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.

Transcriptomic and Metabolomic Profiling Reveals the Protective Effect of Acanthopanax senticosus (Rupr. & Maxim.) Harms Combined With Gastrodia elata Blume on Cerebral Ischemia-Reperfusion Injury.

The effects of current treatment strategies used in ischemic stroke are weakened by cerebral ischemia-reperfusion (CIR) injury. Suitable treatment regimens targeting CIR injury are still lacking. Two herbs, namely, Acanthopanax senticosus (Rupr. & Maxim.) Harms (ASE) and Gastrodia elata Blume (GEB), have been used as traditional Chinese medicine and are indicated in the treatment of stroke and cerebrovascular diseases. However, there are no studies that report the effects of ASE combined with GEB in the treatment of CIR injury. In this study, we used the Zea Longa method to induce CIR injury in male Wistar rats. Results of the pharmacodynamic studies revealed that co-administration of ASE and GEB may improve neuronal injury and prevent neuronal apoptosis by reducing oxidative stress and inflammation, and also help prevent CIR injury. On the basis of our hypothesis, we combined the results from transcriptomic and metabonomic analyses and found that ASE and GEB could prevent CIR injury by targeting phenylalanine, pyrimidine, methionine, and sphingolipid metabolism. Therefore, our study provides the basis for the compatibility and efficacy of ASE and GEB.

Cichoric Acid Ameliorates Monosodium Urate-Induced Inflammatory Response by Reducing NLRP3 Inflammasome Activation via Inhibition of NF-kB Signaling Pathway.

Gouty arthritis is characterized by the deposition of monosodium urate (MSU) within synovial joints and tissues due to increased urate concentrations. Here, we elucidated the role of the natural compound cichoric acid (CA) on the MSU crystal-stimulated inflammatory response. The THP-1-derived macrophages (THP-Ms) were pretreated with CA and then stimulated with MSU suspensions. The protein levels of p65 and IκBα, the activation of the NF-κB signaling pathway by measuring the expression of its downstream inflammatory cytokines, and the activity of NLRP3 inflammasome were measured by western blotting and ELISA. CA treatment markedly inhibited the degradation of IκBα and the activation of NF-κB signaling pathway and reduced the levels of its downstream inflammatory genes such as IL-1ß, TNF-α, COX-2, and PGE2 in the MSU-stimulated THP-M cells. Therefore, we infer that CA effectively alleviated MSU-induced inflammation by suppressing the degradation of IκBα, thereby reducing the activation of the NF-κB signaling pathway and the NLRP3 inflammasome. These results suggest that CA could be a novel therapeutic strategy in averting acute episodes of gout.