Baicalein Alleviates Iron Overload-Induced Ferroptosis and Osteogenic Blockade in Osteoblasts by Activating the Nrf2/GPX4 Pathway

Silymarin prevents iron overload induced bone loss by inhibiting oxidative stress in an ovariectomized animal model

Dysregulated iron metabolism-induced ferroptosis is considered a key pathological mechanism in the development of osteoporosis (OP). G protein-coupled receptor 30 (GPR30, also known as Gper1) is an estrogen-binding receptor that has shown therapeutic benefits in patients with certain degenerative diseases. Moreover, several studies have demonstrated the anti-ferroptotic effects of estrogen receptor activation. However, its role in the prevention and treatment of OP remains unclear, and there are currently no reports on the anti-ferroptotic function of GPR30 in OP. Therefore, this study aimed to investigate the ferroptosis-related effects and mechanisms of GPR30 in the context of OP. In vivo and in vitro experiments were conducted using wild-type (WT) C57BL/6 female mice and GPR30-knockout (GPR30-KO) C57BL/6J female mice. The microarchitecture of the distal femur was assessed using micro-computed tomography (micro-CT), and histomorphological changes were analyzed via hematoxylin and eosin (H&E) staining. Bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured to establish an iron overload model using ferric ammonium citrate (FAC). Interventions included GPR30 overexpression via transfection and BMP-6 inhibition using LDN-214117. Cell viability was evaluated with the CCK-8 assay, while osteogenic differentiation and mineralization levels were assessed using ALP and Alizarin Red S (ARS) staining. Iron accumulation was detected via Prussian blue staining, oxidative stress levels were evaluated using ROS staining, and mitochondrial membrane potential changes were analyzed using JC-1 staining. Transmission electron microscopy (TEM) was employed to observe mitochondrial ultrastructural changes. Additionally, key gene and protein expression levels were measured using immunofluorescence and Western blotting. The micro-CT analysis revealed significant bone microarchitecture deterioration and bone loss in the GPR30-KO mouse model. At the cellular level, GPR30 overexpression markedly reduced iron accumulation and oxidative stress in BMSCs, restored the mitochondrial membrane potential, and improved the mitochondrial ultrastructure. Furthermore, GPR30 enhanced osteogenic differentiation in BMSCs by promoting the activation of the BMP-6/HEP/FPN signaling pathway, leading to increased expression of osteogenic markers. The protective effects of GPR30 were reversed by the BMP-6 inhibitor LDN-214117, indicating that BMP-6 is a critical mediator in GPR30-regulated iron metabolism and ferroptosis inhibition. GPR30 inhibits ferroptosis in BMSCs and enhances osteogenic differentiation by activating the BMP-6/HEP/FPN signaling pathway. This provides new insights and potential therapeutic targets for the treatment of osteoporosis OP.

Mesenchymal stem cells (MSCs) can differentiate into multiple cell lineages, including osteoblasts and adipocytes. We reported previously that glucocorticoid-induced leucine zipper (GILZ) inhibits peroxisome proliferator-activated receptor gamma-2 (Ppargamma2) expression and blocks adipocyte differentiation. Here we show that overexpression of GILZ in mouse MSCs, but not MC3T3-E1 osteoblasts, increases alkaline phosphatase activity and enhances mineralized bone nodule formation, whereas knockdown of Gilz reduces MSC osteogenic differentiation capacity. Consistent with these observations, real-time reverse transcription-PCR analysis showed that both basal and differentiation-induced transcripts of the lineage commitment gene Runx2/Cbfa1, as well as osteoblast differentiation marker genes including alkaline phosphatase, type I collagen, and osteocalcin, were all increased in GILZ-expressing cells. In contrast, the mRNA levels of adipogenic Ppargamma2 and C/ebpalpha were significantly reduced in GILZ-expressing cells under both osteogenic and adipogenic conditions. Together, our results demonstrate that GILZ functions as a modulator of MSCs and that overexpression of GILZ shifts the balance between osteogenic and adipogenic differentiation of MSCs toward the osteogenic pathway. These data suggest that GILZ may have therapeutic value for stem cell-based therapies of metabolic bone diseases, such as fracture repair.

Inhibition of cytochrome P450 2E1 and activation of transcription factor Nrf2 are renoprotective in myoglobinuric acute kidney injury

Nrf2 alleviates acute ischemic stroke induced ferroptosis via regulating xCT/GPX4 pathway.

Iron overload induced osteocytes apoptosis and led to bone loss in Hepcidin−/− mice through increasing sclerostin and RANKL/OPG

Summary The present investigation found that curculigoside (CUR) can prevent excess-iron-induced bone loss in mice and cells through antioxidation and inhibiting excess-iron-induced phosphorylation of the Akt-FoxO1 pathway. CUR can attenuate the decreasing of cell viability, enhance autophagy, potentiate the antioxidant effect, and reduce apoptosis in MC3T3-E1 cells treated with excess iron through regulating the expression of FoxO1 target gene. Introduction Oxidative stress induced by iron overload is an important factor involved in primary osteoporosis disease and iron overload-related diseases. Curculigoside (CUR), a phenolic glycoside found abundantly in Curculigo orchioides Gaertn., has been demonstrated to possess antioxidant and antiosteoporotic properties. The aim of the present study is to explore the underlying molecular mechanism of CUR on excess-iron-induced bone loss in mice and osteoblastic MC3T3-E1 cells. Methods An iron-overload mice model was used to study the protective effects of CUR on bone loss induced by oxidative stress. Serum bone metabolism markers and antioxidant enzymes were also measured. To explore the antioxidant mechanism of CUR, the MC3T3-E1 osteoblastic cell line was used. Results In vivo studies showed that BMD and microarchitectural parameters were improved after a 3-month administration of CUR. CUR improved the biochemical parameters related to bone metabolism and the expressions of Runx2, OCN, and type 1 collagen and increased the formation of bone-mineralized nodules in vitro. CUR also inhibited ROS generation and increased the activities of antioxidant enzymes both in vivo and in vitro treated with excess iron. CUR can upregulate the level of FoxO1 and Nrf2, downregulate the level of p53 and the phosphorylation level of FoxO1, improve nuclear translocation of FoxO1, probably by inhibiting the IGFR/AKT signaling pathway, then increased cell viability and autophagy, and reduced apoptosis of MC3T3-E1 cells treated with excess iron by regulating the expression of FoxO1 target genes MnSOD, Gadd45a, Bim, FasL, and Rab7. Conclusions These results demonstrated that CUR was able to alleviate bone loss induced by oxidative stress resulting from iron overload, suggesting its potential use for the treatment of primary osteoporosis and bone loss in iron-overload-related diseases.

Exogenous iron caused osteocyte apoptosis, increased RANKL production, and stimulated bone resorption through oxidative stress in a murine model

Iron is an essential element that participates in several metabolic activities of cells; however, excess iron is a major cause of iron-induced oxidative stress and several human diseases. Natural flavonoids, as rutin, are well-known antioxidants and could be efficient protective agents. Therefore, the present study was undertaken to evaluate the protective influence of rutin supplementation to improve rat antioxidant systems against IOL-induced hepatic oxidative stress. Sixty male albino rats were randomly divided to three equal groups. The first group, the control, the second group, iron overload group, the third group was used as iron overload+rutin group. Rats received six doses of ferric hydroxide polymaltose (100 mg kg(-1) b.wt.) as one dose every two days, by intraperitoneal injections (IP) and administrated rutin (50 mg kg(-1) b.wt.) as one daily oral dose until the sacrificed day. Blood samples for serum separation and liver tissue specimens were collected three times, after three, four and five weeks from the onset of the experiment. Serum iron profiles total iron, Total Iron Binding Capacity (TIBC), Unsaturated Iron Binding Capacity (UIBC), transferrin (Tf) and Transferrin Saturation% (TS%)}, ferritin, albumin, total Protein, total cholesterol, triacylglycerols levels and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were determined. Moreover, total iron in the liver, L-malondialdehyde (L-MDA), glutathione (GSH), Nitric Oxide (NO) and Total Nucleic Acid (TNA) levels and glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) activities were also determined. The obtained results revealed that, iron overload (IOL) resulted in significant increase in serum iron, TIBC, Tf, TS% and ferritin levels and AST and ALT activities and also increased liver iron, L-MDA and NO levels. Meanwhile, it decreased serum UIBC, total cholesterol, triacylglycerols, albumin, total protein and liver GSH, TNA levels and Gpx, CAT and SOD activities when compared with the control group. Rutin administration to iron-overloaded rats resulted in significant decrease in serum total iron, TIBC, Tf, TS%, ferritin levels and AST and ALT activities and liver total iron, L-MDA and NO levels with significant increases in serum UIBC, albumin, total protein and total cholesterol levels and in liver GSH, CAT and SOD activities compared with the IOL group. This study provides in vivo evidence that rutin administration can improve the antioxidant defense systems against IOL-induced hepatic oxidative stress in rats. This protective effect in liver of iron-loaded rats may be due to both antioxidant and metal chelation activities.

Differentiation imbalance of bone marrow mesenchymal stem cells (BMSCs) is a key mechanism of osteoporosis (OP). This study sought to elucidate how kinsenoside (Kin) regulates ferroptosis and enhances BMSCs' osteogenic differentiation. Network pharmacology, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, as well as molecular docking were employed for the identification and analysis of the intersection targets of Kin and OP. Differential gene expression analysis was conducted to validate the identified core targets. The culture of BMSCs was carried out in high glucose (HG) cell culture medium containing Kin, the ferroptosis agonist erastin, or the AMP-activated protein kinase (AMPK) inhibitor dorsomorphin (DM). The viability of cells was evaluated using the cell counting kit-8 assay. BMSCs' osteogenic differentiation was assessed through alkaline phosphatase and alizarin red S staining. Additionally, the expression of OP-related proteins, ferroptosis-related proteins, p-AMPK, AMPK, and sirtuin 1 (SIRT1) were detected via Western blot. The ferroptosis of BMSCs was evaluated using 2,7-dichlorofluorescein diacetate staining, along with quantification of malondialdehyde, glutathione, and ferroptosis-related proteins. HG suppressed BMSCs' osteogenic differentiation and enhanced ferroptosis, while Kin promoted BMSCs' osteogenic differentiation and inhibited ferroptosis. Kin and erastin co-treatment reduced osteogenic differentiation and enhanced ferroptosis of BMSCs compared to Kin treatment alone. Network pharmacology, molecular docking, differential gene expression, and KEGG enrichment analyses, suggested AMPK/SIRT1 as a potential key pathway underlying Kin's therapeutic effect on OP. Kin activated AMPK/SIRT1 signaling pathway, while co-treatment with DM reversed this activation, accompanied by decreased osteogenic differentiation and increased ferroptosis in BMSCs. Kin enhances BMSCs' osteogenic differentiation under HG conditions by suppressing ferroptosis via activation of the AMPK/SIRT1 signaling pathway.

Objective To observe the effect of fibroblast growth factor receptor 2 (FGFR2) on the ability of bone marrow mesenchymal stem cells (BMSCs) to differentiate into osteoblasts. Methods The FGFR2 gene of mouse BMSCs was overexpressed, and the proliferation ability at 24, 48 and 72 h was measured by methyl thiazol tetrazolium (MTT) assay. After the FGFR2 gene was overexpressed, the osteogenic differentiation and mineralization ability of the cells was examined by alizarin red staining and alkaline phosphatase (ALP) staining, and Western blotting was used to detect the expression of osteocalcin (OCN), osteopontin (OPN), runt related transcription factor-2 (Runx2), ALP and bone morphogenetic protein-2 (BMP-2) protein related genes of osteogenic differentiation after 14 days of osteogenesis induced differentiation. A mouse model of castrated osteoporosis was established, and the effect of up-regulation of FGFR2 on osteoporosis in mice was studied. The number of bone trabeculae and bone volume fraction were evaluated by mircoCT. Results The proliferation of BMSCs at 24, 48 and 72 h was promoted after up-regulation of FGFR2. The number of calcium nodules in BMSCs (95.7±8.9) in the FGFR2-up-regulated group was significantly greater than that in the control group (26.4±3.9). The results of ALP staining showed that the osteogenic differentiation ability in the FGFR2-up-regulated group was stronger than that in the control group. The quantitative analysis of ALP activity showed that the ALP activity in the FGFR2-up-regulated group [(6.33±1.33) U/L]was significantly stronger than that in the control group [(2.54±0.75) U/L]. The expression levels of OCN, OPN, UNX2, ALP and BMP-2 proteins in the FGFR2-up-regulated group were higher than those in the control group. The trabecular number (Tb.N) of bone trabecula in the FGFR2 up-regulated group [(2.37±0.26)/mm]was greater than that in the control group [(1.54±0.34)/mm], and the tibial bone volume fraction (BV/TV) in the FGFR2-up-regulated group [(9.97±1.77)%] was higher than that in the control group [(5.91±0.98)%]. Conclusion Upregulation of FGFR2 can promote the proliferation and osteogenic differentiation of mouse BMSCs, which may promote the recovery of osteoporosis. Key words: Fibroblast growth factor receptor 2; Bone marrow mesenchymal stem cells; Osteogenic differentiation; Osteoporosis

Protocatechualdehyde inhibits iron overload-induced bone loss by inhibiting inflammation and oxidative stress in senile rats

Protective effect of curcumin on hepatolenticular degeneration through copper excretion and inhibition of ferroptosis

Iron overload disrupts alveolar-capillary tight junctions via oxidative stress in a murine model

Excessive dietary iron exposure increases the susceptibility of largemouth bass (Micropterus salmoides) to Aeromonas hydrophila by interfering with immune response, oxidative stress, and intestinal homeostasis