation, may have potential for treating anemia in aging.
The estrogen receptor (ER) is known to be involved in erythropoiesis [36]. Our results indicate that both E2 and a phytoestrogen diarylheptanoid, ASPP 049, play a role in the regulation of RBC production through ERs. In the present study, HSCs treated with ICI 182,780 (an ER antagonist) exhibited clearly decreased HSC-derived erythroblast numbers and BFU-E colony formation (Figure 4). Based on studies from two independent groups [10,11], activation of ER signaling by a selective agonist of ERα (PPT) but not ER-β (diarylpropionitrile) significantly improved erythropoiesis, suggesting the involvement of ERα during erythropoiesis. Indeed, our previous study reported that ASPP 049 also acted on ERα for the activation of uterotrophic and estrogenic osteogenesis [17,18]. Taken together, our results suggest that ASPP 049 mediated ERα for activation of erythropoiesis in erythroblast-derived human CD34 + HSCs.
Production of RBCs is mainly influenced by EpoR activation and subsequent stimulation of three major signaling pathways: STAT5, MAPK/ERK, and PI3K/AKT [37]. To determine whether ASPP 049 plays a role in the regulation of these pathways in erythroid cells, we treated Epo-sensitive UT-7 cells with ASPP 049, Epo, or combination treatment.
We observed that treatment with ASPP 049 or Epo clearly enhanced phosphorylation of STAT5, MAPK/ERK, and PI3K/AKT in both a time-and concentration-dependent manner, whereas a combination of ASPP 049 with suboptimal Epo prolonged activation of these pathways. These results corroborate well with a previous report showing that iron potentiated the action of Epo in erythroid cells through transferrin receptor functions [38].
Consistently, it has been demonstrated that iron supplementation decreases the requirement for Epo to support erythropoietic differentiation and improves anemia in patients with chronic disease [39]. In the present study, molecular docking analysis revealed that the susceptibility of ASPP 049 to bind to the Epo-EpoR complex, the initiating activator of STAT5, MAPK/ERK, and PI3K/AKT pathways. Thus, it is likely that ASPP 049 exerts its action similar to that of polymeric immunoglobulin A1 or iron on Epo-EpoR-induced erythropoiesis. Moreover, we observed that activation of these pathways correlated to reduced expression of the cell surface form of EpoR. Consistent with our findings, previous studies have shown that control of erythroid cell proliferation was mediated by rapid degradation of EpoR through lysosomal and proteasomal pathways [28,29]. However, the intermolecular link between EpoR-mediated pathways induced by ASPP 049 and EpoR degradation requires further investigation.
Inhibition and induction of cytochrome P450 (CYP) enzymes, which result in clinically significant drug-drug interactions, are commonly found in herbal remedies [40]. It is often a limitation of using herbal medicine. In the present study, although the C. comosa extract and the diarylheptanoid ASPP 049 have therapeutic potential with curative outcomes in anemia, their induction of some CYP enzymes may limit their clinical use. To minimize clinical risks before long-term application, they are required to assess these particular issues of herbdrug interactions. Another limitation is that the stimulation of STAT5, MAPK/ERK, and PI3K/AKT pathways, which are activated by ASPP 049, is also observed in the proliferative tissues including leukemia [41]. In addition, the estrogenic activity of the ASPP 049 may be able to affect other ERα-expressing target organs in the body. Long-term treatment of this compound therefore needs to be warranted. It is worth noting that ASPP 049 possesses a weak estrogenic activity compared with E2 [17], and treatment-related adverse effects of the compounds in both animal models and cellular levels have not been observed. To determine whether C. comosa and ASPP 049 are clinically significant, a study in humans warrants further application.

Conclusions
In conclusion, the diarylheptanoid ASPP 049 enhances the effects of Epo and displays estrogenic activity via ERα-mediated activation of STAT5, MAPK/ERK, and PI3K/AKT signaling pathways. These actions are conserved in both in vitro human erythroid cellderived HSCs and an in vivo mouse model of anemia. Therefore, C. comosa could be used as an innovative therapeutic strategy in combination with physiological concentrations or lower Epo for the treatment of patients with anemia.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/biomedicines10061427/s1, Figure S1: Effects of Curcuma comosa extract on WBCs and platelets in anemic mice; Figure S2: Representative flow cytometry showing effects of ASPP 049 and E2 on immature erythrocyte marker expression; Figure S3: Morphology of erythropoietic response in ASPP 049-treated HSC-derived erythroblasts; Figure S4: Co-treatment of an ER antagonist with ASPP 049 or E2 failed to enhance erythroid precursor cell proliferation; Figure S5: ASPP 049 binds to the Epo-EpoR complex.  Institutional Review Board Statement: The animal experimental protocol was approved by the French Institutional Committee, Institut Imagine, Université Sorbonne Paris Cité, Hôpital Necker, Paris, France (A75-15-34). All animal experiments were performed in accordance with relevant guidelines and regulations. Human CD34 + HSCs were obtained from human cord blood according to the Helsinki Declaration and with approval from the Ethics Committee of Institut Imagine, Université Sorbonne Paris Cité, Hôpital Necker (Comité de Protection des Personnes "Ile-de-France II") and the Ethical Clearance Committee on Human Rights Related to Research Involving Human Subjects at Ramathibodi Hospital, Mahidol University, Bangkok, Thailand (MURA2019/916).

Informed Consent Statement:
Informed consent was obtained from all subjects before performing the experiments.
Data Availability Statement: Not applicable.