Bisphenol S exposure promotes cell apoptosis and mitophagy in murine osteocytes by regulating mtROS signaling.

Bisphenol S (BPS), a safer alternative to bisphenol A, is commonly used as a plasticizer to manufacture various food-packaging materials. The accumulated BPS inhibits osteoblastic bone formation and promotes osteoclastogenesis, thereby accelerating remarkable bone destruction, but it is unclear whether BPS affects osteocytes, comprising over 95% of all bone cells. This study aimed to investigate the biological effect of BPS on osteocytes in vitro, as well as the detailed mechanism. Results showed that BPS (200, 400 µmol/L) exposure caused dose-dependently cell death of osteocytes MLO-Y4, and increased cell apoptosis. BPS induced loss of mitochondrial membrane potential (MMP) and mitochondria impairment. Furthermore, BPS upregulated expressions of mitophagy-related proteins including microtubule-associated protein light chain 3 (LC-3) II and PTEN-induced putative kinase (PINK) 1, accompanied by elevation of autophagy flux and the accumulation of acidic vacuoles; whereas p62 level was downregulated after BPS treatment. Additionally, BPS triggered the production of intracellular reactive oxygen species (ROS) and mitochondrial ROS (mtROS), while it decreased expression levels of nuclear factor E2-related factor 2 (Nrf2) and quinone oxidoreductase 1 (NQO1). The specific mtROS scavenger MitoTEMPO reversed cell apoptosis and mitophagy, suggesting that mtROS contributes to BPS exposure-induced apoptosis and mitophagy in MLO-Y4 cells. Our data first provide novel evidence that apoptosis and mitophagy as cellular mechanisms for the toxic effect of BPS on osteocytes, thereby helping our understanding of the potential role of osteocytes in the adverse effect of BPS and its analogs on bone growth, and supporting strategies targeting bone destruction caused by BPS.

Benzo[a]pyrene exposure promotes RIP1-mediated necroptotic death of osteocytes and the JNK/IL-18 pathway activation via generation of reactive oxygen species.

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon (PAH) of environmental pollutants, readily produced during the processing of petroleum and fatty foods. BaP exposure can cause skeletal deformities. However, whether BaP affects osteocytes, making up over 95% of all the bone cells, remains unknown. This study aimed to investigate the effect of BaP on osteocytes in vivo and in vitro, as well as explore the underlying mechanisms. The in vivo data showed that BaP (50 mg/kg) exposure for 12 weeks could cause bone destruction, and increase osteocytes death in mouse cortical femur. Our in vitro results revealed that BaP (25-100 µmol/L) exposure inhibited cell viability of MLO-Y4 cells, and resulted in cell death in a dose-dependent manner. Furthermore, BaP exposure significantly triggered necroptosis of MLO-Y4 cells, as indicated by increased propidium iodide (PI)-positive cells and up-regulation of necroptosis-related protein expressions of receptor-interacting protein kinase 1 (RIP1), RIP3, and mixed lineage kinase domain-like protein (MLKL). This necrotic effect was reversed by the RIP1 inhibitor necrostatin-1 (Nec-1). Simultaneously, BaP activated the downstream c-Jun N-terminal kinase (JNK)/ interleukin (IL)-18 signaling pathway, which was suppressed after the JNK inhibitor SP600125 or Nec-1 treatment. In addition, BaP exposure promoted the production of intracellular reactive oxygen species (ROS), mitochondrial ROS (mtROS), and elevated malondialdehyde (MDA) levels; while BaP decreased superoxide dismutase (SOD) activity and antioxidant enzymes including nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) levels, leading to oxidative damage. The ROS scavenger N-acetylcysteine (NAC) inhibited this necroptotic death and the JNK/IL-18 pathway activation. Collectively, BaP exposure may cause RIP1-mediated necroptotic death of osteocytes and activate the JNK/IL-18 pathway via ROS generation.

[Effects of fucoidan inducing impairment of human osteosarcoma cell 143B and its mechanism].

Objective: To investigate the effects of fucoidan inducing impairment of human osteosarcoma cell 143B, as well its mechanisms. Methods: After 143B cells were treated with different concentrations of FUC (0, 0.5, 1, 10, 100, 400, 800 µg/ml) for 48 h, the cell viability and dehydrogenase (LDH) level were detected by MTT assay and chemical colorimetry with six multiple wells for each concentration. Based on MTT results, we determined the value of IC50 was 244.5 µg/ml. The follow-up experiments were divided into control group (without FUC), FUC (10 µg/ml)-treated group, FUC (100 µg/ml)-treated group, FUC (400 µg/ml)-treated group and positive group (resveratrol, 40 µmol/L). There were four multiple wells for each concentration, and each experiment was repeated at least three times. Flow cytometry was performed to detect cell apoptosis and intracellular reactive oxygen species (ROS) level; acridine orange (AO) staining and lyso-tracker red staining were used to observe the autophagolysosome formation; chemical colorimetric analysis was performed to determine malondialdehyde (MDA) content and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px); Western blot was used to detect protein expressions of nuclear factor E2-associated factor 2 (Nrf2), heme oxygenase 1 (HO-1) and autophagy-associated proteins including microtubule-associated light chain protein 3 (LC-3), Atg7, Beclin-1 and p62. Results: Compared with control group, the cell viability was decreased significantly in FUC (100～400 µg/ml)-treated groups (P＜0.01); LDH levels in the supernatant (P＜0.05 or P＜0.01), the percentage of cell apoptosis (P＜0.01), intracellular ROS level and MDA content (P＜0.01) were increased remarkably; protein expressions of Atg7 and Beclin-1 were upregulated (P＜0.05 or P＜0.01); the conversion from LC-3I to LC-3II was significant (P＜0.01) together with elevation of autophagolysosome formation (P＜0.05 or P＜0.01); while the activities of SOD and GSH-Px and protein expressions of Nrf2, HO-1 and p62 were decreased remarkably (P＜0.05 or P＜0.01). Conclusion: FUC (100～400 µg/ml) treatment induces oxidative damage and autophagic death in osteosarcoma 143B cells.

Bisphenol A induces pyroptotic cell death via ROS/NLRP3/Caspase-1 pathway in osteocytes MLO-Y4.

Bisphenol A (BPA), a ubiquitous endocrine-disrupting chemical, is commonly used as a plasticizer to manufacture various food packaging materials. Evidence has demonstrated that BPA disturbed bone health. However, few studies focused on the effect of BPA on osteocytes, making up over 95% of all the bone cells. Here, we reported that BPA inhibited the cell viability of MLO-Y4 cells, and increased apoptosis in a dose-dependent manner. Furthermore, BPA up-regulated protein expressions of speck-like protein containing CARD (ASC), NLRP3, cleaved caspase-1 (Casp-1 p20) and cleaved gasdermin D (GSDMD-N), and increased the ratios of interleukin (IL)-1ß/pro-IL-1ß and IL-18/pro-IL-18 in MLO-Y4 cells. BPA enhanced levels of lactate dehydrogenase (LDH), IL-1ß and IL-18 in culture supernatants. This pyroptotic death and the NLPR3 inflammasome activation were reversed by the caspase-1 inhibitor VX765 or the NLRP3 inflammasome inhibitor MCC950. Furthermore, BPA stimulated the production of intracellular reactive oxygen species (ROS), mitochondrial ROS (mtROS), elevated malondialdehyde (MDA) level and decreased superoxide dismutase (SOD) activity, which led to oxidative damage in MLO-Y4 cells. The ROS scavenger N-acetylcysteine (NAC) or the mitochondrial antioxidant Mito-TEMPO inhibited the NLPR3 inflammasome activation and pyroptotic death induced by BPA. Collectively, our data suggest that BPA causes pyroptotic death of osteocytes via ROS/NLRP3/Caspase-1 pathway.