低分子量褐藻糖胶对破骨细胞凋亡的影响

目的探讨低分子量褐藻糖胶(LMWF)对小鼠单核细胞RAW264.7诱导成熟破骨细胞凋亡的影响。方法通过100ng/m L RANKL诱导RAW264.7细胞株分化为破骨细胞,经TRAP特异性染色和骨吸收陷窝对诱导后的细胞进行鉴定。鉴定成功后,用100 ng/m L RANKL诱导RAW264.7细胞株5 d后,使用含有LMWF的培养基继续培养3 d,通过对TRAP阳性细胞计数和分析骨吸收面积来观察低分子量褐藻糖胶对破骨细胞的抑制和骨吸收功能情况;采用流式细胞术检测LMWF对破骨细胞凋亡的影响,capsase-3活性测试试剂盒检测LMWF对capsase-3活性进行测定;RT-PCR检测LMWF对成熟破骨细胞BAX与BCL-2基因表达的影响。结果单纯采用100 ng/m L的RANKL可成功诱导成熟的、有功能的破骨细胞。LMWF可以明显抑制RANKL诱导成熟破骨细胞的形成以及成熟破骨细胞的骨吸收功能;流式细胞术显示LMWF可增加成熟破骨细胞的早期凋亡率;并且能升高capsase-3的活性;PCR显示LMWF可明显下调破骨细胞凋亡相关的BCL-2和上调BAX基因mRNA表达,降低BCL-2/BAX的比值。结论低分子量褐藻糖胶可抑制破骨细胞的活性与骨吸收能力,促进破骨细胞凋亡,其主要机制是通过下调BCL-2和上调BAX mRNA基因表达实现的。     

Imatinib promotes osteoblast differentiation by inhibiting PDGFR signaling and inhibits osteoclastogenesis by both direct and stromal cell-dependent mechanisms.

Several lines of evidence suggest that imatinib may affect skeletal tissue. We show that inhibition by imatinib of PDGFR signaling in osteoblasts activates osteoblast differentiation and inhibits osteoblast proliferation and that imatinib inhibits osteoclastogenesis by both stromal cell-dependent and direct effects on osteoclast precursors. INTRODUCTION: Imatinib mesylate, an orally active inhibitor of the c-abl, c-kit, and platelet-derived growth factor receptor (PDGFR) tyrosine kinases, is in clinical use for the treatment of chronic myeloid leukemia (CML) and gastrointestinal stromal cell tumors. Interruption of both c-kit and c-abl signaling in mice induces osteopenia, suggesting that imatinib might have adverse effects on the skeleton. However, biochemical markers of bone formation increase in patients with CML starting imatinib therapy, whereas bone resorption is unchanged, despite secondary hyperparathyroidism. We assessed the actions of imatinib on bone cells in vitro to study the cellular and molecular mechanism(s) underlying the skeletal effects we observed in imatinib-treated patients. MATERIALS AND METHODS: Osteoblast differentiation was assessed using a mineralization assay, proliferation by [(3)H]thymidine incorporation, and apoptosis by a TUNEL assay. Osteoclastogenesis was assessed using murine bone marrow cultures and RAW 264.7 cells. RT and multiplex PCR were performed on RNA prepared from human bone marrow samples, osteoblastic cells, and murine bone marrow cultures. Osteoprotegerin was measured by ELISA. RESULTS: The molecular targets of imatinib are expressed in bone cells. In vitro, imatinib increases osteoblast differentiation and prevents PDGF-induced inhibition of this process. Imatinib inhibits proliferation of osteoblast-like cells induced by serum and PDGF. In murine bone marrow cultures, imatinib inhibits osteoclastogenesis stimulated by 1,25-dihydroxyvitamin D(3) and partially inhibits osteoclastogenesis induced by RANKL and macrophage-colony stimulating factor. Imatinib partially inhibited osteoclastogenesis in RANKL-stimulated RAW-264.7 cells. Treatment with imatinib increases the expression of osteoprotegerin in bone marrow from patients with CML and osteoblastic cells. CONCLUSIONS: Taken together with recent in vivo data, these results suggest a role for the molecular targets of imatinib in bone cell function, that inhibition by imatinib of PDGFR signaling in osteoblasts activates bone formation, and that the antiresorptive actions of imatinib are mediated by both stromal cell-dependent and direct effects on osteoclast precursors.     

The generation of osteoclasts from RAW 264.7 precursors in defined,serum-free conditions

Osteoclasts are the unique cell type capable of resorbing bone. The discovery of the TNF-ligand family member, RANKL, has allowed more reliable study of these important cells. The mouse monocytic cell line, RAW 264.7, has been shown to readily differentiate into osteoclasts upon exposure to recombinant RANKL. Unlike primary osteoclast precursors, there is no requirement for the addition of macrophage colony stimulating factor (M-CSF). However, to date, their differentiation has always been studied in the context of added foetal calf serum (FCS). FCS is a complex and largely undefined mixture of growth factors and matrix proteins, and varies between batches. For this reason, osteoclastogenesis would ideally be studied in the context of a defined, serum-free medium. RAW 264.7 cells were cultured in serum-replete α-MEM or serum-deprived medium (SDM) shown previously to support the growth of human osteoclasts in a co-culture with normal osteoblasts. In SDM, in the presence of recombinant RANKL, RAW 264.7 cells readily differentiated into tartrate resistant acid phosphatase (TRAP) positive multinucleated osteoclast-like cells, a process that was enhanced with the addition of 1α,25-dihydroxyvitamin D₃ (1,25D). While the osteoclasts grown in SDM were smaller in size compared with those derived in serum-replete media, their resorptive capacity was significantly increased as indicated by a twofold increase in average resorption pit size. In conclusion, we describe a defined model for studying osteoclast differentiation and activity in the absence of serum, which will be ideal for studying the role of agonistic and antagonistic molecules in this process.     

Prostate-specific antigen stimulates osteoprotegerin production and inhibits receptor activator of nuclear factor-kappaB ligand expression by human osteoblasts

BACKGROUND: Prostate cancer cells produce a large amount of prostate-specific antigen (PSA), which is widely used as a marker for this cancer. Even though it is widely used in the diagnosis of prostate cancer, many aspects of the pathophysiologic role of PSA in bone metastasis remain obscure. The receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for the activation of osteoclasts, while osteoprotegerin (OPG) neutralizes the action of RANKL. Various substances that act on bone have been shown to modulate the production of RANKL and OPG by osteoblasts. METHODS: In this study, we investigated the effect of PSA on the expression of OPG and RANKL mRNA and on protein production in human osteoblast-like cells. RESULTS: After addition of PSA and culture for 72 h, OPG mRNA expression and protein secretion by MG-63 and SaOS-2 cells showed a concentration-dependent increase. When osteoblasts were incubated with PSA (100 ng/ml), OPG mRNA expression and protein secretion increased with the passage of time. alpha1 -antichymotrypsin (ACT), which inactivates the serine protease activity of PSA, inhibited the increase of OPG mRNA expression and protein production in response to PSA, and this effect of PSA was also inhibited by anti-transforming growth factor-beta antibody. CONCLUSIONS: Based on our findings, PSA acts on human osteoblast-like cells via its own serine protease activity and promotes osteoblast differentiation. In addition, PSA stimulates OPG production and inhibits RANKL expression of osteoblasts, and inhibits bone resorption by osteoclasts, suggesting that it contributes to the characteristic osteoblastic features of bone metastases of prostate cancer.     

Dissociation of the pro-apoptotic effects of bisphosphonates on osteoclasts from their anti-apoptotic effects on osteoblasts/osteocytes with novel analogs

Bisphosphonates induce osteoclast apoptosis, thereby decreasing bone resorption and reducing the rate of bone remodeling. Earlier work from our group and others has demonstrated that, additionally, bisphosphonates prevent osteoblast and osteocyte apoptosis in vivo and in vitro, raising the possibility that perhaps part of their anti-fracture efficacy may result from preserving the integrity of the osteocyte network and prolonging the working time of bone forming cells. Whereas induction of osteoclast apoptosis results from inhibition of the mevalonate pathway or from conversion to toxic ATP analogs, prevention of osteoblastic cell apoptosis is mediated by connexin43 hemichannel opening and activation of the extracellular signal-regulated kinases (ERKs). We examined here the ability of several bisphosphonates, including novel analogs, to exert these two effects. All 16 bisphosphonates studied inhibited etoposide-induced apoptosis of MLO-Y4 osteocytic cells and osteoblastic cells derived from calvaria, with EC50 between 10(-12) and 10(-10) M. On the other hand, only 10 analogs induced apoptosis of RAW-264.7-cell-derived osteoclasts. Each of the 6 bisphosphonates that lack pro-apoptotic activity in osteoclasts but retain anti-apoptotic activity in osteoblasts and osteocytes has a structural-related analog that is active in both cell types. These findings indicate that the structural prerequisites for the anti-apoptotic effect of bisphosphonates on cells of the osteoblastic lineage are less stringent than the ones required to induce osteoclast apoptosis and confirm that bisphosphonates act on the two cell types by distinct mechanisms. Preservation of osteoblast and osteocyte viability without inducing osteoclast apoptosis by these bisphosphonates analogs opens new possibilities for the treatment of bone fragility in conditions in which a decrease in bone remodeling is not desirable.     

中药复方鹿角胶丸通过PI3-K/AKT信号通路调节破骨细胞凋亡

目的探索中药复方鹿角胶丸促进破骨细胞凋亡机制的实验研究。方法 RAW264.7细胞培养并传代,RANKL+MCSF诱导RAW264.7细胞向破骨细胞分化;MTS检测不同浓度的鹿角胶丸对破骨细胞增殖活性影响;倒置相差显微镜观察鹿角胶丸对破骨细胞形态学的影响;流式细胞仪检测鹿角胶丸及加入PI3K/AKT通路抑制剂LY294002后对破骨细胞凋亡的影响;Western blot检测Bax、Bcl-2、Caspase-3,Cleaved-Caspase-3、p-AKT、T-AKT的蛋白表达情况,以及检测加入LY294002后对凋亡通路蛋白的影响。结果 MTS显示各个浓度的鹿角胶丸(LJJW高、中、低)均能抑制破骨细胞的增殖活性,其中以LJJW(中浓度)作用24 h后具有明显的增殖活性抑制作用。加入鹿角胶丸后,破骨细胞透光度下降,凋亡的细胞皱缩,漂浮于培养基中。流式细胞仪检测结果显示鹿角胶丸促进破骨细胞凋亡,预处理LY294002后,鹿角胶丸的促破骨细胞凋亡作用受到抑制。免疫印迹结果显示,鹿角胶丸促进线粒体Bax表达,抑制Bcl-2的表达,同时促进Caspase-3的裂解。鹿角胶丸促进AKT磷酸化,加入LY294002后AKT的磷酸化受到抑制,预处理LY294002后鹿角胶丸的促AKT磷酸化作用被抑制。结论中药复方鹿角胶丸通过PI3K/AKT通路促进破骨细胞凋亡。     

重组人骨保护素与重组核因子κb活化因子受体蛋白对破骨前体细胞分化的影响

目的:对比重组人骨保护素(rhOPG-Fc)与重组核因子κb活化因子受体蛋白(rhRANK)对破骨前体细胞分化的影响.方法:采用成骨细胞与破骨前体细胞RAW264.7混合培养,在地塞米松、1,25 (OH) 2VitD3诱导下生成破骨细胞的方法.研究分3组:rhRANK组:10-5 g/L;rhOPG-Fc组:10-5 g/L;空白对照组.作用9d后观察破骨细胞数目和形态,抗酒石酸酸性磷酸酶(TRAP)染色阳性细胞个数,骨磨片吸收陷窝计数.结果:在空白对照组,小鼠成骨细胞与破骨前体细胞RAW264.7混合培养6d后,开始出现多核细胞,9d时可见大量成熟多核细胞,经TRAP染色证实为成熟破骨细胞,而rhRANK组及rhOPG-Fc组TRAP染色阳性多核细胞数较对照组均减少,特别是rhRANK组减少更明显.骨片吸收陷窝计数显示rhRANK组及rhOPG-Fc组较对照组也明显减少,而相对来说,rhRANK组较rhOPG-Fc组更少.结论:rhOPG-Fc与rhRANK均可以有效抑制破骨前体细胞分化成为成熟破骨细胞,且rhRANK较rhOPG-Fc抑制效果更明显.     

Oscillatory fluid flow-induced shear stress decreases osteoclastogenesis through RANKL and OPG signaling

Physical activity creates deformation in bone that leads to localized pressure gradients that drive interstitial fluid flow. Due to the cyclic nature of the applied load, this flow is oscillatory by nature. Oscillatory fluid flow (OFF) may lead to positive bone remodeling through effects on both osteoblasts and osteoclasts but its effect on osteoclastogenesis is poorly understood. In this study, the effects of OFF on expression of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG), two important regulators of osteoclast differentiation, were investigated. In addition, its effect on osteoclast formation was quantified. ST-2 murine bone marrow stromal cells were plated on glass slides and cultured with 1,25-dihydroxyvitamin D(3) to express RANKL. Cells were exposed to various durations of OFF resulting in a peak shear stress of 1 Pa. Time course and dose-response studies were performed and real-time RT-PCR was used to quantify levels of RANKL, OPG mRNA. ST-2 cells exposed to OFF were also co-cultured with RAW 264.7 monocytes and osteoclast number quantified. Decrease in RANKL/OPG was maximal immediately after end of flow and there existed a significant increase in OPG and decrease in RANKL with increasing load duration of up to 2 h. OFF resulted in a decrease in osteoclast formation by ST-2 cells co-cultured with RAW 264.7 cells compared to co-culture of control (non-loaded) ST-2 cells with RAW 264.7 cells. These results suggest that indeed OFF is a potent regulator of bone remodeling, and that shift towards positive bone remodeling mediated by loading-induced fluid flow may occur via suppression of the formation of osteoclasts.     

Differential contribution of osteoclast- and osteoblast-lineage cells to CpG-oligodeoxynucleotide (CpG-ODN) modulation of osteoclastogenesis.

CpG-ODNs modulate osteoclast differentiation through Toll-like receptor 9 (TLR9). Using TLR9-deficient mice, we found that activation of TLR9 on both osteoclast precursors and osteoblasts mediate the osteoclastogenic effect of CpG-ODN. Osteoclastic TLR9 is more important for this activity. INTRODUCTION: Bacterial infections cause pathological bone loss by accelerating differentiation and activation of the osteoclast. A variety of bacteria-derived molecules have been shown to enhance osteoclast differentiation through activation of Toll-like receptors (TLRs). We have shown that CpG-oligodeoxynucleotides (CpG-ODNs), mimicking bacterial DNA and exerting their cellular activities through TLR9, modulate osteoclast differentiation in a complex manner: the ODNs inhibit the activity of the physiological osteoclast differentiation factor RANKL in early osteoclast precursors (OCPs) but markedly stimulate osteoclastogenesis in cells primed by RANKL. MATERIALS AND METHODS: Osteoclast precursors and osteoblasts from TLR9-deficient (TLR9-/-) and wildtype (TLR9+/+) mice were used for in vitro analyses of osteoclast differentiation and modulation of signal transduction and gene expression. RESULTS: As expected CpG-ODN did not exert any activity in cells derived from TLR9-/-mice; these cells, however, responded in a normal manner to other stimuli. Using bone marrow/osteoblasts co-cultures from all possible combinations of TLR9-/- and TLR9+/+ mice-derived cells, we showed that TLR9 in the two lineages is required for CpG-ODN induction of osteoclastogenesis. CONCLUSIONS: CpG-ODN modulates osteoclastogenesis in a TLR9-dependent manner. Activation of TLR9 in bone marrow-derived osteoclasts precursors is more crucial to induction of osteoclastogenesis than activation of the osteoblastic TLR9.     

通过骨基质表面培养板检测RANKL诱导破骨细胞骨侵蚀能力

目的探讨以骨基质表面培养板替代骨磨片鉴定破骨细胞骨侵蚀能力的应用方法。方法通过RANKL诱导破骨前体细胞RAW264. 7建立破骨细胞分化模型,运用TRAP染色检测破骨细胞分化程度,并以骨基质表面培养板替代骨磨片行骨陷窝试验检测破骨细胞骨侵蚀能力,以侵蚀面积反映骨侵蚀能力。结果不同浓度的RANKL因子可有效诱导破骨前体细胞RAW264. 7分化为成熟多核破骨细胞,呈浓度依赖性。成熟破骨细胞在骨基质表面培养板上形成不同面积的不规则侵蚀圆环,趋势与破骨细胞分化程度一致。结论使用骨基质表面培养板可有效反映破骨细胞形成及骨侵蚀能力,与TRAP染色结果一致,并且具有操作简便、结果直观、便于统计分析等优点。     

RANKL regulates Fas expression and Fas-mediated apoptosis in osteoclasts.

Osteoclast apoptosis is an influential determinant of osteoclast bone-resorbing activity. RANKL, a critical factor for osteoclastogenesis, is also important in osteoclast survival. However, the mechanisms by which RANKL prevents osteoclast apoptosis remain largely unknown. INTRODUCTION: Fas, a death receptor, mediates apoptosis in multiple types of cells including osteoclasts. Here we report that RANKL acts as a survival factor in osteoclasts by downregulating Fas-mediated apoptosis and Fas expression in mature osteoclasts. MATERIALS AND METHODS: RAW264.7 and mouse bone marrow macrophage/monocyte progenitors and progenitor-derived osteoclasts, in the presence of various concentrations of RANKL, were used in this study. Western blotting, semiquantitative RT-PCR, flow cytometry, nuclear staining, and a fluorescent caspase-3 activity assay were used to assess the effect of RANKL on Fas expression and Fas-mediated apoptosis. The involvement of NF-kappaB in the regulation of Fas by RANKL was analyzed by luciferase assay and EMSA. RESULTS: Mature osteoclasts generated in the presence of a high concentration of RANKL (3.33 nM) failed to respond to Fas-induced apoptosis. The lack of responsiveness in mature osteoclasts is caused by the low level of Fas expression, as detected by both semiquantitative PCR and Western blotting. Fas protein and mRNA expression are inhibited by RANKL in concentration-dependent manners. The downregulation of Fas expression by RANKL is not because of modulation of the stability of Fas protein or mRNA. The regulation of Fas expression by RANKL is biphasic. During the early stage of osteoclastogenesis (1 day) when Fas is expressed at a very low level, RANKL upregulates Fas promoter activity by 2.4 +/- 0.1-fold in a concentration-dependent manner and increases Fas mRNA and protein. This event correlates with regulation of the binding activity of NF-kappaB to the Fas promoter by RANKL, as detected by EMSA. In osteoclast precursors, the induction of Fas promoter activity by RANKL was dramatically reduced when NF-kappaB binding sites on the Fas promoter were mutated. CONCLUSION: RANKL upregulates Fas expression in osteoclast progenitors through NF-kappaB, making osteoclasts targets of Fas-stimulated apoptosis. In differentiated mature osteoclasts, RANKL reduces the levels of Fas expression and Fas-mediated apoptosis, acting as a survival factor.     

Biglycan deficiency increases osteoclast differentiation and activity due to defective osteoblasts

Bone mass is maintained by a fine balance between bone formation by osteoblasts and bone resorption by osteoclasts. Although osteoblasts and osteoclasts have different developmental origins, it is generally believed that the differentiation, function, and survival of osteoclasts are regulated by osteogenic cells. We have previously shown that the extracellular matrix protein, biglycan (Bgn), plays an important role in the differentiation of osteoblast precursors. In this paper, we showed that Bgn is involved in regulating osteoclast differentiation through its effect on osteoblasts and their precursors using both in vivo and in vitro experiments. The in vivo osteolysis experiment showed that LPS (lipopolisaccharide)-induced osteolysis occurred more rapidly and extensively in bgn deficient mice compared to wild type (WT) mice. To further understand the mechanism of action, we determined the effects of Bgn on 1, 25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃)-induced osteoclast differentiation and bone resorption in an co-culture of calvariae-derived pre-osteoblasts and osteoclast precursors derived from spleen or bone marrow. Time course and dose response experiments showed that tartrate-resistant acid phosphatase-positive multinuclear cells appeared earlier and more extensively in the co-cultures containing calvarial cells from bgn deficient mice than WT mice, regardless of the genotype of osteoclast precursors. The osteoblast abnormality that stimulated osteoclast formation appeared to be independent of the differential production of soluble RANKL and OPG and, instead, due to a decrease in osteoblast maturation accompanied by increase in osteoblastic proliferation. In addition to the imbalance between differentiation and proliferation, there was a differential decrease in secretory leukocyte protease inhibitor (slpi) in bgn deficient osteoblasts treated with 1,25-(OH)₂D₃. These findings point to a novel molecular factor made by osteoblasts that could potentially be involved in LPS-induced osteolysis.     

BSP and RANKL induce osteoclastogenesis and bone resorption synergistically.

RANKL and BSP are upregulated in several bone resorptive disorders. However, the mechanisms by which these two factors might induce osteoclastogenesis and bone resorption synergistically under pathological conditions remain largely unknown. INTRODUCTION: RANKL and bone sialoprotein II (BSP) have been shown to be upregulated in the serum of individuals with abnormally high osteoclastogenic and bone resorptive activities. Here we provide experimental evidence that RANKL and BSP induce osteoclastogenesis and bone resorption synergistically but mediate opposite effects in osteoclast survival and apoptosis. MATERIALS AND METHODS: RAW264.7 cells and mouse bone marrow-derived monocytes/macrophages were treated with human recombinant BSP in the presence and absence of RANKL. TRACP stainings, bone resorption assays, Western blotting, immunoprecipitation analyses, and semiquantitative RT-PCR were used to evaluate the effects of BSP in osteoclast differentiation and bone resorption. Survival, DNA condensation, and caspase activity assays were used to determine the putative effects of BSP in osteoclast survival and apoptosis. RESULTS AND CONCLUSIONS: RANKL induced osteoclast differentiation and bone resorption at a higher extent in the presence than in the absence of BSP in RAW264.7 cells and bone marrow-derived monocytes/macrophages. c-Src-dependent c-Cbl phosphorylation was 8-fold higher in RAW264.7 cells treated with BSP and RANKL than in those treated with RANKL alone. Furthermore, BSP and RANKL activated the master regulator of osteoclastogenesis nuclear factor of activated T cells (NFAT)-2 and increased the mRNA expression of other differentiation markers such as cathepsin K or TRACP. Inhibition of c-Src activity or chelating intracellular calcium inhibited the synergistic effects in bone resorption and the phosphorylation of the c-Src substrate c-Cbl. Inhibition of calcineurin or intracellular calcium elevation inhibited the synergistic effects in osteoclastogenesis and decreased NFAT-2 nuclear levels. On the other hand, BSP and RANKL mediated opposite effects in osteoclast survival and apoptosis. Thus, BSP increased survival and decreased apoptosis markers in differentiated RANKL-treated RAW267.5 cells and RANKL/macrophage-colony stimulating factor (M-CSF)-treated bone marrow-derived monocytes/macrophages. In addition, RAW267.5 cells treated with BSP and RANKL exhibited decreased activation of the proapoptotic Jun N-terminal kinase pathway and increased activation of anti-apoptotic AKT pathway than cells treated with RANKL or BSP alone. Taken together, our findings suggest that BSP contributes to RANKL-mediated bone resorption by inducing osteoclastogenesis and osteoclast survival and decreasing osteoclast apoptosis.     

Bergapten prevents lipopolysaccharide mediated osteoclast formation,bone resorption and osteoclast survival

Purpose

This study was designed to investigate the potential effect of bergapten on lipopolysaccharide (LPS)-mediated osteoclast formation, bone resorption and osteoclast survival in vitro.

Methods

After osteoclast precursor RAW264.7 cells were treated with bergapten (5, 20, 40 μmol/L) for 72 hours in the presence of LPS (100 ng/ml), osteoclastogenesis was identified by tartrate-resistant acid phosphatase (TRAP) staining, and the number of TRAP-positive multinucleated cells [TRAP(+)MNCs] per well were counted. To investigate the effect of bergapten on osteoclastic bone resorption, RAW264.7 cells were treated with bergapten for six days in the presence of LPS, and the area of bone resorption was analyzed with Image Pro-Plus. Next, we examined apoptosis of RAW264.7 cells after bergapten incubation for 48 hours by flow cytometer using annexin V/propidium iodide (PI) double labeling. Finally, osteoclast survival was observed by Hoechst 33342 labeling and Western blotting after bergapten treatment for 24 hours.

Results

Data showed that bergapten (5–40 μmol/L) dose-dependently inhibited LPS-induced osteoclast formation and bone resorption. Treatment with bergapten triggered apoptotic death of osteoclast precursor RAW264.7 cells in a dose-dependent manner. Furthermore, bergapten significantly reduced the survival of mature osteoclast, as demonstrated by emergence of apoptotic nuclei and activation of apoptotic protein caspase 3/9.

Conclusions

These findings suggest that bergapten effectively prevents LPS-induced osteoclastogenesis, bone resorption and survival via apoptotic response of osteoclasts and their precursors. The study identifies bergapten as an inhibitor of osteoclast formation and bone resorption and provides evidence that bergapten might be beneficial as an alternative for prevention and treatment of inflammatory bone loss.     

GLP-1RA艾塞那肽调控骨骼代谢的作用研究

目的 为了探讨胰高血糖素样肽-1 (glucagon-like peptide-1,GLP-1)受体激动剂对糖尿病患者的成骨细胞和破骨细胞的影响。方法 利用小鼠成骨细胞MC3T3-E1和小鼠单核巨噬细胞RAW264.7模拟成骨细胞和破骨细胞。在高糖培养基中分别加入不同浓度的艾塞那肽孵育72 h,利用RT-qPCR技术分别检测成骨细胞MC3T3-E1中GLP1-R、ALP、LRP-5和破骨细胞RAW 264.7中GLP1-R、Cathespin K的表达情况。结果 艾塞那肽诱导成骨细胞MC3T3-E1细胞中GLP-1R、ALP、LRP-5基因的表达显著升高,而明显抑制破骨细胞RAW 264.7中GLP1-R、Cathespin K的表达。结论 GLP-1受体激动剂艾塞那肽可能通过影响成骨细胞及破骨细胞的分化,从而对糖尿病引起的骨质疏松起到一定程度的改善。     

高糖及TNF-α对RAW264.7细胞向破骨细胞分化的影响

【摘要】 目的 观察高糖及TNF-α的培养条件对RAW264.7细胞向破骨细胞诱导分化的影响。方法 在正常、高糖(30 mmol/L)及TNF-α(10 μmol/L)条件下培养RAW264.7细胞后,加入浓度为100 ng/mL的细胞核转录因子κB受体激活物的配体(receptor activator of NF-κB ligand, RANKL)为诱导剂,诱导RAW264.7向破骨细胞分化;诱导9天后,抗酒石酸酸性磷酸酶(TRAP)染色,比较各组TRAP+细胞数,RT-PCR及Western Blot检测各组破骨细胞标志基因CTR和MMP-9的表达。结果 不同的培养条件下RANKL均能诱导RAW264.7分化为成熟的破骨细胞,其中TNF-α环境中RAW264.7形成的TRAP+阳性细胞数、CTR和MMP-9的表达最高,而在高糖环境下最低。结论 TNF-α可以促进RAW264.7向破骨细胞分化,而高糖对这个过程可能是抑制作用,这一现象符合Ⅰ型和Ⅱ糖尿病患者骨质破坏的表现;高糖及TNF-α的培养条件下RANKL对RAW264.7的作用可模拟糖尿病足病变微环境中OC的诱导分化的过程。