Chronic hyperglycemia in diabetes elevates oxidative stress, damaging pancreatic β-cells and worsening complications. Oxidative stress also disrupts osteoblast function, suppresses their maturation and triggers apoptosis, thereby contributing to bone fragility. Spironolactone, an aldosterone receptor antagonist prescribed for the treatment of hypertension and heart failure, has potential protective effects on skeletal health. The present study examined whether spironolactone could protect osteoblasts from damage and restore their differentiation under oxidative conditions caused by 2-deoxy-D-ribose (dRib). MC3T3-E1 pre-osteoblastic cells were incubated with spironolactone (0-100 µM) and 15 mM dRib. Cell toxicity (through lactate dehydrogenase release), markers of differentiation (collagen content, alkaline phosphatase activity and mineral deposition), inflammatory cytokine levels (TNF-α and IL-6), reactive oxygen species (ROS), mitochondrial superoxide levels, endoplasmic reticulum (ER) stress markers (activating transcription factor 6 and inositol-requiring enzyme 1), mitochondrial integrity (mitochondrial membrane potential and ATP levels), glyoxalase I activity and reduced glutathione (GSH) levels were analyzed by ELISA. Pretreatment with spironolactone reduced dRib-induced toxicity and improved differentiation markers in MC3T3-E1 cells. It also lowered the elevated cytokine levels, ROS production, mitochondrial oxidative burden and ER stress responses caused by dRib. Furthermore, spironolactone preserved mitochondrial performance and enhanced antioxidant defenses by increasing GSH levels and glyoxalase I activity, thereby promoting the detoxification of harmful byproducts. These findings suggested that spironolactone may protect osteoblasts by mitigating oxidative and inflammatory stress, stabilizing mitochondrial function and enhancing differentiation. Overall, this highlights the possible benefits of spironolactone in the management of diabetes-related bone fragility.

Abstract Disclosure: S. Yun: None. H. Sang: None. S. Park: None. K. Suh: None. H. Kim: None. S. Chin: None. Background: In diabetes, prolonged hyperglycemia causes oxidative stress, resulting in pancreatic beta cells' dysfunction and diabetic complications. Oxidative stress can inhibit osteoblast differentiation and induce dysfunction and apoptosis. Spironolactone is an aldosterone antagonist used to treat hypertension and heart failure. In this study, we observed whether spironolactone can reduce osteoblast cytotoxicity and differentiation caused by oxidative stress induced by 2-deoxy-d-ribose (dRib). Methods: MC3T3-E1 cells were treated with spironolactone (0-100 uM) in the presence of 15 mM dRib. The cytotoxicity (lactate dehydrogenase, LDH) and differentiation (collagen content, alkaline phosphatase [ALP] activity, and mineralization) of osteoblasts, inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin [IL]-6), oxidative stress (reactive oxygen species [ROS], mitochondrial superoxide), endoplasmic reticulum (ER) stress (activating transcription factor 6 [ATF-6], inositol-requiring 1 [IRE1]), mitochondrial function (mitochondrial membrane potential [MMP], adenosine triphosphate [ATP]), glyoxalase I activity, and glutathione (GSH) were measured. Results: Pretreatment of MC3T3-E1 osteoblastic cells with spironolactone prevented the dRib-induced cytotoxicity of osteoblasts and promoted differentiation of osteoblasts suppressed by dRib. Spironolactone significantly reduced dRib-induced inflammatory cytokines, ROS, mitochondrial superoxide, and ER stress. dRib-induced mitochondrial dysfunction was significantly improved by treatment with spironolactone. dRib is detoxified by the glyoxalase system. Pretreatment with spironolactone also increased the level of reduced GSH and the activity of glyoxalase I in dRib. Conclusion: Spironolactone could reduce dRib-induced cytotoxicity and promote differentiation in MC3T3-E1 osteoblastic cells by reducing oxidative stress and inflammatory cytokines, increasing mitochondrial biogenesis, and detoxifying dRib. These results suggest that spironolactone can be useful in the prevention and treatment of diabetic bone disease. Presentation: 6/1/2024

Objective: Dipeptidyl peptidase-4 (DPP-4) inhibitors have been shown to reduce the rate of bone fractures.A newly developed antidiabetic drug, gemigliptin, may improve bone quality.We investigated the effects of gemigliptin on 2-deoxy-D-ribose (dRib)-induced oxidative damage and cellular dysfunction in the MC3T3-E1 osteoblastic cell line.Methods: Osteoblasts were treated with dRib, a strong reducing sugar, in the presence or absence of gemigliptin.Cell viability was evaluated using the CCK-8 assay.Apoptosis and reactive oxygen species (ROS) production were subsequently examined.The effects of gemigliptin on the expression of genes related to osteoblastic differentiation were determined via RT-PCR. Results:We observed that dRib reduced cell survival and markedly increased apoptosis and intracellular levels of ROS.However, pre-treatment with gemigliptin partially attenuated these dRib-induced effects.Additionally, treatment with gemigliptin increased alkaline phosphatase (ALP) activity and collagen production.Gemigliptin increased the expression of the bone-related markers ALP, collagen, osteocalcin, OPN, BMP2 and BMP7.The expression level of PI3K was increased after gemigliptin treatment under dRib condition. Conclusion:Taken together, these results suggest that gemigliptin attenuates dRib-induced cellular damage in osteoblasts.Gemigliptin may improve oxidative conditions in bone.Increased ALP activity and increased expression of genes related to osteoblastic differentiation indicate that gemigliptin treatment can improve the quality of bone formation.Our results suggest that gemigliptin treatment is effective in diminishing oxidative stress and improving bone strength through PI3K/AKT/BMP axis.

Tetrabromobisphenol A (TBBPA) is a reactive brominated flame retardant widely used in various industrial and household products. This compound is persistent in the environment and accumulates in living organisms through the food chain, and is toxic to animals and human beings. Studies have shown that TBBPA is toxic to various human cell lines, including neuronal cells. Apigenin is a dietary flavonoid that exhibits various beneficial health effects on biological activities, including antioxidant, anti-inflammatory, and neuroprotective effects. This study investigated the cytoprotective effects of apigenin against TBBPA-mediated cytotoxicity in SK-N-MC cells. Our results demonstrated that treatment of SK-N-MC cells with apigenin increased the cell viability, which was decreased by TBBPA, and reduced apoptosis and autophagy induced by TBBPA. Although we did not observe any change in the levels of IL-1β and nitrite in cultured cells after TBBPA treatment, apigenin was found to decrease the production of these pro-inflammatory mediators. Apigenin decreased the intracellular Ca2+ concentration, NOX4 level, oxidative stress, and mitochondrial membrane potential loss and increased the mitochondrial biogenesis and nuclear Nrf2 levels that were reduced by TBBPA. Finally, apigenin treatment decreased Akt and ERK induction in cells exposed to TBBPA. Based on these results, apigenin could be a promising candidate for designing natural drugs to treat or prevent TBBPA-related neurological disorders.

The accumulation of advanced glycation end products (AGEs) causes metabolic dysfunction and neuronal cell damage. Methylglyoxal (MG) is a major glycating agent that reacts with basic residues present in proteins and promotes the formation of AGEs. Sciadopitysin, a type of biflavonoid, exerts protective effects against neuronal cell damage; however, the underlying mechanisms have not been studied. This study aimed to investigate the mechanisms underlying the protective effects of sciadopitysin against MG-mediated cytotoxicity in SK-N-MC neuroblastoma cells. Our results demonstrated that pretreatment of SK-N-MC cells with sciadopitysin improved the cell viability that was inhibited by MG and inhibited the apoptosis induced by MG. Sciadopitysin attenuated intracellular Ca2+ , NOX4 levels, oxidative stress, and MG-protein adduct levels, and increased nuclear Nrf2 and glyoxalase 1 levels in the presence of MG. These results suggest that sciadopitysin exerts neuroprotective effects against MG-induced death of human SK-N-MC cells via its antioxidative action. This study highlights sciadopitysin as a promising candidate for antioxidant therapy and designing natural drugs against AGE-induced neurodegenerative disorders.

Background Methylglyoxal (MG) is associated with the pathogenesis of age- and diabetes-related complications. Spironolactone is a competitive antagonist of aldosterone that is widely employed in the treatment of hypertension and heart failure. This study examined the effects of spironolactone on MG-induced cellular dysfunction in MC3T3-E1 osteoblastic cells. Methods MC3T3-E1 cells were treated with spironolactone in the presence of MG. The mitochondrial function, bone formation activity, oxidative damage, inflammatory cytokines, glyoxalase I activity, and glutathione (GSH) were measured. Results Pretreatment of MC3T3-E1 osteoblastic cells with spironolactone prevented MG-induced cell death, and improved bone formation activity. Spironolactone reduced MG-induced endoplasmic reticulum stress, production of intracellular reactive oxygen species, mitochondrial superoxides, cardiolipin peroxidation, and inflammatory cytokines. Pretreatment with spironolactone also increased the level of reduced GSH and the activity of glyoxalase I. MG induced mitochondrial dysfunction, but markers of mitochondrial biogenesis such as mitochondrial membrane potential, adenosine triphosphate, proliferator-activated receptor gamma coactivator 1α, and nitric oxide were significantly improved by treatment of spironolactone. Conclusion Spironolactone could prevent MG-induced cytotoxicity in MC3T3-E1 osteoblastic cells by reduction of oxidative stress. The oxidative stress reduction was explained by spironolactone's inhibition of advanced glycation end-product formation, restoring mitochondrial dysfunction, and anti-inflammatory effect.

Obesity is caused by excessive fat cells and the overgrowth of adipocytes and is a major risk factor for several chronic illnesses. Aronia melanocarpa fruit is rich in anthocyanins and polyphenols and has protective effects against various diseases. In this study, we examined the effect of Aronia extract (Aronia bioactive fraction, ABF®) on the biomarkers of the adipogenic pathway during adipocyte differentiation of 3T3-L1 cells. Lipid accumulation was verified by Oil Red O staining. mRNA and protein expression of lipoprotein lipase (LPL), CCAAT/enhancer-binding protein α (C/EBPα), peroxisome proliferator-activated receptor γ (PPARγ), fatty acid-binding protein 2 (FABP2), and fatty acid synthase (FAS) were assayed by RT-qPCR and Western blot analyses. Adiponectin and leptin secretion were measured using enzyme-linked immunosorbent assays. ABF® treatment downregulated lipid accumulation based on Oil Red O staining. ABF®-treated cells exhibited decreased mRNA and protein expression of LPL, C/EBPα, PPARγ, FABP2, and FAS. Moreover, ABF® treatment significantly increased adiponectin secretion and decreased leptin secretion. In conclusion, ABF® has anti-adipogenic effects on the differentiation of 3T3-L1 cells and may be used as an anti-obesity nutraceutical.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an endocrine disrupting compound and persistent organic pollutant that has been associated with diabetes in several epidemiological studies. Oleuropein, a major phenolic compound in olive fruit, is a superior antioxidant and radical scavenger. This study aimed to examine the effects of oleuropein against TCDD-induced stress response in a pancreatic beta cell line, INS-1 cells. Cells were pre-incubated with various concentrations of oleuropein and then stimulated with TCDD (10 nM) for 48 hrs. When treated with TCDD, INS-1 cells produced robust amounts of prostaglandin E-2 (PGE(2)) compared to the untreated control, and this increase was inhibited by oleuropein treatment. TCDD increased Ca2+-independent phospholipase A(2) (iPLA(2)beta) level, but had no effect on Group 10 secretory phospholipase A(2) (PLA(2)G10) level, while oleuropein deceased the levels of iPLA(2)beta and PLA(2)G10 in the presence of TCDD. Cyclooxygenase-1 (COX-1) was significantly increased by TCDD treatment and attenuated with oleuropein pretreatment. Oleuropein decreased TCDD-mediated production of JNK, TNF-alpha, and ROS. In addition, oleuropein increased Akt and GLUT2 levels suppressed by TCDD in INS-1 cells. Thus, the results suggest that oleuropein prevents pancreatic beta cell impairment by TCDD.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) accumulates in human body, probably influencing adipocyte differentiation and causing various toxic effects, including wasting syndrome. Recently, orientin, a phenolic compound abundant in natural health products, has been shown to have antioxidant properties. We investigated the protective effects of orientin against TCDD-induced adipocyte dysfunction and its underlying mechanisms. In this study, orientin suppressed TCDD-induced loss of lipid accumulation. Orientin inhibited TCDD-driven decreases in the levels of peroxisome proliferator-activated receptor γ and adiponectin. Orientin also reduced TCDD-induced prostaglandin E2, and cytosolic phospholipase A2α levels, and increased TCDD-inhibited peroxisome proliferator-activated receptor gamma coactivator 1-alpha levels in 3T3-L1 adipocytes. TCDD reduced the levels of insulin receptor substrate 1 and glucose transporter 4, and decreased insulin-stimulated glucose uptake activity; however, orientin diminished these TCDD-induced effects. These results suggest that orientin may have beneficial effects on the prevention of TCDD-induced wasting syndrome and type II diabetes mellitus accompanied by insulin resistance.

Methylglyoxal (MG), a highly reactive dicarbonyl compound, is a major cell-permeant precursor of advanced glycation end-products, which are associated with several conditions, including diabetes and degenerative diseases. Crocin, a constituent of saffron, is involved in many pharmacological activities. Recent studies have reported that crocin exerts protective effects against bone diseases. Osteoclasts are multinucleated cells derived from hematopoietic stem cells that are responsible for bone resorption. The up- or down-regulation of their proliferation and differentiation is often associated with many bone-related diseases. The present study aimed to investigate the effects of crocin on osteoclast differentiation and to clarify its mechanism of action in the presence of MG. We demonstrated that crocin reversed MG-induced inhibition of tartrate-resistant acid phosphatase activity and bone resorption activity in osteoclasts. Quantitative reverse transcription-polymerase chain reaction analysis indicated that crocin treatment decreased the expression of TNF receptor-associated factor-6 (TRAF6), Akt2, extracellular-signal-regulated kinase-1 (ERK1), osteopetrosis-associated transmembrane protein 1 (OSTM1), and matrix metalloproteinase 9 (MMP-9) genes in the presence of MG. Crocin pretreatment also reversed MG-induced changes in mitochondrial mass, mitochondrial membrane potential, mitochondrial superoxide, and glyoxalase I levels. Taken together, our data suggest that crocin may be a useful therapeutic agent for the treatment of diabetic bone disorders.

Background Tetrabromobisphenol A (TBBPA), one of the most widely used brominated flame-retardants, is a representative persistent organic pollutants group. Studies on TBBPA toxicity have been conducted using various target cells; however, few studies have investigated TBBPA toxicity in bone cells. Therefore, this study investigated the in vitro effects of TBBPA on osteoclasts, a cell type involved in bone metabolism. Methods RAW264.7 cells were cultured in medium containing 50 ng/mL receptor activator of nuclear factor kappa B ligand (RANKL) and varying concentrations of TBBPA. To evaluate the effects of TBBPA on the differentiation and function of osteoclasts, osteoclast-specific gene expression, tartrate-resistant acid phosphatase (TRAP) activity, bone resorbing activity, mitochondrial membrane potential (MMP) and mitochondrial superoxide were measured. Results The presence of 20 μM TBBPA significantly increased TRAP activity in RANKL-stimulated RAW264.7 cells, the bone resorbing activity of osteoclasts, and the gene expression of Akt2, nuclear factor of activated T-cells cytoplasmic 1, and chloride channel voltage-sensitive 7. However, TBBPA treatment caused no change in the expression of carbonic anhydrase II, cathepsin K, osteopetrosis-associated transmembrane protein 1, Src, extracellular signal-related kinase, GAB2, c-Fos, or matrix metalloproteinase 9. Furthermore, 20 μM TBBPA caused a significant decrease in MMP and a significant increase in mitochondrial superoxide production. Conclusion This study suggests that TBBPA promotes osteoclast differentiation and activity. The mechanism of TBBPA-stimulated osteoclastogenesis might include increased expression of several genes involved in osteoclast differentiation and reactive oxygen species production.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a well-known environmental contaminant that produces a wide variety of adverse effects in humans. Catalpol, a major bioactive compound enriched in the dried root of Rehmannia glutinosa, is a major iridoid glycoside that alleviates bone loss. However, the detailed mechanisms underlying the effects of catalpol remain unclear. The present study evaluated the effects of catalpol on TCDD-induced cytotoxicity in osteoblastic MC3T3-E1 cells. Catalpol inhibited TCDD-induced reduction in cell viability and increases in apoptosis and autophagic activity in osteoblastic MC3T3-E1 cells. Additionally, pretreatment with catalpol significantly decreased the nitric oxide and nitrite levels compared with a control in TCDD-treated cells and significantly inhibited TCDD-induced increases in the levels of cytochrome P450 1A1 and extracellular signal-regulated kinase. Pretreatment with catalpol also effectively restored the expression of superoxide dismutase and extracellular signal-regulated kinase 1 and significantly enhanced the expression of glutathione peroxidase 4 and osteoblast differentiation markers, including alkaline phosphatase and osterix. Taken together, these findings demonstrate that catalpol has preventive effects against TCDD-induced damage in MC3T3-E1 osteoblastic cells.

Methylglyoxal (MG), a highly reactive dicarbonyl compound, is a major precursor in the formation of advanced glycation end products, which are associated with diabetes-related diseases. Bergenin, an active constituent of plants of the genus Bergenia, exhibits multiple biological activities. This study evaluated the effect of bergenin on osteoclast differentiation and determined its mechanism of action. Bergenin reversed MG-inhibited tartrate-resistant acid phosphatase (TRAP) activity and decreased the bone resorption activity of osteoclasts. Quantitative RT-PCR revealed that bergenin decreased the expression of ERK1, Akt2, MMP-9, and OSTM1 genes in the presence of MG. Bergenin pretreatment yielded significant increases in intracellular calcium concentration, mitochondrial mass, mitochondrial membrane potential, and glyoxalase I reduced by MG. Additionally, bergenin decreased the formation of mitochondrial superoxide induced by MG. Detoxification of MG by bergenin may be a viable treatment for bone disorders in patients with diabetes.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental pollutant. TCDD accumulates in the food chain, mainly in the fatty tissues of the human body where it causes various toxic effects. Biochanin A is a natural organic compound in the class of phytochemicals known as flavonoids. We investigated whether biochanin A suppresses TCDD-induced loss of adipogenic action using 3T3-L1 adipocytes as a cell culture model of wasting syndrome. In the present study, biochanin A suppressed TCDD-induced loss of lipid accumulation. Pretreating the cells with biochanin A increased the levels of the adipogenesis-associated factors peroxisome proliferator-activated receptor γ and adiponectin, which were inhibited by TCDD. TCDD decreased insulin-stimulated glucose uptake, which was effectively restored by pretreatment with biochanin A. Biochanin A also inhibited the TCDD-driven decrease in production of insulin receptor substrate-1 and glucose transporter 4. These results suggest a preventive effect of biochanin A against TCDD in the development of insulin resistance and diabetes. TCDD increased production of intracellular calcium ([Ca2+]i), prostaglandin E2, cytosolic phospholipase A2, and cyclooxygenase-1, while reducing the level of peroxisome proliferator-activated receptor gamma coactivator 1-alpha. However, biochanin A inhibited these TCDD-induced effects. We conclude that biochanin A is an attractive compound for preventing TCDD-induced wasting syndrome.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a well-known environmental contaminant that exerts its toxicity through a variety of signaling mechanisms. The present study evaluated the effects of 27-deoxyactein, one of the major constituents isolated from Cimicifuga racemosa, on TCDD-induced toxicity in osteoblastic MC3T3-E1 cells. TCDD reduced cell survival, markedly increased apoptosis, and enhanced autophagy activity. However, pre-treatment with 27-deoxyactein attenuated all TCDD-induced effects and significantly decreased intracellular calcium (Ca2+) concentrations, the collapse of the mitochondrial membrane potential (MMP), the level of reactive oxygen species (ROS), and cardiolipin peroxidation compared to the TCDD-treated controls. Additionally, TCDD-induced increases in the levels of aryl hydrocarbon receptor (AhR), cytochrome P450 1A1 (CYP1A1), and extracellular signal-regulated kinase (ERK) were significantly inhibited by 27-deoxyactein. The mRNA levels of superoxide dismutase (SOD), ERK1, and nuclear factor kappa B (NF-κB) were also effectively restored by pre-treatment with 27-deoxyactein. Furthermore, 27-deoxyactein significantly increased the expressions of genes associated with osteoblast differentiation, including alkaline phosphatase (ALP), osteocalcin, bone sialoprotein (BSP), and osterix. Taken together, the present findings demonstrate the preventive effects of 27-deoxyactein on TCDD-induced damage in osteoblasts.

Bergenin, an active component of plants in the genus Bergenia, has multiple biological activities, including anti-inflammatory and immunomodulatory properties. We investigated the effects of bergenin on MC3T3-E1 osteoblasts. Bergenin treatment significantly elevated collagen synthesis, alkaline phosphatase activity, osteocalcin synthesis, and mineralization in the cells (p < 0.05). Additionally, bergenin increased the ratio of osteoprotegerin to receptor activator of nuclear factor kappa-B ligand, and cyclophilin B release. Methylglyoxal (MG), a highly reactive dicarbonyl compound, is the major precursor in the formation of advanced glycation end products. Pretreatment of MC3T3-E1 cells with bergenin prevented MG-induced cell death. Furthermore, bergenin treatment significantly reduced the induction of activating transcription factor 6 and autophagy by MG. These results indicate that bergenin may have positive effects on critical osteoblastic cell functions.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has various toxicological effects in adipose tissue. Evidence is accumulating that glabridin, a flavonoid extracted from licorice, has beneficial effects on the regulation of glucose homeostasis. In this study, we investigated whether glabridin suppresses TCDD-induced loss of adipogenic action using 3T3-L1 adipocytes as a cell culture model of wasting syndrome. Glabridin effectively suppressed TCDD-induced loss of lipid accumulation in this model. Pretreating cells with glabridin increased the gene expression of not only the adipogenesis-associated key transcription factors peroxisome proliferator-activated receptor gamma (PPAR) and CCAAT/enhancer binding protein alpha, but also lipoprotein lipase in the presence of TCDD. TCDD decreased insulin-stimulated glucose uptake, which was effectively restored by pretreatment with glabridin. Glabridin also inhibited the TCDD-driven decreased production of insulin receptor substrate 1 and glucose transporter 4. TCDD increased the production of mitochondrial superoxides, prostaglandin E-2, phospholipase A(2), cyclooxygenase-1 and intracellular calcium concentrations, while reducing the production of PPAR coactivator 1 alpha and glycolysis. However, glabridin treatment reduced these TCDD-induced effects. We conclude that glabridin suppresses the TCDD-induced loss of lipid accumulation in 3T3-L1 adipocytes by regulating the levels of PPAR, CCAAT/enhancer binding protein alpha, lipoprotein lipase, glucose uptake, prostaglandin E-2 and energy metabolism. These results also provide in vitro evidence of the effects of glabridin on adipocyte metabolism, which suggests a protective effect against dioxin exposure in the development of insulin resistance and diabetes. Glabridin suppresses the TCDD-induced loss of lipid accumulation in 3T3-L1 adipocytes by regulating the levels of PPAR, CCAAT/enhancer binding protein alpha, lipoprotein lipase, glucose uptake, prostaglandin E2 and energy metabolism. These results provide in vitro evidence of the effects of glabridin on adipocyte metabolism, which suggests a protective effect against TCDD exposure in the development of insulin resistance and diabetes.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an environmental contaminant. Xanthohumol is a prenylated flavonoid found in hops (Humulus lupulus) and beer. The aim of the current study was to explore the role of xanthohumol in modulating the toxicity of TCDD in MC3T3-E1 osteoblastic cells. In cells treated with TCDD alone, intracellular Ca2+ concentrations, mitochondrial membrane potential disruption, reactive oxygen species production, cardiolipin peroxidation, nitric oxide release and cytochrome P450 1A1 expression were significantly increased. TCDD treatment increased the mRNA levels of extracellular signal-regulated kinase 1 and nuclear factor kappa B, and significantly decreased the level of protein kinase B (AKT) in MC3T3-E1 osteoblastic cells. However, the presence of xanthohumol alleviated the pathological effects of TCDD. In addition, xanthohumol treatment significantly increased the expression of genes associated with osteoblast differentiation (alkaline phosphatase, osteocalcin, osteoprotegerin and osterix). We conclude that xanthohumol has a beneficial influence and may antagonize TCDD toxicity in osteoblastic cells.

Methylglyoxal (MG) is a reactive dicarbonyl compound produced by glycolytic processing, which has been identified as a precursor of advanced glycation end products. Elevated MG levels in patients with diabetes are believed to contribute to diabetic complications, including bone defects. The objective of this study was to evaluate the effect of MG on RANKL-induced osteoclast differentiation in RAW264.7 cells, a murine macrophage cell line. RAW264.7 cells were cultured in medium containing 50 ng/mL RANKL and different concentrations of MG. Tartrate-resistant acid phosphatase (TRAP) activity and osteoclast bone resorbing activity were assessed and changes in intracellular calcium concentration, mitochondrial mass, mitochondrial membrane potential, and glyoxalase I level were examined. In addition, real-time RT-PCR assay was used to analyse osteoclast-associated genes. MG markedly inhibited RANKL-induced TRAP activity. MG treatment resulted in a significant decrease in intracellular calcium concentration, mitochondrial mass, mitochondrial membrane potential, and glyoxalase I level during osteoclastogenesis. In addition, MG increased the formation of mitochondrial superoxide. Quantitative reverse transcriptase-polymerase chain reaction revealed increased expression of the TRAF6, GAB2, ERK1, c-Fos, NFATc1, CLCN7, and OSTM1 genes, decreased expression of TCIRG and carbonic anhydrase II, and unchanged expression of cathepsin K and MMP-9 upon MG treatment. MG had no effect on the bone resorbing activity of osteoclasts. Our findings indicate that MG inhibits TRAP and glyoxalase I activity and impairs mitochondrial function in osteoclasts. Further validation of the underlying pathway is necessary.