Microtubule destabilization caused by silicate via HDAC6 activation contributes to autophagic dysfunction in bone mesenchymal stem cells.

BACKGROUND: Silicon-modified biomaterials have been extensively studied in bone tissue engineering. In recent years, the toxicity of silicon-doped biomaterials has gradually attracted attention but requires further elucidation. This study was designed to explore whether high-dose silicate can induce a cytotoxicity effect in bone mesenchymal stem cells (BMSCs) and the role of autophagy in its cytotoxicity and mechanism. METHODS: Morphologic changes and cell viability of BMSCs were detected after different doses of silicate exposure. Autophagic proteins (LC3, p62), LC3 turnover assay, and RFP-GFP-LC3 assay were applied to detect the changes of autophagic flux following silicate treatment. Furthermore, to identify the potential mechanism of autophagic dysfunction, we tested the acetyl-α-tubulin protein level and histone deacetylase 6 (HDAC6) activity after high-dose silicate exposure as well as the changes in microtubule and autophagic activity after HDAC6 siRNA was applied. RESULTS: It was found that a high dose of silicate could induce a decrease in cell viability; LC3-II and p62 simultaneously increased after high-dose silicate exposure. A high concentration of silicate could induce autophagic dysfunction and cause autophagosomes to accumulate via microtubule destabilization. Results showed that acetyl-α-tubulin decreased significantly with high-dose silicate treatment, and inhibition of HDAC6 activity can restore microtubule structure and autophagic flux. CONCLUSIONS: Microtubule destabilization caused by a high concentration of silicate via HDAC6 activation contributed to autophagic dysfunction in BMSCs, and inhibition of HDAC6 exerted a cytoprotection effect through restoration of the microtubule structure and autophagic flux.

Red Yeast Rice Protects Circulating Bone Marrow-Derived Proangiogenic Cells against High-Glucose-Induced Senescence and Oxidative Stress: The Role of Heme Oxygenase-1.

The inflammation and oxidative stress of bone marrow-derived proangiogenic cells (PACs), also named endothelial progenitor cells, triggered by hyperglycemia contributes significantly to vascular dysfunction. There is supporting evidence that the consumption of red yeast rice (RYR; Monascus purpureus-fermented rice) reduces the vascular complications of diabetes; however, the underlying mechanism remains unclear. This study aimed to elucidate the effects of RYR extract in PACs, focusing particularly on the role of a potent antioxidative enzyme, heme oxygenase-1 (HO-1). We found that treatment with RYR extract induced nuclear factor erythroid-2-related factor nuclear translocation and HO-1 mRNA and protein levels in PACs. RYR extract inhibited high-glucose-induced (30 mM) PAC senescence and the development of reactive oxygen species (ROS) in a dose-dependent manner. The HO-1 inducer cobalt protoporphyrin IX also decreased high-glucose-induced cell senescence and oxidative stress, whereas the HO-1 enzyme inhibitor zinc protoporphyrin IX and HO-1 small interfering RNA significantly reversed RYR extract-caused inhibition of senescence and reduction of oxidative stress in high-glucose-treated PACs. These results suggest that RYR extract serves as alternative and complementary medicine in the treatment of these diseases, by inducing HO-1, thereby decreasing the vascular complications of diabetes.

Melatonin reverses flow shear stress-induced injury in bone marrow mesenchymal stem cells via activation of AMP-activated protein kinase signaling.

Tissue-engineered heart valves (TEHVs) are a promising treatment for valvular heart disease, although their application is limited by high flow shear stress (FSS). Melatonin has a wide range of physiological functions and is currently under clinical investigation for expanded applications; moreover, extensive protective effects on the cardiovascular system have been reported. In this study, we investigated the protection conferred by melatonin supplementation against FSS-induced injury in bone marrow mesenchymal stem cells (BMSCs) and elucidated the potential mechanism in this process. Melatonin markedly reduced BMSC apoptotic death in a concentration-dependent manner while increasing the levels of transforming growth factor ß (TGF-ß), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and B-cell lymphoma 2 (Bcl2), and decreasing those of Bcl-2-associated X protein (Bax), p53 upregulated modulator of apoptosis (PUMA), and caspase 3. Notably, melatonin exerted its protective effects by upregulating the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), which promotes acetyl-CoA carboxylase (ACC) phosphorylation. Further molecular experiments revealed that luzindole, a nonselective antagonist of melatonin receptors, blocked the anti-FSS injury (anti-FSSI) effects of melatonin. Inhibition of AMPK by Compound C also counteracted the protective effects of melatonin, suggesting that melatonin reverses FSSI in BMSCs through the AMPK-dependent pathway. Overall, our findings indicate that melatonin contributes to the amelioration of FSS-induced BMSC injury by activating melatonin receptors and AMPK/ACC signaling. Our findings may provide a basis for the design of more effective strategies that promote the use of TEHCs in patients.

Immunostimulant and chemoprotective effect of vivartana, a polyherbal formulation against cyclophosphamide induced toxicity in Swiss albino mice.

The aim of the study is to develop a technology for cost effective immunomodulator from natural products to combat adverse effects during cancer chemotherapy. In the present study, the immunomodulatory efficacy of Vivartana, a poly herbal formulation in immunosuppressed animal model induced by cyclophosphamide (CTX) and its comparison with standard herbal immunostimulators Chyawanprash and Brahma Rasayana was investigated. The effect of Vivartana (500 mg/kg x bw) (p.o.), Chyawanprash (20 mg/kg.bw) (p.o.) and Brahma Rasayana (20 mg/kg x bw) (p.o.) on hematological parameters, relative organ weight, Bone marrow cellularity and α-esterase activity were determined in Swiss albino mice by using the standard methods. Among the herbal formulations Vivartana showed the maximum number of leukocytes (13150 cells/mm3) on the 15th day. The leukocyte count in Vivartana treated CTX induced group shows significant increase (5375 cells/mm3) when compared with CTX alone induced group (3358 cells/mm3) on the same day. The Vivartana treated CTX induced group shows increase in the hemoglobin level compared with the CTX induced group. Moreover, Vivartana treatment prevented the loss of organ weight in the CTX induced group by the enhancement of spleenocytes on the 7th day and thymocytes on the 11th day. Similarly the lowered bone marrow cellularity and number of α-esterase positive cells in CTX induced group were restored in the Vivartana treatment. Treatment with vivartana also exhibits hepatoprotective activity by regulating the SGOT and SGPT levels in CTX induced group. The study indicates that Vivartana has the considerable potential as an immunostimulant and chemoprotectant against CTX induced immunosuppression in Swiss albino mice.

Atorvastatin at reperfusion reduces myocardial infarct size in mice by activating eNOS in bone marrow-derived cells.

BACKGROUND: The current study was designed to test our hypothesis that atorvastatin could reduce infarct size in intact mice by activating eNOS, specifically the eNOS in bone marrow-derived cells. C57BL/6J mice (B6) and congenic eNOS knockout (KO) mice underwent 45 min LAD occlusion and 60 min reperfusion. Chimeric mice, created by bone marrow transplantation between B6 and eNOS KO mice, underwent 40 min LAD occlusion and 60 min reperfusion. Mice were treated either with vehicle or atorvastatin in 5% ethanol at a dose of 10 mg/kg IV 5 min before initiating reperfusion. Infarct size was evaluated by TTC and Phthalo blue staining. RESULTS: Atorvastatin treatment reduced infarct size in B6 mice by 19% (p<0.05). In eNOS KO vehicle-control mice, infarct size was comparable to that of B6 vehicle-control mice (p = NS). Atorvastatin treatment had no effect on infarct size in eNOS KO mice (p = NS). In chimeras, atorvastatin significantly reduced infarct size in B6/B6 (donor/recipient) mice and B6/KO mice (p<0.05), but not in KO/KO mice or KO/B6 mice (p = NS). CONCLUSIONS: The results demonstrate that acute administration of atorvastatin significantly reduces myocardial ischemia/reperfusion injury in an eNOS-dependent manner, probably through the post-transcriptional activation of eNOS in bone marrow-derived cells.

Effects of Ti, PMMA, UHMWPE, and Co-Cr wear particles on differentiation and functions of bone marrow stromal cells.

This study investigates the roles of orthopedic biomaterial particles [Ti-alloy, poly(methyl methacrylate) (PMMA), ultrahigh-molecular-weight polyethylene (UHMWPE), Co-Cr alloy] on the differentiation and functions of bone marrow stromal cells (BMSCs). Cells were isolated from femurs of BALB/c mice and cultured in complete osteoblast-induction medium in presence of micron-sized biomaterial particles at various doses. 3-(4,5)-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and lactate dehydrogenase assay were performed for cell proliferation and cytotoxicity. Differentiation and function of osteoblasts were evaluated by alkaline phosphatase (ALP), osteocalcin, RANKL, OSX, and Runx2 expressions. Murine interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α in culture media were determined by enzyme-linked immunosorbent assay. Challenge with low doses of Ti, UHMWPE, or Co-Cr particles markedly promoted the bone marrow cell proliferation while high dose of Co-Cr significantly inhibited cell growth (p < 0.05). Cells challenged with low dose of PMMA or UHMWPE particles (0.63 mg/mL) exhibited strong ALP activity, whereas Ti and Co-Cr groups showed minimal effects (p < 0.05). UHMWPE and Ti particles also promoted higher expression of proinflammatory cytokines. Real-time polymerase chain reaction data suggested that cells treated with low dose (0.5 mg/mL) particles resulted in distinctly diminished RANKL expression compared to those exposed to high concentrated (3 mg/mL) particles. In conclusion, various types of wear debris particles behaved differently in the differentiation, maturation, and functions of osteogenic cells; and the particulate debris-interacted BMSCs may play an important role in the pathogenesis and process of the debris-associated aseptic prosthetic loosening.

Severe hyperhomocysteinemia promotes bone marrow-derived and resident inflammatory monocyte differentiation and atherosclerosis in LDLr/CBS-deficient mice.

RATIONALE: Hyperhomocysteinemia (HHcy) accelerates atherosclerosis and increases inflammatory monocytes (MC) in peripheral tissues. However, its causative role in atherosclerosis is not well established and its effect on vascular inflammation has not been studied. The underlying mechanism is unknown. OBJECTIVE: This study examined the causative role of HHcy in atherogenesis and its effect on inflammatory MC differentiation. METHODS AND RESULTS: We generated a novel HHcy and hyperlipidemia mouse model, in which cystathionine ß-synthase (CBS) and low-density lipoprotein receptor (LDLr) genes were deficient (Ldlr(-/-) Cbs(-/+)). Severe HHcy (plasma homocysteine (Hcy)=275 µmol/L) was induced by a high methionine diet containing sufficient basal levels of B vitamins. Plasma Hcy levels were lowered to 46 µmol/L from 244 µmol/L by vitamin supplementation, which elevated plasma folate levels. Bone marrow (BM)-derived cells were traced by the transplantation of BM cells from enhanced green fluorescent protein (EGFP) transgenic mice after sublethal irradiation of the recipient. HHcy accelerated atherosclerosis and promoted Ly6C(high) inflammatory MC differentiation of both BM and tissue origins in the aortas and peripheral tissues. It also elevated plasma levels of TNF-α, IL-6, and MCP-1; increased vessel wall MC accumulation; and increased macrophage maturation. Hcy-lowering therapy reversed HHcy-induced lesion formation, plasma cytokine increase, and blood and vessel inflammatory MC (Ly6C(high+middle)) accumulation. Plasma Hcy levels were positively correlated with plasma levels of proinflammatory cytokines. In primary mouse splenocytes, L-Hcy promoted rIFNγ-induced inflammatory MC differentiation, as well as increased TNF-α, IL-6, and superoxide anion production in inflammatory MC subsets. Antioxidants and folic acid reversed L-Hcy-induced inflammatory MC differentiation and oxidative stress in inflammatory MC subsets. CONCLUSIONS: HHcy causes vessel wall inflammatory MC differentiation and macrophage maturation of both BM and tissue origins, leading to atherosclerosis via an oxidative stress-related mechanism.

Effects of micropitted/nanotubular titania topographies on bone mesenchymal stem cell osteogenic differentiation.

Micro/nanotopographical modification of biomaterials constitutes a promising approach to direct stem cell osteogenic differentiation to promote osseointegration. In this work, titania nanotubes (NTs) 25 and 80 nm in size with the acid-etched Ti topography (AcidTi) and hierarchical hybrid micropitted/nanotubular topographies (Micro/5VNT and Micro/20VNT) are produced to mimic the structure of the natural bone extracellular matrix (ECM). The effects on bone mesenchymal stem cell (MSC) osteogenic differentiation are studied systematically by various microscopic and biological characterization techniques. Cell adhesion is assayed by nucleus fluorescence staining and cell proliferation is studied by CCK-8 assay and flow cytometry. Osteogenic differentiation is assayed by alkaline phosphatase (ALP) expression, collagen secretion, matrix mineralization, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis on the osteogenesis related gene expression. All the topographies are observed to induce MSC osteogenic differentiation in the absence of osteogenic supplements. The nanotube surfaces significantly promote cell attachment and spread, collagen secretion and ECM mineralization, as well as osteogenesis-related gene expression. Among them, Micro/20VNT shows the best ability to simultaneously promote MSC proliferation and osteogenic differentiation. Our results unambiguously demonstrate their excellent ability to support MSC proliferation and induce MSC osteogenic differentiation, especially those with the micropitted topography.

A novel nanoparticle-enhanced photoacoustic stimulus for bone tissue engineering.

In this study, we introduce a novel nanoparticle-enhanced biophysical stimulus based on the photoacoustic (PA) effect. We demonstrate that the PA effect differentiates bone marrow-derived marrow stromal cells (MSCs) grown on poly(lactic-co-glycolic acid) (PLGA) polymer films toward osteoblasts. We further show that the osteodifferentiation of the MSCs due to PA stimulation is significantly enhanced by the presence of single-walled carbon nanotubes (SWCNTs) in the polymer. MSCs, without the osteogenic culture supplements (0.01 M ß-glycerophosphate, 50 mg/L ascorbic acid, 10(-8) M dexamethasone), were seeded onto plain glass slides, glass slides coated with PLGA, or glass slides coated with SWCNT-PLGA films and photoacoustically stimulated by a 527 nm Nd:YLF pulse laser, with a 200 ns pulse duration, and 10 Hz pulse frequency for 10 min a day for 15 consecutive days. The study had four control groups; three baseline controls similar to the three experimental groups but without PA stimulation, and one positive control where MSCs were grown on glass slides without PA stimulation but with osteogenic culture supplements. The osteogenic differentiation of all the groups was evaluated using quantitative assays (alkaline phosphatase, calcium, osteopontin) and qualitative staining (alizarin red). After 15 days, the PA stimulated groups showed up to a 350% increase in calcium content when compared with the non-PA stimulated positive control. Further, within the PA stimulated group, the PLGA-SWCNT group had 130% higher calcium values than the PLGA film without SWCNTs. These results were further corroborated by the analysis of osteopontin secretion, alkaline phosphatase expression, and qualitative alizarin red staining of extracellular matrix calcification. The results indicate that PA stimulation holds promise for bone tissue engineering and that the nanomaterials which enhance the PA effect should allow the development of biophysical rather than biochemical strategies to induce osteoinductive properties into tissue engineering scaffolds.

Molecular pathobiology of human cervical high-grade lesions: paracrine STAT3 activation in tumor-instructed myeloid cells drives local MMP-9 expression.

In many tumors, the switch from precancerous lesions to malignancy critically relies on expression of the matrix-metalloprotease MMP-9, which is predominantly provided by infiltrating inflammatory cells. Our study defines a novel molecular cascade, how human neoplastic cells instruct tumor-associated myeloid cells to produce MMP-9. In biopsies of human papillomavirus-associated precancerous cervical intraepithelial neoplasia (CIN III lesions), we show broad activation of the transcription factor STAT3 and coexpression of MMP-9 in perivascular inflammatory cells. For the first time, we establish a causative link between tumor-mediated paracrine STAT3 activation and MMP-9 production by human tumor-instructed monocytes, whereas NF-κB activation is dispensable for this response. Our data provide evidence that STAT3 does not directly induce MMP-9 but first leads to a strong production of the monocyte chemoattractant protein-1 (CCL2) in the nanogram range. In a second phase, autocrine stimulation of the CCR2 receptor in the tumor-instructed monocytes amplifies MMP-9 expression via intracellular Ca(2+) signaling. These findings elucidate a critical mechanism in the molecular pathobiology of cervical carcinogenesis at the switch to malignancy. Particularly in tumors, which are associated with infectious agents, STAT3-driven inflammation may be pivotal to promote carcinogenesis, while at the same time limit NF-κB-dependent immune responses and thus rejection of the infected preneoplastic cells. The molecular cascade defined in this study provides the basis for a rational design of future adjuvant therapies of cervical precancerous lesions.

Protective effects and mechanism of Panax Notoginseng saponins on oxidative stress-induced damage and apoptosis of rabbit bone marrow stromal cells.

OBJECTIVE: To investigate the effects and possible mechanism of Panax Notoginseng saponins (PNS) on oxidative stress-induced damage and apoptosis in bone marrow stromal cells (BMSCs). METHODS: BMSCs were isolated and cultured from 2-month-old New Zealand rabbits by the density gradient centrifugation combined with adherent method. The third passage cells were used for subsequent experiments. Oxidative stress was induced in cultured BMSCs by H(2)O(2) (0.1 mmol/L). BMSCs were pretreated with 25-200 µg/mL PNS for 4 h before H(2)O(2) treatment. Proliferation of BMSCs was observed using MTT assay. Alkaline phosphatase (ALP) activity, as an index of early osteoblastic differentiation, was determined with an ALP assay kit. Flow cytometry was used to observe the apoptosis of BMSCs by staining with annexinV-FITC/propidium iodide. Oxidative stress level was examined by reactive oxygen species (ROS) assay. The protein expressions of Bax, Bcl-2 and Caspase-3 in BMSCs were analyzed by Western blotting. RESULTS: PNS had different concentration-dependent effects on proliferation and osteoblast differentiation of BMSCs induced by H(2)O(2). A PNS concentration of 100 µg/mL was determined as the optimal effective concentration. PNS markedly attenuated H(2)O(2)-induced apoptosis rate from 41.91% to 14.67% (P<0.01). PNS significantly decreased ROS level induced by H(2)O(2) (P<0.01). Furthermore, pretreatment with PNS significantly reversed H(2)O(2)-induced inhibition of Bcl-2 expression and augmentation of Bax and Caspase-3 expression (P<0.01). CONCLUSION: PNS had a protective effect on oxidative stress-induced damage and apoptosis in cultured rabbit BMSCs through scavenging ROS and regulating the Bcl-2/Bax pathway.

The role of CaMKII in calcium-activated death pathways in bone marrow B cells.

Calcium is an essential signaling molecule in developing B cells, thus altering calcium dynamics represents a potential target for toxicant effects. GW7845, a tyrosine analog and potent peroxisome proliferator-activated receptor γ agonist, induces rapid mitogen-activated protein kinase (MAPK)-dependent apoptosis in bone marrow B cells. Changes in calcium dynamics are capable of mediating rapid initiation of cell death; therefore, we investigated the contribution of calcium to GW7845-induced apoptosis. Treatment of a nontransformed murine pro/pre-B cell line (BU-11) with GW7845 (40 µM) resulted in intracellular calcium release. Multiple features of GW7845-induced cell death were suppressed by the calcium chelator BAPTA, including MAPK activation, loss of mitochondrial membrane potential, cytochrome c release, caspase-3 activation, and DNA fragmentation. A likely mechanism for the calcium-mediated effects is activation of CaMKII, a calcium-dependent MAP4K. We observed that three CaMKII isoforms (ß, γ, and δ) are expressed in lymphoid tissues and bone marrow B cells. Treatment with GW7845 increased CaMKII activity. All features of GW7845-induced cell death, except loss of mitochondrial membrane potential, were suppressed by CaMKII inhibitors (KN93 and AIP-II), suggesting the activation of multiple calcium-driven pathways. To determine if CaMKII activation is a common feature of early B cell death following perturbation of Ca(2+) flux, we dissected tributyltin (TBT)-induced death signaling. High-dose TBT (1 µM) is known to activate calcium-dependent death. TBT induced rapid apoptosis that was associated with intracellular calcium release, CaMKII activation and MAPK activation, and was inhibited by AIP-II. Thus, we show that early B cells are susceptible to calcium-triggered cell death through a CaMKII/MAPK-dependent pathway.

Glucocerebrosidase deficiency dramatically impairs human bone marrow haematopoiesis in an in vitro model of Gaucher disease.

One of the cardinal symptoms of type 1 Gaucher Disease (GD) is cytopenia, usually explained by bone marrow (BM) infiltration by Gaucher cells and hypersplenism. However, some cases of cytopenia in splenectomized or treated patients suggest possible other mechanisms. To evaluate intra-cellular glucocerebrosidase (GlcC) activity in immature progenitors and to prove the conduritol B epoxide (CBE)-induced inhibition of the enzyme, we used an adapted flow cytometric technique before assessing the direct effect of GlcC deficiency in functional assays. Among haematopoietic cells from healthy donors, monocytes showed the highest GlcC activity but immature CD34(+) and mesenchymal cells also had significant GlcC activity. CBE greatly inhibited the enzyme activity of all cell categories. GlcC-deficient CD34(+) cells showed impaired ability to proliferate and differentiate in the expansion assay and had lower frequency of erythroid burst-forming units, granulocyte colony-forming units (CFU) and macrophage CFU progenitors, but the effect of GlcC deficiency on megakaryocyte CFU lineage was not significant. GlcC deficiency strongly impaired primitive haematopoiesis in long-term culture. Furthermore, GlcC deficiency progressively impaired proliferation of mesenchymal progenitors. These data suggest an intrinsic effect of GlcC deficiency on BM immature cells that supplements the pathophysiology of GD and opens new perspectives of therapeutic approach.

Infection with arginase-deficient Leishmania major reveals a parasite number-dependent and cytokine-independent regulation of host cellular arginase activity and disease pathogenesis.

The balance between the products of L-arginine metabolism in macrophages regulates the outcome of Leishmania major infection. L-arginine can be oxidized by host inducible NO synthase to produce NO, which contributes to parasite killing. In contrast, L-arginine hydrolysis by host arginase blocks NO generation and provides polyamines, which can support parasite proliferation. Additionally, Leishmania encode their own arginase which has considerable potential to modulate infectivity and disease pathogenesis. In this study, we compared the infectivity and impact on host cellular immune response in vitro and in vivo of wild-type (WT) L. major with that of a parasite arginase null mutant (arg(-)) L. major. We found that arg(-) L. major are impaired in their macrophage infectivity in vitro independent of host inducible NO synthase activities. As with in vitro results, the proliferation of arg(-) L. major in animal infections was also significantly impaired in vivo, resulting in delayed onset of lesion development, attenuated pathology, and low parasite burden. Despite this attenuated pathology, the production of cytokines by cells from the draining lymph node of mice infected with WT and arg(-) L. major was similar at all times tested. Interestingly, in vitro and in vivo arginase levels were significantly lower in arg(-) than in WT-infected cases and were directly correlated with parasite numbers inside infected cells. These results suggest that Leishmania-encoded arginase enhances disease pathogenesis by augmenting host cellular arginase activities and that contrary to previous in vitro studies, the host cytokine response does not influence host arginase activity.

[Effects of acupuncture and moxibustion on DNA excision repair-related proteins of bone marrow cell in cyclophosphamide-induced mice].

OBJECTIVE: To explore the molecular biological mechanism of acupuncture and moxibustion for relieving myelosuppression and increasing white blood cells. METHODS: Two hundred and twenty-four clean male Kunming mice were randomly divided into a control group, a model group, an acupuncture group and a moxibustion group, 56 mice in each group. The model of myelosuppression was made with Cyclophosphamide. In the acupuncture group and the moxibustion group, acupoints "Dazhui" (GV 14), "Geshu" (BL 17), "Shenshu" (BL 23) and "Zusanli" (ST 36) were used for treatment with acupuncture and moxibustion, respectively, while, in the control group and the model group, there were no treatment carried out except catching and fixing. The changes of bone marrow cell DNA pol beta and XPD between the 2nd and 7th day were examined with immunohistochemical method. RESULTS: Acupuncture and moxibustion markedly up-regulated the expression of bone marrow cell DNA pol beta and XPD, and promoted the base excision repair and nucleotide excision repair, which leads to the relieving Cyclophosphamide-induced myelosuppression and increasing the number of white blood cells. CONCLUSION: For acupuncture and moxibustion, one of the bone major mechanisms in relieving post-chemotherapy myelosuppression, protecting hemopoietic function and increasing the white blood cells is that it can promote the repair of the bone marrow cell DNA excision and protect hemopoietic cells from injury by chemical drugs.

Effect of ibuprofen on osteoblast differentiation of porcine bone marrow-derived progenitor cells.

PURPOSE: Nonsteroidal anti-inflammatory drugs are commonly prescribed to reduce inflammation and pain. However, little is known about the direct effect of these drugs on the differentiation of bone marrow-derived progenitor cells into osteoblasts. The purpose of this study was to determine the effect of ibuprofen on osteoblast differentiation and proliferation in a minipig model. MATERIALS AND METHODS: Bone marrow was aspirated from the minipig ilium, and porcine bone marrow-derived progenitor cells (pBMPCs) were isolated and expanded in standard culture medium. The pBMPCs were replated and differentiated into osteoblasts by use of osteogenic supplements (OS). Five groups were studied: negative control--pBMPCs in standard medium only; positive control--pBMPCs, standard culture medium, and OS; and 3 experimental groups--pBMPCs, standard culture medium, OS, and ibuprofen added in doses of 0.1, 1.0, and 3.0 mmol/L. Cell cultures were evaluated quantitatively by alkaline phosphatase (ALP) stain, von Kossa stain, and deoxyribonucleic acid (DNA) content. RESULTS: pBMPCs cultured with OS and low-dose ibuprofen (0.1 mmol/L) showed ALP stain, von Kossa stain, and DNA content similar to pBMPCs cultured in OS (positive control). pBMPCs cultured in higher doses of ibuprofen (1.0 and 3.0 mmol/L) produced significantly less positive staining of ALP and von Kossa and decreased DNA content. CONCLUSION: The results indicate that high-dose ibuprofen has a deleterious effect on pBMPC differentiation into osteoblasts whereas low-dose ibuprofen does not. The low dose of 0.1 mmol/L is the typical serum level when prescribed for clinical use.

Anti-inflammatory properties of a novel N-phenyl pyridinone inhibitor of p38 mitogen-activated protein kinase: preclinical-to-clinical translation.

Signal transduction through the p38 mitogen-activated protein (MAP) kinase pathway is central to the transcriptional and translational control of cytokine and inflammatory mediator production. p38 MAP kinase inhibition hence constitutes a promising therapeutic strategy for treatment of chronic inflammatory diseases, based upon its potential to inhibit key pathways driving the inflammatory and destructive processes in these debilitating diseases. The present study describes the pharmacological properties of the N-phenyl pyridinone p38 MAP kinase inhibitor benzamide [3- [3-bromo-4-[(2,4-difluorophenyl)methoxy]-6-methyl-2- oxo-1(2H)-pyridinyl]-N,4-dimethyl-, (-)-(9CI); PH-797804]. PH-797804 is an ATP-competitive, readily reversible inhibitor of the alpha isoform of human p38 MAP kinase, exhibiting a K(i) = 5.8 nM. In human monocyte and synovial fibroblast cell systems, PH-797804 blocks inflammation-induced production of cytokines and proinflammatory mediators, such as prostaglandin E(2), at concentrations that parallel inhibition of cell-associated p38 MAP kinase. After oral dosing, PH-797804 effectively inhibits acute inflammatory responses induced by systemically administered endotoxin in both rat and cynomolgus monkeys. Furthermore, PH-797804 demonstrates robust anti-inflammatory activity in chronic disease models, significantly reducing both joint inflammation and associated bone loss in streptococcal cell wall-induced arthritis in rats and mouse collagen-induced arthritis. Finally, PH-797804 reduced tumor necrosis factor-alpha and interleukin-6 production in clinical studies after endotoxin administration in a dose-dependent manner, paralleling inhibition of the target enzyme. Low-nanomolar biochemical enzyme inhibition potency correlated with p38 MAP kinase inhibition in human cells and in vivo studies. In addition, a direct correspondence between p38 MAP kinase inhibition and anti-inflammatory activity was observed with PH-797804, thus providing confidence in dose projections for further human studies in chronic inflammatory disease.

Bone marrow-derived aldehyde dehydrogenase-bright stem and progenitor cells for ischemic repair.

Adult stem cell populations selected for use in cardiovascular clinical trials typically are mononuclear cell fractions of bone marrow and peripheral blood or cells of specific cell lineages selected by surface markers such as CD34 or CD133. This article describes a potent stem and progenitor cell population identified by an intracellular marker of "stemness" that crosses multiple lineages. Aldehyde dehydrogenase (ALDH)-bright (ALDH(br)) populations isolated from bone marrow contain potent stem and progenitor cells representing all cell types thought to be needed for ischemic repair and include hematopoietic, endothelial, mesenchymal, and neural progenitor cells. An animal model of hindlimb ischemia demonstrated that the ALDH(br) population was highly effective in restoring blood flow to ischemic limbs. Based upon the accumulating evidence for a potential therapeutic effect of bone marrow-derived cells in ischemic disease in humans and the vascular regenerative potential of ALDH(br) cells in the hindlimb model, clinical trials to investigate the use of autologous bone marrow-derived ALDH(br) cells in patients with ischemic heart failure and critical limb ischemia were initiated. Study designs are described. Results of the completed study in patients with critical limb ischemia (CLI) are encouraging and are summarized. Results of 6-month follow-up for the study in ischemic heart failure are pending.

Chemical constituents of Melandrium firmum Rohrbach and their anti-inflammatory activity.

In our ongoing search for anti-inflammatory agents originating from Korean medicinal plants, we found that the hexane and BuOH fractions of the MeOH extract from the whole plants of Melandrium firmum Rohrbach inhibited 5-lipoxygenase (5-LOX) activity. By activity-guided fractionation, eleven compounds, alpha-spinaterol (1), ursolic acid (2), ergosterol peroxide (3), alpha-spinaterol glucoside (4), 2-methoxy-9-beta-D-ribofuranosyl purine (5), aristeromycin (6), ecdysteron (7), polypodoaurein (8), (-)-bornesitol (9), mannitol (10) and cytisoside (11) were isolated from the hexane and BuOH fractions using column chromatography. Compounds 2, 5, 6, 8, 9, 10 and 11 were isolated for the first time from this plant. Compounds 1, 3, 4 and 7 inhibited 5-LOX activity with IC50 values of 21.04 microM, 42.30 microM, 32.82 microM, and 17.18 microM, respectively.

Preventive effects of puerarin on alcohol-induced osteonecrosis.

Alcohol can induce adipogenesis by bone marrow stromal cells and may cause osteonecrosis of the femoral head. Currently, there are no medications available to prevent alcohol-induced osteonecrosis. We hypothesized puerarin, a Chinese herbal medicine with antioxidative and antithrombotic effects, can prevent alcohol-induced adipogenesis and osteonecrosis. Both bone marrow stromal cells (in vitro) and mice (in vivo) were treated either with ethanol or with ethanol and puerarin, with an untreated group serving as a control. In the in vitro study, the number of adipocytes, contents of triglycerides, and levels of PPAR gamma mRNA expression were decreased and alkaline phosphatase activity, contents of osteocalcin, and levels of osteocalcin mRNA expression were increased in cells treated with both alcohol and puerarin, compared with cells treated with alcohol only. In the in vivo study, marrow necrosis, fat cell hypertrophy and proliferation, thinner and sparse trabeculae, diminished hematopoiesis, and increased empty osteocyte lacunae in the subchondral region of the femoral head were observed in mice treated with alcohol. However, no such changes were seen in femoral heads of mice treated with alcohol and puerarin. The data suggest puerarin can inhibit adipogenic differentiation by bone marrow stromal cells both in vitro and in vivo and prevents alcohol-induced osteonecrosis in this model.