Effects of the ferroptosis inducer erastin on osteogenic differentiation and biological pathways of primary osteoblasts.

BACKGROUND: Periodontitis is a chronic destructive inflammatory disease exacerbated by osteoblast dysfunction. Ferroptosis has emerged as a significant factor that could contribute to the pathological changes observed in periodontitis. However, the impact of ferroptosis on osteogenic differentiation and gene expression patterns of primary osteoblasts remain elusive. METHODS: In this study, osteoblasts were osteogenically induced for specific durations with and without the ferroptosis inducer erastin. Subsequently, cell proliferation, ferroptosis-related molecules, and osteogenic differentiation capacity were assessed. Furthermore, the differences in transcriptome expression following erastin treatment were analyzed by RNA sequencing. RESULTS: The results demonstrated that erastin treatment induced ferroptosis, resulting in suppressed cell proliferation and impaired osteogenic differentiation. Transcriptomic analysis revealed significant alterations in processes such as hydrogen peroxide catabolism, response to lipid peroxidation, and metal iron binding, as well as BMP receptor activity and collagen type XI trimer. CONCLUSION: The ferroptosis inducer erastin inhibited osteoblast proliferation and differentiation. Our study provides novel insights into the effect of ferroptosis on osteogenesis, suggesting that targeting ferroptosis may present a promising approach in the treatment of periodontitis.

Interleukin-17 alleviates erastin-induced alveolar bone loss by suppressing ferroptosis via interaction between NRF2 and p-STAT3.

AIM: To investigate the relationship between interleukin-17 (IL-17), ferroptosis and osteogenic differentiation. MATERIALS AND METHODS: We first analysed the changes in ferroptosis-related molecules in experimental periodontitis models. The effects of erastin, a small-molecule ferroptosis inducer, and IL-17 on alveolar bone loss and repair in animal models were then investigated. Primary mouse mandibular osteoblasts were exposed to erastin and IL-17 in vitro. Ferroptosis- and osteogenesis-related genes and proteins were detected. Further, siRNA, immunofluorescence co-localization and immunoprecipitation were used to confirm the roles of the nuclear factor erythroid-2-related factor 2 (NRF2) and phosphorylated signal transducer and activator of transcription 3 (p-STAT3), as well as their interaction. RESULTS: The levels of NRF2, glutathione peroxidase 4 and solute carrier family 7 member 11 were lower in the ligated tissues than in normal periodontal tissues. Alveolar bone loss in an in vivo experimental periodontitis model was aggravated by erastin and alleviated by IL-17. In vitro, IL-17 ameliorated erastin-inhibited osteogenic differentiation by reversing ferroptosis. Altered NRF2 expression correlated with changes in ferroptosis-related molecules and osteogenesis. Furthermore, the physical interaction between NRF2 and p-STAT3 was confirmed in the nucleus. In IL-17 + erastin-stimulated osteoblasts, the p-STAT3-NRF2 complex might actively participate in the downstream transcription of ferroptosis- and osteogenesis-related genes. CONCLUSIONS: IL-17 administration conferred resistance to erastin-induced osteoblast ferroptosis and osteogenesis. The possible mechanism may involve p-STAT3 directly interacting with NRF2.

Ferritin was involved in interleukin-17A enhanced osteogenesis through autophagy activation.

Periodontitis is a prevalent inflammatory immune disease that involves tissue inflammation and excessive bone loss. In murine periodontitis models and periodontitis patients, upregulated interleukin-17A (IL-17A) expression was observed, and its level seemed to correlate with the disease severity. In this study, we intended to investigate the specific role of ferritin, a critical iron storage protein, in IL-17A enhanced osteogenic differentiation as well as the underlying mechanism. Under osteogenic induction, IL-17A stimulation promoted differentiation and mineralization of murine calvarial osteoblasts. In addition, increased iron accumulation and ferritin expression were detected in osteoblasts treated with IL-17A, indicating an alteration in iron metabolism during osteogenesis. Administration of iron chelator deferoxamine (DFO) and transfection with small interfering RNA (siRNA) targeting ferritin heavy chain (FTH) further revealed that ferritin suppression consequently inhibited osteoblast differentiation. Autophagy activation was also found upon IL-17A stimulation, which played a positive role in osteogenic differentiation and was subsequently suppressed by DFO or siRNA targeting FTH. In conclusion, IL-17A induced ferritin expression in osteoblasts, which further enhanced osteogenic differentiation via autophagy activation. These findings may provide further insight into the role of IL-17A in osteoblast differentiation and demonstrate ferritin as a potential target in modulating alveolar bone homeostasis.

Determination of ng rivanol in human plasma by SPE-HPLC method.

A high-performance liquid chromatography assay is described for the determination of rivanol in human plasma. Solid-phase extraction cartridges are used to extract plasma samples. Separation is done by using a C18 column. The mobile phase is a mixture of methanol-0.05% sodium dodecylsulfonate (70:30, v/v, pH 3), with the flow rate at 1.0 mL/min. UV detection of rivanol is at 272 nm. The calibration curve is linear in the concentration range of 1x10(-8) mol/L to 1x10(-5) mol/L with linear correlation coefficient r equal to 0.9998. The limit of detection for the assay is 3x10(-9) mol/L, corresponding to 1.1 ng/mL. Precision, expressed as the within- and between-day coefficient of variation, is 3.3-8.1% and 4.1-9.5%, respectively, at plasma control samples of 5x10(-8), 5x10(-7), and 5x10(-6) mol/L. And the recovery ranges from 94.8% to 107.2%. The selectivity of the method is confirmed. Plasma samples are stable for at least 15 days if they are stored lightproof at -20 degrees C. This method is simple, sensitive, and accurate, and it allows for the determination ng rivanol in human plasma. It could be applied to assessing its plasma level in women receiving an intra-amniotic injection of rivanol.

An HPLC method for the determination of ethacridine lactate in human urine.

An HPLC method was developed and validated for the determination of ethacridine lactate in human urine. Solid-phase extraction cartridges were used to extract urine samples. Separation was carried out on a C(18) column maintained at 30 degrees C with methanol-0.05% sodium dodecylsulfonate (70:30, v/v, pH 3) as mobile phase at a flow rate of 1.0 mL/min. Detection was at UV 272 nm. The calibration curve was linear in the concentration range of 4-4000 ng/mL, with linear correlation coefficient r equal to 0.9998. The limit of detection for the assay was 1.1 ng/mL. The within-day accuracy ranged from 94.8 to 101.6% and precision from 2.3 to 5.4%. The between-day accuracy ranged from 96.8 to 102.6% and precision from 4.0 to 5.3%. The absolute recovery was 95.4-101.2%. Urine samples were stable for at least 15 days if stored in the dark at -20 degrees C. This simple and accurate method allows the sensitive determination of ethacridine lactate in human urine. It was successfully applied to assess the urine level of ethacridine lactate in women received intra-amniotic injection.