Osteoclast-like cells are known to inhibit arterial calcification. Receptor activator of NF-κB ligand (RANKL) is likely to act as an inducer of osteoclast-like cell differentiation. However, several studies have shown that RANKL promotes arterial calcification rather than inhibiting arterial calcification. The present study was conducted in order to investigate and elucidate this paradox. Firstly, RANKL was added into the media, and the monocyte precursor cells were cultured. Morphological observation and Tartrate resistant acid phosphatase (TRAP) staining were used to assess whether RANKL could induce the monocyte precursor cells to differentiate into osteoclast-like cells. During arterial calcification, in vivo and in vitro expression of RANKL and its inhibitor, osteoprotegerin (OPG), was detected by real-time PCR. The extent of osteoclast-like cell differentiation was also assessed. It was found RANKL could induce osteoclast-like cell differentiation. There was no in vivo or in vitro expression of osteoclast-like cells in the early stage of calcification. At that time, the ratio of RANKL to OPG was very low. In the late stage of calcification, a small amount of osteoclast-like cell expression coincided with a relatively high ratio of RANKL to OPG. According to the results, the ratio of RANKL to OPG was very low during most of the arterial calcification period. This made it possible for OPG to completely inhibit RANKL-induced osteoclast-like cell differentiation. This likely explains why RANKL had the ability to induce osteoclast-like cell differentiation but acted as a promoter of calcification instead.
Acid Phosphatase ; genetics ; metabolism ; Animals ; Aorta ; drug effects ; metabolism ; pathology ; Cell Differentiation ; Coculture Techniques ; Gene Expression Regulation ; Isoenzymes ; genetics ; metabolism ; Male ; Monocytes ; cytology ; drug effects ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; pathology ; Osteoclasts ; drug effects ; metabolism ; pathology ; Osteoprotegerin ; genetics ; metabolism ; RANK Ligand ; genetics ; metabolism ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Tartrate-Resistant Acid Phosphatase ; Vascular Calcification ; genetics ; metabolism ; pathology

OBJECTIVETo discuss the effect of Drynariae Rhizoma's naringin on osteoclasts induced by mouse monocyte RAW264.7.

METHODRAW264.7 cells were induced by 100 μg x L(-1) nuclear factor-κB receptor activator ligand (RANKL) and became mature osteoclasts, which were identified through TRAP specific staining and bone resorption. MTT method was sued to screen and inhibit and the highest concentration of osteoclasts. After being cultured with the screened medium containing naringin for 5 days, positive TRAP cell counting and bone absorption area analysis were adopted to observe the effect of naringin on the formation of osteoclast sells and the bone absorption function. The osteoclast proliferation was measured by flow cytometry. The effects of RANK, TRAP, MMP-9, NFATc1 and C-fos mRNA expressions on nuclear factor-κB were detected by RT-PCR.

RESULTNaringin could inhibit osteoclast differentiation, bone absorption function and proliferation activity of osteoclasts, significantly down-regulate RANK, TRAP, MMP-9 and NFATc1 mRNA expressions in the osteoclast differentiation process, and up-regulate the C-fos mRNA expression.

CONCLUSIONNaringin could inhibit osteoclast differentiation, proliferation and bone absorption function. Its mechanism may be achieved by inhibiting the specific gene expression during the osteoclast differentiation process.

Acid Phosphatase ; metabolism ; Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Flavanones ; pharmacology ; Isoenzymes ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; Mice ; NFATC Transcription Factors ; genetics ; Osteoclasts ; cytology ; drug effects ; Tartrate-Resistant Acid Phosphatase

Recent studies have shown that Er-Zhi-Wan (EZW), a traditional Chinese medicine consisting of Herba Ecliptae (HE) and Fructus Ligustri Lucidi (FLL), had a definite antiosteoporotic effect on osteoporotic femur, but its effect on osteoporosis of alveolar bone remains unknown. In the present study, we investigated the effects of Er-Zhi-Wan (EZW) on the microarchitecture and the regulation of Wnt/β-catenin signaling pathway in the alveolar bone of ovariectomized rats. Thirty Sprague-Dawley rats were randomly divided into three groups: sham operation group (sham, n=10), ovariectomy (OVX) group (n=10), and OVX with EZW treatment group (EZW group, n=10). From one week after ovariectomy, EZW (100 mg/mL) or vehicle (distilled water) was fed (1 mL/100 g) once per day for 12 weeks until the sacrifice of the rats. The body weights were measured weekly. After sacrifice, the sera and mandible were collected and routinely prepared for the measurement of alveolar trabecular microarchitecture, serum levels of E2, bone-specific alkaline phosphatase (BALP) and tartrate-resistant acid phosphatase 5b (TRAP5b), as well as mandibular mRNA expression of Wnt/β-catenin signaling pathway molecules wnt3a, low-density lipoprotein receptor-related protein 5 (LRP5), β-catenin and dickkopf homolog 1 (DKK1). The results showed that EZW treatment significantly prevented the body weight gain, degradation of alveolar trabecular microarchitecture and alveolar bone loss in the OVX rats. Furthermore, we observed that EZW could increase the serum levels of E2 and BALP, and decrease levels of serum TRAP5b in EZW group compared with vehicle group. In addition, RT-PCR results revealed that EZW upregulated the expression levels of wnt3a, LRP5 and β-catenin, and reduced the expression of DKK1 in OVX rats. Taken together, our results suggested that EZW may have potential anti-osteoporotic effects on osteoporotic alveolar bone by stimulating Wnt/LRP5/β-catenin signaling pathway.
Acid Phosphatase ; blood ; Alkaline Phosphatase ; blood ; Alveolar Process ; drug effects ; metabolism ; Animals ; Body Weight ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Estradiol ; blood ; Female ; Gene Expression ; drug effects ; Intercellular Signaling Peptides and Proteins ; genetics ; Isoenzymes ; blood ; Low Density Lipoprotein Receptor-Related Protein-5 ; genetics ; Mandible ; drug effects ; metabolism ; Medicine, Chinese Traditional ; methods ; Organ Size ; drug effects ; Ovariectomy ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Tartrate-Resistant Acid Phosphatase ; Time Factors ; Up-Regulation ; drug effects ; Uterus ; drug effects ; growth & development ; Wnt Signaling Pathway ; drug effects ; genetics ; Wnt3A Protein ; genetics ; beta Catenin ; genetics

To investigate 1alpha,25-(OH)2D3 regulation of matrix metalloproteinase-9 (MMP-9) protein expression during osteoclast formation and differentiation, receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were administered to induce the differentiation of RAW264.7 cells into osteoclasts. The cells were incubated with different concentrations of 1alpha,25-(OH)2D3 during culturing, and cell proliferation was measured using the methylthiazol tetrazolium method. Osteoclast formation was confirmed using tartrate-resistant acid phosphatase (TRAP) staining and assessing bone lacunar resorption. MMP-9 protein expression levels were measured with Western blotting. We showed that 1alpha,25-(OH)2D3 inhibited RAW264.7 cell proliferation induced by RANKL and M-CSF, increased the numbers of TRAP-positive osteoclasts and their nuclei, enhanced osteoclast bone resorption, and promoted MMP-9 protein expression in a concentration-dependent manner. These findings indicate that 1alpha,25-(OH)2D3 administered at a physiological relevant concentration promoted osteoclast formation and could regulate osteoclast bone metabolism by increasing MMP-9 protein expression during osteoclast differentiation.
Acid Phosphatase/metabolism ; Animals ; Blotting, Western ; Calcitriol/*pharmacology ; Calcium Channel Agonists/pharmacology ; *Cell Differentiation ; Cell Line ; Cell Proliferation ; Gene Expression Regulation, Enzymologic/*drug effects ; Isoenzymes/metabolism ; Matrix Metalloproteinase 9/*genetics/metabolism ; Mice ; Osteoclasts/*cytology/*enzymology ; Tetrazolium Salts ; Thiazoles

This study is to investigate the effect of 8-prenylnaringenin (8-PNG) on osteoclastogensis of bone marrow cells and bone resorption activity of osteoclasts. Osteoclasts were separated from long bone marrow of newborn rabbits and cultured in alpha-MEM containing 10% FBS. 8-PNG was added into culture media at 1 x 10(-7), 1 x 10(-6), 1 x 10(-5) mol xL(-1), separately. 17beta-Estradiol (E2, 1 x 10(-7) mol x L(-7)) was used as positive control. T RAP staining and TRAP activity measurement were performed after 5 days, and the bone resorption pits were analyzed after 7 days. Annexin V staining for the detection of apoptotic osteoclasts was performed after 2, 4, 8, 12, 24, 36 and 48 h separately. The mRNA expression level of TRAP and cathepsin K (CTSK) was measured by real-time RT-PCR. 8-PNG significantly reduced the number of osteoclasts which was TRAP staining positive and with more than three nucleus, the area and number of bone resorption pits decreased obviously in 8-PNG-supplemented groups. The apoptosis rate peaked earlier in the 8-PNG-supplemented groups and the mRNA expression level of TRAP and CTSK decreased significantly. All these inhibitory effects were in a dose dependent manner, the highest effect was obtained by 1 x 10(-5) mol x L(-1) 8-PNG. 8-PNG inhibits bone resorption activity of osteoclasts by inducing osteoclast apoptosis and inhibiting the gene expression and enzyme activity including TRAP and CTSK, and restrains bone marrow cells to osteoclast differentiation.
Acid Phosphatase ; genetics ; metabolism ; Animals ; Apoptosis ; drug effects ; Bone Marrow Cells ; cytology ; Bone Resorption ; Cathepsin K ; genetics ; metabolism ; Cells, Cultured ; Dose-Response Relationship, Drug ; Flavanones ; administration & dosage ; pharmacology ; Isoenzymes ; genetics ; metabolism ; Osteoclasts ; cytology ; metabolism ; RNA, Messenger ; metabolism ; Rabbits ; Tartrate-Resistant Acid Phosphatase

The purpose of this study was to determine whether osteoprotegerin (OPG) could affect osteoclat differentiation and activation under serum-free conditions. Both duck embryo bone marrow cells and RAW264.7 cells were incubated with macrophage colony stimulatory factor (M-CSF) and receptor activator for nuclear factor kappaB ligand (RANKL) in serum-free medium to promote osteoclastogenesis. During cultivation, 0, 10, 20, 50, and 100 ng/mL OPG were added to various groups of cells. Osteoclast differentiation and activation were monitored via tartrate-resistant acid phosphatase (TRAP) staining, filamentous-actin rings analysis, and a bone resorption assay. Furthermore, the expression osteoclast-related genes, such as TRAP and receptor activator for nuclear factor kappaB (RANK), that was influenced by OPG in RAW264.7 cells was examined using real-time polymerase chain reaction. In summary, findings from the present study suggested that M-CSF with RANKL can promote osteoclast differentiation and activation, and enhance the expression of TRAP and RANK mRNA in osteoclasts. In contrast, OPG inhibited these activities under serum-free conditions.
Acid Phosphatase/genetics/metabolism ; Animals ; Avian Proteins/*pharmacology ; Bone Marrow Cells/drug effects/*metabolism ; Cells, Cultured ; Ducks ; Embryo, Nonmammalian/drug effects/metabolism ; Isoenzymes/genetics/metabolism ; Macrophage Colony-Stimulating Factor/metabolism ; Osteoclasts/cytology/*drug effects/*metabolism ; Osteoprotegerin/*pharmacology ; RANK Ligand/metabolism ; Real-Time Polymerase Chain Reaction ; Receptor Activator of Nuclear Factor-kappa B/genetics/metabolism

OBJECTIVETo investigate the effect of osteoclast bone resorption supernatants on the osteogenic activity of mouse MC3T3-E1 cell line.

METHODSMouse RAW264.7 cell line was induced to osteoclast which was identified with tartrate resistant acid phosphatase (TRAP) staining and osteoclast specific gene detection. The differentiated RAW264.7 osteoclast was co-cultured with bovine milling bone specimen followed by toluidine blue staining. Then mouse MC3T3-E1 cell was cultured with supernatant from the osteoclast bone absorbent model. Methyl thiazolyl tetrazolium (MTT) method, alizarin red S staining, enzyme-linked immunosorbent assay detection of osteocalcin, and reverse transcriptase polymerase chain reaction detection were adopted to investigate the proliferation, calcification and osteogenic activity of MC3T3-E1 cells.

RESULTSTRAP staining, osteoclast specific gene detection and toluidine blue staining all indicated that RAW264.7 cell could be differentiated into functioning osteoclast. The supernatant from the osteoclast bone absorbent model could inhibit the proliferation of MC3T3-E1 cells, with the A value between 0.062 ± 0.004 and 0.405 ± 0.033 (P < 0.05). It could also increase the formation of calcification nods, promote the osteocalcin level which peaked with the tenth day's supernatant at a level of (2.965 ± 0.047) µg/L, as well as enhance the transcription of the alkaline phosphatase and Runt related transcription factor 2 gene.

CONCLUSIONSRAW264.7 osteoclast bone absorbent supernatant might influence the osteogenic activity of osteoblast-like cell by inhibiting proliferation, promoting differentiation and calcification.

Acid Phosphatase ; metabolism ; Alkaline Phosphatase ; genetics ; metabolism ; Animals ; Bone Resorption ; Calcification, Physiologic ; Cathepsin K ; metabolism ; Cell Differentiation ; drug effects ; Cell Line ; Cell Proliferation ; drug effects ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Culture Media, Conditioned ; pharmacology ; Gene Expression ; Isoenzymes ; metabolism ; Mice ; Osteoblasts ; cytology ; metabolism ; Osteocalcin ; metabolism ; Osteoclasts ; cytology ; enzymology ; Tartrate-Resistant Acid Phosphatase ; Transcription, Genetic

Osteoclasts, together with osteoblasts, control the amount of bone tissue and regulate bone remodeling. Osteoclast differentiation is an important factor related to the pathogenesis of bone-loss related diseases. Reactive oxygen species (ROS) acts as a signal mediator in osteoclast differentiation. Simvastatin, which inhibits 3-hydroxy-3-methylglutaryl coenzyme A, is a hypolipidemic drug which is known to affect bone metabolism and suppresses osteoclastogenesis induced by receptor activator of nuclear factor-kappaB ligand (RANKL). In this study, we analyzed whether simvastatin can inhibit RANKL-induced osteoclastogenesis through suppression of the subsequently formed ROS and investigated whether simvastatin can inhibit H2O2-induced signaling pathways in osteoclast differentiation. We found that simvastatin decreased expression of tartrate-resistant acid phosphatase (TRAP), a genetic marker of osteoclast differentiation, and inhibited intracellular ROS generation in RAW 264.7 cell lines. ROS generation activated NF-kappaB, protein kinases B (AKT), mitogen-activated protein kinases signaling pathways such as c-JUN N-terminal kinases, p38 MAP kinases as well as extracellular signal-regulated kinase. Simvastatin was found to suppress these H2O2-induced signaling pathways in osteoclastogenesis. Together, these results indicate that simvastatin acts as an osteoclastogenesis inhibitor through suppression of ROS-mediated signaling pathways. This indicates that simvastatin has potential usefulness for osteoporosis and pathological bone resorption.
Acid Phosphatase/genetics/metabolism ; Animals ; Anticholesteremic Agents/*pharmacology ; Blotting, Western ; *Cell Differentiation ; Cells, Cultured ; Hydrogen Peroxide/pharmacology ; Isoenzymes/genetics/metabolism ; Macrophages/cytology/drug effects/metabolism ; Mice ; Mitogen-Activated Protein Kinases/genetics/metabolism ; NF-kappa B/genetics/metabolism ; Osteoclasts/*cytology/*drug effects/metabolism ; RANK Ligand/metabolism ; RNA, Messenger/genetics ; Reactive Oxygen Species/*metabolism ; Real-Time Polymerase Chain Reaction ; Simvastatin/*pharmacology

BACKGROUNDLactate dehydrogenase (LDH) is a crucial regulator of energy metabolism in many organs including the heart. Lovastatin is widely used in prevention and treatment of coronary heart disease and is a drug with substantial metabolic influences. Our study aimed to determine the activities of the lactate dehydrogenase A and B (LDHA and LDHB) genes following lovastatin treatment.

METHODSThe rat myocardial cell line H9c2(2-1) in culture was exposed to 100 nmol/L lovastatin for 24 hours or for five days. The functions of the LDHA and LDHB genes were examined at the transcriptional (mRNA) level with quantitative real-time polymerase chain reaction (Q-RT-PCR), and at the translational (protein) level with immunoblotting.

RESULTSWhen compared with control levels, the LDHA mRNA went up by (151.65 ± 16.72)% (P = 0.0132) after 24 hours and by (175.28 ± 56.54)% (P = 0.0366) after five days of lovastatin treatment. Although 24 hours of lovastatin treatment had no significant effects on LDHB mRNA levels, when the treatment was extended to five days, LDHB mRNA levels were significantly down-regulated to (63.65 ± 15.21)% of control levels (P = 0.0117). After 24 hours of treatment with lovastatin, there were no significant changes in protein levels of either LDHA or LDHB. When treatment time was extended to five days, the protein levels of LDHA were up-regulated by (148.65 ± 11.81)% (P = 0.00969), while the protein levels of LDHB were down-regulated to (64.91 ± 5.47)% of control levels (P = 0.0192).

CONCLUSIONSLovastatin affects gene activities of LDHA and LDHB differently, which may reveal novel pharmacological effects of lovastatin.

Animals ; Anticholesteremic Agents ; pharmacology ; Blotting, Western ; Cell Line ; Isoenzymes ; genetics ; metabolism ; L-Lactate Dehydrogenase ; genetics ; metabolism ; Lovastatin ; pharmacology ; Myocytes, Cardiac ; drug effects ; enzymology ; Rats ; Reverse Transcriptase Polymerase Chain Reaction

Aging ; metabolism ; Amidohydrolases ; metabolism ; Animals ; Caenorhabditis elegans ; cytology ; drug effects ; growth & development ; metabolism ; Caenorhabditis elegans Proteins ; genetics ; metabolism ; Caloric Restriction ; Ethanolamines ; metabolism ; pharmacology ; Forkhead Transcription Factors ; Gene Expression Regulation, Developmental ; Isoenzymes ; genetics ; metabolism ; Signal Transduction ; Superoxide Dismutase ; genetics ; metabolism ; Trans-Activators ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism