Curcumin has been proven to have a weight-loss effect in a menopausal rat model induced by ovariectomy. However, the effects of curcumin on gut microfloral communities of ovariectomized (OVX) rats remains unclear. Here, we used high-throughput 16S rDNA sequencing to explore the effects of curcumin on microbial diversity in the gut of OVX rats. Female Wistar rats were subjected to either ovariectomy or a sham operation (SHAM group). The OVX rats were treated with vehicle (OVX group) or curcumin (CUR group) by oral gavage. After 12-week treatments, the weights of the bodies and uteri of rats were recorded, the levels of estradiol in the serum were assayed by electrochemiluminescence immunoassay (ECLIA). Then, the fragments encompassing V3–V4 16S rDNA hypervariable regions were PCR amplified from fecal samples, and the PCR products of V3–V4 were sequenced on an Illumina MiSeq for characterization of the gut microbiota. Our results showed that, compared to rats in the SHAM group, rats in the OVX group had more weight gain and lower levels of estradiol in the serum, and curcumin could cause significant weight loss in OVX rats but did not increase the levels of estradiol. Sequencing results revealed the presence of 1120, 1114, and 1119 operational taxonomic units (OTUs) found in the SHAM, OVX, and CUR groups, respectively. The percentage of shared OTUs was 86.1603%. Gut microbiota of rats from the SHAM or CUR group had higher levels of biodiversity and unevenness estimations than those from the OVX group. At the phyla level, compared to rats in SHAM group, rats in the OVX group had a higher ratio of phyla Firmicutes and Bacteroidetes in the gut; at the genus level, four differential gut microbiota (Incertae_Sedis, Anaerovorax, Anaerotruncus, and Helicobacter) between SHAM and OVX groups were found, whereas seven differential gut microbiota (Serratia, Anaerotruncus, Shewanella, Pseudomonas, Papillibacter, Exiguobacterium, and Helicobacter) between OVX and CUR groups were found. In conclusion, estrogen deficiency induced by ovariectomy caused changes in the distribution and structure of intestinal microflora in rats, and curcumin could partially reverse changes in the diversity of gut microbiota.

Osteoclasts are the only multinucleated cellsin vivoresponsible 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 2×10⁴cells/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.

Background: The present study aimed to assess the perturbation in circular RNA (circRNA)/mRNA expression profiles and a circRNA-miRNA-mRNA coexpression network involved in the potential protective effect of diosgenin (DIO) on alveolar bone loss in rats subjected to ovariectomy (OVX).Methods: The Wistar rats (female) manipulated with sham operation were classified as the SHAM group and the grouping of OVX rats administered with DIO, estradiol valerate or vehicle for 12 weeks was DIO group, EV group and OVX group respectively. Following treatments, the plasmatic levels of osteocalcin and tumor necrosis factor-alpha and the microstructure of alveolar bone were assayed. Based on microarray analyses, we identified differentially expressed (DE) circRNAs and mRNAs in alveolar bone of rats in both OVX and DIO group. The DE circRNAs and DE mRNAs involved in the bone metabolism pathway validated by RT-qPCR were considered key circRNAs/mRNAs. On the basis of these key circRNAs/mRNAs, we predicted the overlapping relative miRNAs of key circRNAs/mRNAs, and a circRNA-miRNA-mRNA network was built.Results: DIO showed an anti-osteopenic effect on the rat alveolar bone loss induced by OVX. In total, we found 10 DE circRNAs (6 downregulated and 4 upregulated) and 614 DE mRNAs (314 downregulated and 300 upregulated) in samples of the DIO group compared with those of the OVX group. However, only one circRNA (rno_circRNA_016717) and seven mRNAs (Sfrp1,Csf1,Il1rl1,Nfatc4,Tnfrsf1a,Pik3c2g, andWnt9b) were validated by qRT-PCR and therefore considered key circRNA/mRNAs. According to these key circRNA/mRNAs and overlapping predicted miRNAs, a coexpression network was constructed. After network analysis, one circRNA-miRNA-mRNA axis (circRNA_016717/miR-501-5p/Sfrp1) was identified.Conclusion: The mechanism of DIO inhibiting alveolar bone loss after OVX is possibly relevant to the simultaneous inhibition of osteogenesis and osteoclastogenesis by mediating the expression of important molecules in the Wnt, PI3K, RANK/RANKL or osteoclastogenic cytokine pathways. The circRNA_016717/miR-501-5p/Sfrp1axis may play important roles in these processes.

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.