Necroptosis occurs in osteoblasts during tumor necrosis factor-α stimulation and caspase-8 inhibition

Necroptosis is a regulated cell death mechanism. However, it is unknown whether necroptosis is involved in the death of tumor necrosis factor-α (TNF-α)-treated osteoblasts. Therefore, we conducted the study with TNF-α, Nec-1 (a specific inhibitor of necroptosis), and Z-IETD-FMK (a specific inhibitor of apoptosis) to determine whether necroptosis plays a role in the death of TNF-α-treated osteoblast cell line MC3T3-E1. Cell viability, cell death, and lactate dehydrogenase (LDH) release were assayed to evaluate cytotoxicity. Specific marker proteins receptor interacting protein kinase (RIPK3) and phosphorylated mixed lineage kinase domain-like protein (p-MLKL) for necroptosis, and cleaved caspase 3 for apoptosis were detected by western blot, and mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). We found that TNF-α inhibited cell proliferation in a dose- and time-dependent manner. Nec-1 plus Z-IETD-FMK restored cell viability and significantly decreased LDH release. In addition, TNF-α alone increased the cell population of AV+PI−, while Z-IETD-FMK caused a shift in the cell population from AV+PI− to AV+PI+. Furthermore, TNF-α significantly increased protein cleaved caspase 3. TNF-α plus Z-IETD-FMK significantly increased the proteins RIPK3 and MLKL phosphorylation in MC3T3-E1 cells, while the changes in mRNA levels of RIPK3, MLKL, and caspase 3 were not consistent with the changes in the corresponding protein expression levels. In conclusion, TNF-α induced preferentially apoptosis in osteoblast cell line and necroptosis played a decisive role when TNF-α-induced death was inhibited by the inhibitor of apoptosis. Combined treatment with Nec-1 and Z-IETD-FMK protected mouse osteoblasts from death induced by TNF-α.

Influence of nickel chloride on bone marrow of iron-deficient rats

Alteraçöes no peso corporal, hamoglobina, proteina sérica, ferro, fóforo e em atividades de enzizmas no soro e medula óssea foram investigadas em ratos consumindo uma dieta carente em ferro, em presença e ausencia de cloreto de niqueo (NiCl2). O grau de deficiencia em ferro, no presente trabalho foi suficiente para induzir anemia moderada, sem produzir alteraçöes nas atividade da desidrogenase-láctica total e suas isoenzimas no cérebro de ratos. Anemia moderada ocorreu somente em ratos deficientes em ferro em ausencia de cloreto de níquel. Moderada anemia ferropriva induziu elevaçäo nas actividades da desidrogenase-láctica na medula óssea, provavelmente devido a diminuiçäo na produçäo de energia através de mecanismos oxidativos. Cloreto de níquel, aparentemente por sua capacidade de alterara absorçäo de ferro e pala manutençäo do metabolismo da medula ósseas, inibiu as alteraçöes na biosíntese de hemoglobina e nas atividades da desidrogenase-láctica da medula óssea de ratos deficientes em ferro