Kaempferol, a flavonoid compound, is derived from the rhizome of Kaempferia galanga L., which is used in traditional medicine in Asia. Autophagy has pleiotropic functions that are involved in cell growth, survival, nutrient supply under starvation, defense against pathogens, and antigen presentation. There are many studies dealing with the inhibitory effects of natural flavonoids in bone resorption. However, no studies have explained the relationship between the autophagic and inhibitory processes of osteoclastogenesis by natural flavonoids. The present study was undertaken to investigate the inhibitory effects of osteoclastogenesis through the autophagy inhibition process stimulated by kaempferol in murin macrophage (RAW 264.7) cells. The cytotoxic effect of Kaempferol was investigated by MTT assay. The osteoclast differentiation and autophagic process were confirmed via tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay, western blot, and real-time PCR. Kaempferol controlled the expression of autophagy-related factors and in particular, it strongly inhibited the expression of p62/SQSTM1. In the western blot and real time-PCR analysis, when autophagy was suppressed with the application of 3-Methyladenine (3-MA) only, osteoclast and apoptosis related factors were not significantly affected. However, we found that after cells were treated with kaempferol, these factors inhibited autophagy and activated apoptosis. Therefore, we presume that kaempferol-inhibited autophagy activated apoptosis by degradation of p62/SQSTM1. Further study of the p62/SQSTM1 gene as a target in the autophagy mechanism, may help to delineate the potential role of kaempferol in the treatment of bone metabolism disorders.

Kaempferol, a kind of flavonol, has been reported to possess various osteogenic biological activities, such as inhibiting bone resorption of osteoclasts and promoting the differentiation and mineralization of preosteoblasts. However, the precise cellular mechanism of action of kaempferol in osteogenesis is elusive. Autophagy is a major intracellular degradation system, which plays an important role in cell growth, survival, differentiation and homeostasis in mammals. Recent studies showed that autophagy appeared to be involved in the degradation of osteoclasts, osteoblasts and osteocytes, potentially pointing to a new pathogenic mechanism of bone homeostasis and bone marrow disease. The potential correlation between autophagy, osteogenesis and flavonoids is unclear. The present study verified that kaempferol promoted osteogenic differentiation and mineralization and that it elevated osteogenic gene expression based on alkaline phosphatase (ALP) activity, alizarin red staining and quantitative PCR. And then we found that kaempferol induced autophagy by acridine orange (AO) and monodansylcadaverine (MDC) staining and autophagy-related protein expression. The correlation between kaempferol-induced autophagy and the osteogenic process was confirmed by the autophagy inhibitor 3-methyladenine (3-MA). Kaempferol promoted the proliferation, differentiation and mineralization of osteoblasts at a concentration of 10 μM. Kaempferol showed cytotoxic properties at concentrations above 50 μM. Concentrations above 10 μM decreased ALP activity, whereas those up to 10 μM increased ALP activity. Kaempferol at concentrations up to 10 μM also increased the expression of the osteoblast- activated factors RUNX-2, osterix, BMP-2 and collagen I according to RT-PCR analyses. 10 μM or less, the higher of the concentration and over time, kaempferol promoted the activity of osteoblasts. Kaempferol induced autophagy. It also increased the expression of the autophagy-related factors beclin-1, SQSTM1/p62 and the conversion of LC3-II from LC3-I. The application of 3-MA decreased the activity of ALP and the autophagy induced by kaempferol. In the RT-PCR analysis, the expression of RUNX-2, osterix, BMP-2 and collagen I was decreased. The present study showed that kaempferol stimulated the osteogenic differentiation of cultured osteoblasts by inducing autophagy.

This study aimed to utilize micro-computed tomography (micro-CT) analysis to compare new bone formation in rat calvarial defects using chitosan/fibroin–hydroxyapatite (CFB–HAP) or collagen (Bio-Gide) membranes. Fifty-four (54) rats were studied. A circular bony defect (8 mm diameter) was formed in the centre of the calvaria using a trephine bur. The CFB–HAP membrane was prepared by thermally induced phase separation. In the experimental group (n=18), the CFB–HAP membrane was used to cover the bony defect, and in the control group (n=18), a resorbable collagen membrane (Bio-Gide) was used. In the negative control group (n=18), no membrane was used. In each group, six animals were euthanized at 2, 4 and 8 weeks after surgery. The specimens were then analysed using micro-CT. There were significant differences in bone volume (BV) and bone mineral density (BMD) (P<0.05) between the negative control group and the membrane groups. However, there were no significant differences between the CFB–HAP group and the collagen group. We concluded that the CFB–HAP membrane has significant potential as a guided bone regeneration (GBR) membrane.

Background and Objectives: Malignant glioblastoma (GBM) is caused by abnormal proliferation of glial cells, which are found in the brain. The therapeutic effects of surgical treatment, radiation therapy, and chemo-therapy against GBM are relatively poor compared with their effects against other tumors. Luteolin is abundant in peanut shells and is also found in herbs and other plants, such as thyme, green pepper, and celery. Luteolin is known to be effective against obesity and metabolic syndrome. The anti-inflammatory, and anti-cancer activities of luteolin have been investigated. Most studies have focused on the antioxidant and anti-inflammatory effects of luteolin, which is a natural flavonoid. However, the association between the induction of apoptosis by luteolin in GBM and autophagy has not yet been investigated. This study thus aimed to confirm the occurrence of luteolin-induced apoptosis and autophagy in GBM cells and to assess their relationship. Materials and Methods: A172 and U-373MG glioblastoma cell lines were used for this experiment. We confirmed the apoptosis effect of Luteolin on GBM cells using methods such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, immunofluorescence, Flow cytometry (FACS) western blot, and real-time quantitative PCR (qPCR). Results: In the luteolin-treated A172 and U-373MG cells, cell viability decreased in a concentration- and time-dependent manner. In addition, in A172 and U-373MG cells treated with luteolin at concentrations greater than 100 μM, nuclear fragmentation, which is a typical morphological change characterizing apoptosis, as well as fragmentation of caspase-3 and Poly (ADP-ribose) polymerase (PARP), which are apoptosis-related factors, were observed. Autophagy was induced after treatment with at least 50 μM luteolin. Inhibition of autophagy using 3MA allowed for a low concentration of luteolin to more effectively induce apoptosis in A172 and U-373MG cells. Conclusions: Results showed that luteolin induces apoptosis and autophagy and that the luteolin-induced autophagy promotes cell survival. Therefore, an appropriate combination therapy involving luteolin and an autophagy inhibitor is expected to improve the prognosis of GBM treatment.

Bisphosphonates are one of the most widely used synthetic pyrophosphate analogues for the treatment of bone resorbing diseases such as osteoporosis, multiple myeloma, and bone metastases. Although the therapeutic usefulness of bisphosphonates mainly depends on their anti-osteoclastogenic effect, a severe side-effect of bisphosphonates called bisphosphonate-related osteonecrosis of the jaw (BRONJ) could not be explained by the anti-osteoclastogenic effect of bisphosphonates. In the present study, we have evaluated the changes in osteoclastogenesis- or osteoblastogenesis-supporting activities of osteocytes induced by bisphosphonates. Zoledronate, a nitrogen-containing bisphosphonate, markedly increased both the receptor activator of nuclear factor kB ligand (RANKL) as well as sclerostin in osteocyte-like MLO-Y4 cells, which were functionally revalidated by osteoclast/osteoblast generating activities of the conditioned medium obtained from zoledronate-treated MLO-Y4 cells. Of note, the zoledronate treatment-induced upregulation of the RANKL expression was mediated by autocrine interleukin-6 (IL-6) and subsequent activation of the signal transducer and activator of transcription 3 (STAT3) pathway. These results were evidenced by the blunted RANKL expression in the presence of a Janus activated kinase (JAK2)/STAT3 inhibitor, AG490. Also, the osteoclastogenesis-supporting activity was significantly decreased in zoledronate-treated MLO-Y4 cells in the presence of IL-6 neutralizing IgG compared to that of the control IgG. Thus, our results show previously unanticipated effects of anti-bone resorptive bisphosphonate and suggest a potential clinical importance of osteocytes in BRONJ development.

Background: This study aimed to investigate new bone formation using recombinant human bone morphogenetic protein 2 (rhBMP-2) and locally applied bisphosphonate in rat calvarial defects.Methods: Thirty-six rats were studied. Two circular 5 mm diameter bony defect were formed in the calvaria using a trephine bur. The bony defect were grafted with Bio-Oss® only (group 1, n = 9), Bio-Oss® wetted with rhBMP-2 (group 2, n = 9), Bio-Oss® wetted with rhBMP-2 and 1 mM alendronate (group 3, n = 9) and Bio-Oss® wetted with rhBMP-2 and 10 mM alendronate (group 4, n = 9). In each group, three animals were euthanized at 2, 4 and 8 weeks after surgery, respectively. The specimens were then analyzed by histology, histomorphometry and immunohistochemistry analysis.Results: There were significant decrease of bone formation area (p < 0.05) between group 4 and group 2, 3. Group 3 showed increase of new bone formation compared to group 2. In immunohistochemistry, collagen type I and osteoprotegerin (OPG) didn’t show any difference. However, receptor activator of nuclear factor κB ligand (RANKL) decreased with time dependent except group 4.Conclusion: Low concentration bisphosphonate and rhBMP-2 have synergic effect on bone regeneration and this is result from the decreased activity of RANKL of osteoblast.