Introduction

As we are aware, the life of a woman consists of different stages; from birth to death, some physiological risk factors are threatening her life and health1,2; the menopause situation could be one of those stages that could potentially affect a woman’s life quality3,4. According to the World Health Organization (WHO), menopause state is defined as a natural biological aging process caused by the loss of ovarian follicular function and a decline in circulating blood estrogen levels5. It is believed that averagely every woman experiences 40% of her life span in the menopause stage6. The age of menopause also is different from one country to another; it is reported that the natural menopause age in Western countries equals 51.4 while in Asian communities is about 49–50 years7,8. Also, it is estimated that 47 million women annually enter the course of menopause all around the world9.

During the menopause process, the body undergoes a large spectrum of intensive endocrine fluctuations. Among all, a significant decline in estrogen levels might be considered the most important cause. Recently, multiple studies have shown that the changes in iron and estrogen levels are opposite to each other so that a decrease in estrogen hormone accompanied to increase of iron levels which could potentially lead to iron overload3,10. As a result, the iron accumulation could trigger cytotoxic reactions like Fenton and Haber–Weiss which leads to a destructive condition called: Oxidative stress11. Now a series of reactive species oxygens (ROS) generated by oxidative stress could harm any cellular organ, tissue or more importantly, a vital biological process like hematopoiesis12,13,14,15.

Hematopoiesis is a life-extended process to produce any sort of blood cells which is performed by Hematopoietic Stem Cells (HSCs), mainly located in bone marrow (B.M); notably, any disruptions caused by oxidative stress could lead to suppression of hematopoiesis in bone marrow; at this situation, as a compensatory mechanism, other extramedullary centers like liver and spleen are responsible for meeting the needs of the body16,17.

To manage iron accumulation, as the main reason for oxidative stress in menopausal women, the use of iron chelators is widely prescribed18. Deferasirox (DFX), under the brand name of Exjade & Asunra in injectable form and Oleptiss in tablet formulation approved by the Food and Drug Administration (FDA), is one of those iron chelators that bind to free ions of iron in the blood and so assist the iron homeostasis19; alongside bio-compatibility, DFX also has anti-oxidant and anti-inflammatory activities which make it a suitable choice to confront oxidative stress and control iron homeostasis20.

However, despite many studies have focused on the beneficial effects of DFX on iron accumulation, there is still no shred of evidence that DFX could manage iron homeostasis and hematopoiesis altogether in menopause situations and so suppress the following oxidative stress; this lack of knowledge made the basis of this research to investigate the effects of DFX on iron homeostasis and hematopoiesis in ovariectomized rats with iron accumulation.

Results

Histopathological alterations on the uterus tissue (Macroscopic/Microscopic manner)

The histopathological effects of DFX on uterine tissues in the Sham and OVX groups are shown in Fig. 1. Macroscopically, the Sham group exhibited intact, uniform uterine structures, while the OVX group demonstrated severe atrophy, reduced size, and irregular texture, consistent with estrogen deficiency. Iron administration in OVX rats further worsened these changes, producing darker and coarser tissues. Microscopically, the Sham group displayed a mixture of cylindrical and cubic endometrial cells with active mitotic divisions and sparse but intact uterine glands. In contrast, the OVX group showed inactive cubic cells, a high frequency of apoptotic bodies, and atrophied, irregularly shaped glands. Iron administration intensified these effects, increasing apoptotic activity, lymphocytic infiltration, and reducing glandular density. Treatment with DFX at 50 mg/kg partially restored uterine architecture in iron-treated Sham rats, improving glandular density and reducing apoptotic markers. In OVX rats treated with iron and DFX, slight improvements in endometrial structure were observed, though glandular atrophy and lymphocytic infiltration persisted. At 100 mg/kg, DFX nearly normalized uterine structure in Sham rats, while in OVX rats, apoptotic bodies decreased slightly, but other histological changes showed minimal improvement, indicating a dose-dependent but limited effect in the OVX model.

Fig. 1
figure 1

Deferasirox effects on uterus tissue in Sham and OVX groups. (a) Macroscopic image. (b) Microscopic image. (c) Endometrium thickness. (d) Myometrium thickness. Slides were stained with hematoxylin and eosin (H&E) (100x magnification). Values are reported as mean ± standard deviation (n = 3). **** P < 0.0001; #### P < 0.0001 (as compared with Sham); ! P < 0.05 and !!!! P < 0.0001 (as compared with Sham + Iron); ££ P < 0.01, ££££ P < 0.0001 (as compared with OVX); ¥¥ P < 0.01, ¥¥¥ P < 0.001 and ¥¥¥¥ P < 0.0001 (as compared with OVX + Iron). OVX, ovariectomy; DFX, Deferasirox.

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Iron accumulation and the role of DFX

Examining how DFX affected the concentrations of iron, ferritin, transferrin saturation percentage, and TIBC in the serum of OVX and Sham groups, as shown in Fig. 2, provided several new findings. The OVX group significantly raised Fe, ferritin, TIBC, and transferrin saturation percentage, above those observed in the Sham group (except for transferrin saturation percentage, the increase in other parameters was statistically significant). Furthermore, the administration of FAC to the Sham and OVX groups led to an additional increase in ferritin, transferrin saturation percentage, and iron levels. Iron administration, however, only increased TIBC in the Sham group; it had no discernible effect on TIBC in the OVX group, even though the difference was not statistically significant. Four iron-related parameters decreased in the OVX group when DFX at doses of 50 mg/kg and 100 mg/kg was administered; the lower dose proved to be more effective. In contrast, iron levels, TIBC, and ferritin were all reduced in the Sham group by DFX treatment at both doses, with the exception of ferritin at the 100 mg/kg dose, which did not show statistical significance. Furthermore, neither of the DFX doses significantly changed the transferrin saturation percentage in the Sham group.

This research, alongside assessing the iron profile in serum, delved into how DFX influences iron accumulation in the B.M, liver, and spleen, as illustrated in Fig. 3. No iron deposits were detected in any of these three tissues within the Sham group; on the other hand, the OVX group showed iron deposits only in the spleen and bone marrow and not in the liver. Iron accumulation was visible in all three tissues after iron injections were given to both groups. The OVX group exhibited a more noticeable deposition. As opposed to this, the administration of DFX reduced iron deposition in a way that was contingent upon the dose given to the Sham and OVX groups; however, neither dose was able to bring iron deposition back to the initial levels observed in the corresponding groups.

Fig. 2
figure 2

Deferasirox effects on serum iron accumulation in Sham and OVX groups. (a) Serum iron level. (b) Total Iron-Binding Capacity (TIBC). (c) Transferrin Saturation percentage. (d) Serum ferritin level. Values are reported as mean ± standard deviation (n = 8). ns (not significant); * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001; # P < 0.05, #### P < 0.0001 (as compared with Sham); ! P < 0.05, !!! P < 0.001 and !!!! P < 0.0001 (as compared with Sham + Iron); ££ P < 0.01, ££££ P < 0.0001 (as compared with OVX); ¥ P < 0.05, ¥¥¥ P < 0.001 and ¥¥¥¥ P < 0.0001 (as compared with OVX + Iron). OVX, ovariectomy; DFX, Deferasirox.

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Fig. 3
figure 3

Deferasirox effects on iron deposition in bone marrow, liver, and spleen in Sham and OVX groups. Slides were stained with Prussian blue (bone marrow: 1000x magnification, liver and spleen: 100x magnification). OVX, ovariectomy; DFX, Defrasirox.

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Oxidative stress and antioxidant activity caused by DFX

The impact of DFX on the serum levels of MDA and FRAP in both OVX and Sham rats is shown in Fig. 4. Compared to the Sham group, OVX produced higher MDA levels and a lower serum antioxidant capacity. Iron injections exacerbated these effects, though the MDA levels in the OVX group did not differ significantly. Additionally, the administration of FAC resulted in a significant drop in serum FRAP levels and an increase in MDA levels in the Sham group. Additional research revealed that DFX reduced oxidative stress and increased antioxidant function in the Sham group at all dosages. On the other hand, only the higher dose (100 mg/kg) in the OVX group showed promise in increasing antioxidant metrics and reducing oxidative stress. Significantly, in both the Sham and iron-administered groups, DFX was able to normalize serum FRAP levels to those seen in the Sham group at this dose. Furthermore, in OVX and iron-injected groups, DFX at 100 mg/kg effectively decreased serum MDA levels below those of the Sham group.

Fig. 4
figure 4

Deferasirox effects on serum antioxidant activity and oxidative stress in sham and OVX groups. (a) Ferric Reducing Antioxidant Power (FRAP). (b) Malondialdehyde (MDA). Values are reported as mean ± standard deviation (n = 8). ns (not significant); * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001; #### P < 0.0001 (as compared with Sham); !! P < 0.01, !!!! P < 0.0001 (as compared with Sham + Iron); ££££ P < 0.0001 (as compared with OVX); ¥¥¥ P < 0.001, ¥¥¥¥ P < 0.0001 (as compared with OVX + Iron). OVX, ovariectomy; DFX, Deferasirox.

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Hematological changes caused by DFX

Figure 5 shows how the rats in the Sham and OVX groups’ hematological parameters responded to DFX. Comparing the OVX group to the Sham group, the results showed that there was an increase in WBCs, a drop in RBCs, and a decrease in HCT. Oddly enough, there were no discernible variations found between the Hb level and the number of PLTs. WBC, RBC, and PLT counts, as well as Hb and HCT levels, decreased in both the Sham and OVX groups after receiving FAC; however, this parameter decline was only statistically significant in the Sham group. The two groups were given DFX at different doses (50 and 100 mg/kg), and only the 100 mg/kg dose was able to raise the PLT count in the Sham group (not the OVX group). Nevertheless, both groups had an increase in WBCs. After receiving DFX treatment, no additional statistically significant changes in hematological parameters were noted.

Fig. 5
figure 5

Deferasirox effects on hematological parameters in Sham and OVX groups. (a) White blood cell (WBC) count. (b) Red blood cell (RBC) count. (c) Platelet count (PLT). (d) Hemoglobin (Hb) level. (e) Hematocrit level (HCT). Values are reported as mean ± standard deviation (n = 8). ns (not significant); * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001; # P < 0.05, ## P < 0.01, ### P < 0.001 and #### P < 0.0001 (as compared with Sham); !!!! P < 0.0001 (as compared with Sham + Iron); ¥ P < 0.05, ¥¥¥ P < 0.001 (as compared with OVX + Iron). OVX, ovariectomy; DFX, Deferasirox.

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Proliferative role of DFX on hematopoietic stem cells (HSCs) in the B.M

The effect of DFX on the percentage of HSCs in the B.M of Sham and OVX rats is shown in Fig. 6. When compared to the Sham group, the OVX group showed a statistically significant increase in the percentage of CD34- and CD45-positive cells. When iron was administered to the Sham group, compared to the baseline group, the percentage of these cells increased significantly. Notably, the OVX group’s percentage of these cells significantly decreased as a result of iron administration. The percentage of HSCs increased in both the Sham and OVX groups after receiving treatment with DFX at a dose of 50 mg/kg; the increase in the Sham group was highly significant. Nevertheless, treatment at a dose of 100 mg/kg resulted in a reduction of CD34- and CD45-positive cells in both groups. Although in the Sham group, the decrease was such that the number of HSCs equaled that of the Sham group, the OVX group experienced a slight decrease compared to the OVX and iron group.

Fig. 6
figure 6

Deferasirox effects on the percentage of hematopoietic stem cells (HSCs) in the B.M in sham and OVX groups. B.M-derived cell suspensions were stained with two monoclonal antibodies (PE anti-rat CD34 Antibody and FITC anti-rat CD45 Antibody) and analyzed by flow cytometry (FL1: FITC, FL2: PE). Values are reported as mean ± standard deviation (n = 3). ns (not significant); * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001; #### P < 0.0001 (as compared with Sham); !!!! P < 0.0001 (as compared with Sham + Iron); ££££ P < 0.0001 (as compared with OVX); ¥¥¥ P < 0.001, ¥¥¥¥ P < 0.0001 (as compared with OVX + Iron). OVX, ovariectomy; DFX, Deferasirox.

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Relative expression of Hamp, Pu.1, Gata1, and Gdf11 genes

In the liver tissue of Sham and OVX rats, the effect of DFX on the relative expression of the Hamp gene is shown in Fig. 7a. When compared to the Sham group, the OVX group’s expression of this gene was notably higher. Additionally, this gene’s expression was elevated above baseline levels in both the Sham and OVX groups after receiving iron administration. On the other hand, under DFX treatment, both groups’ liver tissue showed a dose-dependent decrease in Hamp gene expression. Notably, for both groups, a dose of 100 mg/kg successfully reduced the gene’s expression to a level similar to that seen in the Sham group.

Fig. 7
figure 7

Deferasirox effects on the relative expression of Hamp (in liver tissue), Pu.1, Gata1, and Gdf11 (in bone marrow) genes in Sham and OVX groups. (a) Relative expression analysis of Hamp gene. (b) Relative expression analysis of Pu.1 gene. (c) Relative expression analysis of Gata1 gene. (d) Relative expression analysis of Gdf11 gene. Values are reported as mean ± standard deviation (n = 3). ns (not significant); * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001; #### P < 0.0001 (as compared with Sham); !! P < 0.01, !!! P < 0.001 and !!!! P < 0.0001 (as compared with Sham + Iron); ££££ P < 0.0001 (as compared with OVX); ¥¥¥¥ P < 0.0001 (as compared with OVX + Iron). OVX, ovariectomy; DFX, Deferasirox.

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The B.M of Sham and OVX rats was used to demonstrate the relative expression of the Pu.1, Gata1, and Gdf11 genes (Fig. 7b and c, and 7d; respectively). When the OVX group was compared to the Sham group, it showed decreased expression of the Gata1 gene and increased expression of the Pu.1 and Gdf11 genes. All three genes showed a significant increase in expression following iron administration to the Sham and OVX groups. After 50 mg/kg of DFX treatment, the Pu.1 gene’s relative expression increased and the Gata1 gene’s expression decreased in the Sham group; these effects were reversed in the OVX group. Notably, after receiving DFX at a dose of 50 mg/kg, the expression of the Gdf11 gene was significantly decreased in both groups. It is interesting to note that administering a higher dose of DFX (100 mg/kg) to the Sham group produced different results from those obtained with a lower dose. Specifically, the Gata1 gene’s expression increased and the Pu.1 gene’s relative expression decreased, with the Gata1 gene’s expression surpassing that of the Sham group. In contrast, the OVX group experienced a significant reduction in the relative expression of all three genes in the B.M following a dose of 100 mg/kg of DFX. This brought the expression levels of these genes almost to, if not lower than, those of the Sham and OVX groups.

Histopathological observations on femur bone and the role of DFX

Investigations were conducted on the impact of DFX on the femur’s structural integrity in Sham and OVX rats (Fig. 8). Haversian canals and osteoblasts were unaffected, but there was a notable decrease in osteocytes in the OVX group when compared to the Sham group. When iron was administered to the Sham group, there was a decrease in Haversian canals and osteocytes; however, in the OVX group, there was an additional reduction in Haversian canals and osteocytes, as well as a decrease in osteoblasts. In the Sham group, DFX treatment at both doses increased these cellular components and channels, though there was no discernible difference between the two dosages. In contrast, only the higher dose (100 mg/kg) in OVX group showed improvement in the femoral bone structure, specifically in the number of osteocytes.

Fig. 8
figure 8

Deferasirox effects on the femur bone in Sham and OVX groups. Slides were stained with hematoxylin and eosin (H&E) (100x magnification). OVX, ovariectomy; DFX, Deferasirox.

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Local hematopoiesis and adipose tissue regeneration in B.M and the role of DFX

The effect of DFX on the volumetric changes of adipose and hematopoietic tissues in the B.M of Sham and OVX rats is depicted in Fig. 9. Comparative study within the OVX group showed decreased hematopoietic tissue volume and increased adipose tissue volume, along with an increase in the number of adipocytes without changing their size. The hematopoietic and adipose tissues within rat’s B.M underwent a similar pattern of volumetric changes upon iron administration to the Sham and OVX groups. In particular, compared to the OVX and Sham + iron groups, there was a significant increase in the quantity and size of adipocytes in the OVX and iron-treated groups. These changes were successfully reversed by oral DFX administration at doses of 50 and 100 mg/kg, which affected the number and size of adipocytes. Importantly, in the OVX group, the hematopoietic tissue volume changed very little at the 50 mg/kg dose. Moreover, it is noteworthy to emphasize that there was no statistically significant variation in the quantity of megakaryocytes across all groups under investigation.

Fig. 9
figure 9

Deferasirox effects on the volume alterations of hematopoietic and adipose tissues in the B.M in Sham and OVX groups. (a) Slides were stained with Wright-Giemsa stain (1000× magnification). (b) Hematopoietic number. (c) Adipocyte number. Values are reported as mean ± standard deviation (n = 3). **** P < 0.0001; #### P < 0.0001 (as compared with Sham); !!!! P < 0.0001 (as compared with Sham + Iron); ££££ P < 0.0001 (as compared with OVX); ¥ P < 0.05 and ¥¥¥¥ P < 0.0001 (as compared with OVX + Iron). OVX, ovariectomy; DFX, Deferasirox.

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Extramedullary hematopoiesis (EMH) and the role of DFX

The effect of DFX on EMH in Sham and OVX rats is shown in Fig. 10. The liver and spleen tissues of the Sham group showed normal architecture, in contrast to the EMH in the OVX rats, which showed up as clusters and colonies mostly in the liver tissue. Remarkably, EMH in OVX group’s liver and spleen tissues did not change when FAC was administered; in fact, the OVX group’s liver tissue’s EMH was found to be lower than its own. On the other hand, after the Sham group received an iron injection, EMH was visible in every section, and blood cells appeared as colonies. In both the Sham and OVX groups of rats treated with DFX, EMH manifested as scattered cells and small colonies. There was a notable decrease in EMH in the Sham group, and there was no difference in the two administered doses. On the other hand, the colony and blood cell count in the OVX group decreased in a dose-dependent manner, suggesting a stronger effect at higher doses.

Fig. 10
figure 10

Deferasirox effects on extramedullary hematopoiesis (EMH) in liver and spleen tissues in sham and OVX groups. Slides were stained with hematoxylin and eosin (H&E) (400x magnification). OVX, ovariectomy; DFX, Deferasirox.

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Discussion

The OVX rat is an animal model utilized in the medical field for menopause research; the uterus of ovariectomized rats exhibited histological changes resembling menopause in this study. The end of menstruation during menopause frequently results in an increase in iron stores21,22. This overabundance of iron buildup may worsen oxidative stress, promote the generation of reactive oxygen species, and aggravate chronic conditions like diabetes, heart disease, and neurological disorders. Apart from these conditions, hematopoiesis also faces challenges in menopause21,23. In this study for the first time, the effects of an iron chelator known as DFX were examined under lab settings resembling menopause, which is associated with an increase in iron levels. DFX has been shown in numerous studies to be a helpful therapeutic strategy for treating conditions associated with iron overload. This compound affects iron levels, reduces oxidative stress, improves hematological responses, and treats several chronic illnesses. DFX has been shown in numerous studies to be beneficial in lowering iron levels in patients who suffer from chronic iron overload24,25,26. Thus, the purpose of this study is to examine how DFX affects hematopoiesis and iron homeostasis in ovariectomized rats with iron accumulation.

This study demonstrated that DFX, at doses of 50 and 100 mg/kg, was capable of significantly reducing iron levels, the percentage of transferrin saturation, and TIBC. Chen et al. observed a significant decrease in transferrin saturation (TSAT) and ferritin levels, accompanied by a corresponding increase in TIBC in hemodialysis patients treated with DFX27. Additionally, Banerjee et al. discovered that DFX treatment in aged mice regulated iron levels28.

This study reveals a greater iron deposition in the B.M, liver, and spleen of the OVX group compared to the Sham group after iron injection. Notably, in the OVX group, iron deposition was seen in B.M. and spleen, but there was no evidence of iron deposition in the liver. This disparity may be explained by the OVX rats decreased ovarian hormone levels, which may affect the distribution and storage of iron. Moreover, the observed reduction in Fe levels and the elevated ferritin concentration in the OVX group following Fe supplementation, compared to the control, warrant further attention. In the OVX group, the administered iron appears to have been sequestered into tissues, resulting in diminished serum Fe levels, while the elevated ferritin likely reflects enhanced tissue iron storage. These discrepancies may be attributed to the relatively brief duration of iron administration in this experimental model, which contrasts with the prolonged period of iron accumulation typically observed in postmenopausal women over several years. Furthermore, administration of DFX reduces iron deposits in both the Sham and OVX groups in a dose-dependent manner, indicating that DFX is effective even in more complicated metabolic states like menopause. Nevertheless, even at high doses, DFX failed to induce iron deposition to the level of the sham group. This could be because of current therapeutic constraints or physiological variations brought on by the OVX condition. These findings align with those of a study by Quarta et al., which showed that DFX can reduce liver iron levels in patients following B.M transplantation and restore ferritin levels to normal, demonstrating DFX’s efficacy in reducing iron burden across various tissues and potentially protecting tissues from iron overload damage29.

The findings of the present study indicate that DFX significantly reduces oxidative stress in animal models. Notably, in the Sham group, DFX at both doses resulted in a rise in FRAP and a fall in MDA; in the OVX group, however, only the higher dose was able to significantly change these two parameters. This could be related to the complicated hormonal effects of menopause, where DFX’s antioxidant properties may be influenced by the drop in hormones, especially estrogen. Jimenez-Solas et al. indicated that iron chelators like DFX reduce DNA oxidation and double-strand breaks in patients with MDS30, indicating that DFX can enhance antioxidant activity and decrease oxidative stress under various physiological and pathological conditions. However, the effective dosage needs to be determined for each condition. Furthermore, our study’s results on MDA levels align with those of Katsuyasu Saigo et al., demonstrating that DFX lowers oxidative stress by blocking the synthesis of ROS and changing signal transduction pathways in addition to iron chelation31. An intriguing parallel to our study can be found in Tianze Xu’s work on the interaction between iron accumulation, estrogen receptor expression, and atherosclerosis in postmenopausal models32. The underlying mechanism of reduced oxidative stress in a different physiological context, which is related to iron and is regulated by iron chelation, is consistent with our findings, indicating a broad role of iron in age-related conditions. Cheon studied the effects of ferroptosis inhibitors (DFO and frustatin-1) on salivary gland function in postmenopausal dry mouth. DFO and frustatin were shown to enhance GPX4 activity, lower lipid peroxidation and iron accumulation, and enhance salivary gland function, according to the study’s findings33. Our knowledge of the function of iron chelators in the prevention and treatment of disorders associated with menopause can be improved by these discoveries.

The results of the study reveal the intricate interactions among iron metabolism, oxidative stress, and gene expression regulation, which are indicative of the diverse reactions of various tissues to both DFX and iron administration. Significant alterations in hepatic Hamp gene expression were found in this study after iron was administered to some groups, suggesting that the liver responds normally to high iron levels. Crucially, in the sham group, DFX administration was able to reverse these changes to almost baseline levels. Comparable outcomes were noted in the OVX group, where DFX decreased Hamp gene expression even when iron administration was present. Furthermore, compared to the Sham group, the OVX group’s expression of the Hamp gene increased significantly. This increase was probably caused by higher serum iron levels as well as iron deposition in the B.M and spleen as a result of lower estrogen hormone levels. Notably, the OVX group’s liver tissue did not exhibit iron deposition, but the Hamp gene’s expression was impacted. This may be explained by metabolic variations brought on by the OVX rats changed hormonal status, which have an immediate effect on the distribution of iron and the functionality of different tissues. A study conducted by Masaratana et al. examined how Hamp-/- mice responded to an iron-deficient diet and found that these mice used hepcidin-independent mechanisms to maintain iron homeostasis even in the absence of hepcidin34. These results highlight the versatility and complexity of iron regulatory pathways that go beyond the traditional hepcidin axis. They may also help to explain the differing effects of varying dosages of DFX on iron deposition in different tissues, iron profiles in serum, and the expression of genes involved in iron metabolism. Additionally, a study by Dzikaite et al. investigated the impact of interleukin-6 and tumor necrosis factor-alpha on the expression of the Hamp gene in rat hepatocytes loaded with iron. According to the findings, these cytokines raised Hamp expression, which DFO-based iron chelation was able to regulate35.

In the current study, after FAC was administered, the effect of DFX on blood parameters was evaluated in groups of OVX and sham rats. A distinct role for iron in hematopoiesis in the presence and absence of estrogen is indicated by the fact that FAC significantly decreased WBCs, RBCs, PLTs, Hb, and HCT in the Sham group but did not significantly decrease RBC-related parameters in the OVX group or increase WBC. Increase in WBC count in the OVX group was also observed in the research by Li et al.36. Since estrogen is known to support erythropoiesis and guard against oxidative damage, its lack in the OVX model probably makes the effects of iron load on the blood worse. Both the Sham and OVX groups’ WBC counts rose after receiving DFX treatment, suggesting an immunomodulatory or compensatory immune response brought on by iron deficiency. The number of PLTs in the Sham group rose in response to the higher dose of DFX, but the OVX group did not significantly alter as Wu et al. showed that in mice with aplastic anemia and iron overload, DFX increases the number of PLTs37. Estrogen deficiency and its intricate physiological conditions may be connected to the OVX group’s lack of significant change in PLT numbers36. Furthermore, this study confirmed Wu et al.‘s findings by showing that DFX had no effect on RBCs, Hb, or HCT levels in either the Sham or OVX group37. Wang asserted that there is a complex interaction between iron metabolism and bone health that may indirectly affect hematological parameters in a study on ammonium ferric citrate and its effects on iron accumulation, bone turnover/density in ovariectomized rat models. The correlation between this relationship and the observed rise in WBC count and fall in RBC and HCT counts in the OVX group suggests that B.M is affected by estrogen deficiency dynamics and highlights how estrogen supports erythropoiesis and guards against oxidative damage38,39.

Iron injection significantly increased the percentage of CD34-CD45-positive cells in sham groups, according to the current study’s findings. This rise may be explained by the negative effects of oxidative stress and iron on the B.M environment, which may increase cell activity. The OVX group had a significant increase in CD34-CD45-positive cells, suggesting that the ovariectomy had an impact on stem cell activity. According to Nakada et al. Estrogen/ERα signaling promotes HSC self-renewal through elevated estrogen levels during pregnancy40. EMH in the liver and spleen tissues of the study’s OVX group might have resulted from a decline in HSC activity in the B.M17. Notably, iron administration to the OVX group resulted in a decrease in CD34 and CD45-positive cells in the B.M, but did not cause EMH in the tissues of the liver and spleen. The findings of this study indicate that DFX has the potential to enhance HSC activity within the B.M and promote EMH under conditions such as ovariectomy. The differentiation between leukocyte infiltrations and EMH was achieved through histological evaluation, wherein EMH was discerned as structured clusters of hematopoietic cells, while leukocyte infiltration was defined by a diffuse presence of inflammatory cells41. According to earlier research, DFX increases the number of CD34-positive hematopoietic progenitors in MDS42. In line with Tataranni et al. DFX treatment produces ROS, which activates genes necessary for stem cell reprogramming and decreases the expression of important hematopoietic regulatory proteins. This implies that DFX affects stem cell differentiation and maintenance by means other than its iron-chelating capabilities43. Agriesti et al. highlight that, in contrast to other chelators, DFX specifically raises the generation of ROS, resulting in changed expression of proteins essential to stem cell function and producing a unique hematological effect44.

In the B.M, DFX appears to have more complex effects on the expression of the Pu.1, Gata1, and Gdf11 genes. After receiving iron, the expression of these genes increased significantly in both the OVX and sham groups. However, the effects of DFX differed based on the dosage. In summary, treatment with DFX resulted in decreased expression of the PU.1 gene and increased expression of the GATA1 gene in the sham group, and decreased expression of both genes in the OVX group. Furthermore, in comparison to the sham group, the OVX group displayed a significant increase in PU.1 and GDF11 and a decrease in GATA1. This could be explained by the fact that DFX-induced iron reduction has not been able to reverse the effect of lower estrogen levels on the regulation of hematopoietic gene expression, specifically Gata1. These findings might also point to differences in how B.M cells react to environmental changes and adjustments in their regulatory functions. According to Du et al. Estrogen can activate the estrogen receptor, which increases the expression of the Gata1 gene and causes polyploidization and maturation of megakaryocytes through a dosage-dependent mechanism Gdf11 gene expression was significantly reduced by both DFX doses, which may point to DFX’s direct effects on associated signaling pathways involved in cell survival and differentiation. The significance of DFX in regulating important genetic networks and biochemical pathways has been highlighted by recent research. In myeloid leukemia cells, DFX has been shown to suppress the mTOR signaling pathway by upregulating the expression of REDD1, a crucial regulatory protein that impedes mTOR activity. This result raises the possibility of a mechanism through which DFX inhibits the proliferation of cancer cells45. The therapeutic effects of DFX on B.M gene expression of Gata1, Pu.1, and Gdf11 indicate its role in hematopoietic differentiation and regulation. Gata1 and Pu.1 are crucial for the differentiation of erythroid cells and cells of the myeloid lineage, respectively, while Gdf11 is involved in senescence and the regulation of HSCs46.

The current study’s findings, which are consistent with those of Zhu et al.‘s study, which discovered that OVX rats had higher adipose tissue volume and decreased BMD, highlighted the significance of estrogen circulation in the composition of B.M and hematopoiesis. The OVX group’s hematopoietic tissue volume and osteocyte count both significantly decreased when compared to the Sham group47. This study demonstrated that DFX counteracts the effects of iron injections, which generally result in an increase in adipose tissue volume and a decrease in hematopoietic tissue volume in the Sham and OVX groups. These results hold significance as they demonstrate that DFX also mitigates histological alterations resulting from oxidative stress in the femoral bone tissue, with a more notable reduction in the OVX and iron groups in comparison to the Sham and iron group. The combined effects of elevated iron and decreased estrogen are probably correlated with this. These findings highlight the close relationship between inappropriate changes in B.M composition and estrogen deficiency. The significance of this relationship stems from the possibility that treatments such as DFX could concurrently address B.M composition changes and bone density changes, a dual strategy similar to what Chen et al. stated that Flavonoids improved bone health and decreased B.M adipose tissue in this study48.

To deepen our understanding of DFX’s effects on iron homeostasis and hematopoiesis across diverse clinical and experimental contexts, future research should prioritize evaluating its long-term impact in various disease models associated with iron overload. Additionally, investigations should aim to directly examine prolonged iron accumulation following OVX without external iron supplementation, addressing the inherent challenges posed by extended experimental timelines in rodent models. Furthermore, a comprehensive investigation into the epigenetic changes induced by deferasirox treatment could yield valuable insights into how deferasirox influences the activity of genes and proteins involved in iron metabolism and hematopoiesis. Additionally, comparing the effects of different doses of deferasirox with other iron chelators, as well as evaluating the combined treatment of iron chelators with hormonal drugs in various cell and animal models, could assist in determining the most precise and effective treatment strategies for conditions such as menopause.

This study explored the substantial impacts of deferasirox on iron homeostasis and hematopoietic responses in ovariectomized rats exhibiting iron accumulation. The outcomes demonstrated that deferasirox can adjust the parameters and gene expression levels associated with iron metabolism and hematopoiesis, potentially offering a therapeutic approach for conditions linked to iron imbalances, including menopause, thalassemia, and myelodysplastic syndromes. Furthermore, deferasirox’s efficacy in reducing iron levels may contribute to mitigating oxidative stress and enhancing cellular function and viability. These discoveries emphasize the promise of deferasirox as a potent therapeutic tool in managing hematological disorders stemming from iron excess, particularly in addressing menopausal symptoms, and could pave the way for innovative treatment methods yielding superior patient outcomes.

Materials and methods

Animals

Sixty-four adult female Wistar rats (240–260 g and 12–14 weeks old) were used in this research; The animals were grouped in 8 cages and kept under the artificial circumstances of 12 h of dark/light cycle and temperature of 22 ± 2 ℃; during the intervention, the animals were fed with nutritional pellets and plumbing water.

Ethics declarations

The ethical approval of this research was admitted by the ethical research committee of Birjand University of Medical Sciences (IR.BUMS.REC.1401.445). Moreover, this study is reported in accordance with The ARRIVE guidelines 2.0. Also, the experiments were conducted in accordance with the accepted principles outlined in the guide for the “Care and Use of Laboratory Animals’’ prepared by the National Academy of Sciences and published by the National Institutes of Health49.

Experimental design

Sixty-four female Wistar rats were randomly classified into 8 groups: (i) Sham (mimic; without ovarian deletion), (ii) Sham + Iron, (iii) Sham + Iron + DFX 50 mg/kg, (iv) Sham + Iron + DFX 100 mg/kg, (v) OVX (ovariectomy), (vi) OVX + Iron, (vii) OVX + Iron + DFX 50 mg/kg, (viii) OVX + Iron + DFX 100 mg/kg. After grouping, rats were enrolled in the study; first of all, in order to induce a menopause mimic situation, an ovariectomy surgery was applied. Hence, the animals were anesthetized via intraperitoneal injection (IP) of Ketamine (80 mg/kg) and Xylazine (10 mg/kg) mixture; after that, a small incision was made in the Linea Alba area in the middle of the abdomen; then, both ovaries were cut out using microsurgery without a significant sign of bleeding (Fig. 11). At the end, the incisional area was stitched, and to prevent any post-surgery infection, penicillin was prescribed through an IP route with a dose of 40,000 U/kg/day for 3 days from surgery’s beginning day (Fig. 12). One-week post-ovariectomy, iron accumulation was elicited in the experimental groups through intraperitoneal (IP) administration of ammonium ferric citrate (FAC) at a dosage of 90 mg/kg, according to their designated classifications. Concurrently, DFX was administered orally at daily doses of 50 and 100 mg/kg, prepared by dissolving in distilled water, for a duration of 35 days. FAC was delivered via IP injection twice weekly throughout the same period. Upon conclusion of the 35-day treatment regimen, the animals were humanely sacrificed using an IP injection of 80 mg/kg ketamine and 10 mg/kg xylazine. Blood samples were subsequently obtained via cardiac puncture and the inferior vena cava, with whole blood allocated for complete blood count (CBC) analysis and the serum fraction reserved for biochemical and antioxidant evaluations. To prevent treatment-related anemia caused by preliminary blood sampling, CBC measurements were not performed prior to the intervention. Instead, all 64 rats were acclimatized under standardized conditions for seven days to ensure baseline uniformity. Additionally, the uterus, liver, spleen, and femur tissues were meticulously collected for histopathological assessments, flow cytometric analysis, and gene expression profiling.

Fig. 11
figure 11

Uterus and ovary in a rat.

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Fig. 12
figure 12

Stages of ovariectomy surgery.

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Iron Profile Assessment

Serum levels of free iron, Total Iron Binding Capacity (TIBC), and transferrin saturation percentage were evaluated using of biochemical autoanalyzer (Prestige-i24, Japan) via commercial diagnostic kits (Pars Azmoon Co, Iran). Ferritin serum level was also measured using a ZellBio rat ferritin ELISA kit (ZellBio GmbH Co, Germany) according to the manufacturer’s instructions.

Antioxidant and oxidative stress assays

The antioxidant profile of blood samples was meticulously assessed through Ferric Reducing Antioxidant Power (FRAP) and Malondialdehyde (MDA) assays. These biomarkers of oxidative stress were quantified utilizing advanced commercial kits provided by Zantox (Kavosh Aryan Azma Co., Iran) and subjected to analysis via enzyme-linked immunosorbent assay (ELISA).

Hematological measurements

Hematological parameters of Complete Blood Count (CBC) including White Blood Cell Counts (WBC), Red Blood Cell counts (RBC), Platelet counts (PLT), Hemoglobin (Hb), and Hematocrit (HCT) were measured by CBC cell counter (Sysmex, Japan).

Flow-cytometry analysis

The rat femur and a syringe pre-filled with sterile phosphate-buffered saline (PBS) were used for B.M aspiration, followed by centrifugation (1500 RPM for 7 min at 4 °C) to separate the cells from the supernatant. Then the cells were fixed with 2% paraformaldehyde (PFA) and after washing, the cells were resuspended in sterile PBS for storage. Labeling of cells was by standard staining procedures according to the manufacturer’s protocols with lineage-specific monoclonal antibodies: PE anti-rat CD34 antibody and FITC anti-rat CD45 antibody (BD Pharmingen, San Jose, CA, USA) which were used at optimized concentration and titration. Flow cytometry analysis was performed on a FACSCalibur™ flow cytometer (BD Biosciences).

Assessment of Gene expression level using qRT-PCR

Total RNA was extracted from B.M. and liver tissues using of the Trizol reagent according to the manufacturer’s instructions (Sinaclon Co, Iran). cDNA synthesis was performed with 2 µg RNA via cDNA Synthesis Kit (Parstous Co, Iran). The primers were also designed and synthesized by Metabion (Metabion International AG, Germany). The primers used were: GAPDH (Forward 5́-AAGTTCAACGGCACAGTCAAGG-3́; Reverse 5́-CATACTCAGCACCAGCATCACC-3́), Hamp (Forward 5́-GAAGGCAAGATGGCACTAAGCA-3́; Reverse 5́-TCTCGTCTGTTGCCGGAGATAG-3́), Gata1 (Forward 5́-CAGAACCGGCCTCTCATCC-3́; Reverse 5́-TAGTGCATTGGGTGCCTGC-3́), Pu.1 (Forward 5́-GAGTTTGAGAGCTTCCCTGAG-3́; Reverse 5́-TGGTAGGTCATCTTCTTGCGG-3́), Gdf11 (Forward 5́-GGACTGGATCATCGCACCTAAGC-3́; Reverse 5́-AGCAGAGCCTCGTGGGTTGG-3́). Then, equal amounts of each cDNA sample were quantitatively measured by a qRT-PCR instrument (Applied Biosystems Real-Time PCR) using the SYBR green master mix (2X) method (Parstous Co, Iran). All reactions were performed in biological (three rats) and technical (three qRT-PCR replicates per biological sample) triplicates. The GAPDH gene was used as an internal reference gene. Finally, relative changes in gene expression were calculated using the ΔΔCT formula.

Histopathological evaluations

Briefly, the uterus, liver, and spleen were dissected and fixed in a 10% neutral buffered formalin solution for at least 48 h and then put into the routine process of tissue sectioning; finally, prepared tissue sections were cut at 5 μm thickness using microtome device (Did Sabz Co, Iran) and were stained with hematoxylin and eosin (H&E) staining method50. The histological preparation of femur tissues was carried out. Samples were carefully excised and promptly fixed in 10% neutral buffered formalin for at least 48 h to ensure optimal preservation of tissue integrity. Decalcification was then performed using an EDTA-based solution, allowing for precise sectioning while maintaining the structural integrity of the bone. After decalcification, the tissues underwent standard histological processing, which included sequential dehydration, clearing, and embedding in paraffin to facilitate further microscopic analysis. The sections were eventually observed for perusing the histopathological changes using a light microscope (Olympus, Japan). The severity of histopathological changes was also examined by Knodell Histology Activity Index (HAI)51. Persian blue along with the Wright-Giemsa staining were used to identify iron deposition and bone marrow hematopoietic tissue, respectively.

Statistical analyses

The statistical analysis was carried out using GraphPad Prism software (V.9). The Kolmogorov-Smirnov test was used in order to determine the normal distribution of data by considering P ≥ 0.05; One-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison tests was used to evaluate the significance between multiple groups in normally distributed data. Kruskal-Wallis, followed by Mann–Whitney U tests also were used to evaluate the significant changes between multiple groups in non-parametric status. P ≤ 0.05 has been considered as the significant value. The results were expressed as mean ± standard deviation (mean ± SD).