Mangiferin attenuates osteoporosis by inhibiting osteoblastic ferroptosis through Keap1/Nrf2/SLC7A11/GPX4 pathway.

BACKGROUND: Ferroptosis is a crucial contributor to impaired osteoblast function in osteoporosis. Mangiferin, a xanthonoid glucoside isolated from mangoes, exhibits anti-osteoporosis effects. However, its potential mechanism is not fully understood. PURPOSE: This study explores the potencies of mangiferin on osteoblastic ferroptosis and deciphers its direct target in the context of solute carrier family 7-member 11 (SLC7A11)/glutathione peroxidases 4 (GPX4) pathway. METHODS: In vivo models include bilateral ovariectomy induced osteoporosis mice, iron-dextran induced iron-overloaded mice, and nuclear factor-erythroid 2-related factor 2 (Nrf2)-knockout mice. Mice are orally administrated mangiferin (10, 50 or 100 mg.kg-1.d-1) for 12 weeks. In vitro osteoblast models include iron-dextran induced iron-overloaded cells, erastin induced ferroptosis cells, and gene knockout cells. RNA sequencing is applied for investigating the underlying mechanisms. The direct target of mangiferin is studied using a cellular thermal shift assay, silico docking, and surface plasmon resonance. RESULTS: Mangiferin promotes bone formation and inhibits ferroptosis in vivo models (osteoporosis mice, iron-overloaded mice) and in vitro models (ferroptosis osteoblast, iron-overloaded osteoblasts). Mechanismly, mangiferin directly binds to the kelch-like ECH-associated protein 1 (Keap1) and activates the downstream Nrf2/SLC7A11/GPX4 pathway in both the in vivo and in vitro models. Mangiferin failed to restore the osteoporosis and ferroptosis in Nrf2-knockout mice. Silencing Nrf2, SLC7A11 or GPX4 abolished the anti-ferroptosis effect of mangiferin in erastin-induced cells. Addition of the ferroptosis agonist RSL-3 also blocked the protective effects of mangiferin on iron-overloaded cells. Furthermore, mangiferin had better effects on osteogenesis than the ferroptosis inhibitor (ferrostatin-1) and the Nrf2 agonists (sulforaphane, dimethyl fumarate, and bardoxolone). CONCLUSIONS: We identify for the first time mangiferin as a ferroptosis inhibitor and a direct Keap1 conjugator that promotes bone formation and alleviates osteoporosis. This work also provides a potentially practical pharmacological approach for treating ferroptosis-driven diseases.

Anti-osteoporosis effects of Anemarrhenae Rhizoma / Phellodendri Chinensis Cortex herb pair and its major active components in diabetic rats and zebrafish.

ETHNOPHARMACOLOGICAL RELEVANCE: Anemarrhenae Rhizoma/Phellodendri Chinensis Cortex (AR/PCC) herb pair has been widely used in traditional Chinese medicines for the treatment of diabetic osteoporosis. However, the anti-diabetic osteoporotic active components of AR/PCC remain unclear. This study aimed to explore the major active ingredients in AR/PCC for its protective effects against bone deterioration induced by diabetes. MATERIALS AND METHODS: The aqueous extracts of AR/PCC with different proportions (AR:PCC = 1:3, 1:2, 1:1, 2:1 and 3:1, w/w) were prepared. Streptozotocin-induced diabetic rats were orally administrated with the AR/PCC extracts. The absorbed phytochemical compounds in serum of diabetic rats were identified by ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry method and their contents in the AR/PCC extracts were determined by high performance liquid chromatography-ultraviolet detector-evaporative light scattering detector method. The absorbed compounds in the extracts were considered as the major potential active components in AR/PCC, and their combination was defined as M-AR/PCC. A component-knockout approach was applied to evaluate the contribution of each compound in M-AR/PCC. The larvae and adults of diabetic zebrafish models were then used to evaluated the anti-diabetic osteoporotic performance of the M-AR/PCC. The real-time reverse transcription polymerase chain reaction technique was applied to study the regulation effects of M-AR/PCC on osteogenesis and osteoclastgensis in diabetic zebrafish models. RESULTS: The phenotypes of diabetic osteoporosis rats induced by streptozotocin were reversed by the oral administration of AR/PCC extracts with different ratios, as evidenced by the increased bone mineral density, bone volume density, trabecular thickness, trabecular number, and decreased trabecular separation of femoral metaphysis. Seven phytochemical compounds were detected in the serum and their contents in AR/PCC varied dramatically with different proportions, including 1 xanthone glycoside and 6 alkaloids. By using diabetic zebrafish larvae model and compound-knockout strategy, each compound in M-AR/PCC were proved to play an indispensable role in the positive regulatory actions in the bone mass of diabetic zebrafish. Furthermore, the herb pair with a ratio of 1:1 and the related M-AR/PCC showed the best therapeutic effects on diabetic osteoporosis. They showed similar performances on the inhibition of the tartrate-resistant acid phosphatase activity and the promotion of the alkaline phosphatase activity in diabetic adult zebrafish model. The M-AR/PCC treatment could decrease the blood glucose, upregulate the mRNA expression levels of osteoblast-related genes (alp, runx2b and opg) and downregulate the expression of osteoclast-related genes (acp5α, rankl and sost) in streptozotocin-induced zebrafish. CONCLUSION: AR/PCC herb pair and its major active components possess potent anti-diabetic osteoporotic effect on streptozotocin-induced in vivo models. The combination of the seven active compounds derived from AR/PCC herbal pair could be a potential agent for protection against osteoporosis associated with diabetes.

Structural elucidation and anti-diabetic osteoporotic activity of an arabinogalactan from Phellodendron chinense Schneid.

Phellodendron chinense Schneid. was widely used as a medicinal herb for the treatment of diabetic osteoporosis in China. In this study, an arabinogalactan, named as PPCP-1, was isolated from the bark of Phellodendron chinense Schneid., and purified by DEAE-cellulose DE52 and Sephacryl S-200 HR column chromatography. The structure of PPCP-1 was characterized as a repeating unit consisting of →3)-ß-d-Galp-(1→, →3,6)-ß-d-Galp-(1→, →5)-α-l-Araf-(1→, →4)-α-d-Glcp-(1→, →3)-α-d-Glcp-(1→, →4)-α-d-Manp-(1→ with branches of →5)-α-l-Araf-(1→, →3,5)-α-l-Araf-(1→ and terminal α-l-Araf. Pharmacologically, the oral administration of PPCP-1 preserved osteoporosis associated with hyperglycemia by inhibiting α-glucosidase activity, improving glucose tolerance, decreasing the accumulation of advanced glycation end products (AGEs), as well as down-regulating the expression of receptor for AGEs in tibias of streptozotocin-induced diabetic rats. Collectively, the present study suggested that the arabinogalactan PPCP-1 from Phellodendron chinense Schneid. might potentially be used as functional foods for bone health and/or developed for drug discovery for alleviating diabetic osteoporosis.

Timosaponin AIII attenuates inflammatory injury in AGEs-induced osteoblast and alloxan-induced diabetic osteoporosis zebrafish by modulating the RAGE/MAPK signaling pathways.

BACKGROUND: Advanced glycation end products (AGEs) deposition causes inflammatory injury in osteoblasts and contributes to diabetic osteoporosis. The receptor for advanced glycation end product/mitogen-activated protein kinase pathway (RAGE/MAPK) signaling pathway is closely linked to the pathogenesis of diabetic osteoporosis. Timosaponin AIII, a steroidal saponin isolated from Anemarrhena asphodeloides Bunge (Asparagaceae), shows anti-inflammatory and anti-osteoporosis effects. PURPOSE: The present study was aimed to investigate the therapeutic effects of timosaponin AIII on diabetic osteoporosis and whether its effect is dependent on protecting osteoblasts against AGEs-induced injury via RAGE/MAPK signaling suppression. METHODS: An alloxan-induced diabetic osteoporosis zebrafish model was applied to investigate the effects of timosaponin AIII in vivo, and alendronate was used as a positive control. Moreover, related mechanisms were explored in primary rat osteoblasts. Molecular docking was applied to investigate the interactions between timosaponin AIII and RAGE. RESULTS: Timosaponin AIII treatment reversed alloxan-induced reduction in the mineralized area of the larvae head skeleton, accompanied by a decreased level of triglyceride and total cholesterol in the zebrafish. Additionally, AGEs significantly influenced RAGE expression, alkaline phosphatase activity, interleukin 1ß expression, interleukin 6 expression, and tumor necrosis factor-α expression, and increased cell apoptosis. Timosaponin AIII significantly downregulated AGEs-induced interleukin 1ß, interleukin 6, and tumor necrosis factor-α levels, and upregulated alkaline phosphatase and osteocalcin levels. Timosaponin AIII also significantly reduced the expression of RAGE and had additive effects on downstream P38, extracellular signal-regulated kinase and c-Jun N-terminal kinase in AGEs-induced osteoblast. Molecular docking predicted that hydrogen and hydrophobic interactions occurred between timosaponin AIII and RAGE. CONCLUSION: These data clarified that timosaponin AIII attenuates diabetic osteoporosis via a novel mechanism involved suppressing the RAGE/MAPK signaling pathway. Our finding highlights the potential value of timosaponin AIII as an anti-diabetic osteoporosis agent.

Preparation and application of polyvinyl alcohol-decorated cell membrane chromatography for screening anti-osteoporosis components from Liuwei Dihuang decoction-containing serum.

Cell membrane chromatography is a powerful tool for screening active components from complex matrices. New cell membrane carriers need to be developed to increase the coverage of cell membranes on the surface of stationary phases, thereby improving cell membrane chromatographic retention. In this work, we prepared polyvinyl alcohol-poly dimethyl diallyl ammonium chloride modified silica gel as a cell membrane carrier. Osteoblast cell was used as cell membrane source, which was widely used to evaluate the osteogenic activity of drugs. The new cell membrane chromatographic stationary phase was used to screen anti-osteoporosis components in Liuwei Dihuang decoction-containing serum. The chemical structures of the new modified materials were characterized by scanning electronic microscopy, Fourier transform infrared spectroscopy, and elemental analysis characterization. Compared with the common cell membrane column, the cell membrane coverage of this modified material was increased by 30%, and thus provided a stronger retention effect in positive drugs. Nineteen metabolites in rat serum samples were retained on the cell membrane chromatography and identified by ultra-high-performance liquid chromatography/time-of-flight mass spectrometry. Among those, four components (morroniside, catalpol, loganin, and acteoside) were selected for in vitro pharmacodynamics validation. They significantly increased the osteoblast proliferation. The new modified material was successfully applied to screen anti-osteoporosis components from Liuwei Dihuang Decoction-containing serum.

Erxian Decoction Attenuates TNF-α Induced Osteoblast Apoptosis by Modulating the Akt/Nrf2/HO-1 Signaling Pathway.

Erxian decoction (EXD), a traditional Chinese medicine formula, has been used for treatment of osteoporosis for many years. The purpose of this study was to investigate the pharmacological effect of EXD in preventing osteoblast apoptosis and the underlying mechanism of prevention. Putative targets of EXD were predicted by network pharmacology, and functional and pathway enrichment analyses were also performed. Evaluations of bone mineral density, serum estradiol level, trabecular area fraction, serum calcium levels, and tumor necrosis factor (TNF)-α levels in ovariectomized rats, as well as cell proliferation assays, apoptosis assays, and western blotting in MC3T3-E1 osteoblasts were performed for further experimental validation. Ninety-three active ingredients in the EXD formula and 259 potential targets were identified. Functional and pathway enrichment analyses indicated that EXD significantly influenced the PI3K-Akt signaling pathway. In vivo experiments indicated that EXD treatment attenuated bone loss and decreased TNF-α levels in rats with osteoporosis. In vitro experiments showed that EXD treatment increased cell viability markedly and decreased levels of caspase-3 and the rate of apoptosis. It also promoted phosphorylation of Akt, nuclear translocation of transcription factor NF-erythroid 2-related factor (Nrf2), and hemeoxygenase-1 (HO-1) expression in TNF-α-induced MC3T3-E1 cells. Our results suggest that EXD exerted profound anti-osteoporosis effects, at least partially by reducing production of TNF-α and attenuating osteoblast apoptosis via Akt/Nrf2/HO-1 signaling pathway.

Integrated pathological cell fishing and network pharmacology approach to investigate main active components of Er-Xian decotion for treating osteoporosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Oxidative damage to osteoblasts was a key factor in the development of osteoporosis. Er-Xian Decotion (EXD) is widely used in China for the treatment of osteoporosis, which has a variety of antioxidant active ingredients. EXD may be an important source of protection against oxidative damage in osteoblasts, but the anti-osteoporotic active components of EXD is currently unclear. AIM OF THE STUDY: This work established an effective and reliable drug screening method to find main active ingredients in EXD (M-EXD) that can protect osteoblasts against oxidative stress and achieve anti-osteoporosis effects. MATERIALS AND METHODS: H2O2-induced osteoblast cell fishing with UHPLC-QTOF/MS was firstly used to discover the potential active components from EXD. Afterword, the EXD compound-osteoporosis target network was constructed using network pharmacology, thus potentially anti-osteoporosis ingredients were founded, and their combination were defined as the M-EXD. Finally, pharmacology effects of M-EXD was evaluated by ovariectomized rats, prednisolone induced-zebrafish and H2O2-induced osteoblasts. RESULTS: 40 candidate active ingredients in EXD were initially screened out via pathological cell fishing. According to network pharmacology result, M-EXD consisted of 13 ingredients since they had a close relationship with 65 osteoporosis-related targets. Pharmacological evaluation showed that M-EXD significantly ameliorated oxidative stress in H2O2-induced osteoblast model, evidently reversed the activity of ALP, ROS, GSH-px, NO and MDA compared with the model group. M-EXD showed better anti-oxidative activities than individual ingredients, presenting obvious synergetic effects. In osteoporosis rat and zebrafish models, M-EXD also demonstrated good anti-osteoporotic properties by mitigating the osteoporosis bone loss and increasing serum bone morphogenetic protein 2, and reversing osteocalcin expression in bone tissue. It significantly ameliorated oxidative stress in the in-vivo models. Moreover, M-EXD and EXD showed similar anti-osteoporotic and anti-oxidative properties, while the rest components of EXD had no satisfactory anti-osteoporotic efficacy. CONCLUSIONS: Our work successfully identified the main active components in EXD, which could represent the efficacy of EXD on treating osteoporosis, and meanwhile, it also provided an effective strategy to investigate active ingredients from natural medicines, which might be helpful for drug development and application.

[Rapid screening of anti-osteoporosis active ingredients from Liuwei Dihuang Decoction by osteoblast membrane chromatography/ultra-high performance liquid chromatography-time of flight mass spectrometry].

A method was established for determination of potential anti-osteoporosis ingredients from Liuwei Dihuang Decoction by osteoblast cell membrane chromatography/ultra-performance liquid chromatography/time-of-flight mass spectrometry (CMC/UPLC-TOF/MS). The osteoblasts were used as the source of cell membrane for the preparation of CMC stationary phase. An osteoblast CMC column (10 mm×2 mm) was prepared by coating silica gel (0.05 g) with cell membrane. The active ingredients in the aqueous extract of Liuwei Dihuang Decoction (90 g/L) were first screened by CMC. Water was used as the mobile phase, and the flow rate was 0.20 mL/min. Then, the eluates of the CMC were qualitatively analyzed by UPLC-TOF/MS using a WATERS ACQUITY UPLC BEH C18 column (10 mm×2 mm). Acetonitrile-water was used as the mobile phase at a flow rate of 0.40 mL/min. This method could quickly and selectively identify 16 potential anti-osteoporotic active ingredients from Liuwei Dihuang Decoction. Due to high catalpol content in Liuwei Dihuang Decoction and its good affinity with CMC column, catapol was selected for in vivo and in vitro pharmacological examinations. It was found that catalpol (1-10 µmol/L) could significantly promote the proliferation of osteoblasts in a dose-dependent manner. It also significantly increased the area of bone staining in osteoporosis zebrafish model. The osteoblast CMC/UPLC-TOF/MS method could quickly screen the anti-osteoporosis active ingredients in complex traditional Chinese medicine prescriptions, and had the advantages of simple operation, high efficiency and high sensitivity.

Preparation of micro-cell membrane chromatographic columns with polyvinyl alcohol-modified polyether ether ketone tube as cellular membrane carrier.

Cell membrane chromatography is a promising technique for screening active components from complex matrices. Unfortunately, the large consumption of cells and low resolutions of analytes limit the applications of this method. Herein, we report polyether ether ketone tube as a novel cellular membrane carrier for cell membrane chromatography. Its inner surface is firstly coated by polyvinyl alcohol and then cell membranes are physically adsorbed onto the polyvinyl alcohol layer. To verify this approach, osteoclast and osteoblast micro-column were prepared and characterized by calcitonin and verapamil, respectively. Comparing with common cell membrane chromatographic column, the micro-cell membrane chromatographic columns showed about 1000-fold decrease of cell consumption and satisfactory retention behavior. The developed column was applied to screen potential active components from Cortex Phellodendri Chinensis. A total of 18 components in Cortex Phellodendri Chinensis extract were observed as having retention property of osteoclast micro-cell membrane chromatographic column, while 10 components retained on osteoblast micro-cell membrane chromatographic column. The results of in vitro assay showed that berberine, obacunoic acid and phellodendrine had an obvious inhibitory effect on osteoclast differentiation and function. Berberine and tetrahydropalmatine increased the osteoblast proliferations and mineralized nodules density. This cell membrane/polyvinyl alcohol column can be applied to various biological chromatography models.

A Network Pharmacology Approach to Determine the Active Components and Potential Targets of Curculigo Orchioides in the Treatment of Osteoporosis.

BACKGROUND Osteoporosis is a complex bone disorder with a genetic predisposition, and is a cause of health problems worldwide. In China, Curculigo orchioides (CO) has been widely used as a herbal medicine in the prevention and treatment of osteoporosis. However, research on the mechanism of action of CO is still lacking. The aim of this study was to identify the absorbable components, potential targets, and associated treatment pathways of CO using a network pharmacology approach. MATERIAL AND METHODS We explored the chemical components of CO and used the five main principles of drug absorption to identify absorbable components. Targets for the therapeutic actions of CO were obtained from the PharmMapper server database. Pathway enrichment analysis was performed using the Comparative Toxicogenomics Database (CTD). Cytoscape was used to visualize the multiple components-multiple target-multiple pathways-multiple disease network for CO. RESULTS We identified 77 chemical components of CO, of which 32 components could be absorbed in the blood. These potential active components of CO regulated 83 targets and affected 58 pathways. Data analysis showed that the genes for estrogen receptor alpha (ESR1) and beta (ESR2), and the gene for 11 beta-hydroxysteroid dehydrogenase type 1, or cortisone reductase (HSD11B1) were the main targets of CO. Endocrine regulatory factors and factors regulating calcium reabsorption, steroid hormone biosynthesis, and metabolic pathways were related to these main targets and to ten corresponding compounds. CONCLUSIONS The network pharmacology approach used in our study has attempted to explain the mechanisms for the effects of CO in the prevention and treatment of osteoporosis, and provides an alternative approach to the investigation of the effects of this complex compound.