Targeted activation of Nrf2/HO-1 pathway by Corynoline alleviates osteoporosis development.

Oxidative stress is preferentially treated as a risk factor for the development and progression of osteoporosis. Corynoline as a component of Corydalis bungeana Turcz presents antioxidative and anti-inflammatory properties. In the present study, the effects of Corynoline on osteoblasts following hydrogen peroxide (H2O2)-induced injury were evaluated accompanied by the investigation of the molecular mechanisms involved. It was found that Corynoline downregulated the intracellular reactive oxygen species (ROS) generation and restored the osteogenic potential of the disrupted osteoblasts by H2O2 exposure. Furthermore, Corynoline was revealed to activate the Nrf2/HO-1 signaling pathway, while ML385 (an Nrf2 inhibitor) would prevent the Corynoline-mediated positive effects on the disrupted osteoblasts. In terms of the animal experiments, Corynoline treatment contributed to a significantly alleviated bone loss. These findings indicate that Corynoline may significantly attenuate the H2O2-induced oxidative damage of osteoblasts via the Nrf2/HO-1 signaling pathway, providing novel insights to the development of treatments for osteoporosis induced by oxidative injury.

A Novel Anti-Osteoporosis Mechanism of VK2: Interfering with Ferroptosis via AMPK/SIRT1 Pathway in Type 2 Diabetic Osteoporosis.

Type 2 diabetic osteoporosis (T2DOP) is a chronic bone metabolic disease. Compared with traditional menopausal osteoporosis, the long-term high glucose (HG) microenvironment increases patients' risk of fracture and osteonecrosis. We were accumulating evidence that implicated ferroptosis as a pivotal mechanism of glucolipotoxicity-mediated death of osteocytes and osteoblast, a novel form of programmed cell death resulting from uncontrolled lipid peroxidation depending on iron. Vitamin K2 (VK2), a fat-soluble vitamin, is clinically applied to prevent osteoporosis and improve coagulation. This study aimed to clarify the role and mechanism of VK2 in HG-mediated ferroptosis. We established the mouse T2DOP model by intraperitoneal injection of streptozotocin solution and a high-fat and high-sugar diet. We also cultured bone marrow mesenchymal stem cells (BMSCs) in HG to simulate the diabetic environment in vitro. Based on our data, VK2 inhibited HG-mediated bone loss and ferroptosis, the latter manifested by decreased levels of mitochondrial reactive oxygen species, lipid peroxidation, and malondialdehyde and increased glutathione in vitro. In addition, VK2 treatment was capable of restoring bone mass and strengthening the expression of SIRT1, GPX4, and osteogenic markers in the distal femurs. As for further mechanism exploration, we found that VK2 could activate AMPK/SIRT1 signaling, and knockdown of SIRT1 by siRNA prevented the VK2-mediated positive effect in HG-cultured BMSCs. Summarily, VK2 could ameliorate T2DOP through the activation of the AMPK/SIRT1 signaling pathway to inhibit ferroptosis.

Application of interpretable machine learning algorithms to predict distant metastasis in osteosarcoma.

BACKGROUND: Osteosarcoma is well-established as the most common bone cancer in children and adolescents. Patients with localized disease have different prognoses and management than those with metastasis at the time of diagnosis. The purpose of this study was to explore potential risk factors for metastatic disease. METHODS: The Surveillance, Epidemiology, and End Results (SEER) Program database was used to identify patients diagnosed with osteosarcoma between 2004 and 2015. We developed prediction models for distant metastasis using six machine learning (ML) techniques, including logistic regression (LR), support vector machine (SVM), Gaussian Naive Bayes (GaussianNB), Extreme Gradient Boosting (XGBoost), random forest (RF), and k-nearest neighbor algorithm (kNN). The adaptive synthetic (ADASYN) technique was used to deal with imbalanced data. The Shapley Additive Explanation (SHAP) analysis generated visualized explanations for each patient. Finally, the average precision (AP), sensitivity, specificity, accuracy, F1 score, precision-recall curves, calibration plots, and decision curve analysis (DCA) were conducted to evaluate the models' effectiveness. RESULTS: The six machine learning algorithms achieved AP of 0.661-0.781 for predicting distant metastasis. The RF model yielded the best performance with an accuracy of 71.8 percent and an AP of 0.781 and was highly dependent on tumor size, primary surgery, and age. SHAP analysis provided model-independent interpretation, highlighting significant clinical factors associated with the risk of metastasis in osteosarcoma patients. CONCLUSIONS: An accurate machine learning-based prediction model was established for metastasis in osteosarcoma patients to help clinicians during clinical decision-making.

Therapeutic effect of SIRT3 on glucocorticoid-induced osteonecrosis of the femoral head via intracellular oxidative suppression.

Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is a serious complication after long-term or excess administration of clinical glucocorticoids intervention, and the pathogenic mechanisms underlying have not been clarified yet. Oxidative stress is considered as a major cause of bone homeostasis disorder. This study is aimed to explore the potential relevance between SIRT3 and GIONFH, as well as the effect of resveratrol, which has been reported for its role in SIRT3 activation, on dexamethasone-induced oxidative stress and mitochondrial compromise in bone marrow stem cells (BMSCs). In this study, our data showed that SIRT3 level was declined in GIONFH rat femoral head, corresponding to a resultant decrease of SIRT3 expression in dexamethasone-treated BMSCs in vitro. We also found that dexamethasone could result in oxidative injury in BMSCs, and resveratrol treatment reduced this deleterious effect via a SIRT3-dependent manner. Moreover, our results demonstrated that rewarding effect of resveratrol on BMSCs osteogenic differentiation was via activation of AMPK/PGC-1α/SIRT3 axis. Meanwhile, resveratrol administration prevented the occurrence of GIONFH, enhanced SIRT3 expression and reduced oxidative level in GIONFH model rats. Therefore, our study provides basic evidence that SIRT3 may be a promising therapeutic target for GIONFH treatment and resveratrol could be an ideal agent for clinical uses.

Apelin-13 induces mitophagy in bone marrow mesenchymal stem cells to suppress intracellular oxidative stress and ameliorate osteoporosis by activation of AMPK signaling pathway.

Osteoporosis is characterized by impaired bone metabolism. Current estimates show that it affects millions of people worldwide and causes a serious socioeconomic burden. Mitophagy plays key roles in bone marrow mesenchymal stem cells (BMSCs) osteoblastic differentiation, mineralization, and survival. Apelin is an endogenous adipokine that participates in bone homeostasis. This study was performed to determine the role of Apelin in the osteoporosis process and whether it affects mitophagy, survival, and osteogenic capacity of BMSCs in in vitro and in vivo models of osteoporosis. Our results demonstrated that Apelin was down-regulated in ovariectomized-induced osteoporosis rats and Apelin-13 treatment activated mitophagy in BMSCs, ameliorating oxidative stress and thereby reviving osteogenic function via AMPK-α phosphorylation. Besides, Apelin-13 administration restored bone mass and microstructure as well as reinstated mitophagy, enhanced osteogenic function in OVX rats. Collectively, our findings reveal the intrinsic mechanisms underlying Apelin-13 regulation in BMSCs and its potential therapeutic values in the treatment of osteoporosis.

Proanthocyanidins-Mediated Nrf2 Activation Ameliorates Glucocorticoid-Induced Oxidative Stress and Mitochondrial Dysfunction in Osteoblasts.

The widespread use of therapeutic glucocorticoids has increased the frequency of glucocorticoid-induced osteoporosis (GIOP). One of the potential pathological processes of GIOP is an increased level of oxidative stress and mitochondrial dysfunction, which eventually leads to osteoblast apoptosis. Proanthocyanidins (PAC) are plant-derived antioxidants that have therapeutic potential against GIOP. In our study, a low dose of PAC was nontoxic to healthy osteoblasts and restored osteogenic function in dexamethasone- (Dex-) treated osteoblasts by suppressing oxidative stress, mitochondrial dysfunction, and apoptosis. Mechanistically, PAC neutralized Dex-induced damage in the osteoblasts by activating the Nrf2 pathway, since silencing Nrf2 partly eliminated the protective effects of PAC. Furthermore, PAC injection restored bone mass and promoted the expression of Nrf2 in the distal femur of Dex-treated osteoporotic rats. In summary, PAC protect osteoblasts against Dex-induced oxidative stress and mitochondrial dysfunction via the Nrf2 pathway activation and may be a promising drug for treating GIOP.

Vitamin K2 Can Rescue the Dexamethasone-Induced Downregulation of Osteoblast Autophagy and Mitophagy Thereby Restoring Osteoblast Function In Vitro and In Vivo.

Chronic long-term glucocorticoids (GC) use is associated with glucocorticoid-induced osteoporosis (GIOP) by inhibiting the survival and impairing the functions of osteoblasts. Autophagy and mitophagy play key roles in osteoblast differentiation, mineralization and survival, and mounting evidence have implicated osteoblast autophagy and mitophagy as a novel mechanism in the pathogenesis of GIOP. Vitamin K2 (VK2) is an essential nutrient supplement that have been shown to exert protective effects against osteoporotic bone loss including GIOP. In this study, we showed that the glucocorticoid dexamethasone (Dex) deregulated osteoblast autophagy and mitophagy by downregulating the expression of autophagic and mitophagic markers LC3-II, PINK1, Parkin. This consequently led to inhibition of osteoblast differentiation and mineralization function in vitro. Interestingly, co-treatment with VK2 significantly attenuated the Dex-induced downregulation of LC3-II, PINK1, Parkin, thereby restoring autophagic and mitophagic processes and normal osteoblastic activity. In addition, using an established rat model of GIOP, we showed that VK2 administration can protect rats against the deleterious effects of Dex on bone by reinstating autophagic and mitophagic activities in bone tissues. Collectively, our results provide new insights into the role of osteoblast autophagy and mitophagy in GIOP. Additionally, the use of VK2 supplementation to augment osteoblast autophagy/mitophagy may significantly improve clinical outcomes of GIOP patients.

Combined treatment with vitamin K2 and PTH enhanced bone formation in ovariectomized rats and increased differentiation of osteoblast in vitro.

Osteoporosis is accompanied by insufficient osteogenic capacity. Several lines of evidence suggested that solutions to enhance osteoblastogenesis were important strategies for osteoporotic bone defect repair. This study investigated the effect of combined treatment with vitamin K2 and PTH on bone formation in calvarial bone defect in osteoporotic rats and its influence on osteoblast in vitro. Bilateral ovariectomy was used in SPF Sprague Dawley rats to generate an osteoporosis model. Subsequently, a calvarial defect model was established and all osteoporotic rats were randomly assigned to the following groups: control, VK (vitamin K2, 30 mg/kg everyday), PTH (recombinant human PTH (1-34), 60 µg/kg, three times a week) or VK + PTH (vitamin K2, 30 mg/kg everyday plus PTH, 60 µg/kg three times a week) for 8 weeks. In vitro, bone marrow-derived stem cells (BMSCs) were cultured and treated with vitamin K2, PTH or vitamin K2+PTH. ALP staining and western blot were performed to observe the influence of combined treatment on BMSCs. Bone formation within calvarial defect were assessed by serum γ-carboxylated osteocalcin (Gla-OC), micro-CT, histological and immunofluorescent labeling. In this study, combined treatment of PTH and vitamin K2 showed positive effects on preventing bone loss in femurs in OVX rats. Combined treatment increased serum Gla-OC and promoted bone formation in osteoporotic calvarial bone defects. Immunohistochemistry showed that OCN and RUNX2 were more highly expressed in the VK + PTH group than in the control groups. In vitro studies results suggested that combined treatment with PTH and vitamin K2 increased expression of ALP, BMP2 and RUNX2 in BMSCs. Our data suggested that the combination of vitamin K2 and PTH increased differentiation of osteoblast and had a synergistic effect on bone formation in osteoporotic calvarial bone defect.

Effects of combined menaquinone-4 and PTH1-34 treatment on osetogenesis and angiogenesis in calvarial defect in osteopenic rats.

PURPOSE: The aim of this study was to evaluate the effect of combining human parathyroid hormone (1-34) (PTH1-34; PTH) and menaquinone-4 (MK-4) on calvarial bone defect repair in osteopenic rats. METHODS: Fourteen week olds were subject to craniotomy for the establishment of osteopenic animal models fed through a chronically low-protein diet. After that, critical calvarial defect model was established and all rats were randomly divided into four groups: sham, MK-4, PTH, and PTH + MK-4. The animals received MK-4 (30 mg/kg/day), PTH1-34 (60 µg/kg, three times a week), or PTH1-34 (60 µg/kg, three times a week) plus MK-4 (30 mg/kg/day) for 8 weeks, respectively. Serum γ-carboxylated osteocalcin (Gla-OC) levels, histological and immunofluorescent labeling were employed to evaluate the bone formation and mineralization in calvarial bone defect. In addition, Microfil perfusion, immunohistochemical, and micro-CT suggested enhanced angiogenesis and bone formation in calvarial bone healing. RESULTS: In this study, treatment with either PTH1-34 or MK-4 promoted bone formation and vascular formation in calvarial bone defects compared with the sham group. In addition, combined treatment of PTH1-34 plus MK-4 increased serum level of Gla-OC, improved vascular number and vascular density, and enhanced bone formation in calvarial bone defect in osteopenic conditions as compared with monotherapy. CONCLUSIONS: In summary, this study indicated that PTH1-34 plus MK-4 combination therapy accelerated bone formation and angiogenesis in calvarial bone defects in presence of osteopenia.

Combined treatment with Cinnamaldehyde and ß-TCP had an additive effect on bone formation and angiogenesis in critical size calvarial defect in ovariectomized rats.

Accumulating evidence suggests that improvements in osteogenesis and angiogenesis play an important role in repairing osteoporotic bone defects. Cinnamomum cassia (C. cassia), a traditional Chinese medicinal herb, is reported to show anabolic effects on osteoblasts. However, whether C. cassia could actually repair bone defects in osteoporotic conditions remains unknown. The purpose of this study was to evaluate the effect of combined treatment with Cinnamaldehyde (main oil isolated from the C. cassia) and ß-tricalcium phosphate (ß-TCP) on bone formation and angiogenesis in critical size calvarial defects in ovariectomized (OVX) rats. Using a previously established OVX model, 5 mm critical size calvarial defect was established in OVX rats. All OVX rats were then randomly divided into OVX group (OVX rats + empty defect), TCP group (OVX rats + ß-TCP), and CTCP group (Cinnamaldehyde 75 mg/kg/day for 12 weeks + ß-TCP). Twelve weeks after treatment, according to Micro-CT and HE staining, combination of Cinnamaldehyde and ß-TCP had an additive effect on bone regeneration compared with other groups (p < 0.05). Based on dynamic fluorochrome-labelling analysis, Cinnamaldehyde+ß-TCP continuously promoted new bone mineralization compared with other groups at each time point (p < 0.05). Microfil perfusion suggested that CTCP group showed more neovascularization compared with other groups (p < 0.05). Immunohistochemical assay supported the findings that Cinnamaldehyde+ß-TCP enhanced expression of OCN, VEGF and CD31. The present study demonstrated that combined treatment with Cinnamaldehyde and ß-TCP promoted bone formation and angiogenesis in osteoporotic bone defects, which provides a promising new strategy for repairing bone defects in osteoporotic conditions.

Chitosan oligosaccharide promotes osteoclast formation by stimulating the activation of MAPK and AKT signaling pathways.

Chitosan Oligosaccharide (COS) has been widely used for the systemic treatment of clinical diseases such as bone tissue engineering. However, its influence on osteoclast formation, which plays a critical role in bone homeostasis, has never been investigated. The aim of this study was to investigate the effect of chitosan oligosaccharide on differentiation of osteoclast. Using cell counting kit-8, tartrate-resistant acid phosphatase staining, reverse transcription­quantitative polymerase chain reaction assay and western blot analysis, we demonstrated that chitosan oligosaccharide cannot inhibit RANKL-induced osteoclast precursor proliferation but does promote osteoclast differentiation by stimulating the activation of p38/mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK)/MAPK, extracellular signal-regulated kinase (ERK)/MAPK and protein kinase B (AKT) without affecting nuclear factor kappaB (NF-kB) signaling pathways. Based on the promoting effect of chitosan oligosaccharide on osteoclast differentiation, we suggest that this property of chitosan oligosaccharide may have potential detrimental effect on bone homeostasis.

Effects of combined human parathyroid hormone (1-34) and menaquinone-4 treatment on the interface of hydroxyapatite-coated titanium implants in the femur of osteoporotic rats.

The objective of this study was to investigate the effects of human parathyroid hormone (1-34) (PTH1-34; PTH) plus menaquinone-4 (vitamin K2; MK) on the osseous integration of hydroxyapatite (HA)-coated implants in osteoporotic rats. Ovariectomized female Sprague-Dawley rats were used for the study. Twelve weeks after bilateral ovariectomy, HA-coated titanium implants were inserted bilaterally in the femoral medullary canal of the remaining 40 ovariectomized rats. All animals were then randomly assigned to four groups: Control, MK, PTH and PTH + MK. The rats from groups MK, PTH and PTH + MK received vitamin K2 (30 mg/kg/day), PTH1-34 (60 µg/kg, three times a week), or both for 12 weeks. Thereafter, serum levels of γ-carboxylated osteocalcin (Gla-OC) were quantitated by ELISA and the bilateral femurs of rats were harvested for evaluation. The combination of PTH and MK clearly increased the serum levels of Gla-OC (a specific marker for bone formation) compared to PTH or MK alone. The results of our study indicated that all treated groups had increased new bone formation around the surface of implants and increased push-out force compared to Control. In addition, PTH + MK treatment showed the strongest effects in histological, micro-computed tomography and biomechanical tests. In summary, our results confirm that treatment with PTH1-34 and MK together may have a therapeutic advantage over PTH or MK monotherapy on bone healing around HA-coated implants in osteoporotic rats.