Anemia is a global health issue that affects a significant portion of the population, particularly preschool-age children.1 It is characterized by low hemoglobin levels and can lead to various symptoms and complications.2 Hemolytic anemia is a specific type of anemia characterized by the destruction of red blood cells. It can be caused by various factors including toxins, infections, and autoimmune reactions.3

Phenylhydrazine (PHZ) is commonly used to induce hemolytic anemia in animal models owing to its ability to cause oxidative damage to red blood cells. In this context, reactive oxygen species (ROS) are harmful molecules that can cause damage to the developing red blood cells.4 Low levels of sulfhydryl groups, a key antioxidant, and elevated levels of ROS support the involvement of oxidative stress in the development of hemolysis and ineffective erythropoiesis.5 Disruption of redox homeostasis can trigger apoptotic signaling pathways and induce erythrocyte death.6,7

In this context, blueberries (Vaccinium myrtillus L.) are known for their health-protective attributes, such as anti-inflammatory, antioxidant, and anticancer properties.8-10 A high phenolic compound content, which is a secondary plant metabolite, has beneficial effects in preventing diseases attributed to the production of free radicals. Turck et al. showed the anti-apoptotic effect of blueberry extract on H9c2 cardiac cells exposed to a norepinephrine-induced oxidative stress model, indicating the cardioprotective action of this compound.11 In view of that, phenolic compounds from blueberries can react with the cell membranes and protect them against oxidative stress, avoiding hemolysis-induced cell death.12

Nevertheless, studies that explore the therapeutic impact of using blueberry extract in anemic conditions are scarce. The aim of this study was to investigate the potential therapeutic effect of blueberries on phenylhydrazine-induced hemolytic anemia in rats, focusing on the impact on hematological parameters and inflammatory markers.

Male Wistar rats with an average age of 21 days, from the Center for Reproduction and Experimentation of Laboratory Animals (CREAL), were used. Animals were kept in the Animal House of the Pharmacology Department of the Federal University of Rio Grande do Sul (UFRGS) at room temperature (22 °C ± 2 °C) with a 12-hour light-dark cycle and free access to water and food. All procedures were approved by the Animal Care and Use Committee at the UFRGS (protocol number: 45500). All experimental groups were excluded from hematological analysis (two animals).

This is a pioneering study exploring the therapeutic role of blueberries on hematological and inflammatory changes induced by phenylhydrazine. The experimental model of acute hemolytic anemia was successful because both hematological data and spleen morphometric evaluation supported a relevant condition of hemolysis. Furthermore, significant increases in leukocytes and reactive lymphocytes were observed, suggesting an inflammatory response induced by phenylhydrazine. The mechanism underlying this improvement may involve an increase in antioxidant defense represented by the augmented sulfhydryl group levels induced by blueberries. In contrast, blueberry treatment partially modified the differential profile of leukocytes and IL-10 levels. However, the administration of blueberry extract was not effective in mitigating anemia (Figure 9).

Figure 9.

Experimental procedure was based on the establishment of acute hemolytic anemia, through the administration of phenylhydrazine (PHZ), preceded by two weeks of daily treatment with blueberry (BB). This experimental model produces important results regarding the cellular inflammatory response to stress induced by the anemic crisis, such as shown by data on IL-10, neutrophil and lymphocyte counts, as well as relevant changes in erythrocyte morphology and iron metabolism. Parts of the figure were drawn by using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).”

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A phenylhydrazine-induced acute anemia model was successfully established in this study as the data show a high degree of hemolysis in 0.85% and 0.55% sodium chloride solutions. Furthermore, when analyzing the morphological characteristics of erythrocytes, significant hemolytic characteristics were observed, such as ghost erythrocytes, polychromatophilia, anisocytosis, and platelet aggregation. Phenylhydrazine is known to induce the production of excess hydrogen peroxide, which oxidizes the sulfhydryl groups of proteins, causing their inactivation, peroxidation of membrane lipids, and formation of the product ferrihemochrome, resulting in loss of membrane flexibility, rupture, and consequent intravascular or extravascular hemolysis in the spleen.18 Indeed, the data of this study show phenylhydrazine-induced ROS production which could be involved with both oxidative stress and hemolysis. However, blueberry treatment augmented sulfhydryl group levels as a response to oxidative stress. In this sense, blueberry extract was tested because of its known antioxidant and anti-inflammatory properties.13 The phenolic compounds from blueberry extract exert a relevant action against oxidative damage in experimental models of pulmonary hypertension and myocardial infarction.19,20 Couto et al.21 showed that pterostilbene, a phenolic compound from blueberries, increased cellular viability in H9c2 cells stressed with hydrogen peroxide. Nonetheless, this study shows that erythrocyte loss due to hemolysis was not reduced by blueberry extract administration. This can be demonstrated by the reduction in the number of erythrocytes observed in both groups in which phenylhydrazine was administered, reinforcing the anemic condition of these animals.

Hemolytic crises cause temporary changes in serum iron transport, which can affect the serum levels of the proteins involved in the transport of this metal. Although in this study, alterations in the serum iron levels were not observed in the PHZ Group, an elevated transferrin concentration and total iron-binding capacity were detected in this group. Transferrin is a glycoprotein that preferentially binds iron in its ferric form, preventing oxidation reactions mediated by this metal and promoting its release into cells.22 The elevations of TIBC and transferrin implies a counterregulatory response to a hemolytic challenge. On the other hand, these parameters, in the PHZ+BB Group, did not show significant differences when compared to the Control Group or the PHZ Group, suggesting, even if partially, a positive interference of blueberry extract against the harmful consequences of hemolysis and iron release. Similar to this study, Kazmierczak et al.12 explored the protective mechanism of polyphenols from blueberry extracts on red blood cells. These authors showed that low molecular weight polyphenols can bind to the polar groups of the erythrocyte membrane and change the chemical properties of the membrane, thereby providing protection against oxidative stress. In this sense, in the PHZ+BB Group, it is possible that there was less iron release due to the protective mechanism of the compounds present in the extract, reducing the need to transport it through the circulation and placing less burden on transferrin function. Further studies are necessary to confirm this hypothesis.

Hemolysis can trigger inflammation because the release of hemoglobin exposes the heme group, which, when free in circulation, can predispose to oxidative and inflammatory processes.23 The heme group has been shown to directly participate in the development of diseases associated with red blood cell damage.24 It is highly hydrophobic, and according to Kato et al.,25 this property is important for activating innate immune cells and inflammatory response. This hypothesis implies that the immune system can detect and respond to hydrophobic substances even if they are not directly associated with pathogens or damage. The concentration of free heme groups arising from hemolysis was not measured directly. In contrast, the total bilirubin level was evaluated as an indirect measure of heme metabolism. According to the data of this study, there was an increase in total bilirubin in both PHZ and PHZ+BB Groups, suggesting augmented levels of circulating heme resulting from hemolysis. Martin-Ventura et al.26 found that heme groups amplify neutrophil activity and lead to proinflammatory cellular responses and oxidative stress.

The absolute lymphocyte and neutrophil counts, as well as the total white blood cell count, were increased in the PHZ Group to approximately four times that of the Control Group. These findings indicate activation of leukopoiesis by the bone marrow or the release of these cells from the vascular marginal pool into the circulation. In this context, this increase indicates an inflammatory response to hemolysis.27 This process can be mediated by the heme group, which activates protein kinase C and induces the expression of pro-inflammatory cytokines (interleukin-8 and tumor necrosis factor-alpha) and the production of ROS through a process called oxidative burst. In addition, the hemolytic process induces the expression of adhesion molecules and increases the chemotactic activity of neutrophils towards heme gradients.23 In contrast, in the PHZ+BB Group, lymphocyte and neutrophil absolute counts showed no significant changes when compared to the Control Group or the PHZ Group. These results suggest that blueberry extract partially attenuates the inflammatory conditions established by hemolysis. Sergazy et al.28 showed, in an experimental model of hepatotoxicity induced by CCl4, an anti-inflammatory effect of blueberry extract in the liver by decreasing interleukin-6, tumor necrosis factor-alpha, and interferon-gamma levels. Surprisingly, the data of this study show an increased anti-inflammatory response in the PHZ Group, since IL-10 levels, which are anti-inflammatory cytokines, were augmented in this group compared to control rats. This increase may represent a counter-regulatory response to the inflammatory conditions imposed by the hemolytic crisis experienced in this experimental protocol. However, in the PHZ+BB Group, the levels of this cytokine were not significantly different in relation to the Control Group. This finding may indicate that the components of the extract may have mitigated inflammation in this group. Therefore, there was no evidence of an increase in the cytokine IL-10 in this group, probably because the extract itself reduced the inflammatory response. This finding corroborates with the lower lymphocyte and neutrophil counts in the group treated with the extract.

In this experimental model, blueberry extract administration decreased neutrophil and lymphocyte counts, and IL-10 levels. This result indicates the mild anti-inflammatory effect of this extract. The data of this study, suggest the antioxidant role of blueberry extract as a mechanism potential. However, the use of blueberries in hemolytic anemia is a non-classical therapeutic approach that requires further study to identify any beneficial effects on blood diseases.

The authors report that the data of this study will be made available without reservation.