Bioactive materials from berberine-treated human bone marrow mesenchymal stem cells promote alveolar bone regeneration by regulating macrophage polarization.

Alveolar bone regeneration has been strongly linked to macrophage polarization. M1 macrophages aggravate alveolar bone loss, whereas M2 macrophages reverse this process. Berberine (BBR), a natural alkaloid isolated and refined from Chinese medicinal plants, has shown therapeutic effects in treating metabolic disorders. In this study, we first discovered that culture supernatant (CS) collected from BBR-treated human bone marrow mesenchymal stem cells (HBMSCs) ameliorated periodontal alveolar bone loss. CS from the BBR-treated HBMSCs contained bioactive materials that suppressed the M1 polarization and induced the M2 polarization of macrophages in vivo and in vitro. To clarify the underlying mechanism, the bioactive materials were applied to different animal models. We discovered macrophage colony-stimulating factor (M-CSF), which regulates macrophage polarization and promotes bone formation, a key macromolecule in the CS. Injection of pure M-CSF attenuated experimental periodontal alveolar bone loss in rats. Colony-stimulating factor 1 receptor (CSF1R) inhibitor or anti-human M-CSF (M-CSF neutralizing antibody, Nab) abolished the therapeutic effects of the CS of BBR-treated HBMSCs. Moreover, AKT phosphorylation in macrophages was activated by the CS, and the AKT activator reversed the negative effect of the CSF1R inhibitor or Nab. These results suggest that the CS of BBR-treated HBMSCs modulates macrophage polarization via the M-CSF/AKT axis. Further studies also showed that CS of BBR-treated HBMSCs accelerated bone formation and M2 polarization in rat teeth extraction sockets. Overall, our findings established an essential role of BBR-treated HBMSCs CS and this might be the first report to show that the products of BBR-treated HBMSCs have active effects on alveolar bone regeneration.

The Modulation of Phospho-Extracellular Signal-Regulated Kinase and Phospho-Protein Kinase B Signaling Pathways plus Activity of Macrophage-Stimulating Protein Contribute to the Protective Effect of Stachydrine on Acetaminophen-Induced Liver Injury.

Stachydrine, a prominent bioactive alkaloid derived from Leonurus heterophyllus, is a significant herb in traditional medicine. It has been noted for its anti-inflammatory and antioxidant characteristics. Consequently, we conducted a study of its hepatoprotective effect and the fundamental mechanisms involved in acetaminophen (APAP)-induced liver injury, utilizing a mouse model. Mice were intraperitoneally administered a hepatotoxic dose of APAP (300 mg/kg). Thirty minutes after APAP administration, mice were treated with different concentrations of stachydrine (0, 2.5, 5, and 10 mg/kg). Animals were sacrificed 16 h after APAP injection for serum and liver tissue assays. APAP overdose significantly elevated the serum alanine transferase levels, hepatic pro-inflammatory cytokines, malondialdehyde activity, phospho-extracellular signal-regulated kinase (ERK), phospho-protein kinase B (AKT), and macrophage-stimulating protein expression. Stachydrine treatment significantly decreased these parameters in mice with APAP-induced liver damage. Our results suggest that stachydrine may be a promising beneficial target in the prevention of APAP-induced liver damage through attenuation of the inflammatory response, inhibition of the ERK and AKT pathways, and expression of macrophage-stimulating proteins.

Decreased melanoma CSF-1 secretion by Cannabigerol treatment reprograms regulatory myeloid cells and reduces tumor progression.

During solid tumor progression, the tumor microenvironment (TME) evolves into a highly immunosuppressive milieu. Key players in the immunosuppressive environment are regulatory myeloid cells, including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), which are recruited and activated via tumor-secreted cytokines such as colony-stimulating factor 1 (CSF-1). Therefore, the depletion of tumor-secreted cytokines is a leading anticancer strategy. Here, we found that CSF-1 secretion by melanoma cells is decreased following treatment with Cannabis extracts. Cannabigerol (CBG) was identified as the bioactive cannabinoid responsible for the effects. Conditioned media from cells treated with pure CBG or the high-CBG extract reduced the expansion and macrophage transition of the monocytic-MDSC subpopulation. Treated MO-MDSCs also expressed lower levels of iNOS, leading to restored CD8+ T-cell activation. Tumor-bearing mice treated with CBG presented reduced tumor progression, lower TAM frequencies and reduced TAM/M1 ratio. A combination of CBG and αPD-L1 was more effective in reducing tumor progression, enhancing survival and increasing the infiltration of activated cytotoxic T-cells than each treatment separately. We show a novel mechanism for CBG in modulating the TME and enhancing immune checkpoint blockade therapy, underlining its promising therapeutic potential for the treatment of a variety of tumors with elevated CSF-1 expression.

[Integrated strategy for biomarkers of stable coronary heart disease with phlegm and blood stasis syndrome based on RNA-seq and network pharmacology].

This study aimed to analyze the biological foundation and biomarkers of stable coronary heart disease(CHD) with phlegm and blood stasis(PBS) syndrome based on RNA-seq and network pharmacology. Peripheral blood nucleated cells from five CHD patients with PBS syndrome, five CHD patients with non-PBS syndrome, and five healthy adults were collected for RNA-seq. The specific targets of CHD with PBS syndrome were determined by differential gene expression analysis and Venn diagram analysis. The active ingredients of Danlou Tablets were screened out from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, and the "component-target" prediction was completed through PubChem and SwissTargetPrediction. The "drug-ingredient-target-signaling pathway" network of Danlou Tablets against CHD with PBS syndrome was optimized by Cytoscape software. After the target biomarkers were identified, 90 participants were enrolled for diagnostic tests, and 30 CHD patients with PBS syndrome were included in before-and-after experiment to determine the therapeutic effect of Danlou Tablets on those targets. As revealed by RNA-seq and Venn diagram analysis, 200 specific genes were identified for CHD with PBS syndrome. A total of 1 118 potential therapeutic targets of Danlou Tablets were predicted through network pharmacology. Through integrated analysis of the two gene sets, 13 key targets of Danlou Tablets in the treatment of CHD with PBS syndrome were screened out, including CSF1, AKR1C2, PDGFRB, ARG1, CNR2, ALOX15B, ALDH1A1, CTSL, PLA2G7, LAP3, AKR1C3, IGFBP3, and CA1. They were presumably the biomarkers of CHD with PBS syndrome. The ELISA test further showed that CSF1 was significantly up-regulated in the peripheral blood of CHD patients with PBS syndrome, and was significantly down-regulated after Danlou Tablets intervention. CSF1 may be a biomarker for CHD with PBS syndrome, and it is positively correlated with the severity of the disease. The diagnostic cut-off of CSF1 for CHD with PBS syndrome was 286 pg·mL~(-1).

Plumbagin, a Biomolecule with (Anti)Osteoclastic Properties.

Plumbagin is a plant-derived naphthoquinone that is widely used in traditional Asian medicine due to its anti-inflammatory and anti-microbial properties. Additionally, plumbagin is cytotoxic for cancer cells due to its ability to trigger reactive oxygen species (ROS) formation and subsequent apoptosis. Since it was reported that plumbagin may inhibit the differentiation of bone resorbing osteoclasts in cancer-related models, we wanted to elucidate whether plumbagin interferes with cytokine-induced osteoclastogenesis. Using C57BL/6 mice, we unexpectedly found that plumbagin treatment enhanced osteoclast formation and that this effect was most pronounced when cells were pre-treated for 24 h with plumbagin before subsequent M-CSF/RANKL stimulation. Plumbagin caused a fast induction of NFATc1 signalling and mTOR-dependent activation of p70S6 kinase which resulted in the initiation of protein translation. In line with this finding, we observed an increase in RANK surface expression after Plumbagin stimulation that enhanced the responsiveness for subsequent RANKL treatment. However, in Balb/c mice and Balb/c-derived RAW264.7 macrophages, these findings could not be corroborated and osteoclastogenesis was inhibited. Our results suggest that the effects of plumbagin depend on the model system used and can therefore either trigger or inhibit osteoclast formation.

Dynamic gene regulation by nuclear colony-stimulating factor 1 receptor in human monocytes and macrophages.

Despite their location at the cell surface, several receptor tyrosine kinases (RTK) are also found in the nucleus, as either intracellular domains or full length proteins. However, their potential nuclear functions remain poorly understood. Here we find that a fraction of full length Colony Stimulating Factor-1 Receptor (CSF-1R), an RTK involved in monocyte/macrophage generation, migrates to the nucleus upon CSF-1 stimulation in human primary monocytes. Chromatin-immunoprecipitation identifies the preferential recruitment of CSF-1R to intergenic regions, where it co-localizes with H3K4me1 and interacts with the transcription factor EGR1. When monocytes are differentiated into macrophages with CSF-1, CSF-1R is redirected to transcription starting sites, colocalizes with H3K4me3, and interacts with ELK and YY1 transcription factors. CSF-1R expression and chromatin recruitment is modulated by small molecule CSF-1R inhibitors and altered in monocytes from chronic myelomonocytic leukemia patients. Unraveling this dynamic non-canonical CSF-1R function suggests new avenues to explore the poorly understood functions of this receptor and its ligands.

Tetrahydroxystilbene Glucoside Regulates Proliferation, Differentiation, and OPG/RANKL/M-CSF Expression in MC3T3-E1 Cells via the PI3K/Akt Pathway.

Tetrahydroxystilbene glucoside (TSG) is a unique component of the bone-reinforcing herb Radix Polygoni Multiflori Preparata (RPMP). It has the ability to promote bone formation and protect osteoblasts. However, the underlying mechanism remains unclear. To better understand its biological function, we determined TSG's effect on murine pre-osteoblastic MC3T3-E1 cells by the MTT assay, flow cytometry, FQ-PCR, Western blot, and ELISA. The results showed that TSG caused an elevation of the MC3T3-E1 cell number, the number of cells in the S phase, and the mRNA levels of the runt-related transcription factor-2 (Runx2), osterix (Osx), and collagen type I α1 (Col1a1). In addition, the osteoprotegerin (OPG) mRNA level was up-regulated, while the nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) mRNA levels were down-regulated by TSG. Furthermore, TSG activated the phosphoinosmde-3-kinase/protein kinase B (also known as PI3K/Akt) pathway, and blocking this pathway by the inhibitor LY-294002 could impair TSG's functions in relation to the MC3T3-E1 cells. In conclusion, TSG could activate the PI3K/Akt pathway and thus promote MC3T3-E1 cell proliferation and differentiation, and influence OPG/RANKL/M-CSF expression. TSG merits further investigation as a potential therapeutic agent for osteoporosis treatment.

Antiinflammatory Activity of Cinnamon (Cinnamomum zeylanicum) Bark Essential Oil in a Human Skin Disease Model.

The effect of cinnamon (Cinnamomum zeylanicum) bark essential oil (CBEO) on human skin cells has not been elucidated. Therefore, we investigated the activity of a commercially available CBEO in a validated human dermal fibroblast system, a model of chronic inflammation and fibrosis. We first evaluated the impact of CBEO on 17 protein biomarkers that play critical roles in inflammation and tissue remodeling. The impact of CBEO on genome-wide gene expression was also evaluated. CBEO showed strong anti-proliferative effects on skin cells and significantly inhibited the production of several inflammatory biomarkers, including vascular cell adhesion molecule-1, intercellular cell adhesion molecule-1, monocyte chemoattractant protein-1, interferon gamma-induced protein 10, interferon-inducible T-cell alpha chemoattractant, and monokine induced by gamma interferon. In addition, CBEO significantly inhibited the production of several tissue remodeling molecules, including epidermal growth factor receptor, matrix metalloproteinase-1, and plasminogen activator inhibitor-1. Macrophage colony-stimulating factor, which is an immunomodulatory protein molecule, was also significantly inhibited by CBEO. Furthermore, CBEO significantly modulated global gene expression and altered signaling pathways, many of which are important in inflammation, tissue remodeling, and cancer biology. The study shows that CBEO is a promising antiinflammatory agent; however, further research is required to clarify its clinical efficacy. © 2017 The Authors. Phytotherapy Research published by John Wiley & Sons Ltd.

Different dynamics of NLRP3 inflammasome-mediated IL­1ß production in GM-CSF- and M-CSF-differentiated human macrophages.

IL-1ß is a "master" cytokine regulating a wide variety of physiologic and immunologic processes. The most frequently studied models for NLRP3 inflammasome-mediated IL-1ß production are the macrophages; however, depending on their microenvironment, they can develop into functionally different cells. Several protocols have been developed to model the diversity of these cells in vitro. Here, we report for the first time, to our knowledge, a comparative study about the dynamics and molecular mechanisms of NLRP3 inflammasome priming and activation in LPS-stimulated, human, monocyte-derived GM- or M-macrophages, differentiated in the presence of GM-CSF or M-CSF, respectively. Our results show that IL-1ß production by LPS-stimulated M-macrophages is a rapid and short event that requires ATP supplementation and is attenuated, in part, by the presence of IL-10, which reduces Akt signaling. However, IL-1ß production by GM-macrophages develops gradually, and these cells produce IL-1ß, even in the absence of ATP supplementation, because of the constitutively active caspase-1 enzyme. We show that the membrane-bound ectonucleotidases have an important regulatory role on the IL-1ß secretion in GM-macrophages. Furthermore, we provide evidence that adenosine treatment enhances LPS-primed IL-1ß secretion by GM-macrophages, but not by M-macrophages. These results show that, because of the different activation status and expression levels of the NLRP3 inflammasome components, as well as the signaling activity of the pathways, the two subtypes of macrophages respond very differently to the same stimuli. For this reason, the molecular composition of the microenvironment that shapes macrophage development should be considered when research or therapeutic methods are planned to control IL-1ß production.

Long-pulse gastric electrical stimulation protects interstitial cells of Cajal in diabetic rats via IGF-1 signaling pathway.

AIM: To investigate the effects of different parameters of gastric electrical stimulation (GES) on interstitial cells of Cajal (ICCs) and changes in the insulin-like growth factor 1 (IGF-1) signal pathway in streptozotocin-induced diabetic rats. METHODS: Male rats were randomized into control, diabetic (DM), diabetic with sham GES (DM + SGES), diabetic with GES1 (5.5 cpm, 100 ms, 4 mA) (DM + GES1), diabetic with GES2 (5.5 cpm, 300 ms, 4 mA) (DM + GES2) and diabetic with GES3 (5.5 cpm, 550 ms, 2 mA) (DM + GES3) groups. The expression levels of c-kit, M-SCF and IGF-1 receptors were evaluated in the gastric antrum using Western blot analysis. The distribution of ICCs was observed using immunolabeling for c-kit, while smooth muscle cells and IGF-1 receptors were identified using α-SMA and IGF-1R antibodies. Serum level of IGF-1 was tested using enzyme-linked immunosorbent assay. RESULTS: Gastric emptying was delayed in the DM group but improved in all GES groups, especially in the GES2 group. The expression levels of c-kit, M-SCF and IGF-1R were decreased in the DM group but increased in all GES groups. More ICCs (c-kit(+)) and smooth muscle cells (α-SMA(+)/IGF-1R(+)) were observed in all GES groups than in the DM group. The average level of IGF-1 in the DM group was markedly decreased, but it was up-regulated in all GES groups, especially in the GES2 group. CONCLUSION: The results suggest that long-pulse GES promotes the regeneration of ICCs. The IGF-1 signaling pathway might be involved in the mechanism underlying this process, which results in improved gastric emptying.

Beneficial effect of refined red palm oil on lipid peroxidation and monocyte tissue factor in HCV-related liver disease: a randomized controlled study.

BACKGROUND: A large amount of endotoxin can be detected in the peripheral venous blood of patients with liver cirrhosis, contributing to the pathogenesis of hepatotoxicity because of its role in oxidative stress. The present study aimed to test the effect of the supplementation with red palm oil (RPO), which is a natural oil obtained from oil palm fruit (Elaeis guineensis) rich in natural fat-soluble tocopherols, tocotrienols and carotenoids, on lipid peroxidation and endotoxemia with plasma endotoxin-inactivating capacity, proinflammatory cytokines profile, and monocyte tissue factor in patients with chronic liver disease. METHODS: The study group consisted of sixty patients (34 males and 26 females; mean age 62 years, range 54-75) with Child A/B, genotype 1 HCV-related cirrhosis without a history of ethanol consumption, randomly enrolled into an 8-week oral daily treatment with either vitamin E or RPO. All patients had undergone an upper gastrointestinal endoscopy 8 months before, and 13 out of them showed esophageal varices. RESULTS: Both treatments significantly decreased erythrocyte malondialdehyde and urinary isoprostane output, only RPO significantly affected macrophage-colony stimulating factor and monocyte tissue factor. Liver ultrasound imaging did not show any change. CONCLUSIONS: RPO beneficially modulates oxidative stress and, not least, downregulates macrophage/monocyte inflammatory parameters. RPO can be safely advised as a valuable nutritional implementation tool in the management of chronic liver diseases.

[Effects of retinol on expressions of epidermal growth factor, stem cell factor, colony-stimulating factor 1 and leukemia inhibitory factor in human umbilical cord-derived mesenchymal stem cells].

OBJECTIVE: To investigate effects of retinol on the expressions of epidermal growth factor (EGF), stem cell factor (SCF), colony-stimulating factor 1 (CSF1) and leukemia inhibitory factor (LIF) in cultured human umbilical-derived mesenchymal stem cells (UCMSCs). METHODS: Human UCMSCs were isolated from human umbilical cord and identified for immunophenotypes. The cells were then cultured in DMEM/F12 media supplemented with 12% fetal bovine serum (FBS), 12% FBS+1 µmol/L retinol, 15% knockout serum replacement (KSR) and 15% KSR+ 1 µmol/L retinol. The expressions of the cytokines EGF, SCF, CSF1 and LIF in the cells were detected using RT-PCR and ELISA. RESULTS: The isolated cells exhibited characteristic immunophenotypes of human UCMSCs and expressed EGF, CSF1 and SCF at both mRNA and protein levels but not LIF protein. Retinol (1 µmol/L) significantly promoted the expressions of SCF and CSF1 at both mRNA and protein levels but did not result in changes of EGF and LIF expressions in human UCMSCs. CONCLUSION: Retinol at the concentration of 1 µmol/L can promote expression of SCF and CSF1 in human UCMSCs in vitro.

Tumour cell derived effects on monocyte/macrophage polarization and function and modulatory potential of Viscum album lipophilic extract in vitro.

BACKGROUND: Macrophages are highly versatile cells that play an important role in tumour microenvironment. Tumour associated macrophages (TAMs) have been linked to both, good or bad prognosis of several cancer types depending on their number, composition and polarization. Viscum album lipophilic extract (VALE) contains several pentacyclic triterpenes known to modulate the activity of monocytes and other immune cells and to exhibit anticancer properties. In our in vitro study, we investigated the effect of tumour cell lines on macrophage polarization and monocyte chemotactic transmigration and examined the modulatory potential of VALE and its predominant triterpene oleanolic acid (OA). METHODS: Human peripheral blood monocytes were differentiated into monocyte derived macrophages (MDM) using M-CSF and polarized into M1 by IFN-γ and LPS and into M2 macrophages by IL-4 and IL-13 or by co-culture with two different tumour cell lines. Polarized macrophages were subsequently treated with VALE or OA. Phenotypic markers and cytokines were assessed by flow cytometry and immunoanalysis. Migration of human peripheral blood monocytes induced by monocyte chemotactic protein-1 (MCP-1) or supernatants of different tumour cell lines under the influence of VALE or OA was measured in a chemotaxis transmigration assay. RESULTS: In vitro polarized M1 and M2 type macrophages revealed specific phenotypic patterns and tumour cell co-cultured MDM displayed ambiguous phenotypes with M1 as well as M2 associated markers. VALE and OA showed modest influence on cell surface marker profile and cytokine expression of tumour cell co-cultured macrophages. All tumour cell supernatants markedly enhanced the migratory activity of monocytes. VALE and OA significantly inhibited MCP-1 induced monocyte transmigration, whereas monocyte migration initiated by tumour cell derived supernatants was not affected. CONCLUSIONS: In our study we reconfirmed that co-culture with different tumour cell lines can result in a mixed macrophage phenotype with M1 as well as M2 patterns, a finding that is important for a better understanding of tumour microenvironment functions. Moreover, we demonstrated that VALE shows slight immunomodulatory effects on tumour cell co-cultured macrophages and modulates monocyte chemotactic transmigration in vitro, indicating promising possibilities of triterpenes from Viscum album L. to contribute in a multimodal concept of anti-cancer therapy in future. Our data contribute to an understanding of monocyte function and macrophage polarization in vitro and of the possibility to influence their behaviour by triterpene containing mistletoe extracts.

(5R)-5-Hydroxytriptolide (LLDT-8) inhibits osteoclastogenesis via RANKL/RANK/OPG signaling pathway.

BACKGROUND: The aim of this study was to investigate the regulative activity of (5R)-5-hydroxytriptolide (LLDT-8) on receptor activator of nuclear factor κ-B ligand (RANKL)/receptor activator of nuclear factor κ-B (RANK)/Osteoprotegerin (OPG) system in rheumatoid arthritis (RA) and its anti-osteoclastogenesis mechanism. METHODS: The expression of OPG, RANK and RANKL in CD3(+) T leukomonocytes in both peripheral blood and synovial fluid of RA patients was evaluated by flow cytometry. The levels of interleukin (IL) 1ß, IL-6, IL-10, IL-21 and IL-23 in the supernatants of peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were assayed by ELISA. Tartaric acid phosphatase (TRAP) staining was used to identify the osteoclast-like cells derived from RAW264.7. Western blotting analysis was used to check the downstream molecules of RANKL. RESULTS: LLDT-8 increased the rate of OPG expression in CD3(+) T leukomonocytes in peripheral blood as well as the ratio of OPG/RANKL in both peripheral blood and synovial fluid. LLDT-8 inhibited IL-1ß, IL-6, IL-21 and IL-23 secretion, but promoted the secretion of IL-10 in the supernatants of PBMCs and SFMCs. In addition, LLDT-8 decreased the number of TRAP-positive cells derived from RAW264.7 in the presence of RANKL and M-CSF. Furthermore, LLDT-8 also inhibited the expression of p-IκB, a key regulator of RANKL signaling pathway. CONCLUSIONS: LLDT-8 exerts its anti-osteoclastogenesis effect in RA probably through regulating RANKL/RANK/OPG system and its downstream signaling pathway as well as cytokine productions.

Obtusifolin suppresses phthalate esters-induced breast cancer bone metastasis by targeting parathyroid hormone-related protein.

This study is the first to demonstrate that parathyroid hormone-related protein (PTHrP), produced by human breast cancer cells after exposure to phthalate esters, contributes to bone metastasis by increasing osteoclastogenesis. This is also the first to reveal that obtusifolin reverses phthalate esters-mediated bone resorption. Human breast cancer cells were treated with dibutyl phthalate (DBP), harvested in conditioned medium, and cultured to osteoblasts or osteoclasts. Cultures of osteoblasts with DBP-MDA-MB-231-CM increased the osteoclastogenesis activator RANKL (receptor activator of nuclear factor κ-B ligand) and M-CSF (macrophage colony-stimulating factor). PTHrP was secreted in MDA-MB-231 cells. DBP-MDA-MB-231-CM reduced osteoblasts to produce osteoprotegerin, an osteoclastogenesis inhibitor, while DBP mediated PTHrP up-regulation, increasing IL-8 secretion in MDA-MB-231 and contributing to breast cancer-mediated osteoclast differentiation and bone resorption. Obtusifolin, a major bioactive compound present in Cassia tora L., suppressed phthalate esters-mediated bone resorption. Therefore, obtusifolin may be a novel anti-breast-cancer bone metastasis agent.

[Effect of Bushen Tongdu Capsule on RANK/RANKL/OPG pathway of collagen induced arthritis rats].

OBJECTIVE: To study the effect of Bushen Tongdu Capsule (BTC) on RANK/RANKL/ OPG pathway of collagen induced arthritis (CIA) rats, thereby laying theoretic evidence for treating rheumatic arthritis (RA) by Chinese medicine. METHODS: RA model was induced by CIA. Totally 42 rats were randomly divided into six groups, i.e., the normal control group, the model group, the low dose BTC (BSL) group, the medium dose BTC (BSM) group, the high dose BTC (BSH) group, and the Tripterygium Glycosides (TG) group, 7 in each group. BTC at the daily dose of 120, 240, and 480 mg/kg was given by gastrogavage to rats in the BSL, BSM, and BSH group respectively from the 13th day of modeling. TG at the daily dose of 24 mg/kg was given by gastrogavage to rats in the TG group. All medication was given once daily, 2 mL each time. Two mL normal saline was administered to rats in the normal control group and the model group. All medication lasted for 18 days. Samples were taken at day 31. The TRAP section of the ankle joint was fixed in 10% formalin for TRAP stain. Serum levels of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), and macrophage colony-stimulating factor (M-CSF) were detected using ELISA. RESULTS: Compared with the normal control group, positive reactions of pathological ankle joint section, inflammation, and osteoclasia degree were significantly improved in the model group, serum levels of RANKL and M-CSF were up-regulated, levels of OPG and OPG/RANKL were significantly lowered (all P < 0.01). Compared with the model group, positive reactions of pathological ankle joint section, inflammation, and osteoclasia degree also significantly decreased in the BSH group and the TG group (all P < 0.01). RANKL and M-CSF were significantly down-regulated in each medicated group, while levels of OPG and OPG/RANKL were significantly up-regulated (all P < 0.01). Compared with the TG group, M-CSF was lower, but levels of OPG and OPG/RANKL were significantly up-regulated in the normal control group (all P < 0.01). RANKL and M-CSF were significantly up-regulated, while levels of OPG and OPG/RANKL were significantly down-regulated in the model group and each BS group (all P < 0.01). CONCLUSION: BTC could relieve bone damage of CIA rats possibly through regulating and controlling osteoclasts.

Labisia pumila regulates bone-related genes expressions in postmenopausal osteoporosis model.

BACKGROUND: Labisia Pumila var. alata (LPva) has shown potential as an alternative to estrogen replacement therapy (ERT) in prevention of estrogen-deficient osteoporosis. In earlier studies using postmenopausal model, LPva was able to reverse the ovariectomy-induced changes in biochemical markers, bone calcium, bone histomorphometric parameters and biomechanical strength. The mechanism behind these protective effects is unclear but LPva may have regulated factors that regulate bone remodeling. The aim of this study is to determine the bone-protective mechanism of LPva by measuring the expressions of several factors involved in bone formative and resorptive activities namely Osteoprotegerin (OPG), Receptor Activator of Nuclear Factor kappa-B Ligand (RANKL), Macrophage-Colony Stimulating Factor (MCSF) and Bone Morphogenetic Protein-2 (BMP-2). METHODS: Thirty-two female Wistar rats were randomly divided into four groups: Sham-operated (Sham), ovariectomized control (OVXC), ovariectomized with Labisia pumila var. alata (LPva) and ovariectomized with ERT (Premarin) (ERT). The LPva and ERT were administered via daily oral gavages at doses of 17.5 mg/kg and 64.5 µg/kg, respectively. Following two months of treatment, the rats were euthanized and the gene expressions of BMP-2, OPG, RANKL and MCSF in the femoral bones were measured using a branch - DNA technique. RESULTS: The RANKL gene expression was increased while the OPG and BMP-2 gene expressions were reduced in the OVXC group compared to the SHAM group. There were no significant changes in the MCSF gene expressions among the groups. Treatment with either LPva or ERT was able to prevent these ovariectomy-induced changes in the gene expressions in ovariectomized rats with similar efficacy. CONCLUSION: LPva may protect bone against estrogen deficiency-induced changes by regulating the RANKL, OPG and BMP-2 gene expressions.

Comparison of the ability of chondroitin sulfate derived from bovine, fish and pigs to suppress human osteoclast activity in vitro.

Chondroitin sulfate (CS) compounds are commonly used to manage OA symptoms. Recent literature has indicated that abnormal subchondral bone metabolism may have a role in the pathogenesis of OA. The aim of this study was to access the effects of chondroitin sulfate obtained from bovine, fish and porcine sources on human osteoclast formation and activity in vitro. Human osteoclasts were generated from blood mononuclear cells. Cells were cultured over 17 days with the addition of macrophage colony stimulating factor (M-CSF) and then stimulated with receptor activator of nuclear factor kappa B ligand from day 7. Cells were treated with the CS commencing from day 7 onwards. To assess effects on osteoclasts, tartrate resistant acid phosphatate (TRAP) expression and resorption of whale dentine assays were used. Bovine-derived CS consistently suppressed osteoclast activity at concentrations as low as 1 µg/ml. Fish and porcine CS was less consistent in their effects varying with different donor cells. All CS compounds had little effect on TRAP activity. mRNA analysis using real-time PCR of bovine CS treated cells indicated that the inhibition of activity was not due to inhibition of the late stage NFATc1 transcription factor (p > 0.05). These results are consistent with CS inhibition of mature osteoclast activity rather than the formation of mature osteoclasts. It would appear that there are differences in activity of the different CS compounds with bovine-derived CS being the most consistently effective inhibitor of osteoclast resorption, but the results need to be confirmed.

Inhibition of human in vitro osteoclastogenesis by Equisetum arvense.

OBJECTIVES: Equisetum arvense has long been used in traditional medicines to treat different disorders, including bone pathologies. In this study a hydromethanolic extract of E. arvense was assessed for its effects on human osteoclastogenesis. MATERIALS AND METHODS: Osteoclast precursors were maintained in non-stimulated and stimulated (presence of M-CSF and RANKL) conditions, or in co-cultures with osteoblasts. Cell cultures were treated with 0.00016-0.5 mg/ml of a hydromethanolic E. arvense extract. RESULTS: The extract did not affect spontaneous osteoclastogenesis. In osteoclast precursors committed to osteoclastogenesis (stimulated or co-cultured with osteoblasts), E. arvense caused dose-dependent inhibitory effect that became statistically significant at concentrations ≥0.004 mg/ml. This was observed using different osteoclast differentiation and activation markers. Cell response was associated with changes in relative contribution of MEK and NFkB signalling pathways, as well as PGE2 production. As there were differences in the response of osteoclast precursors maintained in the presence of inductive factors, or co-cultured with osteoblastic cells, it seems that E. arvense extract had the ability to modulate osteoclastogenesis, either by acting directly on osteoclast precursor cells, and/or via osteoblasts. CONCLUSIONS: Equisetum appeared to have a negative effect on human osteoclastogenesis, which is in line with its putative beneficial role in pathophysiological conditions associated with increased osteoclastic activity, and might suggest potential utility for treatment with bone regeneration strategies.

Osteoblasts stimulate osteoclastogenesis via RANKL expression more strongly than periodontal ligament cells do in response to PGE(2).

OBJECTIVE: Periodontal ligament cells (PDLs) produce prostaglandin E(2) (PGE(2)) in response to orthodontic force. PGE(2) is a potent osteoclast-inducing factor that induces the receptor activator of nuclear factor-κB ligand (RANKL). Some studies reported that PDLs express RANKL in response to mechanical stress, whereas another study reported that they do not. Based on an immunohistochemical study, RANKL expression is localized around the alveolar bone surface 3 days after tooth movement. However, ankylosed teeth cannot be moved by therapeutic mechanical stress, suggesting that PDLs play a major role in alveolar bone resorption. In this study, we compared the functional difference in osteoclastogenesis between human PDLs (HPDLs) and normal human osteoblasts (HOBs) as a direct effect of PGE(2) exposure. DESIGN: We examined the expression of RANKL, osteoprotegerin, and macrophage colony-stimulating factor after 48-h culture with or without PGE(2) (10(-11) to 10(-5)M) in HPDLs and HOBs. Then to confirm whether RANKL produced by PGE(2) treatment induces osteoclastogenesis or not, RAW264.7 cells were co-cultured on HPDLs or HOBs pretreated with 10(-6)M of PGE(2). RESULT: PGE(2) exposure increased significantly RANKL expression in HOBs compared with HPDLs. PGE(2) exposure significantly decreased osteoprotegerin expression in HPDLs compared with HOBs. The number of tartrate-resistant acid phosphatase staining osteoclast-like cells from RAW264.7 cells increased significantly by PGE(2) pretreatment in HOBs and was reduced by small interfering RNA knockdown of RANKL. CONCLUSION: These results suggest that osteoblasts strongly influence the stimulation of osteoclastogenesis via RANKL, induced by PGE(2) in periodontal tissues, compared with PDLs.