Bu-Shen-Zhu-Yun decoction induces PRLR deubiquitination and JAK2/STAT5 activation via CSN5 in vitro.

PURPOSE: To determine the effect of Bu-Shen-Zhu-Yun Decoction (BSZY-D) on the kisspeptin through JAK2/STAT5 signaling pathway in hyperprolactinemia (HPRL) infertility. METHOD: SD rats were treated with BSZY-D for cerebrospinal fluid (CSF) extraction. GT1-7 cells were subjected to different treatments. The phosphorylation levels of JAK2 and STAT5, and the expressions of PRLR and kisspeptin of GT1-7 cells in different groups were detected by western blot, RT-qPCR and immunofluorescence. The expressions of CSN5 and GATA1 and other molecular features were checked by western blot, RT-PCR, co-immunoprecipitation and renilla luciferase activity. RESULTS: The phosphorylation levels of JAK2 and STAT5, and the expressions of PRLR and kisspeptin in the HPRL group were significantly decreased, and these changes could be reversed after BSZY-D treatment. In addition, the presence of PRLR deubiquitination was detected in the HPRL group, which could be reversed by shRNA-CSN5, suggesting that BSZY-D played a role through targeting CSN5. The binding level of GATA1 and CSN5 promoter in the HPRL group was significantly decreased, but elevated in the HPRL (BSZY-D/CSF) group (P < 0.05). CONCLUSION: BSZY-D improved the transcription activity of GATA1 and increased the binding of GATA1 and CSN5. BSZY-D was involved in the deubiquitination of PRLR, which contributes to alleviating the symptoms of HPRL infertility.

Ginseng-Derived Panaxadiol Saponins Promote Hematopoiesis Recovery in Cyclophosphamide-Induced Myelosuppressive Mice: Potential Novel Treatment of Chemotherapy-Induced Cytopenias.

OBJECTIVE: To investigate the potential efficacy of panaxadiol saponins component (PDS-C), a biologically active fraction isolated from total ginsenosides, to reverse chemotherapy-induced myelosuppression and pancytopenia caused by cyclophamide (CTX). METHODS: Mice with myelosuppression induced by CTX were treated with PDS-C at a low- (20 mg/kg), moderate- (40 mg/kg), or high-dose (80 mg/kg) for 7 consecutive days. The level of peripheral white blood cell (WBC), neutrophil (NEU) and platelet (PLT) were measured, the histopathology and colony formation were observed, the protein kinase and transcription factors in hematopoietic cells were determined by immunohistochemical staining and Western blot. RESULTS: In response to PDS-C therapy, the peripheral WBC, NEU and PLT counts of CTX-induced myelosuppressed mice were significantly increased in a dose-dependent manner. Similarly, bone marrow histopathology examination showed reversal of CTX-induced myelosuppression with increase in overall bone marrow cellularity and the number of hematopoietic cells (P<0.01). PDS-C also promoted proliferation of granulocytic and megakaryocyte progenitor cells in CTX-treated mice, as evidenced by significantly increase in colony formation units-granulocytes/monocytes and -megakaryocytes (P<0.01). The enhancement of hematopoiesis by PDS-C appears to be mediated by an intracellular signaling pathway, this was evidenced by the up-regulation of phosphorylated mitogen-activated protein kinase (p-MEK) and extracellular signal-regulated kinases (p-ERK), and receptor tyrosine kinase (C-kit) and globin transcription factor 1 (GATA-1) in hematopoietic cells of CTX-treated mice (P<0.05). CONCLUSIONS: PDS-C possesses hematopoietic growth factor-like activities that promote proliferation and also possibly differentiation of hematopoietic progenitor cells in myelosuppressed mice, probably mediated by a mechanism involving MEK and ERK protein kinases, and C-kit and GATA-1 transcription factors. PDS-C may potentially be a novel treatment of myelosuppression and pancytopenia caused by chemotherapy.

Effects of arsenic disulfide on apoptosis, histone acetylation, toll like receptor 2 activation, and erythropoiesis in bone marrow mononuclear cells of myelodysplastic syndromes patients in vitro.

OBJECTIVE: As the main component of traditional Chinese medicine realgar, arsenic disulfide (As2S2) is widely used in treating myelodysplastic syndromes (MDS). The goal of the current study is to assess the effects of As2S2 on bone marrow mononuclear cells (BMMNC) of MDS. METHODS: BMMNCs were obtained from 10 lower risk MDS patients, 5 higher risk MDS patients, and 3 healthy controls. Then, the cells were treated with As2S2 for 48h, using vorinostat (also known as SAHA) as control. Cell proliferation and apoptosis were detected. mRNA and protein levels of histone deacetylase-1 (HDAC1), Toll-like receptor 2 (TLR2), and erythroid transcription factor (GATA-1) were detected by quantitative real-time PCR and western blot analysis. RESULTS: After As2S2 treatment in concentrations ranging from 3.125 to 100µmol/L, cell proliferation was inhibited in both lower risk and higher risk MDS. Fifty percent inhibitory concentrations were 24.4µmol/L and 23.6µmol/L, respectively, for lower and higher risk MDS. Apoptotic cells significantly increased in both types of MDS. mRNA and protein levels of HDAC1 and TLR2 were reduced, whereas GATA-1 was increased in both types of MDS. CONCLUSIONS: As2S2 could inhibit cell proliferation and induce apoptosis through histone acetylation modulation in MDS. Similar to SAHA, As2S2 could reduce TLR2 activation and increase GATA-1 expression. Current data suggest epigenetic and immunological alternations are involved in therapeutic mechanisms of realgar in the treatment of MDS.

Herbal drug ninjin'yoeito accelerates myelopoiesis but not erythropoiesis in vitro.

Some Kampo medicines that are herbal and traditional in Japan have had beneficial effects when given to patients with anemia. However, molecular mechanisms underlying their effects are unclear. To address this question, four Kampo medicines used to treat anemia-ninjin'yoeito (NYT), shimotsuto (SMT), juzentaihoto (JTT), and daibofuto (DBT)-were tested separately using in vitro cultures of mouse bone marrow mononuclear cells. Among them, NYT was most effective in stimulating cell proliferation and up-regulating Myc expression. Flow cytometry analysis indicated that, among hematopoietic components of those cultures, myeloid cells expressing CD45/Mac-1/Gr-1/F4/80 increased in number, but Ter119/CD71 erythroid cells did not. Accordingly, real-time PCR analysis showed up-regulation of the myeloid gene Pu.1, whereas the erythroid genes Gata1 and Klf1 were down-regulated. Overall, these findings provide molecular evidence that NYT accelerates myelopoiesis but not erythropoiesis in vitro.

Omega-3 fatty acids suppress Th2-associated cytokine gene expressions and GATA transcription factors in mast cells.

Because the interaction between omega-3 fatty acids and mast cells has remained largely unknown in allergies, we investigated whether omega-3 fatty acids affect the activation of mast cells by examining Th2-associated cytokine production and possible molecular mechanisms. Alpha-linolenic acid and its metabolites including eicosapentaenoic acid and decosahexaenoic acid induced a dramatic decrease in the production of interleukin (IL)-4, IL-5 and IL-13 in a dose-dependent manner, as well as mRNA expression of their genes, in activated MC/9 mast cells and bone marrow-derived mast cells. The effects were comparable to those of cyclosporin A (1 µM), a well-known immunosuppressive agent. Nuclear expression of GATA binding protein-1 (GATA-1) and GATA binding protein-2 (GATA-2), essential transcription factors for mast cell activation, was also greatly suppressed. However, their mRNA expressions were not affected. In P815 mast cells, which do not express GATA-1, the suppressive effects on cytokines were abolished. On the contrary, omega-3 fatty acids had less significant effects on IL-4 and IL-5 and resulted in a slight decrease in IL-13 production in EL-4 T cells. Finally, oral administration of fish oil containing high level of omega-3 fatty acids significantly reduced the severity of dermatitis and the thickening of epidermis/dermis in a NC/Nga murine atopic model. The number of cells expressing CD117(+) and FcεRIα(+) was greatly decreased and GATA-1 expression in the cells was also diminished. Taken together, omega-3 fatty acids might target mast cells to a greater extent than T cells to suppress Th2 cytokine expression by inhibiting GATAs for alleviation of allergic disease.

Ginger stimulates hematopoiesis via Bmp pathway in zebrafish.

BACKGROUND: Anemia is a hematologic disorder with decreased number of erythrocytes. Erythropoiesis, the process by which red blood cells differentiate, are conserved in humans, mice and zebrafish. The only known agents available to treat pathological anemia are erythropoietin and its biologic derivatives. However, erythropoietin therapy elicits unwanted side-effects, high cost and intravenous or subcutaneous injection, warranting the development of a more cost effective and non-peptide alternative. Ginger (Zingiber officinale) has been widely used in traditional medicine; however, to date there is no scientific research documenting the potential of ginger to stimulate hematopoiesis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we utilized gata1:dsRed transgenic zebrafish embryos to investigate the effect of ginger extract on hematopoiesis in vivo and we identified its bioactive component, 10-gingerol. We confirmed that ginger and 10-gingerol promote the expression of gata1 in erythroid cells and increase the expression of hematopoietic progenitor markers cmyb and scl. We also demonstrated that ginger and 10-gingerol can promote the hematopoietic recovery from acute hemolytic anemia in zebrafish, by quantifying the number of circulating erythroid cells in the dorsal aorta using video microscopy. We found that ginger and 10-gingerol treatment during gastrulation results in an increase of bmp2b and bmp7a expression, and their downstream effectors, gata2 and eve1. At later stages ginger and 10-gingerol can induce bmp2b/7a, cmyb, scl and lmo2 expression in the caudal hematopoietic tissue area. We further confirmed that Bmp/Smad pathway mediates this hematopoiesis promoting effect of ginger by using the Bmp-activated Bmp type I receptor kinase inhibitors dorsomorphin, LND193189 and DMH1. CONCLUSIONS/SIGNIFICANCE: Our study provides a strong foundation to further evaluate the molecular mechanism of ginger and its bioactive components during hematopoiesis and to investigate their effects in adults. Our results will provide the basis for future research into the effect of ginger during mammalian hematopoiesis to develop novel erythropoiesis promoting agents.

Sequential chromatin immunoprecipitation protocol: ChIP-reChIP.

Chromatin immunoprecipitation has been widely used to determine the status of histone covalent modifications and also to investigate DNA-protein and protein-protein associations to a particular genomic location in vivo. Generally, DNA regulatory elements nucleate the interaction of several transcription factors in conjunction with ubiquitous and/or tissue-specific cofactors in order to regulate gene transcription. Therefore, it has become relevant to determine the cohabitation of several proteins in a particular developmental stage and cell type. Furthermore, multiple post-translational histone modifications can be analyzed on the same genomic location with the aim of deciphering the combinatorial pattern of histone modifications associated to specific transcriptional stages during cell commitment. Here we describe the ChIP-reChIP assay that represents a direct strategy to determine the in vivo colocalization of proteins interacting or in close contact in a chromatinized template on the basis of double and independent rounds of immunoprecipitations with high-quality ChIP grade antibodies.

[Up-regulation of transcription factors GATA-1 and GATA-2 induced by Panax notoginosides in hematopoietic cells].

OBJECTIVE: To observe the role of Panax notoginosides (PNS) in up-regulation of GATA family transcription factors, and explore intracellular signal pathway of PNS in the proliferation of hematopoietic cells. METHODS: Human bone marrow cells were incubated with different concentrations of PNS for colony-forming assay. Human cell lines HL-60, K562, CHRF-288 and Meg-01 were incubated with PNS (10 mg/L) for 14 days. The cell nuclear proteins were extracted and analyzed by Western blot with antibodies against GATA-1, GATA-2. Electrophoretic mobility shift assay (EMSA) and antibody gel supershift assay was performed using (32)P labeled GATA consensus oligonucleotide which contains binding site for GATA transcription factors. RESULTS: PNS could promote the proliferation of CFU-GM and CFU-E and induce the expression of GATA-1, GATA-2. The nuclear proteins of both GATA-1 and GATA-2 in K562, CHRF-288 and Meg-01 cells treated by PNS were increased by (1.5 - 2.8) and (2.0 - 3.1)-fold over untreated cells respectively. GATA binding activity initiated by PNS was apparently elevated to form higher density band of GATA-DNA complex. While there was no detectable change in HL-60 cells before and after PNS treatment. The predominant GATA binding complex was mainly attributable to both GATA-1 and GATA-2 proteins being in phosphorylated status. CONCLUSION: PNS can induce the synthesis of transcription factors GATA-1 and GATA-2 and enhance their DNA binding activity, which could play a role in the up-regulation of the expression genes related to proliferation and differentiation in hematopoietic cells.