Harmine promotes megakaryocyte differentiation and thrombopoiesis by activating the Rac1/Cdc42/JNK pathway through a potential target of 5-HTR2A.

Harmine (HM), a ß-carboline alkaloid extracted from plants, is a crucial component of traditional Chinese medicine (TCM) known for its diverse pharmacological activities. Thrombocytopenia, a common and challenging hematological disorder, often coexists with serious illnesses. Previous research has shown a correlation between HM and thrombocytopenia, but the mechanism needs further elucidation. The aim of this study was to clarify the mechanisms underlying the effects of HM on thrombocytopenia and to develop new therapeutic strategies. Flow cytometry, Giemsa staining, and Phalloidin staining were used to assess HM's impact on Meg-01 and HEL cell differentiation and maturation in vitro. A radiation-induced thrombocytopenic mouse model was employed to evaluate HM's effect on platelet production in vivo. Network pharmacology, molecular docking, and protein blotting were utilized to investigate HM's targets and mechanisms. The results demonstrated that HM dose-dependently promoted Meg-01 and HEL cell differentiation and maturation in vitro and restored platelet levels in irradiated mice in vivo. Subsequently, HM was found to be involved in the biological process of platelet production by upregulating the expressions of Rac1, Cdc42, JNK, and 5-HTR2A. Furthermore, the targeting of HM to 5-HTR2A and its correlation with downstream Rac1/Cdc42/JNK were also confirmed. In conclusion, HM regulates megakaryocyte differentiation and thrombopoiesis through the 5-HTR2A and Rac1/Cdc42/JNK pathways, providing a potential treatment strategy for thrombocytopenia.

UHPLC-Q-TOF/MS Analysis of the Active Components of Total Flavonoids Extracts from Sarcandra glabra in Promoting Megakaryocyte Differentiation / 中药新药与临床药理

Objective To screen the active components of total flavonoid extracts of Sarcandra glabra to promote megakaryocyte differentiation.Methods(1)A model of megakaryocyte differentiation disorder was established by co-culturing human megakaryocytic leukaemia cells(Dami)with human bone marrow stromal cells(HS-5)as an evaluation system,and the experimental groupings were as follows:the Dami group(Dami),the control group(Dami+HS-5),and the PMA group[Dami+HS-5+5 ng·mL-1 foprolol 12-tetradecanoate 13-acetate(PMA)],and model group[Dami+HS-5+1%rabbit anti-rat platelet serum(APS)+5 ng·mL-1 PMA]were cultured for 48 hours.The expressions of megakaryocyte differentiation and maturation surface marker molecules,CD41a and CD61 were detected by flow cytometry.(2)Forty-nine SD male rats were randomly divided into blank plasma group,15-minute group,30-minute group,60-minute group,90-minute group,120-minute group,and 240-minute group,with 7 rats in each group.The rats in each administration group were gavaged with 1.26 g·kg-1 of total flavonoids extracts of Sarcandra glabra,and blood was collected at six set time points(15,30,60,90,120,240 minutes)for the preparation of time-dependent serum-containing plasma of total flavonoids extracts of Sarcandra glabra.(3)Ultra-high performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry(UHPLC-Q-TOF/MS)was used to analyze the plasma of the time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra,and the peak area was used to construct a matrix(X-matrix)of the amount of chemical composition change over time in the time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra.The collected time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra at six different time points was used to intervene in the model of megakaryocyte differentiation and maturation disorder,and the expression of cell surface molecules CD41a and CD61 was detected by flow cytometry to construct the matrix of effect of time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra(Y-matrix).(4)After the data of X and Y matrices were standardized,partial least squares(PLS)was used to calculate and analyze the quantitative and qualitative effect relationship,and variable importance for projection(VIP)>1 was used as the threshold to screen the effect components related to the changes of cell surface molecules CD41a and CD61,and chemical composition identification,as the potential effector components in the total flavonoid extracts of Sarcandra glabra were used to promote the differentiation of megakaryocytes,and finally the regression evaluation system was used to verify the efficacy of its medicinal effect.Results(1)Compared with the Dami group,the expression level of CD41a on the surface of Dami cells in the control group was significantly increased(P＜0.05).Compared with the control group,the expression levels of CD41a and CD61 on the surface of Dami cells in the PMA group were significantly increased(P＜0.01).Compared with the PMA group,the expression levels of CD41a and CD61 on the surface of Dami cells in the model group were significantly reduced(P＜0.01).(2)Compared with the blank plasma group,the expression levels of the molecules CD41a and CD61 on the surface of Dami cells at each time point of 15,30,60,90,120,and 240 minutes were significantly increased(P＜0.01),and the expression levels of CD41a and CD61 were both highest in the 30-minute group.The potential effective components with VIP value greater than 1 were screened out in the positive and negative ion mode,and 540.3638@12.25 and 559.2991@11.53 were selected for pharmacodynamic verification.559.2991@11.53 was identified as daucosterol(Dau),540.3638@12.25 was identified as rosmarinic acid 4-O-β-D-glucoside(Ros).After Ros and Dau intervened in the megakaryocyte differentiation and maturation disorder model respectively,the expression levels of CD41a and CD61 on the surface of Dami cells in the low-,medium-and high-dose groups(40,60 and 80 μg·mL-1)of Ros and Dau were significantly increased compared with the model group(P＜0.05,P＜0.01).Conclusion Ros and Dau may be the active components of the total flavonoids extracts of Sarcandra glabra to promote the differentiation of megakaryocytes.

The role of CREG1 in megakaryocyte maturation and thrombocytopoiesis.

Abnormal megakaryocyte maturation and platelet production lead to platelet-related diseases and impact the dynamic balance between hemostasis and bleeding. Cellular repressor of E1A-stimulated gene 1 (CREG1) is a glycoprotein that promotes tissue differentiation. However, its role in megakaryocytes remains unclear. In this study, we found that CREG1 protein is expressed in platelets and megakaryocytes and was decreased in the platelets of patients with thrombocytopenia. A cytosine arabinoside-induced thrombocytopenia mouse model was established, and the mRNA and protein expression levels of CREG1 were found to be reduced in megakaryocytes. We established megakaryocyte/platelet conditional knockout (Creg1pf4-cre) and transgenic mice (tg-Creg1). Compared to Creg1fl/fl mice, Creg1pf4-cre mice exhibited thrombocytopenia, which was mainly caused by inefficient bone marrow (BM) thrombocytopoiesis, but not by apoptosis of circulating platelets. Cultured Creg1pf4-cre-megakaryocytes exhibited impairment of the actin cytoskeleton, with less filamentous actin, significantly fewer proplatelets, and lower ploidy. CREG1 directly interacts with MEK1/2 and promotes MEK1/2 phosphorylation. Thus, our study uncovered the role of CREG1 in the regulation of megakaryocyte maturation and thrombopoiesis, and it provides a possible theoretical basis for the prevention and treatment of thrombocytopenia.

Xanthotoxin, a novel inducer of platelet formation, promotes thrombocytopoiesis via IL-1R1 and MEK/ERK signaling.

BACKGROUND: Thrombocytopenia is a common hematological disease caused by many factors. It usually complicates critical diseases and increases morbidity and mortality. The treatment of thrombocytopenia remains a great challenge in clinical practice, however, its treatment options are limited. In this study, the active monomer xanthotoxin (XAT) was screened out to explore its medicinal value and provide novel therapeutic strategies for the clinical treatment of thrombocytopenia. METHODS: The effects of XAT on megakaryocyte differentiation and maturation were detected by flow cytometry, Giemsa and phalloidin staining. RNA-seq identified differentially expressed genes and enriched pathways. The signaling pathway and transcription factors were verified through WB and immunofluorescence staining. Tg (cd41: eGFP) transgenic zebrafish and mice with thrombocytopenia were used to evaluate the biological activity of XAT on platelet formation and the related hematopoietic organ index in vivo. RESULTS: XAT promoted the differentiation and maturation of Meg-01 cells in vitro. Meanwhile, XAT could stimulate platelet formation in transgenic zebrafish and recover platelet production and function in irradiation-induced thrombocytopenia mice. Further RNA-seq prediction and WB verification revealed that XAT activates the IL-1R1 target and MEK/ERK signaling pathway, and upregulates the expression of transcription factors related to the hematopoietic lineage to promote megakaryocyte differentiation and platelet formation. CONCLUSION: XAT accelerates megakaryocyte differentiation and maturation to promote platelet production and recovery through triggering IL-1R1 and activating the MEK/ERK signaling pathway, providing a new pharmacotherapy strategy for thrombocytopenia.

The combination of machine learning and transcriptomics reveals a novel megakaryopoiesis inducer, MO-A, that promotes thrombopoiesis by activating FGF1/FGFR1/PI3K/Akt/NF-κB signaling.

Radiation-induced thrombocytopenia (RIT) occurs widely and causes high mortality and morbidity in cancer patients who receive radiotherapy. However, specific drugs for treating RIT remain woefully inadequate. Here, we first developed a drug screening model using naive Bayes, a machine learning (ML) algorithm, to virtually screen the active compounds promoting megakaryopoiesis and thrombopoiesis. A natural product library was screened by the model, and methylophiopogonanone A (MO-A) was identified as the most active compound. The activity of MO-A was then validated in vitro and showed that MO-A could markedly induce megakaryocyte (MK) differentiation of K562 and Meg-01 cells in a concentration-dependent manner. Furthermore, the therapeutic action of MO-A on RIT was evaluated, and MO-A significantly accelerated platelet level recovery, platelet activation, megakaryopoiesis, MK differentiation in RIT mice. Moreover, RNA-sequencing (RNA-seq) indicated that the PI3K cascade was closely related to MK differentiation induced by MO-A. Finally, experimental verification demonstrated that MO-A obviously induced the expression of FGF1 and FGFR1, and increased the phosphorylation of PI3K, Akt and NF-κB. Blocking FGFR1 with its inhibitor dovitinib suppressed MO-A-induced MK differentiation, and PI3K, Akt and NF-κB phosphorylation. Similarly, inhibition of PI3K-Akt signal pathway by its inhibitor LY294002 suppressed MK differentiation, and PI3K, Akt and NF-κB phosphorylation induced by MO-A. Taken together, our study provides an efficient drug discovery strategy for hematological diseases, and demonstrates that MO-A is a novel countermeasure for treating RIT through activation of the FGF1/FGFR1/PI3K/Akt/NF-κB signaling pathway.

Thrombopoietin-independent Megakaryocyte Differentiation of Hematopoietic Progenitor Cells from Patients with Myeloproliferative Neoplasms.

Primary hematopoietic stem and progenitor cell (HSPC)-derived megakaryocytes are a valuable tool for translational research interrogating disease pathogenesis and developing new therapeutic avenues for patients with hematologic disorders including myeloproliferative neoplasms (MPNs). Thrombopoietin (TPO)-independent proliferation and megakaryocyte differentiation play a central role in the pathogenesis of essential thrombocythemia and myelofibrosis, two MPN subtypes that are characterized by increased numbers of bone marrow megakaryocytes and somatic mutations in either JAK2, CALR, or MPL. However, current culture strategies generally use healthy HSPCs for megakaryocyte production and are not optimized for the investigation of TPO-independent or TPO-hypersensitive growth and megakaryocyte-directed differentiation of primary patient-derived HSPCs. Here, we describe a detailed protocol covering all necessary steps for the isolation of CD34+ HSPCs from the peripheral blood of MPN patients and the subsequent TPO-independent differentiation into CD41+ megakaryocytes using both a collagen-based colony assay and a liquid culture assay. This protocol provides a novel, reproducible, and cost-effective approach for investigating megakaryocyte growth and differentiation properties from primary MPN patient cells that can be easily adapted for research on other megakaryocyte-related disorders. This protocol was validated in: EMBO Rep (2022), DOI: 10.15252/embr.202152904 Graphical abstract Schematic representation of the isolation of CD34+ progenitor cells and subsequent TPO-independent megakaryocyte differentiation.

A Novel Antithrombocytopenia Agent, Rhizoma cibotii, Promotes Megakaryopoiesis and Thrombopoiesis through the PI3K/AKT, MEK/ERK, and JAK2/STAT3 Signaling Pathways.

BACKGROUND: Cibotii rhizoma (CR) is a famous traditional Chinese medicine (TCM) used to treat bleeding, rheumatism, lumbago, etc. However, its therapeutic effects and mechanism against thrombocytopenia are still unknown so far. In the study, we investigated the effects of aqueous extracts of Cibotii rhizoma (AECRs) against thrombocytopenia and its molecular mechanism. METHODS: Giemsa staining, phalloidin staining, and flow cytometry were performed to measure the effect of AECRs on the megakaryocyte differentiation in K562 and Meg-01 cells. A radiation-induced thrombocytopenia mouse model was constructed to assess the therapeutic actions of AECRs on thrombocytopenia. Network pharmacology and experimental verification were carried out to clarify its mechanism against thrombocytopenia. RESULTS: AECRs promoted megakaryocyte differentiation in K562 and Meg-01 cells and accelerated platelet recovery and megakaryopoiesis with no systemic toxicity in radiation-induced thrombocytopenia mice. The PI3K/AKT, MEK/ERK, and JAK2/STAT3 signaling pathways contributed to AECR-induced megakaryocyte differentiation. The suppression of the above signaling pathways by their inhibitors blocked AERC-induced megakaryocyte differentiation. CONCLUSIONS: AECRs can promote megakaryopoiesis and thrombopoiesis through activating PI3K/AKT, MEK/ERK, and JAK2/STAT3 signaling pathways, which has the potential to treat radiation-induced thrombocytopenia in the clinic.

Synthetic flavagline derivative 1-chloroacetylrocaglaol promotes apoptosis in K562 erythroleukemia cells through miR-17-92 cluster genes.

Chronic myeloid leukemia accounts for human deaths worldwide and could enhance sevenfold by 2050. Thus, the treatment regimen for this disorder is highly crucial at this time. Flavaglines are a natural class of cyclopentane benzofurans exhibiting various bioactivities like anticancer action. Despite the antiproliferative activity of flavaglines against diverse cancer cells, their roles and mechanism of action in chronic myeloid leukemia (CML) remain poorly understood. Thus, this study examines the antiproliferative effect of a newly synthesized flavagline derivative, 1-chloracetylrocaglaol (A2074), on erythroleukemia K562 cells and the zebrafish xenograft model. The study revealed that A2074 could inhibit proliferation, promote apoptosis, and boost megakaryocyte differentiation of K562 cells. This flavagline downregulated c-MYC and miR-17-92 cluster genes, targeting upregulation of the apoptotic protein Bcl-2-like protein 11 (BIM). The work uncovered a critical role of the c-MYC-miR-17-92-BIM axis in the growth and survival of CML cells.

Discovery of a novel megakaryopoiesis enhancer, ingenol, promoting thrombopoiesis through PI3K-Akt signaling independent of thrombopoietin.

Thrombocytopenia, a most common complication of radiotherapy and chemotherapy, is an important cause of morbidity and mortality in cancer patients. However, there are still no approved agents for the treatment of radiation- and chemotherapy-induced thrombocytopenia (RIT and CIT, respectively). In this study, a drug screening model for predicting compounds with activity in promoting megakaryocyte (MK) differentiation and platelet production was established based on machine learning (ML), and a natural product ingenol was predicted as a potential active compound. Then, in vitro experiments showed that ingenol significantly promoted MK differentiation in K562 and HEL cells. Furthermore, a RIT mice model and c-MPL knock-out (c-MPL-/-) mice constructed by CRISPR/Cas9 technology were used to assess the therapeutic action of ingenol on thrombocytopenia. The results showed that ingenol accelerated megakaryopoiesis and thrombopoiesis both in RIT mice and c-MPL-/- mice. Next, RNA-sequencing (RNA-seq) was carried out to analyze the gene expression profile induced by ingenol during MK differentiation. Finally, through experimental verifications, we demonstrated that the activation of PI3K/Akt signaling pathway was involved in ingenol-induced MK differentiation. Blocking PI3K/Akt signaling pathway abolished the promotion of ingenol on MK differentiation. Nevertheless, inhibition of TPO/c-MPL signaling pathway could not suppress ingenol-induced MK differentiation. In conclusion, our study builds a drug screening model to discover active compounds against thrombocytopenia, reveals the critical roles of ingenol in promoting MK differentiation and platelet production, and provides a promising avenue for the treatment of RIT.

TMEA, a Polyphenol in Sanguisorba officinalis, Promotes Thrombocytopoiesis by Upregulating PI3K/Akt Signaling.

Thrombocytopenia is closely linked with hemorrhagic diseases, for which induction of thrombopoiesis shows promise as an effective treatment. Polyphenols widely exist in plants and manifest antioxidation and antitumour activities. In this study, we investigated the thrombopoietic effect and mechanism of 3,3',4'-trimethylellagic acid (TMEA, a polyphenol in Sanguisorba officinalis L.) using in silico prediction and experimental validation. A KEGG analysis indicated that PI3K/Akt signalling functioned as a crucial pathway. Furthermore, the virtual molecular docking results showed high-affinity binding (a docking score of 6.65) between TMEA and mTOR, suggesting that TMEA might target the mTOR protein to modulate signalling activity. After isolation of TMEA, in vitro and in vivo validation revealed that this compound could promote megakaryocyte differentiation/maturation and platelet formation. In addition, it enhanced the phosphorylation of PI3K, Akt, mTOR, and P70S6K and increased the expression of GATA-1 and NF-E2, which confirmed the mechanism prediction. In conclusion, our findings are the first to demonstrate that TMEA may provide a novel therapeutic strategy that relies on the PI3K/Akt/mTOR pathway to facilitate megakaryocyte differentiation and platelet production.

Involvement of autophagy in diosgenin­induced megakaryocyte differentiation in human erythroleukemia cells.

Natural agents have been used to restart the process of differentiation that is inhibited during leukemic transformation of hematopoietic stem or progenitor cells. Autophagy is a housekeeping pathway that maintains cell homeostasis against stress by recycling macromolecules and organelles and plays an important role in cell differentiation. In the present study, an experimental model was established to investigate the involvement of autophagy in the megakaryocyte differentiation of human erythroleukemia (HEL) cells induced by diosgenin [also known as (25R)­Spirosten­5­en­3b­ol]. It was demonstrated that Atg7 expression was upregulated from day 1 of diosgenin­induced differentiation and was accompanied by a significant elevation in the conversion of light chain 3 A/B (LC3­A/B)­I to LC3­A/B­II. Autophagy was modulated before or after the induction of megakaryocyte differentiation using 3­methyladenine (3­MA, autophagy inhibitor) and metformin (Met, autophagy initiation activator). 3­MA induced a significant accumulation of the LC3 A/B­II form at day 8 of differentiation. It was revealed that 3­MA had a significant repressive effect on the nuclear (polyploidization) and membrane glycoprotein V [(GpV) expression] maturation. On the other hand, autophagy activation increased GpV genomic expression, but did not change the nuclear maturation profile after HEL cells treatment with Met. It was concluded that autophagy inhibition had a more prominent effect on the diosgenin­differentiated cells than autophagy activation.

Thrombopoietic effects of CCAAT/enhancer-binding protein ß on the early-stage differentiation of megakaryocytes.

CCAAT/enhancer-binding protein ß (C/EBPß) is a transcription factor that is involved in adipocytic and monocytic differentiation. However, the physiological role of C/EBPß in megakaryocytes (MKs) is not clear. In this study, we investigated the effects of C/EBPß on the early-stage differentiation of MKs, and explored the potential mechanisms of action. We established a cytosine arabinoside-induced thrombocytopenia mouse model using C57BL/6 mice. In the thrombocytopenia mice, the platelet count was found to be decreased, and the mRNA and protein expression levels of C/EBPß in MKs were also reduced. Furthermore, the maturation of Dami (MKs cell line) cells was induced by phorbol 12-myristate 13-acetate. When C/EBPß was silenced in Dami cells by transfection using C/EBPß-small interfering RNA, the expression of MKs-specific markers CD41 and CD62P, was dramatically decreased, resulting in morphological changes and differentiation retardation in low ploidy, which were evaluated using flow cytometry, real-time polymerase chain reaction, western blot, and confocal microscopy. The mitogen activated protein kinase-extracellular signal-regulated kinase signaling pathway was found to be required for the differentiation of MKs; knockdown of C/EBPß in MEK/ERK1/2 pathway attenuated MKs differentiation. Overexpression of C/EBPß in MEK/ERK1/2 pathway inhibited by U0126 did not promote MKs differentiation. To the best of our knowledge, C/EBPß plays an important role in MKs differentiation and polyploidy cell cycle control. Taken together, C/EBPß may have thrombopoietic effects in the differentiation of MKs, and may assist in the development of treatments for various disorders.

On the Quest for In Vitro Platelet Production by Re-Tailoring the Concepts of Megakaryocyte Differentiation.

The demand of platelet transfusions is steadily growing worldwide, inter-donor variation, donor dependency, or storability/viability being the main contributing factors to the current global, donor-dependent platelet concentrate shortage concern. In vitro platelet production has been proposed as a plausible alternative to cover, at least partially, the increasing demand. However, in practice, such a logical production strategy does not lack complexity, and hence, efforts are focused internationally on developing large scale industrial methods and technologies to provide efficient, viable, and functional platelet production. This would allow obtaining not only sufficient numbers of platelets but also functional ones fit for all clinical purposes and civil scenarios. In this review, we cover the evolution around the in vitro culture and differentiation of megakaryocytes into platelets, the progress made thus far to bring the culture concept from basic research towards good manufacturing practices certified production, and subsequent clinical trial studies. However, little is known about how these in vitro products should be stored or whether any safety measure should be implemented (e.g., pathogen reduction technology), as well as their quality assessment (how to isolate platelets from the rest of the culture cells, debris, microvesicles, or what their molecular and functional profile is). Importantly, we highlight how the scientific community has overcome the old dogmas and how the new perspectives influence the future of platelet-based therapy for transfusion purposes.

In vitro platelet production for transfusion purposes: Where are we now?

Over the last decade there has been a worldwide increase in the demand of platelet concentrates (PCs) for transfusion. This is, to a great extent, due to a growing and aging population with the concomitant increase in the incidence of onco-hematological diseases, which require frequent platelet (PLT) transfusions. Currently, PLTs are sourced uniquely from donations, and their storage time is limited only to a few days. The necessity to store PCs at room temperature (to minimize loss of PLT functional integrity), poses a major risk for bacterial contamination. While the implementation of pathogen reduction treatments (PRTs) and new-generation PLT additive solutions have allowed the extension of the shelf life and a safer PLT transfusion product, the concern of PCs shortage still pressures the scientific community to find alternative solutions with the aim of meeting the PLT transfusion increasing demand. In this concise report, we will focus on the efforts made to produce, in in vitro culture, high yields of viable and functional PLTs for transfusion purposes in a cost-effective manner, meeting not only current Good Manufacturing Practices (cGMPs), but also transfusion safety standards.

Nanocurcumin as a novel stimulator of megakaryopoiesis that ameliorates chemotherapy-induced thrombocytopenia in mice.

AIMS: Platelet production improvement can resolve concerns about the limitations of external platelet supply and platelet transfusion in thrombocytopenia patients. To this end, scientists encourage to induce the generation of megakaryocyte and platelet. Curcumin is a safe ingredient of turmeric that affects various cellular pathways. The effect of this component on platelet production has not been yet reported. MAIN METHODS: Our in vitro experiments include the investigation of the effects of nanocurcumin on megakaryocytes production from K562 cells and hematopoietic stem cells via megakaryocyte markers expression, DNA content, ROS, and morphologic analysis, and CFC assay. The regulatory functions of MAPKs pathways were also determined. In the in vivo study tissue distribution of nanocurcumin was determined and two treatment schedules were used to evaluate the capability of nanocurcumin to prevent the occurrence of Busulfan-induced thrombocytopenia in the mouse model. KEY FINDING: In vitro evidences demonstrated that nanocurcumin can induce MK production from K562 cells and hematopoietic stem cells. Inhibition of ERK1/2 and JNK pathways arrested this activity. In vivo experiments showed the uptake of nanocurcumin by tissues in mice. Administration of nanocurcumin could preserve bone marrow integrity and increase of the number of circulating platelets in the Busulfan-treated mice models. SIGNIFICANCE: Our results have demonstrated that nanocurcumin administration can be useful for the improvement of megakaryocytes and platelet generation in vitro. This component may be exerting these beneficial effects on megakaryopoiesis by modulating ERK1/2 and JNK pathways. As well as nanocurcumin has the potential to prevent thrombocytopenia in chemotherapy threated mice.

Study on apoptosis mechanism of leukemia cells induced by bisindolylmaleimide derivative L6 / 中草药

Objective: To investigate the effect of bisindolylmaleimide derivative L6 on inducing apoptosis of leukemia cells and its molecular mechanism. Methods: MTT assay was used to determinate the killing effect of L6 on HELL, K562, and KG1a cells. Flow cytometry was used to detect the effects of L6 on apoptosis, cell cycle, and differentiation of HEL cells. Western blotting was used to detect the expression of apoptosis-related protein. Finally, the effect of L6 on leukemia mouse was studied in vivo. Results: MTT assay showed that L6 had a stronger inhibitory activity against HEL, K562, and KG1a cell lines than the positive control PKC412 compound, with IC50 of (0.05 ± 0.03), (0.32 ± 0.01), and (0.19 ± 0.10) μmol/L, respectively. L6 could induce the apoptosis, G2/M arrest, megakaryocyte differentiation of HEL cells with a dose effect. Western blotting revealed that L6 mainly performed apoptosis by activating Caspase-3, which is an apoptotic executive protein. Hematoxylin-eosin (HE) staining of liver tissue of mice showed a reduction in HEL cell infiltration, but the more significant reduction in group L6 was observed, indicating that L6 could delay the metastasis of leukemia, and its effect was better than that of PKC412. Conclusion: Bisindolylmaleimide derivative L6 has a strong anti-leukemia activity, providing new hope for the development of new leukemia drugs.

Effects of bone marrow mesenchymal cells from immune thrombocytopenia patients on the biological behaviors of megakaryocytes / 上海交通大学学报（医学版）

Objective • To investigate changes of immune thrombocytopenia (ITP) patients-derived bone marrow mesenchymal cells (BMCs) in cells survival, cytokines expression as well as the effects of BMCs on the biological behaviors of megakaryocytes. Methods • BMCs were collected from 7 ITP patients and 5 normal controls (NC), and cultivated by the whole marrow adherent method. Surface markers and basal apoptosis rate of BMCs were analyzed by flow cytometry (FCM). Proliferation of BMCs was assessed by CCK-8 method. Phorbol 12-myristate 13-acetate (PMA) was used to stimulate differentiation of HEL cells. The induced HEL cells (inHEL) were divided into 3 groups: inHEL cultured alone (group a), inHEL co-cultured with BMCs derived from ITP patients (group b), inHEL co-cultured with BMCs derived from NC (group c). After 72 h incubation, the expression of cell surface proteins (CD41a, CD42b) and cell apoptosis rate were analyzed by FCM. The mRNA and proteins expression levels of cytokines IL6, IL11, TPO, SCF were detected by real-time fluorescent quantitative PCR (RT-qPCR) and enzyme linked immunosorbent assay (ELISA), respectively. Results • Compared with NC, BMCs from ITP patients grew progressively slowly (Day 4, P=0.039; Day 6, 10, P=0.009; Day 8, P=0.007), cell basal apoptosis rates were increased [AV+PI- (early apoptosis rate), P=0.036; AV+PI+ (late apoptosis rate), P=0.003; AV+PI-/+ (total apoptosis rate), P=0.004]. Compared with group a, the expression of CD41a in group c was much higher (P=0.000). The expression of CD41a in group b was higher than that in group a (P=0.015), but still much less than that in group c (P=0.000). Compared with group a, the early and total apoptosis rate in group b, c and the late apoptosis rate in group c were decreased obviously (all P=0.000), whereas there was no obvious change of the late apoptosis rate in group b. However, compared with group c, the late and total apoptosis rate in group b were significantly increased (both P=0.000). The expression levels of IL6, SCF mRNA and IL6 protein were significantly decreased in ITP BMCs (all P=0.000), but there was no obvious difference in the expression levels of IL11 and TPO between ITP BMCs and NC BMCs. Conclusion • BMCs from ITP patients show some defects in supporting megakaryocytic differentiation and survival under co-culture conditions, which mechanisms are related to the reduction of IL6 and SCF expression.

Effects of bone marrow mesenchymal cells from immune thrombocytopenia patients on the biological behaviors of megakaryocytes / 上海交通大学学报（医学版）

Objective·To investigate changes of immune thrombocytopenia (ITP) patients-derived bone marrow mesenchymal cells (BMCs) in cells survival, cytokines expression as well as the effects of BMCs on the biological behaviors of megakaryocytes. Methods?·?BMCs were collected from 7 ITP patients and 5 normal controls (NC), and cultivated by the whole marrow adherent method. Surface markers and basal apoptosis rate of BMCs were analyzed by flow cytometry (FCM). Proliferation of BMCs was assessed by CCK-8 method. Phorbol 12-myristate 13-acetate (PMA) was used to stimulate differentiation of HEL cells. The induced HEL cells (inHEL) were divided into 3 groups: inHEL cultured alone (group a), inHEL co-cultured with BMCs derived from ITP patients (group b), inHEL co-cultured with BMCs derived from NC (group c). After 72 h incubation, the expression of cell surface proteins (CD41a, CD42b) and cell apoptosis rate were analyzed by FCM. The mRNA and proteins expression levels of cytokines IL6, IL11, TPO, SCF were detected by real-time fluorescent quantitative PCR (RT-qPCR) and enzyme linked immunosorbent assay (ELISA), respectively. Results?·?Compared with NC, BMCs from ITP patients grew progressively slowly (Day 4, P=0.039; Day 6, 10, P=0.009; Day 8, P=0.007), cell basal apoptosis rates were increased [AV+PI- (early apoptosis rate), P=0.036; AV+PI+(late apoptosis rate), P=0.003; AV+PI-/+(total apoptosis rate), P=0.004]. Compared with group a, the expression of CD41a in group c was much higher (P=0.000). The expression of CD41a in group b was higher than that in group a (P=0.015), but still much less than that in group c (P=0.000). Compared with group a, the early and total apoptosis rate in group b, c and the late apoptosis rate in group c were decreased obviously (all P=0.000), whereas there was no obvious change of the late apoptosis rate in group b. However, compared with group c, the late and total apoptosis rate in group b were significantly increased (both P=0.000). The expression levels of IL6, SCF mRNA and IL6 protein were significantly decreased in ITP BMCs (all P=0.000), but there was no obvious difference in the expression levels of IL11 and TPO between ITP BMCs and NC BMCs. Conclusion?·?BMCs from ITP patients show some defects in supporting megakaryocytic differentiation and survival under co-culture conditions, which mechanisms are related to the reduction of IL6 and SCF expression.