Aplastic Anemia Mice Research Articles

Chinese herbal formula, modified Guilu Erxian glue, alleviates apoptosis of hematopoietic stem cells by regulating SLAM-SAP signal pathway in aplastic anemia mice model

Ethnopharmacological relevanceGuilu Erxian Glue (GEG) and Danggui Buxue Tang (DBT) are traditional Chinese herbal formulas. According to the theory of traditional Chinese medicine, the combination of those two formulas (Modified Guilu Erxian Glue, MGEG) has the effects of tonifying the kidney and producing blood, was usually used to treat bone marrow failure diseases, including aplastic anemia (AA). Aim of the studyT lymphocytes play a crucial role in the disease pathogenesis and progression of AA. Our preliminary results confirmed that GEG can improve the damage of hematopoietic stem cells in mice, while DBT can reduce the proliferation and differentiation of T lymphocytes and inhibit the production of IFN-γ. We hypothesized that the combination of those two herbal formulas could inhibit immune attack and restore hematopoietic function through multiple mechanisms. In this study, we aim to study the curative effect of MGEG on regulating the expression of Signal lymphocyte activating molecule (SLAM), an activation-related molecule in T lymphocytes, thereby suppressing the immune function of T cells and decelerating the damage to hematopoietic stem cells. Materials and methodsHigh-performance liquid chromatography-electrospray ionization/mass spectrometry system was used to identify the components of the MGEG formulation. Induction of aplastic anemia mouse model by injecting allogeneic lymphocyte suspension into BABL/c mice after ionizing radiation. Cyclosporine A (CsA) was used as a positive control drug. Flow cytometry was used to detect the number and apoptosis rate of hematopoietic stem cells in the bone marrow. Enzyme-linked immunosorbent assay was performed to measure the levels of IFN-γ and TNF-α. Immunofluorescence staining was used to assess the expression of T-bet and SLAM-SAP. Western Blot was conducted to examine the expression of activation-related molecules in T lymphocytes and proteins related to the Fas signal pathway. Hematoxylin-eosin staining was performed to observe pathological changes in the bone marrow tissue. Wright-Giemsa staining was utilized to evaluate alterations in the cellular composition and basic structure of the bone marrow cells (BMCs). Transmission electron microscopy was employed to observe changes in the structure and morphology of hematopoietic stem cells. The hematology analyzer was used to detect peripheral blood parameters. ResultsTwenty-three different components were identified in MGEG. After MGEG treatment, the expression levels of Fyn and SLAM-SAP binding were increased in AA mice, while the expression levels of T-bet were decreased and the secretion of IFN-γ was reduced significantly. Additionally, MGEG also could downregulate the protein levels of Fas, caspase-3, and cleaved caspase-3 in AA mice. ConclusionMGEG could attenuate the production of IFN-γ by promoting the SLAM-SAP signal pathway to regulate the generation and distribution of T-bet in T cells. Additionally, it suppresses apoptosis of HSCs through intervention in the Fas-dependent pathway, thereby mitigating immune-mediated damage to HSCs.

Effect of ginsenoside Rg1 on hematopoietic stem cells in treating aplastic anemia in mice via MAPK pathway.

Aplastic anemia (AA) presents a significant clinical challenge as a life-threatening condition due to failure to produce essential blood cells, with the current therapeutic options being notably limited. To assess the therapeutic potential of ginsenoside Rg1 on AA, specifically its protective effects, while elucidating the mechanism at play. We employed a model of myelosuppression induced by cyclophosphamide (CTX) in C57 mice, followed by administration of ginsenoside Rg1 over 13 d. The investigation included examining the bone marrow, thymus and spleen for pathological changes via hematoxylin-eosin staining. Moreover, orbital blood of mice was collected for blood routine examinations. Flow cytometry was employed to identify the impact of ginsenoside Rg1 on cell apoptosis and cycle in the bone marrow of AA mice. Additionally, the study further evaluated cytokine levels with enzyme-linked immunosorbent assay and analyzed the expression of key proteins in the MAPK signaling pathway via western blot. Administration of CTX led to significant damage to the bone marrow's structural integrity and a reduction in hematopoietic cells, establishing a model of AA. Ginsenoside Rg1 successfully reversed hematopoietic dysfunction in AA mice. In comparison to the AA group, ginsenoside Rg1 provided relief by reducing the induction of cell apoptosis and inflammation factors caused by CTX. Furthermore, it helped alleviate the blockade in the cell cycle. Treatment with ginsenoside Rg1 significantly alleviated myelosuppression in mice by inhibiting the MAPK signaling pathway. This study suggested that ginsenoside Rg1 addresses AA by alleviating myelosuppression, primarily through modulating the MAPK signaling pathway, which paves the way for a novel therapeutic strategy in treating AA, highlighting the potential of ginsenoside Rg1 as a beneficial intervention.

Resolvin E1 improves efferocytosis and rescues severe aplastic anemia in mice

Severe aplastic anemia (SAA) is a rare, fatal disease characterized by severe cytopenias and loss of hematopoietic stem cells (HSCs). Immune-mediated destruction and inflammation are known drivers of SAA, however, the underlying mechanisms driving persistent inflammation are unknown. Current treatments for SAA rely on immunosuppressive therapies or HSC transplantation, however, these treatments are not always effective. Using an established mouse model of SAA, we observed a significant increase in apoptotic cells within the bone marrow (BM) and impaired efferocytosis in SAA mice, relative to radiation controls. Single-cell transcriptomic analysis revealed heterogeneity among BM monocytes and unique populations emerged during SAA characterized by increased inflammatory signatures and significantly increased expression of Sirpa and Cd47. CD47, a “don’t eat me” signal, was increased on both live and apoptotic BM cells, concurrent with markedly increased expression of signal regulatory protein alpha (SIRPα) on monocytes. Functionally, SIRPα blockade improved cell clearance and reduced accumulation of CD47-positive apoptotic cells. Lipidomic analysis revealed a reduction in the precursors of specialized pro-resolving lipid mediators (SPMs) and increased prostaglandins in the BM during SAA, indicative of impaired inflammation resolution. Specifically, 18-HEPE, a precursor of E-series resolvins, was significantly reduced in SAA-induced mice relative to radiation controls. Treatment of SAA mice with Resolvin E1 (RvE1) improved efferocytic function, BM cellularity, platelet output, and survival. Our data suggest that impaired efferocytosis and inflammation resolution contributes to SAA progression and demonstrate that SPMs, such as RvE1, offer new and/or complementary treatments for SAA that do not rely on immune suppression.

Resolvin E1 improves efferocytosis and rescues severe aplastic anemia in mice

Resolvin E1 improves efferocytosis and rescues severe aplastic anemia in mice

Dioscin alleviates aplastic anemia through regulatory T cells promotion

ABSTRACT Objectives: Aplastic anemia (AA) is one of the immune-mediated bone marrow failure disorders caused by multiple factors, including the inability of CD4 + CD25 + regulatory T cells (Tregs) to negatively regulate cytotoxic T lymphocytes (CTLs). Dioscin is a natural steroid saponin that has a similar structure to steroid hormones. The purpose of this study is to look into the effect of Dioscin on the functions of CD4 + CD25+ Tregs in the AA mouse model and explore its underlying mechanism. Methods: To begin with, bone marrow failure was induced through total body irradiation and allogeneic lymphocyte infusion using male Balb/c mice. After 14 consecutive days of Dioscin orally administrated, the AA mouse model was tested for complete blood counts, HE Staining of the femur, Foxp3, IL-10 and TGF-β. Then CD4 + CD25+ Tregs were isolated from splenic lymphocytes of the AA mouse model, Tregs and the biomarkers and cytokines of Tregs were measured after 24 h of Dioscin intervention treatment in vitro. Results: Dioscin promotes the expression of Foxp3, IL-10, IL-35 and TGF-β, indicating its Tregs-promoting properties. Mechanistically, the administration of Dioscin resulted in the alteration of CD152, CD357, Perforin and CD73 on the surface of Tregs, and restored the expression of Foxp3. Conclusion: Dioscin markedly attenuated bone marrow failure, and promoted Tregs differentiation, suggesting the maintenance of theimmune balance effect of Dioscin. Dioscin attenuates pancytopenia and bone marrow failure via its Tregs promotion properties.

PRMT5 regulates epigenetic changes in suppressive Th1-like iTregs in response to IL-12 treatment.

Induced regulatory T cells (iTregs) are a heterogeneous population of immunosuppressive T cells with therapeutic potential. Treg cells show a range of plasticity and can acquire T effector-like capacities, as is the case for T helper 1 (Th1)-like iTregs. Thus, it is important to distinguish between functional plasticity and lineage instability. Aplastic anemia (AA) is an autoimmune disorder characterized by immune-mediated destruction of hematopoietic stem and progenitor cells in the bone marrow (BM). Th1-like 1 iTregs can be potent suppressors of aberrant Th1-mediated immune responses such as those that drive AA disease progression. Here we investigated the function of the epigenetic enzyme, protein arginine methyltransferase 5 (PRMT5), its regulation of the iTreg-destabilizing deacetylase, sirtuin 1 (Sirt1) in suppressive Th1-like iTregs, and the potential for administering Th1-like iTregs as a cell-based therapy for AA. We generated Th1-like iTregs by culturing iTregs with IL-12, then assessed their suppressive capacity, expression of iTreg suppression markers, and enzymatic activity of PRMT5 using histone symmetric arginine di-methylation (H3R2me2s) as a read out. We used ChIP sequencing on Th1 cells, iTregs, and Th1-like iTregs to identify H3R2me2s-bound genes unique to Th1-like iTregs, then validated targets using CHiP-qPCR. We knocked down PRMT5 to validate its contribution to Th1-like iTreg lineage commitment. Finally we tested the therapeutic potential of Th1-like iTregs using a Th1-mediated mouse model of AA. Exposing iTregs to the Th1 cytokine, interleukin-12 (IL-12), during early events of differentiation conveyed increased suppressive function. We observed increased PRMT5 enzymatic activity, as measured by H3R2me2s, in Th1-like iTregs, which was downregulated in iTregs. Using ChIP-sequencing we discovered that H3R2me2s is abundantly bound to the Sirt1 promoter region in Th1-like iTregs to negatively regulate its expression. Furthermore, administering Th1-like iTregs to AA mice provided a survival benefit. Knocking down PRMT5 in Th1-like iTregs concomitantly reduced their suppressive capacity, supporting the notion that PRMT5 is important for the superior suppressive capacity and stability of Th1-like iTregs. Conclusively, therapeutic administration of Th1-like iTregs in a mouse model of AA significantly extended their survival and they may have therapeutic potential.

Umbilical Cord Blood-Derived Cells Can Reconstruct Hematopoiesis in an Aplastic Anemia Animal Model.

To explore the efficacy and the mechanism of the umbilical cord-derived cells combined with cyclosporine A (CsA) in treating aplastic anemia (AA) in mice. Immune-mediated AA model mice were treated with CsA + UC mesenchymal stem cells (UC-MSC), CsA + umbilical cord blood regulatory T cells (UCB-Treg), UC-MSC, UCB-Treg, CsA alone, or blank control, respectively (n = 9 mice/group). CsA and the cell infusion was administered on d0. Routine peripheral blood testing was performed once weekly; bone marrow colony culture, bone marrow cell flow cytometry, peripheral blood T cell subsets, and serum inflammatory cytokines tests were performed on d14. Transcriptome sequencing was performed for cells from CsA + UC-MSC, CsA + UCB-Treg, and CsA groups to detect the possible related genes. Gene function cluster and signal pathway enrichment analysis were also performed. Blank control mice died due to pancytopenia within 21 days, whereas mice in other groups survived for >28 days. On d14, the CsA + UC-MSC and CsA + UCB-Treg groups had higher white blood cell (WBC) counts than the other groups (p < 0.05), along with higher burst-forming unit (BFU) and colony-forming unit-granulocyte, macrophage (CFU-GM) counts (p < 0.01). The CsA + UC-MSC group had the highest BFU count (p < 0.01). The CsA + UC-MSC and CsA + UCB-Treg groups exhibited the highest bone marrow CD34+ cell proportion (9.68% ± 1.35% and 8.17% ± 0.53%, respectively; p < 0.01). Tumor necrosis factor (TNF)-α and interleukin (IL)-2 levels in the CsA + UC-MSC group (p < 0.05) and TNF-α, interleukin-2, and interferon (INF)-γ levels in the CsA + UC-Treg group (p < 0.01) were lower than those in the CsA group. Compared with CsA treatment, CsA + UC-MSC significantly downregulated the histone methylation pathway (p < 0.05), whereas CsA + UCB-Treg significantly upregulated energy metabolism processes (p < 0.05). Treatment with CsA + UC-MSC upregulated superoxide dismutase activity compared with CsA + UCB-Treg treatment. Adding UC-MSC or UCB-Treg to CsA markedly enhanced the reconstruction of hematopoiesis in AA mice, with UC-MSC eliciting greater efficiency than UCB-Treg. Accordingly, the addition of these cells could further improve immune abnormalities.

Impaired Immunosuppressive Effect of Bone Marrow Mesenchymal Stem Cell-Derived Exosomes on T Cells in Aplastic Anemia

Background: Previous studies have verified the dysfunction of mesenchymal stem cells (MSCs) for immunoregulation in acquired aplastic anemia (AA) patients. Exosomes derived from MSCs can partially substitute MSC acting as immune regulator. Dysfunction of exosomes (Exos) derived from AA-MSC ( AA-Exos) may play a key role in immunologic dissonance. Objectives: This study aims to investigate the interaction between exosomes derived from mesenchymal stem cells and T cells of patients with aplastic anemia by examining the immunosuppressive effects of exosomes on T cells both in vitro and in vivo. Besides, this study intends to explore the differently expressed miRNA of exosomes and mRNAs of T cells between patients with AA and healthy donors (HD) to better understand the mechanisms of AA on molecular lever. Method: In this study, CD3 + T cells were collected and cocultured with AA-Exos and exosomes derived from HD-MSC (HD-Exos). The proliferation, differentiation and activation of CD3 + T cells were detected to compare the immunosuppressive effects between AA-Exos and HD-Exos. An immune-mediated murine model of AA was structured to compare the therapeutic effect of AA-Exos and HD-Exos. Furthermore, total RNA including miRNA from exosomes we purified and total RNA of CD3 + T cells were extracted for RNA-seq in order to construct the miRNA-mRNA network for interactions and functional analysis. Results: Compared to HD-Exos, AA-Exos exhibit a weakened ability to inhibit the proliferation of CD3+/CD4+/CD8+ T cells (40.73% vs 32.43%，p = 0.0007；46.20% vs 38.08%，p = 0.0009；36.28% vs 29.73%，p = 0.0077). Furthermore, AA-Exos show reduced capacity to suppress T cell activation compared to HD-Exos. In AA-Exos group, the ability of inhibiting CD4+ T cell differentiation towards Th1 is diminished (12.80 vs 10.17%， p = 0.0047), while the induction of Th17 cell generation is significantly enhanced (3.613% vs 1.153%， p=0.0015). Additionally, AA-Exos demonstrate weaker capabilities to inhibit CD8+ T cell differentiation towards Tc1 compared to HD-Exos (41.93% vs 35.40%，p=0.0007). The capacity of AA-Exos to induce Treg is also weakene (7.78% vs 10.36%，p=0.041).HD-Exos other than AA-Exos can rescued the AA mice. Administration of HD-Exos effectively improves the bone marrow failure, the BMNC counting revealed that HD-Exos had more potency in ameliorating bone marrow hyperplasia than AA-Exos (8.27×106/L vs 0.85×106/L，p=0.0021). The RNA cargo of exosomes was believed to be a key mediator exerting their function, especially the carry of miRNA.Importantly, we identified some differentially expressed miRNA involved in immune response under the standard of adjusted p &amp;lt; 0.05 &amp;| log2 (foldchange) | &amp;gt; 1.5, such as miR-199, miR-128, miR-486 and miR-375. The Gene Ontology analysis of differentially expressed genes (DEGs) revealed involvement of various cellular processes, such as lymphocyte chemotaxis, lymphocyte migration and response to interferon-gamma. The Kyoto Encyclopedia of Genes and Genomes analysis illustrated upregulation of critical pathways associated with T cell function after co-culturing with AA-Exos compared with HD-Exos, such as graft-versus-host disease, Th17 cell differentiation, and JAK-STAT signaling pathway. A miRNA-mRNA network was established to visualize the interaction between them, in which a total of 61 node and 142 edge network pathways were constructed, including 14 miRNAs and 47 mRNAs. To validate the findings, qPCR was performed on four DEGs, and significant differences were observed in three of them, namely MRPL40, TNF and CCL4L2 (p=0.0116, p=0.0016, p=0.0122, respectively). Conclusion: In summary, AA-Exos had impaired immunosuppressive effect on T cells, less ability to rescue AA mice and differently expressed miRNA profile, which might partly account for the pathogenesis of AA as well as provide a new target of AA treatment.

Irisin protected hemopoietic stem cells and improved outcome of severe bone marrow failure

Acquired aplastic anemia (AA) is a bone marrow failure (BMF) disease, characterized by fatty bone marrow (BM) and BM hypocellularity resulted from auto-immune dysregulated T cells-mediated destruction of BM haemopoietic stem cells (HPSC). The objective of this study was to investigate potential therapeutic effect of irisin, a molecule involved in adipose tissue transition, on AA mouse model. Our results showed that the concentration of irisin in serum was lower in AA patients than in healthy controls, suggesting a role of irisin in the pathogenesis of AA. In the AA mice, irisin administration prolonged the survival rate, prevented or attenuated peripheral pancytopenia, and preserved HPSC in the BM. Moreover, irisin also markedly reduced BM adipogenesis. In vitro results showed that irisin increased both cell proliferation and colony numbers of HPSC. Furthermore, our results demonstrated that irisin upregulated the expression of mitochondrial ATPase Inhibitory Factor 1 (IF1) in HPSC, inhibited the activation of mitochondrial fission protein (DRP1) and enhanced aerobic glycolysis. Taken together, our findings indicate novel roles of irisin in the pathogenesis of AA, and in the protection of HPSC through stimulation of proliferation and regulation of mitochondria function, which provides a proof-of-concept for the application of irisin in AA therapy.

Network pharmacology and experimental verification of the potential mechanism of Er-Xian decoction in aplastic anemia

To investigate the potential mechanism of Er-Xian decoction (EXD) in treating aplastic anemia (AA), the active components of EXD were screened by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and the targets of the components were predicted by the Swiss Target Prediction database. AA targets were collected from the GeneCards, OMIM, DisGeNET, PharmGKB, DrugBank, and TTD databases, the intersection of AA targets and EXD targets was calculated, and an herb-component-target network was constructed by Cytoscape 3.7.2 software. The STRING database was used for protein‒protein interaction (PPI) analysis, and Cytoscape 3.7.2 software was used to construct a PPI network and perform topology analysis. The core targets were imported into the DAVID database for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The molecular docking software AutoDock was used to measure the affinity between active components and key targets. Finally, we established a mouse model of AA and verified the key targets and signaling pathways of EXD by RT‒PCR, ELISA and Western blot analysis. A total of 53 active components were screened from EXD, 2516 AA-related targets were collected, and 195 common targets were obtained. An herb-component-target network and a PPI network were successfully constructed, and 36 core targets were selected from the PPI network. The main active components of EXD include luteolin, kaempferol, berberine, etc., and key targets include PIK3CA, AKT1, STAT3, etc. GO functional enrichment analysis showed that cell components, molecular functions and biological processes with significant correlations were macromolecular complexes, protein serine/threonine/tyrosine kinase activity and protein phosphorylation, respectively. KEGG pathway analysis showed that the pathways with significant correlations included the PI3K-Akt signaling pathway and JAK-STAT signaling pathway. Molecular docking results showed that the tested key targets had good affinity for the corresponding active components. In AA mice, we found that EXD significantly increased white blood cell count, red blood cell count, platelet count and hemoglobin levels, increased mRNA levels of PIK3CA, PIK3CD, AKT1, JAK2, STAT3 and MAPK1, and promoted phosphorylation of PI3K, AKT, ERK1/2 and STAT3. In summary, EXD acts on PI3K, AKT, STAT3 and other targets through berberine, luteolin, quercetin and other components to regulate the PI3K-Akt pathway, JAK-STAT pathway and other pathways, thus exerting its therapeutic effect on AA. This study explained the Chinese medicine theory of treating AA with EXD by tonifying kidney-yang and provides a scientific basis for the use of EXD in treating AA.

Impaired immunosuppressive effect of bone marrow mesenchymal stem cell-derived exosomes on T cells in aplastic anemia

BackgroundPrevious studies have verified the dysfunction of mesenchymal stem cells (MSCs) for immunoregulation in acquired aplastic anemia (AA) patients. Exosomes derived from MSCs can partially substitute MSCs acting as immune regulator. Dysfunction of exosomes (Exos) derived from AA-MSC (AA-Exos) may play a key role in immunologic dissonance.MethodIn this study, CD3 + T cells were collected and cocultured with AA-Exos and exosomes derived from HD-MSC (HD-Exos). The proliferation, differentiation and activation of CD3 + T cells were detected to compare the immunosuppressive effects between AA-Exos and HD-Exos. An immune-mediated murine model of AA was structured to compare the therapeutic effect of AA-Exos and HD-Exos. Furthermore, total RNA including miRNA from exosomes we purified and total RNA of CD3 + T cells were extracted for RNA-seq in order to construct the miRNA–mRNA network for interactions and functional analysis.ResultsAA-Exos had impaired inhibition effects on CD3 + T cells in terms of cell proliferation, activation and differentiation compared with exosomes from HD-Exos. HD-Exos showed a more effective rescue of AA mice compared to AA-Exos. Importantly, we found some differentially expressed miRNA involved in immune response, such as miR-199, miR-128 and miR-486. The Gene Ontology analysis of differentially expressed genes (DEGs) revealed involvement of various cellular processes, such as lymphocyte chemotaxis, lymphocyte migration and response to interferon-gamma. The Kyoto Encyclopedia of Genes and Genomes analysis illustrated upregulation of critical pathways associated with T cell function after coculturing with AA-Exos compared with HD-Exos, such as graft-versus-host disease, Th17 cell differentiation and JAK-STAT signaling pathway. A miRNA–mRNA network was established to visualize the interaction between them.ConclusionIn summary, AA-Exos had impaired immunosuppressive effect on T cells, less ability to rescue AA mice and differently expressed miRNA profile, which might partly account for the pathogenesis of AA as well as provide a new target of AA treatment.

Umbilical cord-derived mesenchymal stem cells cultured in the MCL medium for aplastic anemia therapy

BackgroundMesenchymal stem cells (MSCs) are a class of adult stem cells with self-renewal and multidirectional differentiation potential that may be a treatment for aplastic anemia (AA).MethodUmbilical cord-derived MSCs were cultured in three media (Mesencult-XF, MCL, and StemPro MSC SFM CTS). HGF, PGE2, ANG-1, TGF-β1, IFN-γ, and TNF-α were detected using ELISA. The AA mouse model was built via post-irradiation lymphocyte infusion. After different treatments, routine blood, VEGF, and Tregs were detected every week. On day 28, all mice were killed, and their femurs were stained with HE.ResultsUmbilical cord-derived MSCs cultured in the three media all conformed to the general characteristics of MSCs. HGF secreted by MSCs in the Mesencult-XF, and MCL was greater than that in the StemPro MSC SFM CTS; ANG-1 and TGF-β1 in the MCL were more than that in Mesencult-XF and StemPro MSC SFM CTS; PGE2 in the MCL and StemPro MSC SFM CTS was more than that in the Mesencult-XF. MSCs in the MCL and StemPro MSC SFM CTS inhibited IFN-γ and TNF-α more than those in the Mesencult-XF. The peripheral blood cell in the AA groups was at a low level while that in the MSC group recovered rapidly. The Treg ratio and VEGF level in the MSC group were higher than those in the AA group. The bone marrow (BM) recovered significantly after MSC infusion.ConclusionMSCs in the MCL were advantageous in supporting hematopoiesis and modulating immunity and had the potential for effective treatment of AA.

The effectiveness of a novel treatment of TIM-3(-) NK cells infusion in murine models of immune-mediated bone marrow failure.

T-cell immunoglobulin and mucin-containing domain (TIM)-3 exerts its inhibitory effect on NK cells and participates in the immune pathogenesis of SAA. In this study, we aimed to explore a novel treatment method of TIM-3(+) NK or TIM-3(-) NK cell infusion in combination with immunosuppressive therapy for bone marrow failure (BMF)/aplastic anemia (AA) mice. BMF/AA mouse model was constructed. The TIM-3 expression and functional molecules on TIM-3(+) and TIM-3(-) NK cells of the BMF group, total body irradiation (TBI) group, and normal control (NC) group mice were detected by flow cytometry. After treatment, the general condition, whole blood cell and bone marrow cell (BMC) count, and immune condition of mice from each group were compared. TIM-3 expression in the peripheral blood NK cells of BMF mice was significantly lower than that of the TBI and NC group mice. TIM-3(-) NK cells expressed more NKG2D receptors than TIM-3(+) NK cells. The levels of P-Akt and PI3K in TIM-3(-) NK cells were higher than those in TIM-3(+) NK cells. On the 17th day after BMF induction, the weight, peripheral whole blood cell count, and BMC count of BMF mice decreased significantly compared with that of the NC group mice. The therapeutic effect in the TIM-3(-) NK cell treatment group was better than that in the TIM-3(+) NK cell treatment and CsA treatment groups. Concurrently, the ratio of CD4+ T and CD8+ T cells of BMF mice was significantly lower than that of the NC group mice. The therapeutic effect in CsA + TIM-3(-) NK group was more significant than that of the CsA treatment and the CsA + TIM-3(+) NK groups. In this study, we found that the general condition, peripheral whole blood cell and BMC count, and immune status of BMF mice improved significantly after CsA + TIM-3(-) NK cell treatment. These results may provide further insights into the immune pathogenesis of SAA and novel therapeutic ideas for improving SAA treatment.

Repair of dysfunctional bone marrow endothelial cells alleviates aplastic anemia.

Aplastic anemia (AA) is a life-threatening disease characterized by bone marrow (BM) failure and pancytopenia. As an important component of the BM microenvironment, endothelial cells (ECs) play a crucial role in supporting hematopoiesis and regulating immunity. However, whether impaired BM ECs are involved in the occurrence of AA and whether repairing BM ECs could improve hematopoiesis and immune status in AA remain unknown. In this study, a classical AA mouse model and VE-cadherin blocking antibody that could antagonize the function of ECs were used to validate the role of BM ECs in the occurrence of AA. N-acetyl-L-cysteine (NAC, a reactive oxygen species scavenger) or exogenous EC infusion was administered to AA mice. Furthermore, the frequency and functions of BM ECs from AA patients and healthy donors were evaluated. BM ECs from AA patients were treated with NAC in vitro, and then the functions of BM ECs were evaluated. We found that BM ECs were significantly decreased and damaged in AA mice. Hematopoietic failure and immune imbalance became more severe when the function of BM ECs was antagonized, whereas NAC or EC infusion improved hematopoietic and immunological status by repairing BM ECs in AA mice. Consistently, BM ECs in AA patients were decreased and dysfunctional. Furthermore, dysfunctional BM ECs in AA patients led to their impaired ability to support hematopoiesis and dysregulate T cell differentiation toward proinflammatory phenotypes, which could be repaired by NAC in vitro. The reactive oxygen species pathway was activated, and hematopoiesis- and immune-related signaling pathways were enriched in BM ECs of AA patients. In conclusion, our data indicate that dysfunctional BM ECs with impaired hematopoiesis-supporting and immunomodulatory abilities are involved in the occurrence of AA, suggesting that repairing dysfunctional BM ECs may be a potential therapeutic approach for AA patients.

Panaxadiol saponin ameliorates ferroptosis in iron-overload aplastic anemia mice and Meg-01 cells by activating Nrf2/HO-1 and PI3K/AKT/mTOR signaling pathway

Panaxadiol saponin (PND) is a latent targeted drug for the treatment of aplastic anemia (AA). In this study, we examined the effects of PND on ferroptosis in iron-overload AA and Meg-01 cells. We utilized RNA-seq to analyze differentially expressed genes in iron-induced Meg-01 cells treated with PND. The effects of PND or combined with deferasirox (DFS) on iron deposition, labile iron pool (LIP), several ferroptosis events, apoptosis, mitochondrial structure, as well as ferroptosis-, Nrf2/HO-1-, and PI3K/AKT/mTOR pathway-related markers in iron-induced Meg-01 cells were examined by Prussian-blue staining, flow cytometer, ELISA, Hoechst 33342 staining, transmission electron microscope, and Western blot assays, respectively. Additionally, an AA mice model with iron overload was established. Then, the blood routine was assessed, and the number of bone marrow-derived mononuclear cells (BMMNCs) in mice was counted. Also, serum iron, ferroptosis events, apoptosis, histology, T lymphocyte percentage, ferroptosis-, Nrf2/HO-1-, and PI3K/AKT/mTOR signaling-related targets in primary megakaryocytes of AA mice with iron overload were assessed by commercial kits, TUNEL staining, hematoxylin and eosin (H&E) staining, Prussian blue staining, flow cytometer, and qRT-PCR analysis, respectively. PND suppressed iron-triggered iron overload, and apoptosis, and ameliorated mitochondrial morphology in Meg-01 cells. Importantly, PND ameliorated ferroptosis-, Nrf2/HO-1-, and PI3K/AKT/mTOR signaling-related marker expressions in iron-induced Meg-01 cells or primary megakaryocytes of AA mice with iron overload. Moreover, PND ameliorated body weight, peripheral blood cell counts, the number of BMMNCs, and histological injury in the iron-overload AA mice. Also, PND improved the percentage of T lymphocytes in the iron-overload AA mice. PND attenuates ferroptosis against iron-overload AA mice and Meg-01 cells via activating Nrf2/HO-1 and PI3K/AKT/mTOR pathway and is a promising novel therapeutic candidate for AA.

Evaluation of Unrestricted Somatic Stem Cells Therapy in an Ex-Vivo and in-Vivo Acquired Severe Aplastic Anemia Model

Background: In acquired severe aplastic anemia (aSAA), oligoclonal expansion of dysregulated CD8+ cytotoxic T cells, abnormal function of CD4+ T helper cells, along with elevated production of IFN-γ and TNF-α have been associated with the apoptosis of hematopoietic stem and progenitor cells (HSPC) (Young, N Engl J Med, 2018). Liao and Cairo et al (Cell Transplantation, 2014) have demonstrated that cord blood-derived unrestricted somatic stem cells (USSCs) could significantly reverse the abnormal/inverted ratio of CD4:CD8 T cells, increases the number of FOXP3A positive Treg cells and decrease the level of IFN-γ in an animal model of recessive dystrophic epidermolysis bullosa (RDEB). Alternative therapies are in great need for patients with aSAA as immunosuppressive therapy (IST) overall responses are sub-optimal. Objectives:To determine the effect of USSCs on HSPC survival, differentiation and phosphorylation pathways in an ex-vivo culture of aSAA with human CD34+ cells.To determine the effect of USSCs administration on blood cell count recovery, bone marrow (BM) cellularity and survival in an in-vivo inflammatory cytokine mediated mice model of aSAA. Design/Methods: Human HSPC were cultured with SCF, FLT3 and human TPO. HSPCs alone, with and without IFN-γ and TNF-α served as controls. USSCs (transwell and direct) were added to the culture. HSPC were harvested and assayed for their survival at day 7 and 14. Annexin-V+/PI+ were referred to as late apoptotic cells. Each experimental condition was set up in triplicate. The cells were cultured at 37°C with 5% CO2. Multi-lineage differentiation capacity was assessed with a selective colony forming units (CFU) assay and compared between experimental groups. Signaling pathways were determined using phosphor-flow cytometric analysis of pSTAT1. For the immune mediated mouse model of aSAA, 8-week-old C57BL/6 mice were irradiated with 6.5Gy TBI. Spleen lymphocytes were collected from FVB donors, and injected 4 hours post-radiation into B6 recipient mice via the retro-orbital vein, at 1-1.5 × 106 cells/recipient in 100μL PBS. Each experimental group (PBS control and USSCs) included 20 mice per group. Mice were continuously monitored for their survival. Peripheral blood (PB) was analyzed using a VETSCAN® HM5 Analyzer. Mean ± SEM of the blood counts was compared between experimental groups. Mice femur bones were extracted and cleaned of soft tissue, fixed and sectioned. Paraffin sections were stained with Hematoxylin and Eosin (H&E) to assess morphology. Results: The myelosuppressive effect of IFN-γ and TNF-α was significantly rescued (4-fold increase) by the addition of USSCs (transwell) at day 7. After 28 days direct co-culture with USSCs increased CD34+ survival as well (p<0.001) from 5 to 35 cells/µl. A significantly higher percent of viable cells (73% versus 63%) was observed among the USSCs treated cells (P<0.05). Phosphor-flow cytometric analysis demonstrated an almost 50% decrease in pSTAT1 in the presence of USSCs (p<0.05). Based on CFU analysis, CD34+ cells cultured in the presence of USSCs were able to produce more CFU (52, SD±3.7) at day 7 (p<0.01) compared to IFN-γ and TNF-α treated cells (16, SD±2.8). Also, USSCs were associated with more BFU (p<0.05) at day 14. In our immune mediated aplastic anemia mice model, treatment with USSCs was associated with substantial prolonged mice survival (p=0.02) (Figure 1a). Evaluation of PB counts demonstrated increased hemoglobin levels at day 14 in the USSCs treated mice (p<0.05) 5.6g/dL (SD±0.6), compared to PBS treated mice 2.1g/dL (SD±0.1). BM evaluation of USSCs treated mice versus PBS control displayed higher cellularity in the USSCs treated sections (Figure 1b). Conclusions: Cellular therapy, such as USSCs, may be instrumental in the treatment of aSAA, specifically by increasing BFU-E and eventually hemoglobin levels. Future studies will need to be done to investigate the exact mechanisms of action and the clinical efficacy of this novel cellular therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Let-7e-5p, a promising novel biomarker for benzene toxicity, is involved in benzene-induced hematopoietic toxicity through targeting caspase-3 and p21

Benzene is a common industrial chemical and environmental pollutant. However, the mechanism of hematotoxicity caused by exposure to low doses of benzene is unknown. Let-7e-5p pathway regulatory networks were constructed by bioinformatics analysis using a benzene-induced aplastic anemia (BIAA) mouse model. The MTT assay, EdU staining, flow cytometric analysis, dual luciferase reporter gene assay, and RIP assay were utilized to evaluate the effects of benzoquinone (1,4-BQ) on let-7e-5p pathway. This study consisted of 159 workers with a history of low-level benzene exposure and 159 workers with no history of benzene exposure. After the confounding factors were identified, the associations between let-7e-5p expression and hematotoxicity were assessed by multiple linear regression. Furthermore, we used four machine learning algorithms (decision trees, neural network, Bayesian network, and support vector machines) to construct a predictive model for detecting benzene-causing hematotoxicity in workers. In this study, compared with respective controls, let-7e-5p expression was decreased in BIAA mice and benzene-exposed workers. After 1,4-BQ exposure, let-7e-5p overexpression negatively regulated caspase-3 and p21 expression, protected cells from apoptosis, and facilitated cell proliferation. RIP assays, and dual luciferase reporter gene assays confirmed that let-7e-5p could target p21 and caspase-3 and regulate the cell cycle and apoptosis. The support vector machines classifier achieved the best prediction of benzene-induced hematotoxicity (prediction accuracy = 88.27, AUC = 0.83) by statistically characterizing the internal dose of benzene exposure and the oxidative stress index, as well as the expression levels of let-7e-5p pathway-related genes in benzene-exposed workers. Let-7e-5p may be a potential therapeutic target of benzene-induced hematotoxicity, provide a basis for evaluating the health hazards of long-term and low-dose benzene exposure in workers, and supply a reference for revising occupational health standards.

Dioscin Regulating Bone Marrow Apoptosis in Aplastic Anemia.

BackgroundAplastic anemia (AA), a disease of bone marrow failure, is caused by CD8+T mediated apoptosis of hematopoietic cells. However, traditional immunosuppressive therapy (IST) has severe liver and kidney toxicity and even cannot achieve the expected therapeutic effect in some patients.PurposeOur study is aimed to investigate the effect and mechanism of dioscin (DNS) for treating AA.MethodsBriefly, we established and evaluated the AA mouse model, DNS and positive control drugs were used for intervention treatment. After 14 days of intervention, femoral bone marrow pathology, bone marrow mononuclear cells (BMMCs) apoptosis rate, bone marrow CD34+ cell surface Fas (CD95) expression and Fas signaling pathway key proteins were detected.ResultsAfter the establishment of the AA mouse model, the number of peripheral blood cells including granulocytes, erythrocytes, hemoglobin, platelets and reticulocytes in the AA group model was significantly decreased compared with the group control (P < 0.01). The degree of bone marrow hyperplasia in the sternum and femur is extremely low. After different drug interventions, compared with the group model, the number of peripheral blood cells in the AA mice rebounded significantly in group DNS (P < 0.01). Not only that the apoptosis rate of BM-MCs decreased (P < 0.01), meanwhile, the CD95 molecule expressed on the CD34+ bone marrow cells had a significant decline (P < 0.01), and the expression level of the key proteins of Fas signaling pathway was also significantly decreased (P < 0.01).ConclusionDNS recovered the peripheral pancytopenia and bone marrow failure in AA mice. DNS reduced the key protein of Fas signaling pathway level to inhibit apoptosis of bone marrow cells to treat AA.

A mouse model of irradiation and spleen-thymus lymphocyte infusion induced aplastic anemia

ABSTRACT Objectives The immune-induced aplastic anemia (AA) mouse model has been used for the study of AA. However, there were no uniform conditions for establishing a model and no assessment of immunological homeostasis. Our study aimed to identify the conditions of establishing a model and assess the AA model in immunology and pathology. Methods We induced an AA mouse model by the combination between sublethal irradiation and spleen-thymus lymphocyte infusion. The success of establishing the AA model was identified by blood routine tests and pathology of bone marrow. The frequency of Th17 and Treg cells was measured by flow cytometry. The frequency of CD34+ and CD41+ cells was detected by immunohistochemical technique.IL-6, IL-8, IL-17, TNF-α and IFN-γ were evaluated by ELISA. Results The 137Cs sublethal irradiation (5 Gy) and spleen-thymus lymphocyte infusion (5 106) induced the AA mouse model successfully. The AA mice had a long lifetime and manifested pancytopenia and bone marrow failure. The percentage of Th17 cells increased and the percentage of Treg cells decreased distinctly in AA mice. The area of hematopoietic tissues and count of CD34+ cells and CD41+ cells were significantly reduced in AA mice.The level of cytokines, IL-6, IL-8, IL-17, TNF-α and IFN-γ, was increased significantly in peripheral blood and bone marrow. Conclusion Our data suggest that the improved AA mouse model conforms to the diagnosis standard of AA and simulates the immune internal environment of human AA. The AA mouse model has a longer lifetime and unbalances of Th17/Treg cells caused the destruction of CD34+ cells and CD41+ cells, which was immune-mediated pathogenesis to adapt to long-term research.

Kinetics of immune activated T cells in aplastic anemia mouse model

目的探索再生障碍性贫血（AA）小鼠模型中供者来源T细胞不同时间点动力学变化。方法构建AA小鼠模型，分别于不同时间点采用流式细胞术测定模型小鼠脾脏与骨髓内供者T细胞比例、活化分子表达、细胞周期及功能亚群，评估不同时期T细胞功能状态。结果①半致死剂量照射联合主要组织相容性抗原（MHC）半相合的淋巴结细胞输注成功构建T细胞免疫介导的AA小鼠模型。②AA小鼠脾脏中供者T细胞从移植第3天后开始明显浸润，并逐渐出现CD4+/CD8+比例倒置，第5天开始进入骨髓，以CD8+细胞浸润为主。③CD69在供者CD4+细胞中表达高峰晚于CD8+细胞，CD25在CD4+细胞与CD8+细胞中表达水平的变化趋势相同，但在CD8+细胞中的表达高于CD4+细胞。④脾脏内供者CD4+细胞S/G2/M期比例在移植后第3天即达高峰，约12％，而CD8+细胞中S/G2/M期比例在移植后第5天达高峰，约20％，且两者在进入骨髓后S/G2/M期比例均再次升高，但移植第3天后其比例在脾脏与骨髓CD8+细胞中持续高于CD4+细胞。⑤脾脏内免疫活化的T细胞经历短暂的中枢记忆T细胞（TCM）阶段后迅速分化为效应记忆T细胞（TEM），进入骨髓后部分TEM分化为效应细胞进一步发挥效应功能。结论AA小鼠模型中供者T细胞进入异体后迅速活化，5天内达增殖高峰，并完成向TEM细胞的分化，5天后开始进入骨髓进一步增殖损伤造血。