Aplastic Anemia Mice Research Articles

Comparatively Evaluating the Role of Herb Pairs Containing Angelicae Sinensis Radix in Xin-Sheng-Hua Granule by Withdrawal Analysis.

The present study aims to investigate the roles of herb pairs containing Angelicae Sinensis Radix (Danggui) in Xin-Sheng-Hua Granule (XSHG) on hemolytic and aplastic anemia (HAA) mice. HAA model mice were induced by acetyl phenylhydrazine and cyclophosphamide; then the samples of XSHG and its decomposed recipes (DY, DC, DT, DH, DJ, and DZ) were orally administrated to these mice. Indicators of peripheral blood routine, organ index, and ATPase activities were tested. Moreover, the main effective components in these samples were also analyzed by UHPLC-TQ-MS/MS. Clear separation between the control and model groups from score plot of principal component analysis (PCA) was easily seen, indicating that HAA model was successfully conducted. Afterwards, relative distance calculation method between dose groups and control group from PCA score plot was adopted to evaluate the integrated effects of hematinic function of different samples. And the orders of hematinic effects were as follows: XHSG > DJ > DT > DZ > DH > DC > DY. Further analysis of these samples by UHPLC-TQ-MS/MS revealed that XSHG underwent complicated changes when herb pairs containing Danggui were excluded from XSHG, respectively. Compared with XSHG, the vast majority of active compounds in sample DY (formula minus herb pair Danggui-Yimucao) decreased significantly, which could partly explain why herb pair Danggui-Yimucao made great contribution to XSHG. These findings showed that withdrawal analysis method is a valuable tool to analyze the impacts of herb pairs containing Danggui on XSHG, which could lay foundation to reveal the compatibility rules of this formula.

Assessing the Immunomodulatory Activity of Ethanol Extract of Sambucus javanica Berries and Leaves in Chloramphenicol-Induced Aplastic Anemia Mouse Model.

Aplastic anemia, life-threatened disease, is a hematologic disorder characterised by bone marrow hypoplasia. Multiple modalities such as bone marrow transplantation and immunosuppression treatment have been proposed to ameliorate this entity, however it remains ineffective. Sambucus, a group of herb plants, possesses a broad spectrum of medicinal properties such as antioxidant, insulin-like activity, anticancer and antiviral. However, the study about its activity toward aplastic anemia incidence is based on limited data. Thus, the research aim of this study was to evaluate the immunomodulatory activities of Sambucus javanica in chloramphenicol-induced anemia aplastic mouse model. In this present study, BALB/c mice were administrated with chloramphenicol (CMP) to induce aplastic anemia then followed by S. javanica extracts treatment. Additionally, cellular and molecular aspects were evaluated by flow cytometry and Hematoxylin-Eosin staining. Further analysis showed that S. javanica extracts could promote the population number of regulatory T-cells and naive cytotoxic T-cells. Moreover, those extract also reduced the inflammation and necrotic incidence in CMP-induced mouse aplastic anemia model. Together, these results suggest that S. javanica has therapeutically effect to aplastic anemia by altering the immune system as an immunomodulatory agent.

Gadofullerene nanoparticles for robust treatment of aplastic anemia induced by chemotherapy drugs.

Aplastic anemia (AA) is characterized as hypoplasia of bone marrow hematopoietic cells and hematopenia of peripheral blood cells. Though the supplement of exogenous erythropoietin (EPO) has been clinically approved for AA treatment, the side-effects hinder its further application. Here a robust treatment for AA induced by chemotherapy drugs is explored using gadofullerene nanoparticles (GFNPs).Methods: The gadofullerene were modified with hydrogen peroxide under alkaline conditions to become the water-soluble nanoparticles (GFNPs). The physicochemical properties, in vitro chemical construction, stability, hydroxyl radical scavenging ability, in vitro cytotoxicity, antioxidant activity, in vivo treatment efficacy, therapeutic mechanism and biological distribution, metabolism, toxicity of GFNPs were examined.Results: GFNPs with great stability and high-efficiency antioxidant activity could observably increase the number of red blood cells (RBC) in the peripheral blood of AA mice and relieve the abnormal pathological state of bone marrow. The erythropoiesis mainly includes hemopoietic stem cells (HSCs) differentiation, erythrocyte development in bone marrow and erythrocyte maturation in peripheral blood. The positive control-EPO promotes erythropoiesis by regulating HSCs differentiation and erythrocyte development in bone marrow. Different from the anti-AA mechanism of EPO, GFNPs have little impact on both the differentiation of HSCs and the myeloid erythrocyte development, but notably improve the erythrocyte maturation. Besides, GFNPs can notably decrease the excessive reactive oxygen species (ROS) and inhibit apoptosis of hemocytes in blood. In addition, GFNPs are mostly excreted from the living body and cause no serious toxicity.Conclusion: Our work provides an insight into the advanced nanoparticles to powerfully treat AA through ameliorating the erythrocyte maturation during erythropoiesis.

Panaxdiol Saponins Component Promotes Hematopoiesis and Modulates T Lymphocyte Dysregulation in Aplastic Anemia Model Mice.

To investigate the potential efficacy of panaxadiol saponins component (PDS-C) in the treatment of aplastic anemia (AA) model mice. Totally 70 mice were divided into 7 groups as follows: normal, model, low-, medium-, high-dose PDS-C (20, 40, 80 mg/kg, namely L-, M-, H-PDS-C), cyclosporine (40 mg/kg), and andriol (25 mg/kg) groups, respectively. An immune-mediated AA mouse model was established in BALB/c mice by exposing to 5.0 Gy total body irradiation at 1.0 Gy/min, and injecting with lymphocytes from DBA mice. On day 4 after establishment of AA model, all drugs were intragastrically administered daily for 15 days, respectively, while the mice in the normal and model groups were administered with saline solution. After treatment, the peripheral blood counts, bone marrow pathological examination, colony forming assay of bone marrow culture, T lymphocyte subpopulation analysis, as well as T-bet, GATA-3 and FoxP3 proteins were detected by flow cytometry and Western blot. The peripheral blood of white blood cell (WBC), platelet, neutrophil counts and hemoglobin (Hb) concentration were significantly decreased in the model group compared with the normal group (all P<0.01). In response to 3 dose PDS-C treatment, the WBC, platelet, neutrophil counts were significantly increased at a dose-dependent manner compared with the model group (all P<0.01). The myelosuppression status of AA was significantly reduced in M-, H-PDS-C groups, and hematopoietic cell quantity of bone marrow was more abundant than the model group. The colony numbers of myeloid, erythroid and megakaryocytic progenitor cells in the model group were less than those of the normal mice in bone marrow culture, while, PDS-C therapy enhanced proliferation of hematopoietic progenitor cells by significantly increasing colony numbers (all P<0.01). Furthermore, PDS-C therapy increased peripheral blood CD3+ and CD3+CD4+ cells and reduced CD3+CD8+ cells (P<0.05 or P<0.01). Meanwhile, PDS-C treatment at medium- and high doses groups also increased CD4+CD25+FoxP3+ cells, downregulated T-bet protein expression, and upregulated GATA-3 and FoxP3 protein expressions in spleen cells (P<0.05). PDS-C possesses dual activities, promoting proliferation hematopoietic progenitor cells and modulating T lymphocyte immune functions in the treatment of AA model mice.

Monocytic Myeloid-Derived Suppress Cells Restore Adipogenic Bone Marrow Microenvironment in Aplastic Anemia By Inhibiting Intra-BM CD8+ T Cells Proliferation

Aplastic anemia (AA) is a hematopoietic disorder resulted from immune-related hypocellular hematopoiesis in bone marrow (BM). It has been clearly addressed that the activated T cells contribute to the exhaustion of hematopoietic progenitors and hypo-hematopoiesis. The adipogenic BM is one of the characteristics to make AA diagnosis. However, little is known about the relationship of intra-BM immune imbalance and hematopoietic microenvironment abnormity in this disease entity. Functional hematopoiesis relies on not only abundant hematopoietic stem cells (HSCs) but also the balanced supportive hematopoietic niche. Intra-BM immune balance, at either cellular or cytokine level, is one of the key footstones to maintain hematopoietic microenvironment. Various intra-BM immune cellular components play both sides of one coin. Among them, myeloid-derived suppressive cells (MDSCs) are heterogeneous myeloid progenitor cells characterized by the negative immune response in cancers and other inflammatory diseases. In BM aspiration and biopsy samples from the patients who were diagnosed as AA in our study, massive activated lymphocytes infiltration and adipocytes accumulation were observed. Interestingly, the absolute numbers of immune modulatory MDSCs either in AA patients' PB or in BM of immune-related AA mice were reduced, indicating a potential link between polarized BM adipo-osteogenic microenvironment and immune disorder under AA circumstance. We thus adopted AA mice model to look into the embedded details both in vivo and in vitro. We clarified that BM components were more vulnerable to the attack of CD8+ T cells than that of CD4+ T cells. Taking into the fact that BM adipocytes are more abundant either in AA patients or in AA mice models, we differentiated mesenchymal stromal cells (MSCs), the major BM stroma cells, into osteoblastic or adipogenic lineages to mimic the osteo-adipogenic differentiation in BM microenvironment. Interestingly, CD8+ T cells and interferon-γ(IFN-γ) exerted dramatically adipocytic stimulation on BM-MSCs either in vitro or in vivo, by determination of increasing expression of adipogenetic genes including Ap2, Perilipin, Pparg and Cebpα, as well as staining of Oil Red O and perilipin. To dissect intra-BM cellular immune balance, MDSCs were isolated as representative immune regulating population to investigate their function on osteo-adipogenic balance. Interestingly, not CD11b+Ly6G+Ly6C-granulocytic-MDSCs (gMDSCs) but CD11b+Ly6G-Ly6C+monocytic-MDSCs (mMDSCs) inhibited both T cell proliferation and IFN-γ production. Addition of L-NMMA, the antagonist of iNOS pathway in mMDSCs-containing system restored T cell proliferative curve and cell numbers, whereas Nor-NOHA, the antagonist of Arg-1 pathway didn't abrogate mMDSCs' immune-regulation properties, indicating that mMDSCs inhibited T cell proliferation via iNOS pathway. We then performed single dose or multi-dose injection of mMDSCs in AA mice to see whether mMDSCs are able to reconstitute the impacted hematopoiesis. Single injection of mMDSCs was able to prevent from CTL infiltration in a very short term. However, multi-injection of mMDSCs showed significant benefit in overall survival rate compared to AA mice. We further detected the function of mMDSCs on polarized BM-MSCs adipo-osteogenic differentiation potential. To detect sequential BM adipogenetic progression in AA microenvironment, we performed in vivo fluorescent microscopy on AP2 (Fabp4)-Cre×mT/mG reporting mice at different transfusion time points of T cells and mMDSCs. GFP-expressing AP2+ adipocytes accumulated adjacently to perivascular niches whose boarders were labelled by Dextran-CY5 in a time-dependent manner after T cell infusion. Monocytic MDSCs transfused AA mice showed decreased GFP+ adipocytes which was coincident with our in vitro findings. In conclusion, intra-BM immune balance is one of the environmental factors seesawing by activating and suppressive ends to support functional hematopoiesis. Adoptive transfusion of mMDSCs, the immune-suppressive population might be a novel immune-regulating strategy to treat AA, relying on not only restoring the intra-BM immune balance but also improving stroma's multi-differentiating microenvironment. Figure Disclosures No relevant conflicts of interest to declare.

Stanozolol and Danazol Have Different Effects on Hematopoiesis in Murine Model of Immune-Mediated Bone Marrow Failure

Background: Stanozolol and danazol are widely used in the treatment of aplastic anemia (AA), however, they may have different effects on the recovery of hematopoiesis with the detail mechanism unclear. Methods: Bone marrow mononuclear cells from 5 newly diagnosed AA patients and 5 healthy volunteers were collected for marrow colony assays and cultured together with stanozolol, danazol or blank control, separately. After incubated for 14 days, colonies of different lineage were calculated, and erythroid or megakaryocytic differentiation was also identified by the mean fluorescence intensity (MFI) of CD235a or CD41 expressed on the harvest cells. Meanwhile, CB6F1/Crl mice were injected with 1×106 C57BL/6 donor originated lymphocytes after irradiated with 5Gy total body irradiation to setup a model for immune-mediated bone marrow failure (AA mice model). AA mice were treated with CsA monotherapy, CsA combined with stanozolol, CsA combined with danazol for 30 days, respectively. Peripheral blood cell counts once a week, bone marrow colony assays at the end of one month were performed. Proportion of T cell subsets, level of inflammatory factors, EPO and TPO were detected before and after treatment. Level of EPO receptor on the progenitor cells after treatment were tested by western blot. Results: For ex vivo experiment, although the number of BFU-E, CFU-GM and CFU-GEMM colonies of AA patients were significantly lower than that of the normal controls (P&lt;0.05), the number of colonies and MFI of CD235a or CD41 expression of the harvested cultured cells had no significant difference among different treatment groups, either in AA patients or in normal controls, showing no direct hematopoietic stimulating effects of stanozolol and danazol on progenitor cells. However, in the in vivo experiment, AA mice treated with CsA and danazol showed the most rapid recovery of megakaryopoiesis, with the platelet count returned to normal level after three weeks' treatment, at least one week earlier than the other groups. Whereas mice treated with CsA and stanozolol had the best hemoglobin level at the end of treatment (P&lt;0.05). Bone marrow colony assays at the 30 days showed that the number of BFU-E was the highest in mice treated with CsA and stanozolol while the number of CFU-GM was the highest in those with CsA and danazol. Compared to CsA monotherapy, additional stanozolol and danazol can both increase the level of regulatory T cells and up-regulate interleukin-10 (P&lt;0.05). But interferon-α and tumor necrosis factor-α were more effectively reduced by danazol than stanozolol (P&lt;0.05). CsA and stanozolol- treated mice showed higher serum EPO (corrected by HGB level)and higher EPO receptor(EPOR) level on the hematopoietic precursor cells compared with other groups (P&lt;0.05). Conclusions: Neither stanozolol or danazol directly stimulated hematopoiesis in vitro. But in vivo, stanozolol may have advantage in improving erythropoiesis while danazol may has more effects on white cells or platelet. Danazol had more comprehensive immunosuppressive roles compared with stanozolol. Stanozolol can enhance the expression of EPO, probably by increasing EPOR expression on hematopoietic precursor cells. Disclosures No relevant conflicts of interest to declare.

Rapamycin ameliorates immune-mediated aplastic anemia by inhibiting the proliferation and metabolism of T cells

Aplastic anemia (AA) is a serious blood system disease that threatens human health. At present, the main cause of this disease is believed to be immune hyperfunction. However, the specific metabolic mode involved in the occurrence of lymphocytes in AA is still unknown. In addition, whether rapamycin, a specific blocker of the mTOR signaling pathway, plays a therapeutic role by inhibiting lymphocyte metabolism remains unclear. We induced an AA mouse model through the classical immune-mediated pathway and simultaneously administered rapamycin intervention therapy. First, the AA-associated phenotypic changes and the efficacy of rapamycin in the treatment of AA were discussed. Second, the proliferation and metabolic pathway of bone marrow (BM) lymphocytes in AA and the effect of rapamycin on this process were determined. Finally, the expression levels of mTOR pathway-related proteins were analyzed. By inhibiting the mTOR signaling pathway, rapamycin could ameliorate the phenotype of the immune-mediated AA model and inhibit the proliferation of T cells by preventing cell cycle transition from G0 to G1 phase. Moreover, we found that mitochondrial oxidative phosphorylation is involved in the metabolic reprogramming of T cells in AA and that rapamycin can inhibit this process. We confirmed that mitochondrial oxidative phosphorylation is involved in the metabolic reprogramming of T cells in AA and further extended the mechanism of rapamycin in treating AA by inhibiting the mTOR signaling pathway. This viewpoint may provide a new therapeutic idea for clinical applications.

Development and application of the immune-mediated aplastic anemia mouse model

再生障碍性贫血（AA）的发病机制主要是免疫异常，免疫介导的AA小鼠模型的建立有利于AA发病机制的深入研究。制备免疫介导的AA小鼠模型有病毒感染、异基因淋巴细胞输注、全身辐照（TBI）加异基因淋巴细胞输注等方法。我们分别以干扰素-γ（IFN-γ）与白消安合用及TBI加异基因淋巴结细胞输注方法成功制备了两种AA小鼠模型，检测结果显示这两种模型更接近人类AA的特点，其中改良的杂交一代（F1）AA小鼠的制备方法在国内实验条件下易于操作，更适合用于推广，有望成为新的AA基础研究平台。.

Altered expression of leptin and leptin receptor in the development of immune-mediated aplastic anemia in mice.

The current study aimed to explore the levels of leptin (LEP) and LEP receptor (LEP-R) on the progression of aplastic anemia (AA) with bone marrow fat conversion. An AA model was developed by infusing C57BL/6 lymph node cells into BALB/c mice. At 0, 3, 6, 9, 12, 15 and 18 days after modeling, routine blood counts, bone marrow biopsy slides, lymphocyte subsets (CD4+ and CD8+ T cells) and cytokine levels [including interleukin (IL)-2, IL-4, IL-5 and interferon-γ] were assessed. LEP and LEP-R levels in peripheral blood serum, mesenchymal stem cells (MSCs) and bone marrow were also analyzed by enzyme-linked immunosorbent assay, polymerase chain reaction and immunohistochemistry. The relevance of LEP, LEP-R and other factors was analyzed by Pearson's correlation analysis. Peripheral pancytopenia (reduced count of white blood cells, red blood cells, hemoglobin and platelets), abnormal immune factor levels and histological changes in bone marrow sections were detected in the AA model mice, suggesting that these mice mimicked the pathological changes commonly observed in AA. In addition, following the establishment of AA, the LEP level was gradually increased and the LEP-R level was reduced in the mice over time (P<0.05). The expression of adipogenic genes, including CCAAT/enhancer-binding protein (C/EBP)α, C/EBPβ and peroxisome proliferator-activated receptor γ, was markedly increased, while the expression of the osteogenic gene runt-related transcription factor 2 was reduced compared with the levels in the control group (P<0.05). Taken together, damage to LEP-R may lead to dysregulation of LEP and the enhancement of MSCs to differentiate into adipocytes, resulting in excessive fat in bone marrow of AA patients.

PF459 ANGELICA SINENSIS POLYSACCHARIDE MAINTAINS THE FUNCTION OF HEMATOPOIETIC STEM CELLS BY REGULATING THE TREG/TH17 RATIO AND THEREBY INDUCES ANTI‐MITOCHONDRIAL APOPTOSIS IN APLASTIC ANEMIA

Background:Aplastic anemia (AA) is considered a bone marrow failure syndrome because it is characterized by peripheral pancytopenia and marrow hypoplasia. Several studies have confirmed that T helper (Th) cells, such as IL‐17‐producing (Th17) CD4+ T cells, play a negative role in the pathogenesis of AA (Hoyer et al., 2009), whereas regulatory T cells (Tregs) maintain immunological self‐tolerance and immunosuppression (Campbell and Koch, 2011), which play a critical role in protection of the hematopoietic function in AA (Shi et al., 2012). However, whether mitochondrial apoptosis influences hematopoietic stem cells by regulating immunity in AA remains unknown.Aims:To investigate whether mitochondrial apoptosis induced by abnormal regulatory T cell (Treg)/Th17 cell ratios could be corrected by Angelica sinensis polysaccharide (ASP) in a murine model of aplastic anemia (AA).Methods:Abnormal mitochondrial function and mitochondrial apoptosis were first confirmed in AA patients. In the in vivo experiment, ICR mice were randomly divided into five groups: normal control group, AA model control group and three groups treated with different doses. AA mice (the AA model control and treated groups) were treated with 60Co γ‐radiation and then administered daily intraperitoneal injections of cyclophosphamide and chloramphenicol for 3 days. The mice in the various groups were fed saline or ASP for 2 weeks. To confirm that the AA model was successfully established, blood cells and bone marrow cells were obtained from the various groups of animals on day 14, the peripheral blood cell and bone marrow nucleated cells (BMNCs) counts were counted, and a marrow biopsy was performed. The Lin–Sca‐1+c‐Kit+ (LSK) cell populations were then sorted from the bone marrow cells of AA mice and cultured in medium for testing. The mitochondrial function, including the outer membrane integrity and mitochondrial membrane potential (MMP), was tested. The accumulation of reactive oxygen species (ROS), the degree of apoptosis, and the levels of mitochondria‐associated apoptosis‐related proteins, including the Bcl‐2/Bax ratio, the caspase‐3, caspase‐9 and p‐P38/P38 levels, were measured in the groups of AA mice. The Treg/Th17 cell ratio was also tested.Results:The numbers of peripheral blood cells and BMNCs and the hematopoietic tissue area in the bone marrow biopsies from AA mice were notably decreased (P &lt; 0.05), which indicated successful establishment of the AA mouse model. The mice treated with the medium dose of ASP for 14 days showed increased WBC, RBC, PLT and BMNC counts compared with the mice in the AA group mice. The data showed that ASP decreased damage to the mitochondrial outer membrane, improved the stabilization of the mitochondrial membrane, corrected the abnormal levels of ROS and mitochondrial‐associated apoptosis proteins, including the Bcl‐2/Bax ratio and caspase‐3 and caspase‐9 expression, in the AA mice. The changes to the p‐P38/P38 and Treg/Th17 ratios induced by AA were also recovered by the medium dose of ASP.Summary/Conclusion:The indicators of mitochondrial apoptosis and the Treg/Th17 cell ratio in the ASP‐treated group, particularly the medium‐dose group, were significantly better than those in the AA group, which indicates that ASP might induce anti‐mitochondrial apoptosis to restore the function of hematopoietic stem cells by suppressing abnormal T‐cell immunity in AA.image

Iron chelation effect of curcumin and baicalein on aplastic anemia mouse model with iron overload.

Objective(s):The current work aimed to assess whether curcumin and baicalein can chelate iron in aplastic anemia (AA) complicated with iron overload, exploring the potential mechanisms.Materials and Methods:A mouse model of AA with iron overload complication was firstly established. Low and high-dose curcumin or baicalein treatment groups were set up, as well as the deferoxamine positive control, normal and model groups (n=8). Hemogram and bone marrow mononuclear cell detection were performed, and TUNEL and immunohistochemical staining were used to evaluate hematopoiesis and apoptosis in the marrow. ELISA, Western blot, and qRT-PCR were employed to assess serum iron (SI), serum ferritin (SF), bone morphogenetic protein 6 (BMP-6), SMAD family member4 (SMAD4) and transferrin receptor 2 (TfR2) amounts. Results:Both curcumin and baicalein improved white blood cell (increase of 0.28-0.64×109/l in high-dose groups) and hemoglobin (increase of around 10 g/l) amounts significantly, which may related to decreased apoptosis (nearly 30%-50% of that in the model group) in the bone marrow, while their effects on platelet recovery were limited and inferior to that of deferoxamine (DFO). Both test compounds up-regulated hepcidin and its regulators (BMP-6, SMAD, and TfR2) at the protein and mRNA levels; high dosage treatment may be beneficial, being better than DFO administration in lessening iron deposition in the bone marrow.Conclusion:Curcumin and baicalein protect hematopoiesis from immune and iron overload-induced apoptosis, exerting iron chelation effects in vivo.

Sirt1 in the Regulation of Interferon Gamma in Severe Aplastic Anemia

Recent studies have indicated that Sirt1 plays critical roles in the suppression of inflammation, T cell activation, and differentiation of hematopoietic progenitor cells. Severe aplastic anemia (SAA) is an immune-mediated disease that is characterized by elevated cytotoxic lymphocytes and type 1 cytokines. As a negative effector cytokine, interferon gamma (IFNγ) takes part in aplastic anemia through its inhibitory effect on hematopoiesis. In this study, we investigated the role of Sirt1 in the regulation of IFNγ in patients with SAA. A significant decrease in relative SIRT1 (p< 0.05) and increase in IFNG (p< 0.05) expression levels was observed in the sorted CD8+T cells of SAA patients compared to the controls. There was a significant negative correlation (r = –0.53, p < 0.05) between SIRT1 and IFNG expression in SAA patients. SRT3025, a Sirt1 activator, was shown to significantly reduce IFNγ (p < 0.01) and elevate Sirt1 (p < 0.05) expression in the CD8+T cells of SAA patients, and also showed a therapeutic role in an aplastic anemia mouse model. In conclusion, the defective Sirt1 may be correlated to the abnormal IFNγ expression in SAA patients, and activation of Sirt1 signaling may help improve the inflammatory status of SAA.

Exosomal sphingosine 1-phosphate secreted by mesenchymal stem cells regulated Treg/Th17 balance in aplastic anemia.

This study was designed to explore whether exosomal sphingosine 1-phosphate (S1P) from mesenchymal stem cells (MSCs) regulate the Treg/Th17 balance in aplastic anemia (AA) patients and to validate the underlying mechanism. To address this, exosomes from human bone marrow MSCs (MSCs-Exos) were co-cultured with CD4+ T cells from AA patients (AA CD4+ T cells), which were transfected with si-S1PR1, si-S1PR3, or not. The proportion of Th17 and Treg was evaluated by flow cytometry. The levels of Th17-associated interleukin-17 (IL-17), Treg-associated IL-10, and transforming growth factor-β were determined by ELISA. S1P content in MSCs-Exos isolated from control, si-SphK1, or si-SphK2 transfected MSCs was examined by LC-MS/MS. Hematoxylin and eosin staining of bone marrow tissues was performed to evaluate the effect of MSCs-Exos in AA mice. Our results showed that MSCs-Exos reversed the increased Th17/Treg in AA through SphK1-mediated exosomal S1P enrichment. Furthermore, the promotion of Treg differentiation by exosomal S1P from MSCs was mediated through the receptor S1PR1 expressed on CD4+ T cells. Further in vivo experiments showed that MSCs-Exos reversed the increased Th17/Treg and alleviated AA progression in AA mice. In summary, SphK1-mediated enrichment of exosomal S1P secreted by MSCs reversed the increased Treg/Th17 ratio via the receptor S1PR1 in AA patients. © 2019 IUBMB Life, 71(9):1284-1292, 2019.

Hematopoiesis Activity of Sambucus javanica on Chloramphenicol-induced Aplastic Anemia Mouse Model

Hematopoiesis has a pivotal role in the maintenance of body homeostasis. Ironically, several hematological disorder caused by chemicals, drugs, and other environmental factors lead to severe bone marrow failure. Current treatments like stem cell transplantation and immunosuppression remain ineffective to ameliorate this diseases. Therefore, a newtreatment to overcome this entity is necessary, one of them by promoting the usage of medicinal plants. Thus, this study aimed to evaluate the hematopoiesis potency of S. javanica berries and leaves extracts in chloramphenicol (CMP)-induced aplastic anemia mice model. In this present study, several types of blood progenitor cell such as TER-119+VLA-4+ erythrocytes lineage, Gr-1+ granulocytes, and B220+ Bcell progenitor cells were evaluated by flow cytometry analysis. Accordingly, we revealed that S. javanica berries and leaves extracts significantly promoted TER-119+VLA-4+ erythrocytes lineage and Gr-1+ granulocytes after exposed by CMP. Thus, these results suggested that S. javanica berries and leaves extracts might have hematopoiesis activity in CMP-induced aplastic anemia mice model.

Nrf2-mediated metabolic reprogramming of tolerogenic dendritic cells is protective against aplastic anemia

Aplastic anemia (AA) is a rare disease characterized by immune-mediated suppression of bone marrow (BM) function resulting in progressive pancytopenia. Stem cell transplant and immunosuppressive therapies remain the major treatment choices for AA patients with limited benefit and undesired side effects. Here, we report for the first time the therapeutic utility of Nrf2-induced metabolically reprogrammed tolerogenic dendritic cells (TolDCs) in the suppression of AA in mice. CDDO-DFPA-induced Nrf2 activation resulted in a TolDC phenotype as evidenced by induction of IL-4, IL-10, and TGF-β and suppression of TNFα, IFN-γ, and IL-12 levels in Nrf2+/+ but not Nrf2−/− DCs. Cellular metabolism holds the key to determining DC immunogenic or tolerogenic cell fate. Although immature and LPS-induced (mature) Nrf2+/+ and Nrf2−/− DCs exhibited similar patterns of oxidative phosphorylation (OXPHOS) and glycolysis, only Nrf2+/+ DCs partially restored OXPHOS and reduced glycolysis during CDDO-DFPA-induced Nrf2 activation. These results were further confirmed by altered glucose uptake and lactate production. We observed significantly enhanced HO-1 and reduced iNOS/NO production in Nrf2+/+ compared to Nrf2−/− DCs, suggesting Nrf2-dependent TolDC induction is linked to suppression of the inhibitory effect of NO on OXPHOS. Furthermore, Nrf2−/− DCs demonstrated higher antigen-specific T cell proliferation. Lastly, TolDC administration improved hematopoiesis and survival in AA murine model, with decreased Th17 and increased Treg cells. Concomitantly, immunohistochemical analysis of AA patient BM biopsies displayed higher DCs, T cells, and iNOS expression accompanied with lower Nrf2 and HO-1 expression when compared to normal subjects. These results provide new insight into the therapeutic utility of metabolically reprogrammed TolDCs by CDDO-DFPA induced Nrf2 signaling in the treatment of AA.

INDUCTION EFFECT OF MICROALGAE SCENEDESMUS DIMORPHUS AGAINST HEMATOLOGY ON MICE (MUS MUSCULUS) SUFFERING ANEMIA DISEASES

Objective: This study aims to investigate the potential of Scenedesmus dimorphus as a cure for aplastic anemia in mice (Mus musculus).Methods: Fifty male mice divided into five groups, mice suffering anemia due to induction of chloramphenicol. Oral administrated of S. dimorphus for 28 days with a dose of 2.5 g/50 ml, 5.0 g/50 ml, and 7.5 g/50 ml, respectively, to cure aplastic anemia in mice and samcobion as standard drug.Results: Induction of chloramphenicol caused the mice to suffering aplastic anemia and weight loss in mice. Biomass of S. dimorphus has an effect of against hematological parameters and total blood count estimation. The result shows significant (p&lt;0.05 ANOVA) on weight gain, an increase of erythrocyte counts, and hemoglobin level, whereas an increase of hematocrit and reticulocyte values have non-significant, and a dose of S. dimorphus 5.0 g/50 ml were the treatment most effective for anemia in mice.Conclusion: Based on the results of this study, S. dimorphus biomass has the healing benefit of aplastic anemia due to the administration of chloramphenicol.

Human GMSCs Treat Murine Model with Aplastic Anemia Through Regulating the Balance between Treg and Th17 Cells

Severe aplastic anemia (AA) is a rare autoimmune disease characterized by severe pancytopenia and bone marrow failure. The recent finding of an abnormal quantity and function of T helper (Th) and regulatory T (Treg) cells in peripheral blood of patients has emerged as the major cause of the condition. Accumulating evidence has revealed that human gingiva-derived mesenchymal stem cells (GMSCs) are emerging as a new line of mesenchymal stem cells may have the potential to control or even treat autoimmune diseases through correcting the balance between Th and Treg cells. Given that GMSCs have a robust immune regulatory function and regenerative ability, we investigated the effect of GMSCs on preventing T cell-mediated bone marrow failure (BMF) in an AA mouse model. We observed that infusion of GMSCs markedly attenuated histological BM damage, controlled immunologically-mediated disorders in AA mice, significantly improved AA mice survival by significantly reducing cell infiltration of both CD8 cells and CD4 T cells (including Th1 and Th17 cells) in lymph nodes and decreased pro-inflammatory cytokines levels in lymph nodes and plasma. Moreover, GMSCs up-regulated the levels of Tregs and increased Foxp3 expression in lymph nodes. Our results demonstrate that the predominant Th1 and Th17 cell infiltration in the AA model could be significantly reduced while Treg cells are increased by GMSCs treatment. Collectively, our data demonstrate that GMSCs can attenuate T cell-mediated bone marrow failure through targeting the balance of Th17 and Treg cells, implicating that the use of GMSCs many have potential clinical application in prevention and treatment of patients with AA. Funding Statement: This study was supported by the National Key R&D Program of China (2017YFA0105800), the National Natural Science Foundation of China (No. 81671611), the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2016ZT06S252), the National Natural Science Foundation of Guangdong Province (No. 2014A030313131) and NIH R61 AR073409. Declaration of Interests: All authors have no conflict of interest. Ethics Approval Statement: This study was carried out in accordance with the recommendations of the animal use protocol, which was approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University (Approve number: [2018]02-195-01). Human gingiva samples were collected following routine dental procedures at the Division of Dentistry in the Third Hospital at the Sun Yat-sen University, which were approved by the medical ethics committees of Institutional Review Boards (IRB) at the Third Hospital at the Sun Yat-sen University.

Saponins from Panax notoginseng leaves improve the symptoms of aplastic anemia and aberrant immunity in mice

Aplastic anemia (AA) is usually treated with immunosuppressive agents, but their efficacy and safety are not satisfactory. Panax notoginseng saponins (PNS) promote the proliferation of hematopoietic stem/progenitor cells. This study aimed to examine the effects of leaf PNS (LPNS) on hematopoiesis and T cells in mouse models of AA. The experiments were performed in normal mice and AA mice (controls, cyclosporine, and low, medium, and high doses of LPNS). Hematopoietic cells were counted using colony formation assays. The proportions of T cells were measured by flow cytometry. The ERK1/2, T-bet, GATA-3, FOXP3, and RORγ proteins were assessed by western blotting. Cytokines were measured using a cytometric bead array. AA mice showed impaired hematopoiesis, high activation of T cells, and decreased expression of T-bet, GATA-3, and FOXP3. LPNS attenuated the inflammation observed in AA mice, and significantly increased the number of hematopoietic progenitor cells. The proportions of Th2 and regulatory T cells and the protein levels of P-ERK1/2, GATA-3, and FOXP3 were increased in the AA + LPNS mice compared with the AA mice. In contrast, LPNS decreased the proportions of Th1 and Th17 cells and the protein expression of T-bet. LPNS and cyclosporine had similar effects, but of different amplitudes. These results suggest that LPNS have dual activities in AA: 1) promoting the proliferation of hematopoietic progenitor cells; and 2) modulating T cell immune functions, an activity similar to that of cyclosporine. Additional studies are necessary to confirm those results before clinical use.

The herbal decoction modified Danggui Buxue Tang attenuates immune-mediated bone marrow failure by regulating the differentiation of T lymphocytes in an immune-induced aplastic anemia mouse model.

Angelicae Sinensis, Radix Astragali and Rhizoma Coptidis are all herbs of modified Danggui Buxue Tang (DGBX) and are extensively applied herbs in traditional Chinese medicine for the treatment of anemia and inflammation. In this study, immune-induced AA mice were used as an animal model, and the immunosuppressive agent, Ciclosporin A (CsA), was used as a positive control. Multiple pro-inflammatory cytokines were examined by bead-based multiplex flow cytometry. The T-cell subsets were assessed using a fluorescence-activated cell sorter (FACS). Western blot analysis was used to estimate the protein expression levels of specific transcription factors for T helper cells (Th1, Th2 and Th17) and key molecules of the Janus-activated kinase (Jak)/signal transducer and activator of transcription (Stat3) signaling pathway. DGBX treatment could significantly increase the production of whole blood cells in peripheral blood (PB); inhibit the expansion of Th1 and Th17 cells; increase the differentiation of Th2 and Tregs cells; regulate the expression levels of T-bet, GATA-3, RORγ and proinflammatory cytokines; and decrease the expression levels of key molecules in the Jak/Stat signaling pathway. These results indicate that DGBX can regulate the differentiation of T lymphocytes, resulting in immunosuppressive and hematogenic functions on AA mice. DGBX might be a good candidate for inclusion in a randomized study for AA with more data on the possible side effects and doses used in humans. Ultimately, it may be used for applications of traditional medicine against AA in modern complementary and alternative immunosuppressive therapeutics.

The mechanism of bone marrow-derived mesenchymal stem cells excessive senescence in severe aplastic anemia mouse model

目的探讨重型再生障碍性贫血（SAA）模型小鼠骨髓间充质干细胞（BM-MSC）过度衰老的机制。方法选择BALB/c小鼠40只，随机分为正常组20只、SAA组20只。应用白消安灌胃联合IFN-γ腹腔注射诱导建立SAA小鼠模型；分离培养两组小鼠的BM-MSC。观察BM-MSC细胞形态和细胞骨架；CCK-8和流式细胞术检测细胞增殖与细胞周期情况；比较衰老相关β-半乳糖苷酶（Senescence-associated β-Galactosidase, SA-β-gal）的染色阳性细胞比例；Western blot法检测mTOR蛋白的表达水平。结果正常组小鼠的BM-MSC细胞呈纺锤状、边界清晰，应力纤维平行排列、整齐；而SAA小鼠的BM-MSC细胞体积增大，呈平铺、边界不清状，应力纤维紊乱模糊。CCK-8结果显示，SAA小鼠的BM-MSC的增殖速率较正常组缓慢，在第2天开始差异有统计学意义（P<0.05）。SAA组小鼠BM-MSC的G0/G1期细胞比率[（77.461±1.567）%对（46.045±2.055）%，t=−34.384，P<0.001]、SA-β-gal阳性细胞比例[（75±11）%对（28±8）%，t=15.454，P<0.001]较正常组升高；SAA小鼠BM-MSC的mTOR表达水平高于正常小鼠。结论SAA小鼠模型中BM-MSC是衰老的MSC，且mTOR通路激活可能是导致其衰老发生的机制。