Hesperetin affects osteoclast differentiation via MAPK signaling pathway.

BACKGROUND: The number and activity of osteoblasts and osteoclasts play an important role in skeletal biology, especially in bone reconstruction. Scientific and rational regulation of osteoclast formation and activity has become a critical strategy aimed at inhibiting the loss of bone mass in the body and alleviating the occurrence of bone diseases. Currently, there are only a few reports related to hesperetin-regulated osteoclast differentiation. OBJECTIVES: To investigate the influence of hesperetin on osteoclast-like cell differentiation and formation, and determine whether the MAPK signaling pathway is involved in the differentiation process. MATERIAL AND METHODS: The RAW264.7 cells were induced and cultured in vitro to promote their differentiation into osteoclast-like cells. Tetrazolium bromide was utilized to determine the effects of different concentrations (100, 200, 400, and 600 µM) of hesperetin on the proliferation of osteoclast-like cell precursors. Osteoclast-like cell differentiation was conducted using tartrate-resistant acid phosphatase (TRAP) staining assay. The status of nuclei and actin filaments of differentiated osteoclast-like cells was observed with the use of 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and actin-tracker green staining experiments. Changes in key proteins of the MAPK signaling pathway were detected using western blot. RESULTS: The results of TRAP staining experiments showed that the number of osteoclast-like cells decreased with the increase in hesperetin concentration. The DAPI and actin-tracker green staining demonstrated that the nuclei of differentiated osteoclast-like cells reduced in size with the increase in hesperetin concentration, and the osteoclast-like cells became smaller. Western blot for key MAPK signaling pathway proteins revealed that phospho-ERK and phospho-p38 protein levels were not significantly inhibited, but phospho-JNK protein levels were reduced. CONCLUSIONS: Hesperetin inhibits the differentiation of osteoclast-like cells. Further studies revealed that hesperetin also affects the activation level of phospho-JNK, a key signaling protein of the MAPK signaling pathway, in the induced differentiation of osteoclast-like cells.

miR-338-3p regulates osteoclastogenesis via targeting IKKß gene.

This study determined the effects of miR-338-3p on osteoclast (OC) differentiation and activation. The change levels of miR-338-3p in differentiated OCs were investigated by microRNA microarray assay and quantitative real-time PCR analysis. The effects of miR-338-3p on the differentiation and activation of OCs were determined by tartrate-resistant acid phosphatase staining resorption activity assay and Western blot. Target genes of miR-338-3p were identified by target gene prediction and dual-luciferase reporter gene detection assay as well as Western blot. Results showed that miR-338-3p was markedly downregulated in differentiated OCs. miR-338-3p could inhibit the formation and absorption activity of OCs. Western blot showed that miR-338-3p could influence the change levels of OC differentiation-related proteins. Dual-luciferase reporter gene detection assay and Western blot both showed that miR-338-3p directly targeted IKKß gene. In conclusion, miR-338-3p may affect the formation and activity of OCs by targeting the IKKß gene.

3-(4-methoxyl)-1-(2-(4-coumarin)prop)-2-en-1-one inhibits the differentiation of Gaoyou duck embryonic osteoclasts in vitro.

This study determined the influence of 3-(4-methoxyl)-1-(2-(4-coumarin)prop)-2-en-1-one (MCPEO) on the differentiation of Gaoyou duck embryo osteoclasts cultured in vitro. Bone marrow mononuclear cells (BM-MNCs) were harvested from 23-day-old Gaoyou duck embryos and induced by receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) in the presence of MCPEO at different concentrations (i.e., 1, 5, 10, 20, and 40 µM). Cell viability measurement, tartrate-resistant acid phosphatase (TRAP) staining, resorption activity assay, and co-staining with Tetramethylrhodamine (TRITC)-conjugated phalloidin and Hoechst 33,258 were conducted. Results indicated that MCPEO influenced the cell viability of the M-CSF + RANKL-induced BM-MNCs in a concentration-dependent manner, reduced the formation of positive multinucleated cells, and restrained the resorption capability of osteoclasts. Microfilament and nuclear staining indicated that MCPEO restricted the differentiation of BM-MNCs into large multinucleated osteoclasts. In short, MCPEO can inhibit the differentiation of BM-MNCs into mature osteoclasts in duck embryos. Therefore, MCPEO is a promising agent for the treatment of poultry osteoporosis.

Effects of Ca2+/calmodulin­dependent protein kinase pathway inhibitor KN93 on osteoclastogenesis.

The aim of the present study was to determine the effects of the Ca2+/calmodulin­dependent protein kinase pathway inhibitor KN93 on osteoclastogenesis. RAW264.7 cells were incubated with macrophage colony­stimulating factor (M­CSF) + receptor activator of nuclear factor kappa­light­chain­enhancer of activated B cells ligand (RANKL) to stimulate osteoclastogenesis and then treated with 10 µM KN93. The methods included tartrate­resistant acid phosphatase (TRAP) staining, bone resorption activity assays, filamentous (F)­actin staining, determination of intracellular calcium ([Ca2+]i) levels, monitoring of osteoclast­specific gene expression levels and measurement of key transcription factors protein levels. The results suggested that KN93 inhibited the formation of TRAP­positive multinucleated cells, shaping of F­actin rings and resorption activity of the cells. In addition, KN93 decreased the concentration of [Ca2+]i, expression levels of osteoclast specific genes and protein levels of critical transcription factors in the M­CSF + RANKL­induced osteoclast model. In summary, KN93 may directly affect the differentiation and activation of osteoclasts, potentially through the Ca2+/calmodulin­dependent protein kinase signaling pathway.

Anticancer Effects of Resveratrol-Loaded Solid Lipid Nanoparticles on Human Breast Cancer Cells.

In this study, resveratrol-loaded solid lipid nanoparticles (Res-SLNs) were successfully designed to treat MDA-MB-231 cells. The Res-SLNs were prepared using emulsification and low-temperature solidification method. The Res-SLNs were spherical, with small size, negative charge, and narrow size distribution. Compared with free resveratrol, the Res-SLNs displayed a superior ability in inhibiting the proliferation of MDA-MB-231 cells. In addition, Res-SLNs exhibited much stronger inhibitory effects on the invasion and migration of MDA-MB-231 cells. Western blot analysis revealed that Res-SLNs could promote the ratio of Bax/Bcl-2 but decreased the expression of cyclinD1 and c-Myc. These results indicate that the Res-SLN may have great potential for breast cancer treatment.

4-Anilinoquinazoline Derivatives with Epidermal Growth Factor Receptor Inhibitor Activity.

4-Anilinoquinazoline derivatives possess high anti-cancer activities. Many of them are highly selective tyrosine kinase inhibitors (TKI), particularly against epidermal growth factor receptor (EGFR). EGFRs are overexpressed or mutated in most carcinomas and are required for tumor progression. The efficacy of EGFR-targeted anti-tumor drugs is impaired by drug-induced acquired resistance. Therefore, there is urgency to find better anti-cancer agents with novel effects on EGFR. 4-Anilinoquinazolines are small molecule EGFR inhibitors that have been synthesized and assessed for their anti-tumor bioactivity. In this paper, we review the 4-anilinoquinazoline derivatives with EGFR inhibitor activity reported in recent years.