Isolation of (+)-catechin and (-)-epicatechin from Actinidia arguta as bone marrow cell proliferation promoting compounds

The MeOH extract of stems of Actinidia arguta promoted proliferation of cultured bone marrow cells and stimulated formation of myeloid colonies from bone marrow cells. (+)-Catechin ( 1) and (-)-epicatechin ( 2) were isolated as active compounds from the MeOH extract. Compounds 1 and 2 stimulated the cell proliferation in a concentration-dependent manner in the range of 1 to 100 mg/mL. Compounds 1 and 2 also stimulated formation of myeloid colonies and enhanced the effect of interleukin-3 (IL-3) to increase the number of colony forming-units in culture (CFU-c). In an ex vivo experiment using a model mouse of decreasing bone marrow functions, orally administrated 1 (100 mg/kg/day) stimulated IL-3-induced CFU-c formation of the bone marrow cells.     

Effects of a selective cyclooxygenase-2 inhibitor, celecoxib, on bone resorption and osteoclastogenesis in vitro

The effects of an important new anti-inflammatory agent, the selective cyclooxygenase-2 inhibitor celecoxib, on bone resorption and osteoclastogenesis elicited by the inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), the endotoxin lipopolysaccharide (LPS), and the systemic hormones 1alpha,25-dihydroxyvitamin D(3) and parathyroid hormone were examined in vitro. Bone resorption was evaluated by measuring calcium released into the culture medium in a neonatal mouse calvarial bone organ culture. Osteoclastogenesis was evaluated by measuring tartrate-resistant acid phosphatase activity in the cells in cocultures of bone marrow cells and osteoblastic cells and in macrophage-colony-stimulating factor-dependent bone marrow cell cultures. Celecoxib (0.1 microM) completely inhibited the calcium release induced by IL-1beta, TNF-alpha, and LPS. The resorptive effect of 1alpha,25-dihydroxyvitamin D(3) was inhibited partially by celecoxib. In contrast, celecoxib did not inhibit the calcium release elicited by parathyroid hormone or prostaglandin E(2). Celecoxib (0.1 microM) also markedly inhibited osteoclastogenesis induced by these stimulators of bone resorption except for PGE(2) in the coculture system, whereas it failed to inhibit osteoclastogenesis in macrophage-colony-stimulating factor-dependent bone marrow cell cultures. These results indicate that, under certain conditions, cyclooxygenase-2-dependent prostaglandin synthesis is critical for the bone resorption induced by IL-1beta, TNF-alpha, and LPS, and for the osteoclastogenesis induced by these pro-inflammatory molecules and calciotropic hormones. The prevention of prostaglandin synthesis by inflammatory cytokines in bone cells could contribute to the efficacy of celecoxib in preventing bone loss in rheumatoid arthritis.     

Mercury toxicity to hemopoietic and tumor colony-forming cells and its reversal by selenium in vitro.

Inorganic and organic mercury, in micromolar concentrations, inhibited colony formation in primary cultures of mouse bone marrow and in P815 mouse mastocytoma cultures. When selenium, in the form of selenous acid, was added to cell cultures, it was able to prevent the inhibition of colony formation caused by continuous exposure to inorganic mercury in P815 cell cultures and primary cultures of bone marrow. Selenite was also able to prevent colony inhibition resulting from continuous exposure to methylmercury in P815 cell cultures, but failed to prevent colony inhibition in primary cultures of bone marrow. In consideration of the overall effect of methylmercury, the bone marrow is a potential site of toxicity.     

Enhancement of interleukin 1 and interleukin 2 releases by ubenimex

The effect of ubenimex on the release of interleukin 1 (IL-1) and interleukin 2 (IL-2) from immuno-competent cells was studied. Ubenimex enhanced release of IL-1 from mouse peritoneal macrophages at 1.0 and 100 micrograms/ml in vitro and the release at 1.0 microgram/ml was larger. When ubenimex was administered to mice IL-1-releasing activity of the peritoneal macrophages was enhanced 3 and 5 days after the administration but not enhanced 1 day after the administration. Ubenimex also enhanced IL-2 release from rat spleen cells at 0.1 and 10 micrograms/ml, when concanavalin A (Con A) was added in the IL-2-releasing system. The enhancement was still observed with mouse spleen cells, when serum was further added. Moreover, thymocyte-proliferating activity was attained in the broths which rat spleen cells were incubated with ubenimex from 0.1 to 10 micrograms/ml in the absence and presence of Con A.     

Effects of neopterin on the hematopoietic microenvironment of senescence-accelerated mice (SAM)

The pteridine neopterin (NP) is produced by monocytes and is known to be a useful marker of immunological activation, although, it remains elusive whether neopterin itself exhibits biological functions. Recently, we found that NP stimulates hematopoietic cell proliferation and differentiation by activating bone marrow stromal cell function. In order to elucidate the biological effect of NP on stromal cells, its effects on hematopoiesis was determined in the mouse model of age-related stromal impairment, senescence-accelerated mice (SAMs). An intraperitoneal administration of NP increased the number of peripheral leukocytes and CFU-GM in the bone marrow and spleen of young SAMs, however, no increase of CFU-GM in old SAMs (stromal impairment) was observed when compared with young SAMs. NP also increased the CFU-GM colony formation of bone marrow and spleen cells from young SAMs in a soft agar culture system, but it did not enhance CFU-GM colony formation of cells from old SAMs cultured in this system. Treatment with NP induced the production of hematopoietic stimulating factors, including IL-6 and GM-CSF, by bone marrow stromal cells from young SAMs but stromal cells from old SAMs did not respond to NP stimulation. Further studies will be required to clarify the mechanism by which NP stimulates the production of hematopoietic growth factors from stromal cells, the results of this study indicate that NP is a potent hematopoietic regulatory factor by activating stromal cell function(s).     

重组人白细胞介素-11对化疗动物血小板减少症的影响

目的观察重组人白细胞介素 11(rhIL 11)对化疗药所致血小板减少症的影响。方法采用口服环磷酰胺引起犬和静脉注射卡铂造成食蟹猴急性血小板减少症模型 ,观察rhIL 11增加血小板生成作用和对其它血细胞及骨髓的影响。结果rhIL 11对环磷酰胺所致犬血小板减少症和卡铂诱发的食蟹猴血小板减少症可明显增加血小板生成 ,但不影响血小板聚集功能 ;也能增加外周血网织红细比例 ,而且能刺激骨髓 ,促进巨核细胞和多种细胞集落的形成。结论rhIL 11可刺激受抑制的骨髓 ,促进血小板和巨核细胞及多种细胞集落形成单位的产生     

沙纳唑合并γ射线对在体小鼠骨髓粒系造血祖细胞的影响

目的 :研究沙纳唑直接给药以及合并γ射线照射处理对在体小鼠骨髓粒系造血祖细胞 (CFU -GM )集落形成的影响。方法 :通过对小鼠骨髓CFU -GM培养的检测 ,观察从小鼠尾静脉注射不同剂量沙纳唑对小鼠造血系统的影响 ;并于不同放射剂量照射前30min从小鼠尾静脉注射不同剂量沙纳唑 ,观察沙纳唑合并γ射线照射处理对小鼠造血系统的影响。结果 :静脉注射沙纳唑对在体小鼠骨髓CFU -GM集落形成抑制作用较小 ;沙纳唑合并γ射线照射处理后对小鼠骨髓CFU -GM的抑制作用与单一放射照射处理结果相似。结论 :沙纳唑直接给药对在体小鼠骨髓CFU -GM无明显细胞毒性作用 ,合并放射照射处理对放射照射所致小鼠骨髓CFU -GM的抑制没有明显协同作用。     

Suppression of bone marrow stromal cell function by benzene and hydroquinone is ameliorated by indomethacin

Administration of benzene to mice will inhibit bone marrow stromal cell-supported hemopoiesis in culture. Hydroquinone, a major metabolite of benzene, will cause a similar inhibition of stromal cell function in vitro. Stromal cells produce both an inducer (colony-stimulating factor) and an inhibitor (prostaglandin E2; PGE2) of hemopoiesis. This research was conducted to determine if prostaglandin synthesis is involved in the suppression of stromal cell function by benzene and hydroquinone. Male B6C3F1 mice were administered benzene (100 mg/kg), indomethacin (1 mg/kg), or benzene plus indomethacin twice a day for 4 consecutive days. On Day 5 bone marrow cells were removed to determine the effect of treatment. In a second series of experiments mouse bone marrow stromal cells in culture were treated with hydroquinone (10(-7) to 10(-4) M), indomethacin (10(-6) M), or a combination of hydroquinone plus indomethacin. Stromal cell function was based on the ability of the treated stromal cells to support granulocyte/monocyte colony development in coculture. The results demonstrated that preadministration of indomethacin in vivo ameliorated benzene-induced inhibition of bone marrow stromal cell function. In vitro, indomethacin ameliorated hydroquinone toxicity to stromal cell function. Benzene administration in vivo induced elevated PGE2 in bone marrow samples which were prevented by preadministration of indomethacin. However, hydroquinone in vitro did not induce a consistent increase in PGE2 levels. These results suggested that toxicity to stromal cells was not due solely to increased prostaglandin synthetase activity.     

In vitro effects of benzene metabolites on mouse bone marrow stromal cells

Benzene exposure can result in bone marrow myelotoxicity. We examined the effects of benzene metabolites on bone marrow stromal cells of the hemopoietic microenvironment. Male B6C3F1 mouse bone marrow adherent stromal cells were plated at 4 X 10(6) cells per 2 ml of DMEM medium in 35-mm tissue culture dishes. The growing stromal cell cultures were exposed to log 2 doses of five benzene metabolites: hydroquinone, benzoquinone, phenol, catechol, or benzenetriol for 7 days. The dose which caused a 50% decrease in colony formation (TD50) was 2.5 X 10(-6) M for hydroquinone, 17.8 X 10(-6) M for benzoquinone, 60 X 10(-6) M for benzenetriol, 125 X 10(-6) M for catechol, and 190 X 10(-6) M for phenol. We next examined the effect of benzene metabolites on the ability of stromal cells to influence granulocyte/monocyte colony growth (G/M-CFU-C) in a coculture system. Adherent stromal cells were plated and incubated for 14 days and then exposed to a benzene metabolite. After 3 days the medium and metabolite were removed and an agar:RPMI layer containing 10(6) fresh bone marrow cells was placed over the stromal layer. After incubation for 7 days the cultures were scored for G/M colony formation. Hydroquinone and benzoquinone were most toxic, while catechol and benzenetriol inhibited colony growth only at high doses. These results indicate that injured bone marrow stromal cells may be a significant factor in benzene-induced hemotoxicity.     

Quercetin suppresses bone resorption by inhibiting the differentiation and activation of osteoclasts

Although quercetin has suppressed bone resorption in several animal studies, its target cells and the mechanism of its action related to bone resorption has not been fully elucidated. We investigated the effect of quercetin on the differentiation and activation of osteoclasts. We used cocultures of mouse spleen cells and ST2 cells, and cultures of osteoclast progenitor cells [M-CSF-dependent (MD) cells from mouse bone marrow and murine monocytic RAW 264 (RAW) cells]. Quercetin dose-dependently inhibited osteoclast-like (OCL) cell formation at 2-5 microM concentration in both the coculture and MD cell culture. Quercetin inhibited the increase of tartrate-resistant acid phosphatase (TRAP) activity of mononuclear preosteoclasts (pOCs) induced by receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) in both MD and RAW cell cultures. Quercetin reversely induced the disruption of actin rings in OCLs. Quercetin also suppressed both pit formation induced by osteoclasts on dentine slices and PTH-stimulated (45)Ca release in mouse long bone cultures. These results suggest that osteoclast progenitors as well as mature osteoclasts, are quercetin's target cells in relation to bone resorption, and that quercetin's suppressive effect on bone resorption results from both its inhibitory effect on the differentiation of osteoclast progenitor cells into pOCs and from its disruptive effect on actin rings in mature osteoclasts.     

The effect of kampo formulae on bone resorption in vitro and in vivo. II. Detailed study of berberine.

We previously isolated berberine from aqueous extracts of tsu-kan-gan, a Kampo formula used for the treatment of osteoporosis. Berberine caused an inhibitory effect on parathyroid hormone (PTH)-stimulated bone resorption in neonatal mouse bone. In this report we describe the inhibitory effect of berberine on the formation of osteoclast-like multinucleated cells (OCLs) in the co-culture of mouse osteoblastic cells and bone marrow cells in the presence of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3], PTH and interleukin-1alpha (IL-1alpha). Berberine dose-dependently inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive OCLs induced by 1alpha25(OH)2D3, PTH and IL-1alpha. We prepared OCLs in the co-culture of osteoblastic cells and bone marrow cells. The effect of berberine on pit formation by OCLs was examined using dentin slices. As OCLs are terminally differentiated multinucleated cells, the survival of OCLs affects the bone-resorbing activity of OCLs. This prompted us to count the number of TRAP-positive OCLs on the slices. Berberine dose-dependently inhibited pit formation and caused a decrease in the number of TRAP-positive OCLs. Calcitonin (CT) inhibited pit formation without affecting the number of OCLs. Berberine accelerated the cell death in OCLs cultivated on a culture plate, but CT did not affect the cell death of OCLs. This suggests that the decrease in the number of OCLs on dentin slices may be due to apoptotic cell death in OCLs. In fact, Hoechst 33258 staining revealed that the treatment of OCLs with berberine resulted in condensed nuclei and a decrease in cell size. Oral administration of the berberine (30 and 50 mg/kg/d) to ovariectomized rats prevented a decrease in bone mineral density (BMD) of the lumbar vertebra without affecting the weight of the uterus and plasma concentration of estradiol. These results suggested that berberine prevented a decrease in BMD in vivo by inhibiting osteoclastic bone resorption.     

地黄寡糖对SAMP8小鼠造血祖细胞增殖的作用

采用造血祖细胞体外克隆培养等实验血液学技术, 研究了地黄寡糖对快速老化模型P系小鼠(SAMP8)骨髓造血祖细胞增殖作用的影响. 结果表明地黄寡糖可促进SAMP8小鼠骨髓粒系巨噬系祖细胞, 早期和晚期红系祖细胞的增殖, 其脾细胞条件液也可使造血祖细胞克隆集落数明显增加, 地黄寡糖还可使其基质细胞层上粒系巨噬系祖细胞集落的产率明显增多. 提示地黄寡糖可能通过多种途径激活机体组织, 特别是造血微环境中的某些细胞, 促进其分泌多种造血生长因子而增强造血祖细胞的增殖.     

Pathogenesis of aplastic anemia

Several assays were used to study myelopoiesis in 10 patients with aplastic anemia: the soft agar colony assay for granulocyte-monocyte progenitors; colony forming assay after removal of T lymphocytes; coculture of normal marrow with lymphocytes from normal individuals and patients; coculture of normal marrow with bone marrow fibroblasts from normal subjects and patients in the presence or absence of colony-stimulating factor. All patient assays revealed low colony formation. In two patients, colony formation by normal marrow cells in coculture with lymphocytes was suppressed with a colony count increase following T lymphocyte removal from marrow. Suppressor cells may have caused the aplasia in these patients. Most of the fibroblasts from normal individuals enhanced granulopoiesis in the absence of the colony-stimulating factor, while those from all patients failed to do so. When the colony-stimulating factor was present in the cultures, the degree of suppression of colony formation by fibroblasts derived from the patients was far greater than that by those from the healthy subjects. These results indicate that most aplastic anemias arise from defects of the stem cells and the bone marrow fibroblasts which hold major responsibilities in creating a microenvironment inductive to hematopoiesis.     

The effect of cytotoxic drugs on the development of murine granulocyte-macrophage (GM) progenitor cells.

The effect of cytotoxic drugs was tested on the hematopoietic system by assay of the ability of granulocyte-macrophage colony forming cell (GM-CFC) in mice to create GM colonies. The in vivo ability of bone marrow progenitor cells to granulocyte-macrophage colony formation, was tested after long-term peritoneal cytotoxic drug administration. The direct effect of these agents on cells in vitro culture was evaluated also. It was found that cytotoxic drugs inhibit the GM colony formation. The degree of damage to the bone marrow progenitor cells by assaying in vivo colony formation inhibition, depends on the drug dosage and length of therapy (after 25-30 days of treatment the colony growth was below 50%). The in vitro inhibition of granulocyte-macrophage colony formation depends on the concentration of drug (10(-7)-10(-5) M is critical for GM colony growth). The results suggest the possibility of the GM-CFC growth testing as an indicator of the progress or side effects of cytotoxic therapy.     

Inhibition of osteoclast differentiation and bone resorption by a novel lysophosphatidylcholine derivative, SCOH

Osteoclasts are multinucleated cells formed by multiple steps of cell differentiation from progenitor cells of hematopoietic origin. Intervention in osteoclast differentiation is considered as an effective therapeutic approach to the treatment for bone diseases involving osteoclasts. In this study, we found that the organic compound (S)-1-lyso-2-stearoylamino-2-deoxy-sn-glycero-3-phosphatidylcholine (SCOH) inhibited osteoclast differentiation. The inhibitory effect of SCOH was observed in mouse bone marrow cell cultures supported either by coculturing with osteoblasts or by adding macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL). M-CSF and RANKL activate the ERK, Akt, and NF-kappaB signal transduction pathways, and SCOH suppressed this activation. SCOH also inhibited the bone resorptive activity of differentiated osteoclasts. It attenuated bone resorption, actin ring formation, and survival of mature osteoclasts. Reduced activation of Akt and NF-kappaB and decreased induction of XIAP were observed in mature osteoclasts treated with SCOH. Thus, this novel phosphatidylcholine derivative may be useful for treating bone-resorption diseases.     

Acute toxicity of benzene inhalation to hemopoietic precursor cells

When BDF₁ mice were submitted to inhalation of benzene vapors (4680 ppm) for 8 hr, a significant depletion in bone marrow colony forming cells (CFC) was observed in cultures in vitro assayed 1 day after drug inhalation; however, femoral CFC returned to steady state values by Day 7. The return of femoral CFC values was not due to an increase in bone marrow cellularity. Although spleen cellularity increased significantly by Days 4 and 7, the splenic CFC value was not significantly different from controls. This acute toxicity to femoral CFC was enhanced by multiple sessions of benzene inhalation over a period of 3 and 3.5 days. After three and one-half 8-hr sessions of benzene vapor inhalation (4680 ppm), CFC content was dramatically reduced (13% of controls), in addition to significantly lowered values for body and spleen weights and bone marrow cellularity. Splenic colony forming unit (CFU) formation in irradiated mice which received bone marrow cells from benzene-treated mice corresponded well with in vitro femoral CFC formation from mice treated with the identical multiple benzene inhalation regimen as the donor mice; CFU and CFC production was reduced to the same extent, about 40–45% of controls. In summary, precursor cells of the hemic cell renewal system are sensitive to benzene inhalation in mice.     

Pesticide induced alterations in marrow physiology and depletion of stem and stromal progenitor population: An experimental model to study the toxic effects of pesticide

Long‐term exposure of agriculturally used organochloride and organophosphate pesticides have been shown to cause long‐lasting hematotoxicity and increased incidence of aplastic anemia in humans. The mechanisms involved in pesticide induced hematotoxicity and the features of toxicity that may play a major role in bone marrow suppression are not known. The aim of the present study was to investigate the hematological consequences of pesticide exposure in swiss albino mice exposed to aqueous mixture of common agriculturally used pesticides for 6 h/day, 5 days/week for 13 weeks. After the end of last exposure, without a recovery period, the strong hematotoxic effect of pesticide was assessed in mice with long‐term bone marrow explant culture (LTBMC‐Ex) system and cell colony forming assays. Bone marrow explant culture from the pesticide exposed group of mice failed to generate a supportive stromal matrix and did not produce adequate number of hematopoietic cells and found to contain largely the adipogenic precursors. The decreased cell colony numbers in the pesticide exposed group indicated defective maturational and functional status of different marrow cell lineages. As a whole, exposure of mice to the mixture of pesticides reduced the total number of bone marrow cells (granulocytes are the major targets of pesticide toxicity), hematopoietic, and non‐hematopoietic progenitor cells and most of the hematological parameters. Replication of primitive stem/progenitor cells in the marrow was decreased following pesticide exposure with G0/G1‐phase arrest of most of the cells. The progenitor cells showed decreased percentage of cells in S/G2/M‐phase. The increased apoptosis profile of the marrow progenitors (Increased CD95 expression) and primitive stem cells (High Annexin‐V positivity on Sca1+ cells) with an elevated intracellular cleaved caspase‐3 level on the Sca1+ bone marrow cells provided the base necessary for explaining the deranged bone marrow microenvironmental structure which was evident from scanning electron micrographs. These results clearly indicate a strong, long lasting toxic effect of pesticides on the bone marrow microenvironment and different microenvironmental components which ultimately leads to the formation of a degenerative disease like aplastic anemia. © 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 84–97, 2014.     

Biological activity of the main metabolites of ubenimex in humans

The biological activity of the two main metabolites of ubenimex in humans, (-)-N-[(2S,3R)-3-amino-2-hydroxy-4-(4'-hydroxy)phenylbutyryl]-L-leucine (OH-ubenimex) and (2S,3R)-3-amino-2-hydroxy-4-phenylbutyric acid [2S,3R)-AHPA) was examined. OH-Ubenimex was almost identical in inhibitory activity against mouse peritoneal resident macrophage aminopeptidases (APases) and the growth of IMC carcinoma in mice to ubenimex. In contrast, the inhibition of; mouse spleen cell APase activities in vitro, blastogenesis of mouse T and B cells in vitro, delayed cutaneous hypersensitivity in mice, and the growth of C1498 leukemia and HeLa S3 cells in vitro was weaker than ubenimex. Macrophage APase activity was only slightly inhibited by (2S,3R)-AHPA which also had practically no activity in the other biological assays.