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[ ABSTRACT] AIM:To observe the effects of total saponins of Panax ginseng ( TSPG) on the promotion of hema-topoiesis and to explore the underlying mechanism in treating aplastic anemia ( AA) in a mouse model.METHODS:For preparation of AA model, BALB/c mice were exposed to sublethal dose of 5.0 Gyγradiation, followed by transplantation of lymphocytes from DBA/2 donor mice.The experiment was divided into 6 groups, including normal control group, AA model group, TSPG treatment groups with low, medium and high doses, and positive control group with cyclosporine A ( CsA) .Both TSPG and CsA were administered by gastrogavage.After 15 d treatment, the peripheral hemogram was test-ed, the cytokine contents in serum were measured, and bone marrow semisolid culture of colony-forming assay was conduc-ted for determining hematopoietic progenitor cells.The protein levels of extracellular signal-regulated kinase 1/2 ( ERK1/2) and its phosphorylation status in the bone marrow cells were determined.RESULTS:Curative effect of TSPG in trea-ting AA mice was satisfactory.Peripheral counts of white blood cells and platelet, and concentration of hemoglobin in TSPG groups with medium and high doses were significantly higher than those in model control.TSPG increased the colony num-bers of CFU-E, BFU-E, CFU-GM and CFU-MK derived from hematopoietic progenitor cells.Meanwhile, up-regulation of the phosphorylated ERK1/2, decreased contents of Th1 cytokines and increased contents of Th2 cytokines in the serum were observed.CONCLUSION:TSPG possess the dual efficacies on the promotion of hematopoiesis and immunoregulation in AA mice by regulating the expression of Th1/Th2/Th17 cytokines and increasing the phosphorylation of ERK1/2.
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Aim To observe mesenchymal stem cell differentiation into osteoblast induced by TSPG, and explore how TSPG enhances the promotion of hemato-poiesis of osteoblast differentiation from mesenchymal stem cell. Methods MSCs were cultured by TSPG combined with osteoinductive medium. The cellular vi-ability of proliferation was detected with MTT assay. The content of alkaline phosphatase in the cultural su-pernatant was tested with pNPP assay. The ability of MSCs to form calcium nodes was also observed after a-lizarin red stain. The protein expression of RUNX2 was analyzed with Western blot. The content of cytokines associated with hematopoiesis was tested with Elisa as-say. The ability of promoting hematopoiesis was detec-ted with hematopoietic colony forming assay. Results Both MTT and pNPP assay showed that optical den-sity ( OD) values were increased in response to TSPG treatment in a dose-dependent manner. The mineraliza-tion formation ability was enhanced with TSPG-treat-ment. Meanwhile, the expression of RUNX2 protein was up-regulated in TSPG-treated cell. Moreover, the content of cytokines associated with hematopoiesis and the number of hematopoietic progenitor colony were in-creased by TSPG-treatment compared with the control group. Conclusion TSPG could induce MSCs differ-entiation in to osteoblast and enhance the effect of oste-oblast differentiation from MSCs on promoting hemato-poiesis.
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This study was aimed to optimize the extraction process of ginseng in Shen-Qi Bu-Qi (SQBQ) granules. The orthogonal experiment method was used to optimize the extraction process of ginseng adopting ethanol concentration, ethanol volume, extraction time and extraction times as factors. The content of ginsenoside Rg1, Re, Rb1 content in the extract was determined by HPLC. And the optimum extraction conditions were determined with indexes of three kinds of saponin yield. The results showed that the optimum extraction process of ginseng for SQBZ granules was that adding 6 times amount of 60% ethanol technology for medicinal materials, extract for 2 times, and 3 h for each time. It was concluded that the optimization of ginseng extraction process was stable, reasonable and feasible with high extraction rate.
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Objective:To explore the effects of total saponins of panax ginseng ( TSPG) combined with icariin ( ICA) on learning and memory capability, oxygen free radical and apoptosis of hippocampal CA1 pyramidal cells in vascular dementia ( VD) rats. Meth-ods:The VD rats were obtained by occlusion of bilateral common carotid artery and reperfusion repeatedly combined with intraperitoneal injection of sodium nitroprusside. Spacial learning and memory ability was evaluated by 8-arm radial electric maze test on the 21st day af-ter the drug treatment, the activity of SOD and the level of MDA in the brain were determined, and the adjacent sections were used to de-tect fragmented DNA in apoptotic cells( TUNEL) . Results:The learning and memory ability of the model rats judged by the 8-arm radial electric maze test was significantly decreased compared with that in the sham-operated group (P <0.05). The combination treatment could significantly protect and improve all the evaluated indices of the model rats after the 3-week treatment (P<0. 05) with increased SOD (P<0. 05), and decreased MDA (P<0. 01) and TUNEL positive cells (P<0. 05). Conclusion:The combination drug treatment can significantly protect and improve the spacial learning and memory ability of VD rats, which is related with such effects as improving oxygen free radical metabolism and nerve cell injure in hippocampal CA1 region.
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Objective To study the effects of TSPG on apoptosis of K562 cells and the probable mechanism involved. Methods MTT was used to investigate the proliferation of K562 cells;Flow cytometry(FCM) was used to investigate the effects of TSPG on apoptosis of K562 cells; The expression of Survivin in K562 cells treated with differ-ent concentraction of TSPG were examined by RT-PCR. Results The growth of K562 cells was inhibited by TSPG in the concentration dependent manner (P < 0.05). FCM showed that the apoptosis rates of cells in 100 μg/L (23.78%) and 200μg/L TSPG group(33.98%) were higher than those in 101μg/L TSPG group(16.67%), with significant difference(P <0.01 or P < 0.05). The expression rates of Survivin were decreased by the treatment with the increasing concentrations of TSPG (P < 0.05). Conclusion TSPG can restrain the human leukemic cell growth and induce cell apoptosis,which may be related to the decreased expressions of Survivin.
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Objective To investigate the effects of total saponins of Panax ginseng(TSPG) combined with erythropoietin(EPO) or granulocyte macrophage colony stimulating factor(GM CSF) on the differentiation of K562 cells. Methods Cells were cultured in vitro . Morphologic changes of cells were observed with light microscope and electron microscope. The levels of hemoglobin, peroxidase, non specific lipase ? NAE in cells were detected respectively by benzidine staining, peroxidase(POX) staining and ? NAE cytochemical staining. Levels of cell surface differentiation antigens CD15, HIR2, EPO R and GM CSF R were determined by immunocytochemistry. Results TSPG combined with EPO or GM CSF resulted in more mature morphological features of K562, increased expressions of hemoglobin and myeolperoxidase in K562 cells, and enhanced expressions of cell surface differentiation antigens CD15, HIR2, EPO R and GM CSF R. Conclusion TSPG combined with cytokine EPO or GM CSF can induce more mature differentiation of K562 cells.
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Objective To investgate the signal transduction and regulation in erythropoiesis by total saponins of Panax ginseng(TSPG) to clarify the mechanism for Panax Ginseng promoting hematopoiesis.Methods The colony-forming ability of mononuclear cells(MNCs) from human umbilical cord blood was assayed in methylcellulose assay systems,various dilutions of TSPG(0,10-100mg/L) were then added to each methylcellulose culture mixture;MNC proliferation in response to erythropoietin(Epo) was measured by MTT;To investigate whether the preferential differentiation of MNCs into erythroid lineage cells was caused by an increase in the expression of erythropoietin receptor(EpoR) on the cells,immunoblotting analyses were performed;Immunoprecipitation of JAK_2/STAT_5 was used for the effect of TSPG on Epo/EpoR-induced tyrosine phosphorylation of JAK_2/STAT_5. Results 1.TSPG(10-75mg/L) can promote the colony formation of BFU-E,CFU-E,and the preferential differentiation into erythroid lineage cells was induced from 25mg/L of TSPG;2.Using the colorimetric MTT assay,MNCs exhibited proliferous responses to Epo(2?10~3-5?10~4IU/L) reaching maximum at 5?10~3IU/L Epo;3.The addition of TSPG did not increase the expression of EpoR;4.After MNCs were incubated in the presence of with or without TSPG for 24 h,the cells were stimulated with Epo for 0,2,5,30min,and tyrosine phosphorylation JAK_2/STAT_5 was measured.The pretreatment with TSPG enhanced Epo-induced tyrosine phosphorylation of JAK_2 and STAT_5(STAT_(5a) and STAT_(5b)).Conclusion In the present study,we have shown that the Epo/EpoR-mediated signals are enhanced,and JAK_2/STAT_5 signals plays an essential role in the effect of TSPG on erythropoiesis,whereas the expression level of EpoR remains unchanged.
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Object To study the effect of cryopreservation on the ability of hematopoietic stem cells from human bone marrow and the action of total saponins of Panax ginseng (TSPG) to reduce their freezing injury. Methods Gradual cooling methods were used to place mononuclear cells (MNC) in liquid nitrogen with 5% dimethylsulfoxide (DMSO) for 3—5 months. After thawed, the biological properties of MNC were monitored, which included the mean trypan blue exclusion rate and the mean recovery rate of MNC, CFU-Mix, CD34+ cell, and total cells; Thawed MNC were cultured with various concentrations of TSPG, the effect of TSPG on the recoverable ability from cryopreservation damage were detected by colony-forming assay, colorimetric MTT assay for cell proliferation and flow cytometry. Results A fraction of MNC lost their proliferatory ability after thawed, but the damage was not deteriorated with freezing time. TSPG (25 ?g/mL) could raise the colony production rate of thawed hematopoietic stem cells; TSPG (25—50 ?g/mL) could raise the colony production rate of thawed hematopoietic stem cells; TSPG (25—50 ?g/mL) could improve their proliferation; TSPG (25 ?g/mL) could also promote the entray of them into cell proliferatory cycle. Conclusion TSPG could induce the proliferation of thawed hemato- poietic stem cells and raise the post-freezing recoverable ability of hematopoietic stem cells.
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Object To clarify the mechanism for total saponins of Panax ginseng C.A.Mey. (TSPG) inducing K562 cells to apoptosis, and to provide the theoretical basis and the experimental evidence of TSPG's clinical application. Methods By using in vitro cell culture, morphometry, flow cytometry, morphological investigation and immunocytochemistry, the effects of TSPG on apoptosis in K562 cells were studied. Results The results indicated that TSPG could inhibit the proliferation of K562 cells significantly, and induce K562 cells to apoptosis. It was also showed in the experiments that after induced by TSPG, the ratio of positive K562 cells expressing C-MYC and BCL-2 is decreased. Conclusion The mechanism for TSPG to induce K562 cells to apoptosis may be related to the expression of oncogene in K562 cells.
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Objective To clarify the mechanisms of total saponins of panax ginseng (TSPG) in induction of apoptosis of K562 cells and to provide the theoretical basis and the experimental evidence for its clinical application. Methods The effects of TSPG on apoptosis of K562 cells were studied by morphometry, flow cytometry, morphological observation, and immunocytochemistry. Results The results indicated that TSPG could markedly inhibit the proliferation of K562 cells and induce the apoptosis of K562 cells. Our experiment also showed that after induction by TSPG, the ratio of positive K562 cells with Fas expression increased. Conclusion The mechanisms of TSPG in the induction of K562 cell apoptosis may be related to the increased expression of Fas in K562 cells.
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Objective:To explore the effect of TSPG (total saponins of panax ginseng) on gene expression of IL-3 which is one of the important hematopoietic growth factor at early stage of hematopoiesis.Methods:Techniques of RT-PCR and in situ hybridization were used.Results:A band corresponding to IL-3 was observed after RNA extraction from TSPG stimulated Jurkat cells were subjected to RT-PCR,while such band was not observed after RNA extraction from unstimulated Jurkat cells were subjected to RT-PCR.Comparing with the control group,both quantity and intensity of IL-3mRNA expression in fetus thymocytes and splenocytes induced by TSPG were obviously promoted.Conclusion:TSPG can induce human lymphocytes to express IL-3 mRNA,thus promote human hematopoiesis at early stage.
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Objective: To study the relationship between effect of total saponins of Panax ginseng(TSPG) on expression of GM-CSF in human bone marrow stromal cells and modulation of human granulocytopoiesis and monocytopoiesis.Methods:The techniques of culture of hematopoietic progenitor cells and human bone marrow stromal cells in vitro,immunocytochemistry, nucleic acid in situ hybridization were used.Results:TSPG could markedly promote the colony formation of CFU-GM; human bone marrow stromal cells conditioned media prepared with TSPG could significantly enhance the proliferation and differentiation of CFU-GM,and expression of GM-CSF in protein and mRNA level in human bone marrow stromal cells induced by TSPG were much higher than those of the control group.Conclusion:TSPG may directly and/or indirectly promote stromal cells in hematopoietic inductive microenvironment to produce GM-CSF, which further prowote the proliferation and differentiation of human CFU-GM.
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Objective Panax ginseng is a well-known Chinese traditional medicine functional in “invigorating qi”,and saponins are one of its main effective fractions.Our study investigates the effect of total saponins of panax ginseng(TSPG)on human hematopoiesis at early stage and its possible regulative mechanism to clarify the hematonic mechanism of Panax ginseng. Methods The techniques of culturing hematopoietic progenitor cells in vitro,bioassay of hematopoietic growth factor(HGF),immunocytochemistry and nucleic acid in situ hybridization were used to study the effect of TSPG on IL-3’s expression in hematopoietic stromal cell and its possible mechanism. Results TSPG directly added into culture system in vitro can markedly increase the colony forming yields of CFU-Mix;the different conditioned culture media prepared with TSPG can promote the proliferation and differentiation of CFU-Mix;the protein and mRNA expression of IL-3 in BMSC and cells of EcV304,THP induced by TSPG has been much intensified.Conclusion TSPG may regulate hematopoiesis at early stage by activating IL-3's gene expression in hematopoietic stromal cell,which is the most important element in hematopietic inductive microenvironment(HIM).