RANKL-induced RAW264 Research Articles

Formononetin Derivative for Osteoporosis by Simultaneous Regulating Osteoblast and Osteoclast.

Seven new formononetin derivatives (1-7) were designed and prepared from formononetin (phase II phytoestrogen). The derivatives 9-butyl-3-(4-methoxyphenyl)-9,10-dihydro-4H,8H-chromeno[8,7-e][1,3]oxazin-4-one (2) and 9-(furan-3-ylmethyl)-3-(4-methoxyphenyl)-9,10-dihydro-4H,8H-chromeno[8,7-e][1,3]oxazin-4-one (7) promoted significant osteoblast formation by modulating the BMP/Smad pathway. Compound 7 exhibited potent antiosteoclastogenesis activity in RANKL-induced RAW264.7 cells and ovariectomy (OVX)-induced osteoporosis in mice by regulation of the RANK/RANKL/OPG pathway. Compound 7 regulated osteoblast and osteoclast simultaneously and showed better effect than the well-known drug ipriflavone in vivo, suggesting 7 as a patented antiosteoporosis candidate.

Integrating Network Pharmacology, Quantitative Metabolic Network Analysis, In Vitro Experiments, and Molecular Dynamics to Explore the Mechanism of Angelica Sinensis for Regulating Bone Metabolism.

Bone metabolic diseases are serious health issues worldwide. Angelica sinensis (AS) is traditionally used in Chinese medicine for treating bone metabolism diseases clinically. However, the mechanism of AS in regulating bone metabolism remains uncertain. The current investigation was structured to elucidate the potential mechanisms of AS for modulating bone metabolism. Firstly, targets of AS regulating bone metabolism were collected by network pharmacology. Then, the transcriptional regulation of RUNX2 was enriched as one of the key pathways for AS to regulate bone metabolism, constructing its metabolic network. Secondly, combining molecular docking, network efficiency, and network flux analyses, we conducted a quantitative evaluation of the metabolic network to reveal the potential mechanisms and components of AS regulating bone metabolism. Finally, we explored the effect of AS on the differentiation of osteoclasts from M-CSF and RANKL-induced RAW264.7 cells, as well as its impact on the osteogenic induction of MC3T3-E1 cells. We verified the mechanism and key targets of AS on bone metabolism using qRT-PCR. Furthermore, the key component was preliminarily validated through molecular dynamics simulation. Quantitative metabolic network of the transcriptional regulation of RUNX2 was constructed to illustrate the potential mechanism of AS for regulating bone metabolism, indicating that ferulic acid may be a pharmacological component of AS that interferes with bone metabolism. AS suppressed osteoclast differentiation in M-CSF and RANKL-induced RAW264.7 cells and reversed the expressions of osteoclastic differentiation markers, including RUNX2 and SRC. Additionally, AS induced osteogenic generation in MC3T3-E1 cells and reversed the expressions of markers associated with osteoblastic generation, such as RUNX2 and HDAC4. Molecular dynamics simulation displayed a strong binding affinity among ferulic acid, HDAC4 and SRC. This study reveals a systematic perspective on the intervention bone mechanism of AS by transcriptive regulation by RUNX2, guiding the clinical use of AS in treating diseases of the skeletal system.

Icariin ameliorates osteoporosis in ovariectomized rats by targeting Cullin 3/Nrf2/OH pathway for osteoclast inhibition

Osteoporosis, characterized by low bone mass and bone microarchitecture breakdown, has become a growing public health problem. The increase in oxidative stress could lead to an imbalance between osteoblasts-mediated osteogenesis and osteoclast-mediated bone resorption, which gives rise to osteoporosis. Nrf2 is a master transcription factor that regulates oxidative stress and has recently been reported to take part in the development of osteoporosis. Icariin, a leading active flavonoid in herbal Epimedium pubescens, has significant antioxidant activity in and is widely applied for treating bone diseases. In this study, we aimed to explore the effect of icariin on osteoclastogenesis and its potential mechanism from the perspective of oxidative stress inhibition, using ovariectomized (OVX) rats and RANKL-induced RAW264.7 cells. Our results demonstrated that icariin-treated OVX rats exhibited higher bone density, fewer tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and lower ROS levels in bone tissues than vehicle-treated OVX rats. Also, icariin suppressed osteoclast differentiation and inhibited the expression of osteoclastogenesis-related genes, such as NFATc1, Ctsk, Trap, and c-Fos, in RANKL-induced RAW264.7 cells. Icariin also reduced intracellular ROS levels by increasing the expression of nuclear Nrf2 and HO-1. Further mechanistic studies showed icariin inhibited Cullin 3 expression and could delay Nrf2 degradation by reducing the ubiquitination of endogenous Nrf2 in RANKL-stimulated RAW264.7 cells, and these effects were markedly reversed by cullin three overexpression. These findings suggest icariin alleviated osteoporosis by suppressing osteoclastogenesis via targeting the Cullin 3/Nrf2/OH signaling pathway. Our study implied that icariin may be a potential candidate to treat osteoporosis.

Jiangu formula: A novel osteoclast-osteoblast coupling agent for effective osteoporosis treatment

BackgroundThe discovering of an osteoclast (OC) coupling active agent, capable of suppressing OC-mediated bone resorption while concurrently stimulating osteoblast (OB)-mediated bone formation, presents a promising strategy to overcome limitations associated with existing antiresorptive agents. However, there is a lack of research on active OC coupling agents. PurposeThis study aims to investigate the potential of Jiangu Formula (JGF) in inhibiting OCs while maintaining the OCOB coupling function. MethodsThe anti-osteoporosis efficacy of JGF was evaluated in osteoporosis models induced by ovariectomy in C57BL/6 mouse and SD rats. The effect of JGF on OCs was evaluated by detecting its capacity to inhibit OC differentiation and bone resorption in an in vitro osteoclastogenesis model induced by RANKL. The OCOB coupling activity of JGF was evaluated by measuring the secretion levels of OC-derived coupling factors, OB differentiation activity of MC3T3-E1 interfered with conditioned medium, and the effect of JGF on OC inhibition and OB differentiation in a C3H10T1/2-RAW264.7 co-culture system. The mechanism of JGF was studied by network pharmacology and validated using western blot, immunofluorescence (IF), and ELISA. Following that, the active ingredients of JGF were explored through a chemotype-assembly approach, activity evaluation, and LC-MS/MS analysis. ResultsJGF inhibited bone resorption in murine osteoporosis without compromising the OCOB coupling effect on bone formation. In vitro assays showed that JGF preserved the coupling effect of OC on OB differentiation by maintaining the secretion of OC-derived coupling factors. Network analysis predicted STAT3 as a key regulation point for JGF to exert anti-osteoporosis effect. Further validation assays confirmed that JGF upregulated p-STAT3(Ser727) and its regulatory factors IL-2 in RANKL-induced RAW264.7 cells. Moreover, 23 components in JGF with anti-OC activity identified by chemotype-assembly approach and verification experiments. Notably, six compounds, including ophiopogonin D, ginsenoside Re, ginsenoside Rf, ginsenoside Rg3, ginsenoside Ro, and ononin were identified as OC-coupling compounds. ConclusionThis study first reported JGF as an agent that suppresses bone loss without affecting bone formation. The potential coupling mechanism of JGF involves the upregulation of STAT3 by its regulators IL-2. Additionally, the chemotype-assembly approach elucidated the activity compounds present in JGF, offering a novel strategy for developing an anti-resorption agent that preserves bone formation.

M2 macrophages-derived exosomes regulate osteoclast differentiation by the CSF2/TNF-α axis

BackgroundOsteoclast-mediated bone resorption cause bone loss in several bone diseases. Exosomes have been reported to regulate osteoclast differentiation. M2-polarized macrophages exhibit anti-inflammatory activity. This study aimed to explore the effect of exosomes from M2 polarized macrophages (M2-exos) on osteoclastogenesis and molecular mechanisms.MethodsM2-exos were isolated from IL-4-induced Raw264.7 cells (M2 macrophages) and used to treat osteoclasts (RANKL-induced Raw264.7 cells). Osteoclast differentiation was visualized using tartrate resistant acid phosphatase staining. Quantitative real-time PCR (qPCR) was conducted to measure the levels of osteoclastogenesis-related genes. The underlying mechanisms of M2-exos were evaluated using qPCR and western blotting.ResultsM2-exos suppressed osteoclast differentiation induced by RANKL. Additionally, CSF2 was highly expressed in M2 macrophages, and knockdown of CSF2 further enhanced the effects of M2-exos on osteoclast differentiation. Moreover, CSF2 positively regulated TNF-α signaling, which inhibition promoted differentiation of M2-exo-treated osteoclasts.ConclusionM2-exos inhibited RANKL-induced osteoclast differentiation by downregulating the CSF2 expression through inactivating the TNF-α signaling, suggesting the potential application of exosomes in bone disease therapy.

Dysfunction of poly (ADP-ribose) glycohydrolase suppresses osteoclast differentiation in RANKL-stimulated RAW264 cells

Poly (ADP-ribose) glycohydrolase (PARG) is an enzyme that mainly degrades poly (ADP-ribose) (PAR) synthesized by poly (ADP-ribose) polymerase (PARP) family proteins. Although PARG is involved in many biological phenomena, including DNA repair, cell differentiation, and cell death, little is known about the relationship between osteoclast differentiation and PARG. It has also not been clarified whether PARG is a valuable target for therapeutic agents in the excessive activity of osteoclast-related bone diseases such as osteoporosis. In the present study, we examined the effects of PARG inhibitor PDD00017273 on osteoclast differentiation in RANKL-induced RAW264 cells. PDD00017273 induced the accumulation of intracellular PAR and suppressed the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. PDD00017273 also downregulated osteoclast differentiation marker genes such as Trap, cathepsin K (Ctsk), and dendrocyte expressed seven transmembrane protein (Dcstamp) and protein expression of nuclear factor of activated T cells 1 (NFATc1), a master regulator of osteoclast differentiation. Taken together, our findings suggest that dysfunction of PARG suppresses osteoclast differentiation via the PAR accumulation and partial inactivation of the NFATc1.

Sulforaphene suppresses RANKL-induced osteoclastogenesis and LPS-induced bone erosion by activating Nrf2 signaling pathway

Background and purposeInflammatory disorders have been found to induce bone loss through sustained and persistent activation of osteoclast differentiation, leading to heightened bone resorption. The current pharmacological interventions for combating bone loss to harbor adverse effects or contraindications. There is a pressing need to identify drugs with fewer side effects. Experimental approachThe effect and underlying mechanism of sulforaphene (LFS) on osteoclast differentiation were illustrated in vitro and in vivo with RANKL-induced Raw264.7 cell line osteoclastogenesis and lipopolysaccharide (LPS)-induced bone erosion model. Key resultsIn this study, LFS has been shown to effectively impede the formation of mature osteoclasts induced from both Raw264.7 cell line and bone marrow macrophages (BMMs), mainly at the early stage. Further mechanistic investigations uncovered that LFS suppressed AKT phosphorylation. SC-79, a potent AKT activator, was found to reverse the inhibitory impact of LFS on osteoclast differentiation. Moreover, transcriptome sequencing analysis revealed that treatment with LFS led to a significant upregulation in the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and antioxidant-related genes. Then it's validated that LFS could promote NRF2 expression and nuclear translocation, as well as effectively resist oxidative stress. NRF2 knockdown reversed the suppression effect of LFS on osteoclast differentiation. In vivo experiments provide convincing evidence that LFS is protective against LPS-induced inflammatory osteolysis. Conclusion and implicationsThese well-grounded and promising findings suggest LFS as a promising agent to addressing oxidative-stress related diseases and bone loss disorders.

Liver cancer cell-secreted exosomes promote bone metastasis of liver cancer by facilitating osteoclast differentiation through the miR-574-5p/BMP2 axis.

Bone metastasis of liver cancer leads to a worse prognosis with no appropriate treatment clinically. Exosomes are associated with tumor bone metastasis. This study aimed to investigate the effects of liver cancer cell-derived exosomes on bone metastasis. Exosomes were isolated from Hep3B cells, and the effects of osteoclast differentiation were assessed using TRAP assay. The expression of OPG and RANKL was assessed using qRT-PCR. The interaction of miR-574-5p and BMP2 was analyzed using luciferase reporter analysis, RNA pull-down, and qRT-PCR. We found that Hep3B cells promoted osteoclast differentiation of RANKL-induced Raw264.7 cells by secreting exosomes, with decreased OPG and increased RANKL expression. The exosomes were isolated from Hep3B cells, which promoted osteoclast differentiation. Exosomal miR-574-5p promoted osteoclastogenesis by targeting BMP2. Moreover, exosomes facilitated osteoclast differentiation, promoting bone metastasis by regulating miR-574-3p in vivo. In conclusion, liver cancer cell-derived exosomal miR-574-5p promoted osteoclastogenesis by regulating BMP2, thereby promoting bone metastasis in vivo. The findings suggest that liver cancer cell-released exosomes are the potential therapeutic approach for bone metastatic liver cancer. DATA AVAILABILITY STATEMENT: The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Ramie leaf Extract Alleviates Bone Loss in Ovariectomized Rats-The Involvement of ROS and Its Associated Signalings.

Ramie leaf (Boehmeria nivea L.) has been traditionally used to treat gynecological and bone-related disorders. This study aims to evaluate the effect of Ramie leaf extracts (RLE) against osteoporosis in ovariectomized (OVX) rats. Female SD rats aged seven weeks were randomly assigned into five OVX and a sham-operated (sham) group. OVX subgroups include OVX, vehicle-treated OVX group; E2, OVX with 100 μg/kg 17β-estradiol; and RLE 0.25, 0.5, and 1, OVX rats treated with 0.25, 0.5, and 1 g/kg/day RLE, respectively. Two weeks into the bilateral ovariectomy, all the rats were orally administered with or without RLE daily for 12 weeks. OVX rats administered with RLE showed higher bone density, relatively low tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and lower reactive oxygen species (ROS) within bone tissues compared to vehicle-treated OVX rats. Furthermore, supplementation of RLE improved bone mineral density (BMD) and bone microstructure in the total femur. RLE prevented RANKL-induced osteoclast differentiation and expression of osteoclastogenesis-related genes such as Cal-R, MMP-9, cathepsin K, and TRAP in RANKL-induced RAW264.7 cells. Moreover, RLE administration lowered the intracellular ROS levels by reducing NADPH oxidase 1 (NOX-1) and 4-hydroxynonenal (4HNE). These results suggest that RLE alleviates bone mass loss in the OVX rats by inhibiting osteoclastogenesis, where reduced ROS and its associated signalings were involved.

Lacticaseibacillus rhamnosus MG4706 Suppresses Periodontitis in Osteoclasts, Inflammation-Inducing Cells, and Ligature-Induced Rats.

Periodontitis is a chronic inflammatory disease characterized by tooth loss due to inflammation and the loss of alveolar bone. Periodontitis is closely related to various systemic diseases and is emerging as a global health problem. In this study, we investigated the anti-inflammatory effect of lactic acid bacteria (LAB) in vitro on Porphyromonas gingivalis (P. gingivalis) LPS-activated RAW264.7 and human gingival fibroblasts-1 (HGF-1) cells and the anti-osteoclastogenic effect of LAB on RANKL-induced RAW264.7 cells. All LAB strains (Lacticaseibacillus rhamnosus MG4706, MG4709, and MG4711) inhibited nitric oxide (NO)/inducible nitric oxide synthase (iNOS) in P. gingivalis LPS-activated RAW264.7 cells and pro-inflammatory cytokines (IL-1β and IL-6) and matrix metalloproteinase (MMP-8 and MMP-9) in HGF-1 cells. In addition, LAB treatment inhibited osteoclastogenesis by reducing tartrate-resistant acid phosphatase (TRAP) activity and cathepsin K (CtsK) through the downregulation of nuclear factor of activated T cells cytoplasmic 1 (NFATc1) and c-fos gene expression in RANKL-induced RAW264.7 cells. Administration of MG4706 alleviated alveolar bone loss indices and reduced the gene expression of IL-1β, IL-6, MMP-8, MMP-9, and RANKL/OPG ratio in gingival tissue. In conclusion, L. rhamnosus MG4706 has the potential to alleviate periodontitis.

Phytol Suppresses Osteoclast Differentiation and Oxidative Stress through Nrf2/HO-1 Regulation in RANKL-Induced RAW264.7 Cells.

Osteoporosis is a systemic skeletal disorder where osteoclasts are prevalent among osteoblasts. Oxidative stress is one of the main causes of osteoporosis, and nuclear factor erythroid-2-related factor 2 (Nrf2) is the master regulator of antioxidant responses. Phytol, a diterpene isolated from Stevia rebaudiana leaves, has many biological effects, including antimicrobial, antioxidant, and anti-inflammatory effects. This study investigated the crosstalk between Nrf2 and osteoclast differentiation in the presence of phytol. Phytol inhibited osteoclast differentiation through TRAP-positive and F-actin formation. The expression of anti-nuclear factor of activated T cells-c1 (NFATc1) and c-Fos was suppressed by phytol, as shown using Western blot and RT-PCR analysis. Phytol inhibited oxidative stress by suppressing reactive oxidant species (ROS) accumulation while recovering antioxidant enzymes, including superoxide dismutase and catalase. Additionally, phytol ameliorated osteoclast-specific differentiation, function, and oxidative stress through Nrf2 regulation by siRNA transfection. In conclusion, these data demonstrate the inhibitory effect of phytol on osteoclast differentiation through Nrf2 regulation, suggesting its potential use in oxidative stress-related osteoporosis and bone diseases.

Genistein Modified with 8-Prenyl Group Suppresses Osteoclast Activity Directly via Its Prototype but Not Metabolite by Gut Microbiota.

Postmenopausal osteoporosis is a significant threat to human health globally. Genistein, a soy-derived isoflavone, is regarded as a promising anti-osteoporosis drug with the effects of promoting osteoblastogenesis and suppressing osteoclastogenesis. However, its oral bioavailability (6.8%) is limited by water solubility, intestinal permeability, and biotransformation. Fortunately, 8-prenelylated genistein (8PG), a derivative of genistein found in Erythrina Variegate, presented excellent predicted oral bioavailability (51.64%) with an improved osteoblastogenesis effect, although its effects on osteoclastogenesis and intestinal biotransformation were still unclear. In this study, an in vitro microbial transformation platform and UPLC-QTOF/MS analysis method were developed to explore the functional metabolites of 8PG. RANKL-induced RAW264.7 cells were utilized to evaluate the effects of 8PG on osteoclastogenesis. Our results showed that genistein was transformed into dihydrogenistein and 5-hydroxy equol, while 8PG metabolites were undetectable under the same conditions. The 8PG (10-6 M) was more potent in inhibiting osteoclastogenesis than genistein (10-5 M) and it down-regulated NFATC1, cSRC, MMP-9 and Cathepsin K. It was concluded that 8-prenyl plays an important role in influencing the osteoclast activity and intestinal biotransformation of 8PG, which provides evidence supporting the further development of 8PG as a good anti-osteoporosis agent.

Ginsenoside Rb3 inhibits osteoclastogenesis via ERK/NF-κB signaling pathway in vitro and in vivo.

The objective of the study was to determine the anti-osteoclastogenic potential of ginsenoside Rb3 for the treatment of periodontitis. The anti-osteoclastogenic effect was determined using RANKL-induced RAW264.7 cells and murine bone marrow-derived macrophages followed by TRAP and phalloidin staining. Expression of osteoclastogenesis-related genes and proteins were examined by qPCR and WB. Activation of signaling pathways was detected by WB and IHC techniques. Experimental periodontitis rat model was built up by gingival injections of P.gingivalis LPS. After 21 days of Rb3 treatment, rats were sacrificed for micro-CT, IHC, H&E, and TRAP staining analyses. Rb3 dramatically inhibits RANKL-induced osteoclastogenesis. Nfatc1, Mmp9, Ctsk, Acp5 mRNA, and MMP9, CTSK proteins were dose-dependently downregulated by Rb3 pretreatment. WB results revealed that Rb3 suppressed activations of p38 MAPK, ERK, and p65 NF-κB, and the inhibition of ERK was most pronounced. Consistently, IHC analysis revealed that p-ERK was highly expressed in alveolar bone surface, blood vessels, odontoblasts, and gingival epithelia, which were notably suppressed by Rb3 treatment. H&E staining and micro-CT analyses showed that Rb3 significantly attenuated gingivitis and alveolar bone resorption in rats. Rb3 inhibits RANKL-induced osteoclastogenesis and attenuates P.gingivalis LPS-induced gingivitis and alveolar bone resorption in rats via ERK/NF-κB signaling pathway.

Monascin abrogates RANKL-mediated osteoclastogenesis in RAW264.7 cells via regulating MAPKs signaling pathways.

Osteoclasts (OCs) are multinucleated cells that play a major role in osteolytic diseases such as osteoporosis. Monascin (Ms) is one of the active substances in the traditional Chinese medicine red yeast rice. Studies have found that red yeast rice can maintain bone health. In this study, the anti-osteoclastogenesis effects of Ms on RANKL-induced RAW264.7 cells were assessed, and the underlying mechanism was investigated. Ms exhibited inhibitory effects on OC differentiation and formation in a dose-dependent manner and suppressed the bone-resorbing activity of mature OCs. Ms blocked OCs-typical genes (c-Fos, NFATc1, CSTK, MMP-9, TRAP, ITG-β3, OSCAR and DC-STAMP). Furthermore, Ms treatment considerably inhibited the activation of MAPKs, JNK and p38. Taken together, Ms suppresses RANKL-induced osteoclastogenesis of RAW264.7 cells by restraining MAPKs signaling pathways and is a potential therapeutic option as a novel OC inhibitor to mitigate bone erosion.

Effects and Mechanisms of Rhus chinensis Mill. Fruits on Suppressing RANKL-Induced Osteoclastogenesis by Network Pharmacology and Validation in RAW264.7 Cells.

Rhus chinensis Mill. fruits are a kind of widely distributed edible seasoning, which have been documented to possess a variety of biological activities. However, its inhibitory effect on osteoclast formation has not been determined. The objective of this study was to evaluate the effect of the fruits on osteoclast differentiation of RAW264.7 cells, induced by receptor activator of nuclear factor-κB ligand (RANKL) and to illuminate the potential mechanisms using network pharmacology and western blots. Results showed that the extract containing two organic acids and twelve phenolic substances could effectively inhibit osteoclast differentiation in RANKL-induced RAW264.7 cells. Network pharmacology examination and western blot investigation showed that the concentrate essentially decreased the expression levels of osteoclast-specific proteins, chiefly through nuclear factor kappa-B, protein kinase B, and mitogen-activated protein kinase signaling pathways, particularly protein kinase B α and mitogen-activated protein kinase 1 targets. Moreover, the extract likewise directly down regulated the expression of cellular oncogene Fos and nuclear factor of activated T-cells cytoplasmic 1 proteins. Citric acid, quercetin, myricetin-3-O-galactoside, and quercetin-3-O-rhamnoside were considered as the predominant bioactive ingredients. Results of this work may provide a scientific basis for the development and utilization of R. chinensis fruits as a natural edible material to prevent and/or alleviate osteoporosis-related diseases.

Icariin inhibits RANKL-induced osteoclastogenesis in RAW264.7 cells via inhibition of reactive oxygen species production by reducing the expression of NOX1 and NOX4

Icariin (ICA), isolated from Herba Epimedii, is a natural flavonoid glycoside that possesses antioxidant properties and inhibits osteoclastogenesis. However, the mechanism underlying osteoclastogenesis inhibition by ICA remains unclear. Here, we investigated the effects of ICA on receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells. ICA inhibited the expression of osteoclastogenesis-related genes in RAW264.7 cells induced by RANKL. ICA could inhibit osteoclastogenesis without inhibiting the viability of RAW264.7 cells. In addition, ICA inhibited reactive oxygen species production in RANKL-induced RAW264.7 cells. ICA reduced the expression of nuclear factor in activated T cells, cytoplasmic 1, and tartrate-resistant acid phosphatase, which are osteoclast-related molecules. Moreover, ICA decreased the expression of nicotinamide adenine dinucleotide phosphate oxidase (NOX), specifically NOX1 and NOX4, in RANKL-induced RAW264.7 cells. Our findings suggest that ICA can be used as a potential therapeutic agent for osteolytic diseases such as osteoporosis.

Zanthoxylum bungeanum seed oil inhibits RANKL-induced osteoclastogenesis by suppressing ERK/c-JUN/NFATc1 pathway and regulating cell cycle arrest in RAW264.7 cells

Ethnopharmacological relevanceZanthoxylum bungeanum Maxim (ZBM), a traditional Chinese medicine, is traditionally used for osteoporosis treatment recorded in ancient Chinese medicine work Benjingshuzheng and reported to have the anti-bone loss activity in recent studies. However, the anti-osteoporotic activities of the seed of ZBM have not been elucidated yet. Our previous study found that Zanthoxylum bungeanum Maxim seed oil (ZBSO) was rich in polyunsaturated fatty acids (PUFAs), which were reported to prevent bone loss. Thus, we propose a hypothesis that ZBSO could be a potential natural resource for anti-bone loss. Aim of the studyTo investigate whether ZBSO could prevent bone loss by targeting osteoclastogenesis and investigate the potential mechanisms in receptor-activator of nuclear factor κB ligand (RANKL)-induced RAW264.7 cells. Materials and methodsRAW264.7 cells were treated with RANKL in the presence or absence of ZBSO. The effect of ZBSO on osteoclast differentiation and bone resorption activity of RAW264.7 cells were evaluated by tartrate-resistant acid phosphatase (TRAP) staining, F-actin ring staining, and bone resorption assay. Differentially expression genes (DEGs) and relevant pathways of different cell groups were obtained from RNA sequencing and protein-protein interaction (PPI) network analysis followed by KEGG pathway enrichment analysis. The effect of ZBSO on the RANKL-induced cell cycle change was analyzed by flow cytometry assay, and the expression of genes and proteins related to the selected pathways was further verified by RT-qPCR and western blot analysis. ResultsThe inhibitory effects of ZBSO on osteoclast differentiation and bone resorption activity in a dose-dependent manner were demonstrated by TRAP staining, F-actin ring staining, and bone resorption assay in RANKL-induced RAW264.7 cells. Osteoclast differentiation and cell cycle pathways were the most enriched pathways based on DEGs enrichment analysis among different cell groups. The reversion effect of ZBSO on the RANKL-induced RAW264.7 cell cycle arrest at the G1 phase was observed by flow cytometry assay. Western blot results showed that ZBSO markedly decreased RANKL-induced activation of ERK, as well as the phosphorylation of c-JUN and NFATc1 expression, and subsequently suppressed osteoclast-specific genes, such as Ctsk, Trap, and Dc-stamp. ConclusionsZBSO exhibited an inhibitory effect on osteoclastogenesis via suppressing the ERK/c-JUN/NFATc1 pathway and regulating cell cycle arrest induced by RANKL, suggesting that ZBSO may serve as a promising agent for anti-bone loss.

Eight new phenolic acids from the leaves of Illicium dunnianum and their osteoprotective activities.

Eight previously unreported phenolic acids (1–8), including three new phenylpropenoid glycosides (1–3), and five undescribed shikimic acid derivatives (4–8), together with six known analogues (9–14), were obtained from the dried leaves of Illicium dunnianum. The structures of these new compounds were elucidated by extensive spectroscopic analyses (1D, 2D-NMR, HRESIMS, IR, UV) and chemical methods. Compounds 1, 2, 4, and 5 were tested for their promotion effect on osteoblastogenesis of pre-osteoblastic MC3T3-E1 cells and inhibitory effect on osteoclastogenesis of RANKL-induced RAW264.7 cells. As a result, 1 and 4 exerted a promotion effect on osteoblastogenesis, but without activity on osteoclastogenesis. Our studies not only enrich the structural diversity of phenolic acids in nature, but also discover new lead compounds from folk plants with activities on osteoblastogenesis or osteoclastogenesis.

2E-Decene-4,6-diyn-1-ol-acetate inhibits osteoclastogenesis through mitogen-activated protein kinase-c-Fos-NFATc1 signalling pathways.

An imbalance of osteoclasts and osteoblasts can result in a variety of bone-related diseases, including osteoporosis. Thus, decreasing the activity of osteoclastic bone resorption is the main therapeutic method for treating osteoporosis. 2E-Decene-4, 6-diyn-1-ol-acetate (DDA) is a natural bioactive compound with anti-inflammatory and anti-cancer properties. However, its effects on osteoclastogenesis are unknown. Murine bone marrow-derived macrophages (BMMs) or RAW264.7 cells were treated with DDA, followed by evaluation of cell viability, RANKL-induced osteoclast differentiation, and pit formation assay. Effects of DDA on RANKL-induced phosphorylation of MAPKs were assayed by western blot analysis. Expression of osteoclast-specific genes was examined with reverse transcription-PCR (RT-PCR) and western blot analysis. In this study, DDA significantly inhibited RANKL-induced osteoclast differentiation in RAW264.7 cells as well as in BMMs without cytotoxicity. DDA also strongly blocked the resorbing capacity of BMM on calcium phosphate-coated plates. DDA inhibited RANKL-induced phosphorylation of ERK, JNK and p38MAPKs, as well as expression of c-Fos and NFATc1, which are essential transcription factors for osteoclastogenesis. In addition, DDA decreased expression levels of osteoclastogenesis-specific genes, including matrix metalloproteinase-9 (MMP-9), tartrate-resistant acid phosphatase (TRAP), and receptor activator of NF-κB (RANK) in RANKL-induced RAW264.7 cells. Collectively, these findings indicated that DDA attenuates RANKL-induced osteoclast formation by suppressing the MAPKs-c-Fos-NFATc1signalling pathway and osteoclast-specific genes. These results indicate that DDA may be a potential candidate for bone diseases associated with abnormal osteoclast formation and function.

Fengshi Qutong capsule ameliorates bone destruction of experimental rheumatoid arthritis by inhibiting osteoclastogenesis

Ethnopharmacological relevanceBone destruction plays a key role in damaging the joint function of rheumatoid arthritis (RA). Fengshi Qutong capsule (FSQTC) consisting of 19 traditional Chinese medicines has been used for treating RA in China for many years. Preliminary studies show that FSQTC has analgesic activity and inhibits synovial angiogenesis of collagen-induced arthritis (CIA), but its role on bone destruction of RA is still unclear. Aim of the studyTo explore the effect of FSQTC on bone destruction of RA and the possible mechanism of osteoclastogenesis in vivo and in vitro. Materials and methodsLC-MS system was used to detect the quality control components of FSQTC. The anti-arthritic effect of FSQTC on CIA rats was evaluated by arthritis score, arthritis incidence and histopathology evaluation of inflamed joints. The effect of treatment with FSQTC on bone destruction of joint tissues was determined with X-ray and micro-CT quantification, and on bone resorption marker CTX-I and formation marker osteocalcin in sera were detected by ELISA. Then, osteoclast differentiation and mature were evaluated by TRAP staining, actin ring immunofluorescence and bone resorption assay both in joints and RANKL-induced RAW264.7 cells. In addition, RANKL, OPG, IL-1β and TNFα in sera were evaluated by ELISA. The molecular mechanisms of the inhibitions were elucidated by analyzing the protein and gene expression of osteoclastic markers CTSK, MMP-9 and β3-Integrin, transcriptional factors c-Fos and NFATc1, as well as phosphorylation of ERK1/2, JNK and P38 in joints and in RANKL-induced RAW264.7 cells using western blot and/or qPCR. ResultsIn this study, 12 major quality control components were identified. Our data showed that FSQTC significantly increased bone mineral density, volume fraction, trabecular thickness, and decreased trabecular separation of inflamed joints both at periarticular and extra-articular locations in CIA rats. FSQTC also diminished the level of CTX-I and simultaneously increased osteocalcin in sera of CIA rats. The effects were accompanied by reductions of osteoclast differentiation, bone resorption, and expression of osteoclastic markers (CTSK, MMP-9 and β3-Integrin) in joints. Interestingly, FSQTC treatment could reduce the protein level of RANKL, increase the expression of OPG, and decrease the ratio of RANKL to OPG in inflamed joints and sera of CIA rats. In addition, FSQTC inhibited the levels of pro-inflammatory cytokines implicated in bone resorption, such as IL-1β and TNFα in sera. When RAW264.7 cells were treated with RANKL, FSQTC inhibited the formation of TRAP + multinucleated cells, actin ring and the bone-resorbing activity in dose-dependent manners. Furthermore, FSQTC reduced the RANKL-induced expression of osteoclastic genes and proteins and transcriptional factors (c-Fos and NFATc1), as well as phosphorylation of mitogen-activated protein kinases (MAPKs). ConclusionFSQTC may inhibit bone destruction of RA by its anti-osteoclastogenic activity both in vivo and in vitro.