Phenylhydrazine-induced Hemolytic Anemia Research Articles

Effect of quercetin and mirtazapine on spermatogenesis and testis structure in phenylhydrazine-induced hemolytic anemia mice: An experimental study

Anemia poses a significant healthcare challenge across different socioeconomic groups and can result in reproductive system damage through the generation of free radicals and lipid peroxidation. This study examines the protective effects of quercetin (QUE) and mirtazapine (MIR) against the reproductive damage caused by phenylhydrazine (PHZ) in mice. Fifty NMRI mice, aged 8–10 weeks with an average weight of 27.0 ± 2.0 g, were randomly divided into five groups. The control group (Group 1) received oral administration of 10 mL/kg/day of normal saline. Group 2 (PHZ group) received an initial intraperitoneal dose of 8 mg/100 g body weight of PHZ, followed by subsequent doses of 6 mg/100 g every 48 h. Group 3 received PHZ along with oral QUE at a dosage of 50 mg/kg/day. Group 4 received PHZ along with oral MIR at a dosage of 30 mg/kg/day. Group 5 received PHZ along with oral QUE at a dosage of 50 mg/kg/day and MIR at a dosage of 30 mg/kg/day. The treatment duration was 35 days. Sperm samples were collected from the caudal region of the epididymis post-euthanasia to assess the total mean sperm count, sperm viability, motility, DNA damage, and morphology. Testicular tissue was employed to quantify total antioxidant capacity (TAC), superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA) concentrations, while serum levels of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were analyzed. Additionally, various aspects, including testicular histopathology, oxidative enzyme levels, gene expression related to apoptosis and antiapoptotic pathways, and in vivo fertility index, were evaluated after 35 days. The QUE, MIR, and QUE + MIR groups showed less abnormal morphology and DNA damage, as well as better total and progressive sperm motility, motility characteristics, viability, and plasma membrane function compared to the PHZ group. QUE, MIR, and QUE + MIR administration increased TAC, SOD, and GPx activities in testicular tissue, while reducing MDA levels compared to the PHZ group. Furthermore, QUE, MIR, and QUE + MIR significantly reduced Bax, and caspase-3 expression levels, and increased Bcl-2 expression levels, compared to the PHZ group. Mice treated with QUE, MIR, and QUE + MIR exhibited an increased in vivo fertility index and plasma sex hormone levels compared to the PHZ group. These results show that QUE, MIR, and QUE + MIR might be able to improve the fertility index, boost the testicular antioxidant defense system, and control the death of germ cells. This could mean that they could be used to treat mice with PHZ-induced testicular damage.

Therapeutic efficacy of Punarnavadi mandura against phenylhydrazine-induced hemolytic anemia in rats

Background & aimHemolytic anemia is a blood disorder whose incidence is increasing in the world in recent years especially after the pandemic. Conventional treatments include use of steroids and immunosuppresants that are accompanied by numerous adverse effects. With growing interest in using complex multi-component formulations for multi-targeted therapy, the present study aims to investigate the therapeutic efficacy of a traditional Ayurvedic herbomineral preparation, Punarnavadi Mandura, which has been traditionally used as a supplement in iron-deficiency anemia, against phenylhydrazine-induced hemolytic anemia in rodent models. Experimental approachesWe employ a combination of in vivo and in silico methods in this work to study the therapeutic potential and to understand the possible molecular targets of this traditional formulation. Conventional drugs prednisolone and ferrous sulphate were used for comparison. Results and conclusionThe in vivo studies confirm the ability of Punarnavadi Mandura to reverse pathological changes associated with hemolytic anemia at 100 mg/kg and 200 mg/kg concentration. It restored hemoglobin, bilirubin and white blood cell levels to normal and reduced reticulocytes, hemosiderin and Gamna Gandy bodies in the liver, spleen and kidney. In silico studies suggested that the key constituents in Punarnavadi Mandura interact with high affinity to erythropoietic receptor which could contribute to erythropoiesis. The in silico study also predicted that the phytoconstituents of Punarnavadi Mandura could inhibit TNF-α activity which was validated using gene expression studies.

Protective effects of Helicteres isora L. fruit extract on phenylhydrazine-induced hemolytic anemia in rats

BackgroundAnemia is a serious global public health problem that affects young children and pregnant women with an increased risk of maternal and child mortality. Helicteres isora L. (Family: Malvaceae) has significant antioxidant properties responsible for its medicinal properties. The present study was conducted to investigate the protective effects of aqueous fruit extract of Helicteres isora L. (AEHI) on phenylhydrazine-induced hemolytic anemia in rats. MethodsThe fruits of Helicteres isora L. (H. isora L.) were extracted with distilled water to obtain the AEHI. The AEHI was subjected to preliminary phytochemical analysis, iron estimation and in-vitro antioxidant assay. The HPTLC fingerprinting followed by HR-LCMS analysis of the isolated HPTLC fraction of AEHI was carried out to investigate the phytoconstitutents. Male Wistar albino rats were administered phenylhydrazine (40 mg/kg, i.p.) for two consecutive days to induce anemia. The effects of AEHI were investigated at three oral doses viz. 250, 500 and 750 mg/kg once daily for 13 days by estimating blood parameters and pathological changes in the liver, heart, spleen, kidney, lungs and bone marrow. ResultsThe phytochemical analysis showed the presence of alkaloids, tannins, phenolic compounds, carbohydrates and steroids in the AEHI. The iron content of AEHI was found to be 208 µg/ml. In-vitro antioxidant assays, the IC50 value of AEHI was found to be 90.01 µg/ml. Phenylhydrazine administration caused a significant decrease in red blood cells, hemoglobin, and hematocrit levels. These decreased levels were significantly improved by AEHI in a dose-dependent manner. Pathological changes in the liver, heart, spleen, kidney, lungs and bone marrow were prevented by AEHI treatment. ConclusionIt is concluded that the AEHI provides significant protection against phenylhydrazine-induced hemolytic anemia.

Antioxidant Properties of Pistacia vera against the Effects of Phenylhydrazine-Induced Hemolytic Anemia on Male Fertility in Mice

Objective. The reproductive system can be adversely affected by anemia, characterized by elevated free radical production and lipid peroxidation. Conversely, the pistachio is well-known for its antioxidant capabilities. In this study, a mouse model was employed to investigate the safeguarding effects of the aqueous extract of pistachio (Pistacia vera) against oxidative stress induced by phenylhydrazine (PHZ)-triggered hemolytic anemia. Materials and methods. Forty-eight male mice were distributed into six distinct experimental groups (n = 8): control, PHZ, PHZ + 20Pis, PHZ + 80Pis, PHZ + 160Pis, and 80Pis groups. The experimental groups were administered a PHZ dose of 80 mg/kg/48 hr, along with pistachio doses of 20, 80, and 160 mg/kg/day, delivered intraperitoneally. After a period of 35 days, an assessment of sperm count, motility, morphology, and viability was conducted. Additionally, sperm chromatin quality was evaluated through Chromomycin A3, aniline blue, and toluidine blue staining. Hematoxylin–eosin staining was performed to examine the total diameter of seminiferous tubes and spermatogenic cells. Hormonal levels (FSH, LH, T) and antioxidant levels were analyzed using standard kits. Results. The PHZ groups exhibited a noteworthy decrease in various sperm parameters, as well as diminished chromatin quality assays and reduced levels of sex hormones when compared to the control group. Moreover, the morphometric evaluation indicated a substantial reduction in spermatogonia cell number, spermatid count, tubule count, seminiferous diameters, and seminiferous area in the PHZ group. Notably, the Pis group demonstrated the highest concentration of antioxidant contents. Conclusion. The protective impact of P. vera extract against PHZ-induced anemia on the male reproductive system in mice may be attributed to its abundant antioxidant components, which possess therapeutic properties at elevated levels.

Hematopoietic effect of echinochrome on phenylhydrazine-induced hemolytic anemia in rats

Background Hemolytic anemia (HA) is a serious health condition resulting from reduced erythrocytes’ average life span. Echinochrome (Ech) is a dark-red pigment found in shells and spines of sea urchins. Aim Studying the potential therapeutic effect of Ech on phenylhydrazine (PHZ)-induced HA in rats. Methods Eighteen rats were divided into three groups (n = 6): the control group, the phenylhydrazine-induced HA group and the Ech group, injected intraperitoneally with PHZ and supplemented with oral Ech daily for 6 days. Results Ech resulted in a considerable increase in RBCs, WBCs, and platelets counts, hemoglobin, reduced glutathione, catalase, and glutathione-S-transferase levels, and a significant decrease in aspartate & alanine aminotransferases, alkaline phosphatase, gamma-glutamyl transferase, bilirubin, creatinine, urea, urate, malondialdehyde & nitric oxide levels in anemic rats. Histopathological examination of liver and kidney tissue samples showed marked improvement. Conclusion Ech ameliorated phenylhydrazine-induced HA with a hepatorenal protective effect owing to its anti-inflammatory and antioxidant properties.

A Mediator Switch Tunes Transcription Pausing to Drive Erythropoiesis

The Mediator complex regulates various aspects of hematopoietic development, but whether composition of the Mediator complex undergoes dynamic changes for diversifying transcription and functional outputs is unknown. Here, we found that MED26, a subunit in the core Mediator complex, played a distinctive role in facilitating transcription pausing essential for erythroid development. To address whether composition of the Mediator complex undergoes dynamic changes during erythropoiesis, we detected its expression in primary human erythroblasts derived from CD34 + hematopoietic stem and progenitor cells. We found that while the protein levels of most Mediator subunits decreased substantially in the terminal erythroid differentiation, MED26 remained relatively abundant. A gain-of-function assay in the primary human erythroid culture system showed that MED26 overexpression promoted erythroid differentiation. Furthermore, Med26 conditional knockout (cKO) mice exhibited severe erythroid and hematological defects. Erythroid precursors (especially the Cd71+Ter119+ stage) were absent in the bone marrow of Med26-cKO mice, which phenocopies the cKO mice of Gata1. Prussian blue staining of the spleen section showed more iron deposition in the cKO mice. Moreover, the cKO mice were unable to recover after phenylhydrazine-induced hemolytic anemia. To dissect the molecular basis underlying MED26 function, we analyzed the chromatin occupancy signals of MED26 by CUT&Tag assay and compared them to those of MED1, which is often regarded as a representative subunit of Mediator, in human CD34 + erythroid culture cells on Day 4. Intriguingly, we found that ~60% of chromatin sites with MED1 or MED26 occupancy did not colocalize, suggesting these subunits exert context-dependent gene regulation. Notably, MED26 colocalizes with GATA1 and GATA2 better than MED1. PRO-seq analysis further revealed that MED26-enriched loci were associated with RNA polymerase II (Pol II) pausing. To examine how MED26 might mediate transcription pausing, we performed immunoprecipitation coupled with mass spectrometry (IP-MS) to identify interacting proteins of MED1 and MED26. While MED26 and MED1 similarly coimmunoprecipitated with the majority of other Mediator subunits and the elongation complex, MED26 exhibited a substantially higher interaction with transcription pausing related factors (NELF, DSIF, and PAF complexes) compared to MED1. Knocking out MED26 significantly impairs the recruitment of PAF1 to MED26 occupancy sites, indicating the presence of MED26 facilitates the recruitment of PAF1. Our results also revealed that MED26 enrichment is associated with increased transcription pausing during terminal erythroid stages, which can promote erythroid development. Moreover, we uncovered that MED26 exhibited pronounced biomolecular condensate-forming capability. The intrinsically disordered region (IDR) of MED26 was necessary for its function in promoting erythropoiesis and recruiting pausing-related factors to the MED26-enriched condensates. Taken together, our findings demonstrate that the relative enrichment of MED26 in terminal erythropoiesis leads to a shift of transcription condensates towards a MED26-enriched form. This shift, in turn, facilitates the recruitment of pausing related factors, thereby achieving genome-wide transcription repression during erythropoiesis. This study not only illustrates the indispensable function of MED26 in erythropoiesis but also elucidates the functional coordination of MED1 and MED26 during different erythroid stages. The dynamic switch of Mediator stoichiometry might serve as an important example for understanding the mechanism of the versatile Mediator function during development.

MED16 Negatively Regulates Erythropoiesis and Myelopoiesis through Modulation of Chromatin Accessibility

Tissue-specific transcription factors work in conjunction with numerous coregulators to modulate transcription networks during hematopoiesis; disruptions in this process may lead to hematopoietic disorders, including myelodysplastic syndromes (MDS). While the Mediator complex is known to regulate various aspects of hematopoiesis, the mechanisms underlying subunit-specific functions have not been fully understood. Here, we uncovered that MED16, a core component of Mediator tail module, serves as a crucial negative regulator of erythropoiesis and myelopoiesis by controlling chromatin accessibility at specific target genes. To investigate the function of MED16 in vivo, we transplanted control (shSCR) or MED16-deficient (shMED16) human cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) into immunodeficient mice. Mice receiving shMED16 HSPCs showed a higher proportion of human erythroid and myeloid cells in the bone marrow (BM), while B cell and T cell reconstitution remained unaffected. MED16 deficiency led to an increased number of myeloid colonies and a more pronounced increase in erythroid colonies in the colony-forming assay. During erythroid differentiation, MED16 deficiency resulted in an elevated ratio of CD235a+ cells and increased enucleation. Furthermore, hematopoietic-specific Med16 knockout ( Mx1-Cre × Med16 -/- Med16 KO)mice showed normal BM and spleen cellularity, with an increased frequency of common myeloid progenitors (CMPs). Med16 KO mice exhibited a higher frequency of CD11b+ myeloid cells and Ter119+ erythroid cells in the BM and spleen. A higher percentage of enucleated erythrocytes were observed in the BM of Med16 KO mice. Additionally, in the model of phenylhydrazine-induced hemolytic anemia, Med16 KO mice showed accelerated red blood cell recovery and pronounced splenomegaly, highlighting the crucial role of Med16 in stress erythropoiesis. Our findings demonstrate that MED16 plays a repressive role in hematopoietic development. To elucidate how MED16 regulates gene expression, we conducted single-cell RNA-seq and ATAC-seq of cultured CD34+ cells after MED16 knockdown in primary human CD34+ HSPCs. MED16 deficiency resulted in upregulation of erythroid and innate immunity genes in erythroid and myeloid cells, respectively. Interestingly, MED16 deficiency led to significantly increased chromatin accessibility around the transcription start sites of upregulated genes. Considering the Mediator complex often interacts with transcription factors to convey information, we conducted MED16 chromatin IP sequencing (ChIP-seq). The majority of MED16 occupancy peaks were located at promoters (59.68%). Colocalization analysis of MED16 with chromatin occupancy of hematopoietic-related transcription factors revealed significant overlap with RUNX1. To unveil the mechanism of MED16-mediated transcriptional repression, we conducted immunoprecipitation coupled with mass spectrometry on cultured CD34+ cells to identify MED16-interacting nuclear proteins. We identified the FAcilitates Chromatin Transcription (FACT) complex as a potential candidate. ChIP-seq of the FACT complex subunit SPT16 showed weakened chromatin occupancy signals upon MED16 deficiency. Similar results were observed in mouse BM Ter119+ erythroblasts. These findings indicate that MED16 plays a crucial role in modulating chromatin accessibility and transcriptional repression, potentially through cooperation with RUNX1 and the FACT complex. Furthermore, we found that MED16 overexpression impeded erythroid cell enucleation, similar to MDS erythroid dysplasia. We observed higher MED16 expression in BM-derived CD34+ cells from 183 MDS patients across different subtypes compared to healthy individuals. Interestingly, knocking down MED16 in bone marrow mononuclear cells from MDS patients restored normal erythropoiesis. Transcriptome analysis revealed that MED16 deficiency upregulated innate immunity and erythroid genes. Together, these findings shed light on the previously unknown role of MED16 in hematopoiesis and its association with MDS. Our study provides mechanistic insights into the lineage commitment and developmental progression of hematopoiesis governed by Mediator and may have implications for the treatment of hematological disorders.

Murine Bone Marrow Erythroid Cells Have Two Branches of Differentiation Defined by the Presence of CD45 and a Different Immune Transcriptome Than Fetal Liver Erythroid Cells.

Mouse erythropoiesis is a multifaceted process involving the intricate interplay of proliferation, differentiation, and maturation of erythroid cells, leading to significant changes in their transcriptomic and proteomic profiles. While the immunoregulatory role of murine erythroid cells has been recognized historically, modern investigative techniques have been sparingly applied to decipher their functions. To address this gap, our study sought to comprehensively characterize mouse erythroid cells through contemporary transcriptomic and proteomic approaches. By evaluating CD71 and Ter-119 as sorting markers for murine erythroid cells and employing bulk NanoString transcriptomics, we discerned distinctive gene expression profiles between bone marrow and fetal liver-derived erythroid cells. Additionally, leveraging flow cytometry, we assessed the surface expression of CD44, CD45, CD71, and Ter-119 on normal and phenylhydrazine-induced hemolytic anemia mouse bone marrow and splenic erythroid cells. Key findings emerged: firstly, the utilization of CD71 for cell sorting yielded comparatively impure erythroid cell populations compared to Ter-119; secondly, discernible differences in immunoregulatory molecule expression were evident between erythroid cells from mouse bone marrow and fetal liver; thirdly, two discrete branches of mouse erythropoiesis were identified based on CD45 expression: CD45-negative and CD45-positive, which had been altered differently in response to phenylhydrazine. Our deductions underscore (1) Ter-119's superiority over CD71 as a murine erythroid cell sorting marker, (2) the potential of erythroid cells in murine antimicrobial immunity, and (3) the importance of investigating CD45-positive and CD45-negative murine erythroid cells separately and in further detail in future studies.

Antianemic Activity of Allolobophora caliginosa Extract against Phenylhydrazine-induced Anemia in Rats

Background: Anemia is a global hematological disorder. Earthworms have antimicrobial, antispasmodic, antihypertensive, antiallergic, anti-inflammatory, and anticoagulant properties. background: Anemia is a global hematological disorder. Earthworms have antimicrobial, antispasmodic, antihypertensive, antiallergic, anti-inflammatory, and anticoagulant properties. Objective: Our study aimed to investigate the anti-anemic activity of the earthworm Allolobophora caliginosa extract (AcE) on phenylhydrazine-induced hemolytic anemia in rats. Methods: Eighteen adult male albino rats were randomly divided into 3 groups (n = 6 per group) as follows: Control, PHZ, and AcE-treated groups. Induction of anemia was done by intraperitoneal administration of phenylhydrazine (40 mg/kg) for 2 consecutive days. Treatment of AcE by intraperitoneal injection (45 mg/kg) for six successive days. Results: Treatment of anemic rats with AcE extract caused significant increases in red blood cells (RBCs), hemoglobin (HB), hematocrit (HCT), total protein (TP), albumin, glutathione reduced (GSH), catalase (CAT), and glutathione-S-transferase (GST), as compared to the PHZ group. While, mean corpuscular haemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), malondialdehyde (MDA), and nitric oxide (NO) levels significantly decreased after intraperitoneal administration of AcE, when compared to the PHZ group. Liver sections from the AcEtreated group restored the normal micromorphological features of the liver. The expression of caspase- 3 was weak and/or there was no expression in the liver sections from AcE groups. AcE caused a significant reduction in the incidence of DNA damage, which appeared as less DNA in the tails. Conclusion: The present research sheds light on the antianemic efficacy of AcE in rats. This study reveals that AcE has antianemic activity, which can be linked to its iron and branched-chain amino acid content, as well as antioxidant and anti-apoptotic properties. other: None

Anti-Anemic Effect of Antioxidant-Rich Apple Vinegar against Phenylhydrazine-Induced Hemolytic Anemia in Rats.

This study aims to examine the ability of apple vinegar on phenylhydrazine (PHZ)-induced hemolytic anemia in Wistar rats. In vitro, phenolic and flavonoid content and antioxidant activity were determined. In vivo, phenylhydrazine (10 mg/kg) was injected intravenously into rats for 4 days and then treated with apple vinegar daily by gavage (1 mL/kg) for five weeks. high level of polyphenols and flavonoids (90 ± 1.66 mg GAE/100 mL and 7.29 ± 0.23 mg QE/100 mL, respectively) were found in the apple vinegar which gives it a good ability to scavenge free radicals (TAC = 4.22 ± 0.18 mg AAE/100 mL and DPPH, IC50 = 0.49 ± 0.004 µL/ml). The phytochemical composition of apple vinegar revealed the presence of numerous bioactive compounds including arbutin, apigenin, sinapic, ferulic and trans-ferulic acids. The major antioxidant components in apple vinegar were ferulic and trans-ferulic acids (40% and 43%, respectively). PHZ treatment induced changes in platelets, blood cell count, mean corpuscular volume, hemoglobin concentration and mean capsulated hemoglobin. However, the co-administration of apple vinegar revealed its capacity to ameliorate the changes induced by phenylhydrazine. Therefore, apple vinegar use could have a positive impact on the prevention of hemolytic anemia induced by phenylhydrazine due to the antioxidant properties of its major components.

Ficus glumosa Del. reduces phenylhydrazine-induced hemolytic anaemia and hepatic damage in Wistar rats.

Anemia is a direct or indirect consequence of oxidative stress via free radicals on erythrocytes and subsequently on other tissues like liver. Ficus glumosa constitute a rich pharmacologically compound that can prevent or repair oxidative damage. Therefore, this study seeks to evaluate the effect of F.glumosa on phenylhydrazine-induced hemolytic anemia and hepatic damage in rats. Twenty-four (24) albino Wistar rats were assigned to four (4) experimental groups (n=6) as follows: Group I (non-anemic control) and Group 2 (anemic control) received normal saline, while Group III and IV (test groups) 200 and 400mg/kg of aqueous leaf extract of F.glumosa (ALEFG), respectively. All the groups were treated orally (via a cannula) for seven consecutive days. Intraperitoneal (IP) injection of phenylhydrazine (PHZ) at 40mg/kg for two consecutive days induced hemolytic anemia in group II to IV before treatment. Rats of all groups were anaesthetized and sacrificed 24h after the last treatment. Blood and liver samples were collected for some hematological indices, liver function test, antioxidant parameter and histological analysis. The LD50 of ALEFG was assessed orally in rats and found to be above 5,000mg/kg body weight. Significant (p<0.05) decreases in the level of red blood cell (RBC), hemoglobin (HGB) concentrations and packed cell volume (PCV) by 50% after 2days of PHZ induction, were attenuated by more than 50% after 7days administration of ALEFG at 200 and 400mg/kg. The percentage change in body weight increased significantly (p<0.05) after 7days post PHZ-induced anemia, but those that received oral administration of ALEFG (at 200 and 400mg/kg) for 7days increased significantly (p<0.05) by more than 2%, dose-dependently compared to anemic untreated group. Increased level of serum ALT, AST, ALP and GGT in PHZ-induced anemic animals, were significantly (p<0.05) attenuated in the groups that received oral administration of ALEFG (at 200 and 400mg/kg) for 7days. Decreased level of catalase (CAT) and superoxide dismutase (SOD) activities with concomitant increase in malondialdehyde (MDA) content from PHZ-induced untreated group, were significantly (p<0.05) mitigated in the rats that received oral administration of ALEFG (at 200 and 400mg/kg) for 7days. Histopathological analysis showed that ALEFG could remarkably though not completely mitigated PHZ-induced hepatic damage. Our data suggests that the leaves of F.glumosa contain important antioxidant(s) that could effectively reduce hemolytic anemia and hepatic damage, especially during phenylhydrazine-induced toxicity.

Protective effect of quercetin compared to silymarin against phenylhydrazine induced anemia

This study has developed to evaluate the protecting effect of Quercetin in phenylhydrazine (PHZ) induced hemolytic anemia comparing to traditional used antioxidant (Silymarin). For that 45 male white Albino rats were randomly allocated into 5 groups, control group, phenylhydrazine group (PHZ) which injected by PHZ (20 mg/kg b.w, I/P at day 7th, 8th and 9th), Quercetin+PHZ group (Quercetin, 50 mg/kg b.w per os every day for 16 days), silymarin+PHZ group (Silymarin, 100 mg/kg b.w per os every day for 16 days) and Quercetin group. Serum samples of these groups were collected after 3, 5 and 10 days after injection of PHZ and used for biochemical and oxidative stress analysis. Also, specimens from spleen were collected for histopathologial examination. The results of this study showed that, Quercetin ameliorated phenylhydrazine effect on total protein, total bilirubin, direct and indirect bilirubin, LDH, iron and ferritin levels. Also, Quercetin improved levels of oxidative stress parameters GSH, SOD and MDA, and had protective effect on tissue of spleen. Therefore, Quercetin had protective effect in experimental phenylhydrazine-induced hemolytic anemia. Also it was more potent antioxidant than Silymarin.

2014 – FLUORESCENT CELL CYCLE TIMER ENABLED ANALYSIS OF NORMAL AND INEFFECTIVE ERYTHROPOIESIS

Aberrant cell cycle speed during hematopoiesis defines pathologic conditions, such as the inability to compensate for anemia in diseases of ineffective erythropoiesis like hemolysis or thalassemia. We utilized a unique live-cell reporter of cell cycle speed using a Histone H2B-FT fusion protein containing the color-changing Fluorescent Timer (FT) protein, distinguishing faster cycling cells from slower-cycling ones based on the intracellular ratio between blue and red fluorescence. In transgenic mice expressing this reporter, we characterized the stress erythropoietic response of spleen and bone marrow following phenylhydrazine-induced hemolytic anemia. Using flow cytometric sorting of erythroblast stages, we found that all stages divide more rapidly after the hemolytic insult, not only early progenitors as previously thought. We also observe that stress erythropoiesis in the spleen is stimulated almost immediately after hemolysis. And the last nucleated cell stage, orthochromatophilic erythroblasts, stop dividing much earlier than normal, allowing them to terminally differentiate into reticulocytes much faster to alleviate anemia. Mass spectrometry of sorted populations shows upregulation of cell cycle and metabolic pathway related genes. Using this system in in vitro human CD34+ primary cell culture, we assessed the erythropoietic response to known treatments of β-thalassemia and Diamond Blackfan Anemia, like Epo and dexamethasone. We found an increase in cell cycling rates with an increase in dexamethasone concentration but not that of Epo. These findings shed new light on the normal response to external stress, underscoring the possibility of precise quantification of cell cycle speed in animal models of anemia. This system can help in understanding stress hematopoiesis, allowing identification of cellular factors that facilitate response to external stress.

Physiological and microvascular responses to hemoglobin concentration-targeted hemolytic anemia in rats.

Hemolytic anemia (HA) is reduced blood oxygen-carrying capacity resulting from the depletion of red blood cells. Treatment for severe cases involves transfusion to improve oxygen delivery (Do2), which carries risk. In humans, a total hemoglobin (tHb) concentration of 8 g/dL is severe, and <7 g/dL indicates transfusion. Some evidence suggests that compensatory mechanisms maintaining Do2 are not compromised until <5 g/dL rendering transfusion at 7 g/dL premature. A Sprague-Dawley rat model of phenylhydrazine-induced HA was assessed over decreasing tHb for a Do2 decompensation point. Three groups (100, 50, or 25% tHb, equating to 16.4, 7.4, or 3.2 g/dL) were generated. Cardiopulmonary, blood chemistry, and oxygenation parameters were measured under anesthesia. Vasoconstrictive responsiveness to phenylephrine was assessed in the exteriorized spinotrapezius. For 50% tHb, cardiopulmonary parameters, Do2, and lactate levels were similar to those for 100% tHb. Enhanced vasoconstriction occurred with 50% tHb (P < 0.0001), not 25% tHb. The 25% group showed decreases in cardiopulmonary parameters, Do2, and lactate levels compared with the 100% and 50% groups (P < 0.05). Do2 showed a positive correlation with lactate levels at 25% tHb, but decompensation, defined by peripheral hypoxia, was not reached. This is the first study relating Do2 to tHb in rats. A 50% reduction in tHb was supported by vascular compensation, whereas 25% tHb levied the cardiopulmonary system. A decompensation point was not identified. A rising need for treatment as tHb levels decline below 8 g/dL is evident, but, as compensatory mechanisms remain intact as tHb approaches 3.2 g/dL in rats, a transfusion limit of 5 g/dL in healthy patients is supported.NEW & NOTEWORTHY Early, chronic compensation to severe hemolytic anemia is vascular, switching to cardiopulmonary support as hemoglobin levels decline. Oxygen delivery does not correlate with serum lactate level until total hemoglobin is reduced by 75%.

Protective Effect of Cynara scolymus L. on Blood Biochemical Parameters and Liver Histopathological Changes in Phenylhydrazine-Induced Hemolytic Anemia in Rats

Background: Artichoke (Cynara scolymus) possesses bioactive components with antioxidant effects. This plant has been widely used in traditional medicine.&#x0D; Objectives: The current study aimed to examine the protective activity of Hydroethanolic Extract of Cynara scolymus (HECS) against experimentally-induced hemolytic anemia in rats.&#x0D; Methods: Hemolytic anemia was induced by intraperitoneal injection of Phenylhydrazine (PHZ) 40 mg/kg for 2 days. PHZ induces oxidative stress and reactive oxygen species formation, which causes hemolytic anemia. Thirty male Wistar rats were divided into 5 groups (n=6 for each group). Group 1 (normal control) was injected with normal saline. Group 2 (anemic control) received only PHZ. Groups 3 to 5 were injected with 100, 200, 400 mg/kg of the HECS by gavage, respectively, daily from day 2 to day 15 after PHZ administration. At the end of the treatment period, their blood and liver samples were collected for biochemical and histopathological analysis.&#x0D; Results: The results indicated that serum Alkaline Phosphatase (ALP), alanine aminotransferase (ALT), Aspartate Aminotransferase (AST), and Lactate Dehydrogenase (LDH) levels in the PHZ (anemic) group were significantly higher than those in the control group (P&lt;0.05). A significant decrease in serum liver enzymes was determined in rats treated with HECS at different doses compared with the untreated anemic rats (P&lt;0.05). Also, HECS significantly attenuated body weight loss in the PHZ group (P&lt;0.05). Besides, based on the histopathological evaluation, HECS improved disarrangements of the liver parenchyma due to PHZ-induced hepatotoxicity.&#x0D; Conclusion: HECS has hepatoprotective effects against PHZ-induced toxicity presumably by its antioxidative activity.

The protective effect of vitamin C on phenylhydrazine-induced hemolytic anemia on sperm quality and in-vitro embryo development in mice.

BackgroundPhenylhydrazine (PHZ) induced anemia and was shown to have harmful effects on the male reproductive system. ObjectiveTo investigate the protective effect of vitamin C (Vit C) on sperm parameters quality, in vitro fertilization potential and embryonic development in a mouse model of hemolytic anemia induced by PHZ.Materials and MethodsThirty-two NMRI adult male mice (n = 8/each) were randomly classified into four groups. Group I (control) received normal saline, Group II (PHZ) received 8 mg/100 gr body weight PHZ as initial dose, continued by 6 mg/100 gr intraperitoneally every 48 hr, Group III (Vit C) received Vit C (10 mg/kg, daily, intraperitoneally), and group IV (PHZ + Vit C) received PHZ and Vit C. After 35 days, sperm quality parameters, the percentage of sperm with DNA damage and in vitro fertilization outcomes up to blastocyst stage were evaluated.ResultsA significant (p 0.001) reduction in all of the sperm parameters (count, motility, viability and normal morphology) were observed in group II (PHZ) compared with group I (control). In group IV (PHZ Vit C), these parameters and sperm DNA damage (p 0.001) improved significantly when compared with PHZ-treated mice. Furthermore, PHZ caused a significant (p 0.001) decrease in the fertilization rate and the percentage of pre-implantation embryos' (two cell embryo and blastocyst) formation in comparison to group I (control), and Vit C supplementation in mice of group IV improved significantly the fertilization rate (p = 0.002), but it could not improve the percentage of two cell embryos and blastocyst production.ConclusionThe data from this study indicated that Vit C decreased the adverse effects of PHZ on the quality of sperm parameters and in vitro fertilization rate, but it is insufficient to restore the in-vitro embryonic development and fertility potential.

Hematinic and antioxidant potential of aqueous extract of Sesamum indicum seeds against phenylhydrazine-induced hemolytic anemia in albino rats

Background: More than 2 billion people around the world are suffering from anemia. Majority of populations in developing countries are depending on dietary supplementation and herbal medicine for the management of the anemic condition. The seed of Sesamum indicum L is used for its nutritional, medicinal, and industrial purposes in many parts of the world, which is also used as hematinics agent in the traditional system. Aim and Objective: This study investigated the hematinic and antioxidant potential of aqueous extract of S. indicum L. supplementation therapy in a rodent model of hemolytic anemia induced by phenylhydrazine (PHZ). Materials and Methods: About 36 adult albino rats were selected, of which six were considered to be normal controls. Anemia was induced by intraperitoneal administration of PHZ in remaining 30 rats. On the basis of hemoglobin (Hb) estimation after 2 days of PHZ administration, 24 rats were selected as anemic and randomly classified them into four groups. Groups II and III served as negative and positive control, while Groups IV and V were administered with the aqueous extract of Sesamum for 14 days. Rats were sacrificed at the end of 14th day, and their blood samples were collected. Result: After the injection of PHZ rats developed hemolytic anemia reflected by a significant decrease in red blood cell (RBC) count, Hb concentration, and hematocrit percentage. Interestingly, therapy with S. indicum had significantly reversed these deteriorating effects on PHZ on RBCs, HGB, and HCT. In addition, sesame therapy significantly reversed the decreases in serum levels of total glutathione and activities of superoxide dismutase. Conclusion: The result of this study thus indicated that S. indicum is effective remedy to manage anemia in humans.

Bone Marrow Endothelial Cells Induce Immature and Mature B Cell Egress in Response to Erythropoietin.

Bone marrow stromal cells, including endothelial cells and mesenchymal stromal cells, support the maintenance, differentiation, and retention of hematopoietic stem and precursor cells under steady state conditions. At the onset of an emergency, such as severe blood loss or infection, the status of hematopoiesis in the bone marrow changes rapidly to ensure efficient production of cells of specific lineages; however, the function of stromal cells in emergency hematopoiesis has not been fully elucidated. Here, we unexpectedly found that B precursor, mature B, and T cells were released from the bone marrow into the blood circulation in the early phase of hemorrhagic anemia and phenylhydrazine-induced hemolytic anemia. Administration of erythropoietin, which normally increases in response to anemia, stimulated the egress of IgDlow immature B cells and recirculating mature B cells, which usually reside in the perivascular and intravascular space, from the bone marrow within 24 h. We also observed that endothelial cells in the bone marrow expressed erythropoietin receptor, and the expression levels were higher than those in other tissues. Erythropoietin stimulation of bone marrow endothelial cells induced the phosphorylation of STAT5 in vitro. Moreover, in vivo treatment with erythropoietin decreased surface VCAM1 expression and Cxcl12 transcription in bone marrow endothelial cells, both of which are essential for immature and mature B cell retention in the bone marrow. These results suggest that bone marrow endothelial cells can sense and rapidly respond to erythropoietin increase during anemia, thereby regulating B cell emigration from the bone marrow during emergency hematopoiesis.Key words: erythropoietin, anemia, endothelial cells, B cell, bone marrow microenvironment.

Effect of phenylhydrazine-induced hemolytic anemia on morphological changes of renal cortex in adult mice

Effect of phenylhydrazine-induced hemolytic anemia on morphological changes of renal cortex in adult mice

EPO-dependent induction of erythroferrone drives hepcidin suppression and systematic iron absorption under phenylhydrazine-induced hemolytic anemia

Hemolytic anemia is a common form of anemia due to hemolysis, resulting in disordered iron homeostasis. In this study, a dose of 40mg/kg phenylhydrazine (PHZ) was injected into mice to successfully establish a pronounced anemia animal model, which resulted in stress erythropoiesis and iron absorption. We found that serum erythropoietin (EPO) concentration was dramatically elevated by nearly 5000-fold for the first 2days, and then drop to the basal level on day 6 after PHZ injection. Mirrored with serum EPO concentration, the mRNA expression of erythroferrone (ERFE) was rapidly increased in the bone marrow and spleen 3days after injection of PHZ, and then gradually decreased but was still higher than baseline on day 6. In addition, we also found that the hepcidin mRNA levels were gradually reduced almost up to 8-fold on day 5, and then was ameliorated compared to the untreated control. Mechanistic investigation manifested that the increase of serum EPO essentially determined the induction of ERFE expression particular at the first 3days after PHZ treatment. Lentiviral mediated ERFE knockdown significantly restrained hepcidin suppression under PHZ treatment. Thus, our data unearthed EPO-dependent ERFE expression acts as an erythropoiesis-driven regulator of iron metabolism under PHZ-induced hemolytic anemia.