Adipose-derived stem cell exosomes loaded with icariin attenuated M1 polarization of macrophages via inhibiting the TLR4/Myd88/NF-κB signaling pathway.

Abnormal macrophage polarization is one of the common pathological bases of various inflammatory diseases. The current research focus involves targeting macrophages to remodel their phenotype as a treatment approach for inflammatory diseases. Notably, exosomes can be delivered to specific types of cells or tissues or inflammatory area to realize targeted drug delivery. Although icariin (ICA) exhibits regulatory potential in macrophage polarization, the practical application of ICA is impeded by its water insolubility, poor permeability, and low bioavailability. Exploiting the inherent advantages of exosomes as natural drug carriers, we introduce a novel drug delivery system-adipose-derived stem cells-exosomes (ADSCs-EXO)-ICA. High-performance liquid chromatography analysis confirmed a loading rate of 92.7 ± 0.01 % for ADSCs-EXO-ICA, indicating the successful incorporation of ICA. As demonstrated by cell counting kit-8 assays, ADSCs-EXO exerted a significantly higher promotion effect on macrophage proliferation. The subsequent experimental results revealed the superior anti-inflammatory effect of ADSCs-EXO-ICA compared to individual treatments with EXO or ICA in the lipopolysaccharide + interferon-gamma-induced M1 inflammation model. Additionally, results from enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, and western blot analyses revealed that ADSCs-EXO-ICA effectively inhibited macrophage polarization toward the M1-type and concurrently promoted polarization toward the M2-type. The underlying mechanism involved the modulation of macrophage polarization through inhibition of the Toll-like receptor 4/myeloid differentiation factor 88/nuclear transcription factor-kappa B signaling pathway, thereby mitigating inflammation. These findings underscore the potential therapeutic value of ADSCs-EXO-ICA as a novel intervention for inflammatory diseases.

Diosgenin reduces bone loss through the regulation of gut microbiota in ovariectomized rats.

Diosgenin (DIO) is an aglycone of steroid saponins acquired from plants, including Dioscorea alata, Smilax China, and Trigonella foenum graecum, acting as an anti-osteoporosis, anti-diabetic, anti-hyperlipidemic, anti-inflammatory. Recent studies have demonstrated that DIO reduces bone loss. This study aimed to investigate the effects of DIO on the gut microbiota (GM) of ovariectomized (OVX) osteoporotic rats. Female Sprague-Dawley rats were randomly divided into sham operation (sham + vehicle group) or ovariectomy. For 12 weeks, OVX rats were treated using a vehicle (OVX + vehicle group) and DIO (OVX + DIO group). Subsequently, ELISA was conducted to determine serum estradiol levels, micro-CT scanning was performed to evaluate bone quality, and feces were collected for metagenomics sequencing to examine the structure and function of GM. Raw reads were filtered to remove chimera sequences. Operational taxonomic units (OTUs) were clustered in the filtered reads. A Venn diagram analysis was conducted to study the common and unique OTUs in the sham + vehicle, OVX + vehicle, and OVX + DIO groups. LEfSe analysis was conducted to evaluate the specific GM of the three groups. The GM functions were analyzed using the KEGG and CAZy databases. After a 12-week treatment, DIO administration prevented OVX-induced weight gain and increased the estradiol levels. DIO treatment improved the bone microstructure and structural parameters of rat tibias. Metagenomics sequencing results identified 1139, 1207, and 1235 operational taxonomic units (OTUs) in the sham + vehicle, OVX + vehicle, and OVX + DIO groups, respectively. The percentage of common OTUs was 41.2%. Treatment with DIO restored the composition of GM in OVX rats by increasing the abundance of Coriobacteriia Adlercreutzia, Romboutsia, and Romboutsia_idealis and reducing the abundance of Betaproteobacteria, Gammaproteobacteria, Methanobacteria, Bacteroides, Phocaeicola, Alistipes, Bacteroids_uniformis, Bacteroids_xylanisolvens. The anti-osteoporosis effect of DIO can be regulated through environmental information processing, organismal Systems, Cellular Processes, human diseases, metabolism, and genetic information processing. Meanwhile, treatment with DIO improved GM homeostasis by increasing the metabolism of carbohydrates, other amino acids, and glycans and reducing translation, energy metabolism, and nucleotide metabolism. DIO can reduce bone loss by regulating the structural composition and function of GM, a novel strategy for preventing osteoporosis.

Er-Xian decoction attenuates ovariectomy-induced osteoporosis by modulating fatty acid metabolism and IGF1/PI3K/AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Er-Xian decoction (EXD) is a traditional Chinese medicine (TCM) formula used to treat osteoporosis (OP). However, the anti-OP mechanism of EXD has not yet been fully elucidated. AIM OF THE STUDY: The study aimed to verify the anti-OP effect of EXD and to explore its underlying mechanism. METHODS: The anti-OP targets and mechanisms of EXD were predicted by network pharmacological analysis. Then, an ovariectomized (OVX) rat model was established to validate the key anti-OP mechanism of EXD. Firstly, the therapeutic effect of EXD on OP was confirmed using micro-CT bone analysis, pathological observation, and ELISA detection. Secondly, serum metabolites related to key biological processes were detected using an automatic biochemical analyzer and GC-MS. Finally, ELISA, qRT-PCR, and western blot were utilized to further explore the potential key anti-OP pathway of EXD. RESULTS: A total of 159 anti-OP targets of EXD were identified. Functional annotation revealed that OP treatment using EXD was associated with lipid metabolism, fatty acid (FA) metabolism, and PI3K/AKT signaling pathway. Experimental studies confirmed that EXD ameliorated ovariectomy-induced bone loss and bone microstructure deterioration. EXD treatment also upregulated the level of serum estrogen and downregulated the level of OC, PⅠNP, CTX-1, TC, and LDL-C. Besides, principal component analysis (PCA) and heat map of serum FAs distinguished OVX rats from the SHAM and EXD groups. Serum concentrations of important n-3 FAs, including C20:3N3, C20:5N3, and C22:5N3, were significantly increased in the EXD group. The increased stearoyl-CoA desaturase 1 (SCD1) index 1 and index 2 in the OVX group were reversed by EXD administration. Additionally, EXD reversed the decreased serum IGF1 level and tibia IGF1R, PI3K, and AKT expression in OVX rats. CONCLUSION: EXD ameliorated ovariectomy-induced bone loss by modulating lipid metabolism, FA metabolism, and IGF1/PI3K/AKT pathway.

Evaluation of culture conditions for osteoclastogenesis in RAW264.7 cells.

Osteoclasts are the only multinucleated cells in vivo responsible for bone resorption and are vital for regulating bone remodeling and maintaining bone mass. The RAW264.7 cell line is widely used to study osteoclastic differentiation and biological molecular mechanism. However, protocols for inducing osteoclast formation in RAW264.7 cells vary considerably between laboratories, hindering the replication of results. Therefore, we tested the influence of culture conditions on osteoclast differentiation, including cell density and receptor activator of nuclear factor kappa-B ligand (RANKL) concentrations with or without macrophage colony-stimulating factors (M-CSF). Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the morphology of osteoclasts. qPCR was used to detect gene expression of osteoclast-specific gene marker cathepsin K (CTSK), osteoclast transcription factors c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). The bone resorption function was evaluated by a scanning electron microscope (SEM). RANKL treatment increased multinucleated osteoclasts formation and increased CTSK, c-Fos and NFATc1 gene expression. Compared with RANKL treatment, M-CSF significantly decreased multinucleated osteoclasts formation, reduced CTSK gene expression and had little effect on c-Fos and NFATc1 gene expression. Concerning bone resorption activity, RANKL treatment increased bone resorption pits on bovine bone slices. Significantly higher levels of osteoclastogenesis were observed with RAW264.7-cell density of 2×104 cells/well in 24-well plates. Our results suggest that the addition of 50 ng/ml M-CSF has no positive effect on osteoclastogenesis. RANKL treatment and cell density contribute to osteoclast formation, and the optimal conditions are beneficial when exploring osteoclast function and mechanism.

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-ß3, 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.

Exploring the Potential Mechanism of Artemisinin and Its Derivatives in the Treatment of Osteoporosis Based on Network Pharmacology and Molecular Docking.

Objective: This study is aimed at predicting and contrasting the mechanisms of artemisinin (ARS), dihydroartemisinin (DHA), artesunate (ART), artemether (ARM), and arteether (ARE) in the treatment of osteoporosis (OP) using network pharmacology and molecular docking. Methods: The targets of ARS, DHA, ART, ARM, and ARE were obtained from the SwissTargetPrediction. The targets related to OP were obtained from the TTD, DrugBank, Genecards, and DisGeNet databases. Then, the anti-OP targets of ARS, DHA, ART, ARM, and ARE were obtained and compared using the Venn diagram. Afterward, the protein-protein interaction (PPI) networks were built using the STRING database, and Cytoscape was used to select hub targets. Moreover, molecular docking validated the binding association between five molecules and hub targets. Finally, GO enrichment and KEGG pathway enrichment were conducted using the DAVID database. The common pathways of five molecules were analysed. Results: A total of 28, 37, 36, 27, and 33 anti-OP targets of ARS, DHA, ART, ARM, and ARE were acquired. EGFR, EGFR, CASP3, MAPK8, and CASP3 act as the top 1 anti-OP targets of ARS, DHA, ART, ARM, and ARE, respectively. MAPK14 is the common target of five molecules. All five molecules can bind well with these hubs and common targets. Meanwhile, functional annotation showed that MAPK, Serotonergic synapse, AMPK, prolactin, and prolactin signaling pathways are the top 1 anti-OP pathway of ARS, DHA, ART, ARM, and ARE, respectively. IL-17 signaling pathway and prolactin signaling pathway are common anti-OP pathways of five molecules. Besides, GO enrichment showed five biological processes and three molecular functions are common anti-OP mechanisms of five molecules. Conclusion: ARS, DHA, ART, ARM and ARE can treat OP through multi-targets and multi pathways, respectively. All five molecules can treat OP by targeting MAPK14 and acting on the IL-17 and prolactin signaling pathways.

Rhizoma Dioscoreae extract protects against alveolar bone loss by regulating the cell cycle: A predictive study based on the protein­protein interaction network.

Rhizoma Dioscoreae extract (RDE) exhibits a protective effect on alveolar bone loss in ovariectomized (OVX) rats. The aim of this study was to predict the pathways or targets that are regulated by RDE, by re­assessing our previously reported data and conducting a protein­protein interaction (PPI) network analysis. In total, 383 differentially expressed genes (≥3­fold) between alveolar bone samples from the RDE and OVX group rats were identified, and a PPI network was constructed based on these genes. Furthermore, four molecular clusters (A­D) in the PPI network with the smallest P­values were detected by molecular complex detection (MCODE) algorithm. Using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA) tools, two molecular clusters (A and B) were enriched for biological process in Gene Ontology (GO). Only cluster A was associated with biological pathways in the IPA database. GO and pathway analysis results showed that cluster A, associated with cell cycle regulation, was the most important molecular cluster in the PPI network. In addition, cyclin­dependent kinase 1 (CDK1) may be a key molecule achieving the cell­cycle­regulatory function of cluster A. From the PPI network analysis, it was predicted that delayed cell cycle progression in excessive alveolar bone remodeling via downregulation of CDK1 may be another mechanism underling the anti­osteopenic effect of RDE on alveolar bone.

Rhizoma Dioscoreae extract protects against alveolar bone loss in ovariectomized rats via microRNAs regulation.

The aim of this study was to evaluate the osteoprotective effect of aqueous Rhizoma Dioscoreae extract (RDE) on the alveolar bone of rats with ovariectomy-induced bone loss. Female Wistar rats underwent either ovariectomy or sham operation (SHAM). The ovariectomized (OVX) rats were treated with vehicle (OVX), estradiol valerate (EV), or RDE. After treatments, the bone mineral density (BMD) and the three-dimensional microarchitecture of the alveolar bone were analyzed to assess bone mass. Microarrays were used to evaluate microRNA expression profiles in alveolar bone from RDE-treated and OVX rats. The differential expression of microRNAs was validated using real-time quantitative RT-PCR (qRT-PCR), and the target genes of validated microRNAs were predicted and further analyzed using Ingenuity Pathway Analysis (IPA). The key findings were verified using qRT-PCR. Our results show that RDE inhibits alveolar bone loss in OVX rats. Compared to the OVX rats, the RDE-treated rats showed upregulated expression levels of 8 microRNAs and downregulated expression levels of 8 microRNAs in the alveolar bone in the microarray analysis. qRT-PCR helped validate 13 of 16 differentially expressed microRNAs, and 114 putative target genes of the validated microRNAs were retrieved. The IPA showed that these putative target genes had the potential to code for proteins that were involved in the transforming growth factor (TGF)-ß/bone morphogenetic proteins (BMPs)/Smad signaling pathway (Tgfbr2/Bmpr2, Smad3/4/5, and Bcl-2) and interleukin (IL)-6/oncostatin M (OSM)/Jak1/STAT3 signaling pathway (Jak1, STAT3, and Il6r). These experiments revealed that RDE could inhibit ovariectomy-induced alveolar bone loss in rats. The mechanism of this anti-osteopenic effect in alveolar bone may involve the simultaneous inhibition of bone formation and bone resorption, which is associated with modulation of the TGF-ß/BMPs/Smad and the IL-6/OSM/Jak1/STAT3 signaling pathways via microRNA regulation.

High-dose diosgenin reduces bone loss in ovariectomized rats via attenuation of the RANKL/OPG ratio.

The aim of this study was to evaluate effect of diosgenin (DG) on rats that had osteoporosis-like features induced by ovariectomy (OVX). Seventy-two six-month-old female Wistar rats were subjected to either ovariectomy (n = 60) or Sham operation (SHAM group, n = 12). Beginning at one week post-ovariectomy, the OVX rats were treated with vehicle (OVX group, n = 12), estradiol valerate (EV group, n = 12), or DG at three doses (DG-L, -M, -H group, n = 12, respectively). After a 12-week treatment, administration of EV or DG-H inhibited OVX-induced weight gain, and administration of EV or DG-H or DG-M had a significantly uterotrophic effect. Bone mineral density (BMD) and indices of bone histomorphometry of tibia were measured. Levels of protein and mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) in tibia were evaluated by immunohistochemistry and in situ hybridization. Our results show that DG at a high dose (DG-H) had a significant anti-osteoporotic effect compared to OVX control. DG-H treatment down-regulated expression of RANKL and up-regulated expression of OPG significantly in tibia from OVX rats compared to control, and thus lowered the RANKL/OPG ratio. This suggests that the anti-osteoporotic effect of DG might be associated with modulating the RANKL/OPG ratio and DG had potential to be developed as alternative therapeutic agents of osteoporosis induced by postmenopause.