The influence of radiation-induced bystander effect in osteoblasts mediated by plasma-derived extracellular vesicles (EVs).

OBJECTIVES: Head and neck tumor patients may develop post-radiotherapy diseases after radiotherapy treatment. And radiotherapy can elicit radiation-induced bystander effect, wherein extracellular vesicles (EVs) play a crucial role. For normal parts of the body that have not been directly irradiated, the effect of EVs on them needs to be further explored. This study aims to investigate the functions of plasma-derived EVs in regulating normal osteoblasts during radiation-induced bystander effects. METHODS AND MATERIALS: Rat plasma-derived EVs were isolated and identified firstly, followed by an evaluation of their intracellular biological effects on normal osteoblasts in vitro. Transcriptome sequencing analysis and confirmations were performed to identify potential mechanisms. RESULTS: Irradiated plasma-derived EVs were found to enhance osteoblast proliferation, migration, and cell cycle progression, concurrently suppressing the expression of osteogenesis-related genes and proteins. Furthermore, these EVs attenuated the expression of osteogenesis and oxidative stress resistance related genes, while upregulating the PI3K-AKT pathway and intracellular reactive oxygen species in osteoblasts. CONCLUSIONS: Irradiated plasma-derived EVs could alter the biological effects in osteoblasts, which is closely associated with the levels of GPX1 and the PI3K-AKT signaling pathway. This suggests that plasma-derived EVs serve as a crucial factor contributing to radiation-induced bystander effect in osteoblasts.

Copper-containing titanium alloys promote the coupling of osteogenesis and angiogenesis by releasing copper ions.

Previous investigations have reported on the ability of copper (Cu)-bearing biomaterials to accelerate vascular formation and bone regeneration. However, few studies have explored the effects of Cu-bearing materials on the interactions between angiogenesis and osteogenesis. Therefore, in this study, we prepared Cu-containing alloys using selective laser melting (SLM) technology and investigated the impact of preosteoblasts seeded on Ti6Al4V-4.5Cu alloy on angiogenesis. Our results indicated that Ti6Al4V-4.5Cu alloys increased the expression of proangiogenic genes and proteins in preosteoblasts, which further stimulated vascular formation in endothelial cells. Besides, we discovered that the biological effects of the Ti6Al4V-4.5Cu alloy were partly attributed to the release of Cu ions. In short, our research demonstrated the ability of Ti6Al4V-4.5Cu alloys to promote the coupling of angiogenesis and osteogenesis by releasing Cu ions.

The role of FOXO1-mediated autophagy in the regulation of bone formation.

Autophagy is essential for the maintenance of intracellular homeostasis, implicated in various biological processes. Forkhead box protein O1 (FOXO1) is regarded as a key mediator regulating skeletal development. Recent studies indicate that FOXO1 has a multifaceted role in autophagy regulation and dysregulation. Here, we aimed to elucidate the role of FOXO1-autophagy axis in osteogenesis. Osteoblast conditional Foxo1-knockout mice (Foxo1OB-/-, KO) and FOXO1 lentivirus overexpression (Len-FoxO1) model were constructed in vivo. Primary osteoblasts were isolated from KO and their wild-type (WT) littermates. And we also applied overexpression lentivirus to investigate the effects of FOXO1 in vitro. Using Micro-CT, fluorescence labeling detection, real-time qPCR and western blot analyses, we found that bone formation was promoted in Len-FOXO1 mice, which was impaired in KO group. Similarly, FOXO1 overexpression enhanced proliferation, migration and differentiation of osteoblasts, while FOXO1 ablation resulted in poor biological functions of osteoblasts. Through the investigation of autophagic process using mRFP-GFP-LC3 fluorescence labeling and co-immunoprecipitation, we observed that overexpression of FOXO1 initiated autophagy induction, with enhanced FOXO1 interaction with autophagy-related protein 7 (ATG7). On the contrary, FOXO1 knockout in osteoblasts impeded FOXO1-ATG7 conjugation, leading to impaired autophagic activity. Furthermore, inhibition of autophagy by chloroquine (CQ) could reverse favorable influences in bone formation induced by FOXO1 overexpression. Our findings confirmed that FOXO1 was an important regulator of bone formation and autophagy might be part of the underlying mechanisms, offering a significant avenue for the potential strategy in the treatment of bone-related disorders.

1ɑ,25-Dihydroxyvitamin D3 promotes osteogenesis by down-regulating FGF23 in diabetic mice.

1ɑ,25-dihydroxyvitamin D3 (1,25D) and fibroblast growth factor 23 (FGF23) play important roles in bone metabolism through mutual regulation. However, the underlying mechanism between 1,25D and FGF23 in diabetes-induced bone metabolism disorders has not yet been elucidated. In this study, we investigated the effect of 1,25D on FGF23 under diabetic condition both in vitro and in vivo. The results showed that 1,25D down-regulated the expression of FGF23 in osteoblast significantly though a dose-dependent manner in vitro within high glucose environment. Western blot and immunofluorescence analysis indicated that 1,25D activated PI3K/Akt signalling through binding to vitamin D receptor (VDR), which inhibited the phosphorylation of the transcription factor Forkhead Box O1 (FOXO1). Decreased phosphorylation of FOXO1 down-regulated the expression Dickkopf-1 (DKK1), a well-known inhibitor of Wnt signalling. In addition, we observed that 1,25D remarkably ameliorated osteogenic phenotypic markers such as Ocn and Runx2 and rescued diabetes-induced bone loss in vivo. Our results suggested that 1,25D could promote osteogenesis though down-regulating FOXO1/FGF23 in diabetes.

1α,25-Dihydroxyvitamin D3 ameliorates diabetes-induced bone loss by attenuating FoxO1-mediated autophagy.

Autophagy is vital for maintaining cellular homeostasis through removing impaired organelles. It has recently been found to play pivotal roles in diabetes mellitus (DM), which is associated with increased bone fracture risk and loss of bone density. However, the mechanism whereby autophagy modulates DM-induced bone loss is not fully elucidated. Previous work has shown that 1α,25-Dihydroxyvitamin D3 (1,25D) exerts positive effects on autophagy, thus affecting bone metabolism. Here, we investigated whether autophagy was involved in the regulation of diabetic bone metabolism. Using Micro-CT, Elisa, histology, and histomorphometry analysis, we demonstrated that 1,25D rescues glucose metabolism dysfunction and ameliorates bone loss in diabetic mice. In vitro, 1,25D alleviated primary osteoblast dysfunction and intracellular oxidative stress through reducing prolonged high-glucose-mediated excessive autophagy in primary osteoblasts, reflected by decreased protein level of Beclin1 and LC3. Of note, the autophagy activator rapamycin (RAP) ablated the positive effects of 1,25D in diabetic environment, leading to a marked increase in autolysosomes and autophagosomes, examined by mRFP-GFP-LC3 fluorescence double labeling. The excessive autophagy induced by high glucose was deleterious to proliferation and differentiation of primary osteoblasts. Additionally, biochemical studies identified that PI3K/Akt signaling could be activated by 1,25D, resulting in the inhibition of FoxO1. We confirmed that FoxO1 deficiency alleviated high-glucose-induced autophagy and improved biological functions of primary osteoblasts. Together, our results suggest that the PI3K/Akt/FoxO1 signaling pathway is involved in the osteoprotective effect of 1,25D by attenuating autophagy in diabetes, providing a novel insight for the prevention and treatment of diabetes-caused bone loss.

Resveratrol promotes osteogenesis via activating SIRT1/FoxO1 pathway in osteoporosis mice.

AIMS: This study aimed to investigate the effects of resveratrol (3, 4', 5-trihydroxystilbene, RES) on osteoporosis and the role of SIRT1/FoxO1 pathway in the process. MAIN METHODS: In vivo, mice were divided into 3 groups, Sham, ovariectomized (OVX) and OVX-RES group. Micro-CT, histology and histomorphometry were conducted to detect details of bone mass and microstructure. The expression of osteoblast markers was tested by Real-time qPCR and serum markers which reflected bone formation and resorption were analyzed by enzyme-linked immunosorbent assay (ELISA). Besides, we assayed sirtuin 1 (SIRT1) expression and the concentration of serum superoxide dismutase (SOD). In vitro, osteoblasts were seperated into 3 groups: control, H2O2 (hydrogen peroxide, H2O2) and H2O2-RES group. Cell proliferation, differentiation and apoptosis were detected. In addition, we tested intracellular reactive oxygen species (ROS) formation and SOD activity detection of osteoblasts. The SIRT1, acetylated FoxO1 (Ac-FoxO1) and nuclear FoxO1 (Nu-FoxO1) expression were detected by western blot. KEY FINDINGS: Results revealed that RES could ameliorate bone loss and promote osteogenesis by reinforcing resistance of oxidative stress in OVX mice. RES enhanced proliferation, differentiation and suppressed apoptosis of H2O2-treated osteoblasts. In this process, SIRT1 was upregulated and the level of Nu-FoxO1, which had high transcriptional activity to regulate redox balance, significantly increased. SIGNIFICANCE: Oxidative stress plays a crucial role in osteoporosis. RES can reinforce resistance to oxidative damage and hence promote osteogenesis via the activation of SIRT1/FoxO1 signaling pathway, which provides a new idea for the prevention and treatment of osteoporosis.