Identification of the pyroptosis, apoptosis, and necroptosis (PANoptosis) involved in osteogenic differentiation inhibition impaired by tumor necrosis factor-α

BackgroundInflammatory diseases of the bones often result in excessive release of inflammatory cytokines and impaired osteogenic differentiation. Tumor necrosis factor-alpha (TNF-alpha) is an important inflammatory cytokine in disease, and implicated in developing PANoptosis. Although TNF-alpha has captured much attention in PANoptosis, their relationship in osteogenic differentiation remains elusive. Therefore, it is necessary to explore how TNF-alpha inhibits osteogenic differentiation and the mechanism involved.MethodsRNA sequencing was used to sequence normal and TNF-alpha-treated cells and screen for differentially expressed 43 genes of PANotosis. PCR and Western blotting were performed to validate sequencing results consistent. The degree of osteoblast differentiation was determined by alkaline phosphatase and calcium ion quantification. The extent of cell death was evaluated by cell death staining, lactate dehydrogenase quantification, and propidium iodide staining.ResultsCalcium ion quantification, RUNX2 and ALP confirmed that osteogenic differentiation was inhibited, and related to the cell death increase. More importantly, scanning electron microscopy revealed that multiple PANotosis morphologies appeared in the same field of view. We also demonstrated that by interdict NLRP3, cells could be rescued from PANoptosis and the inhibition of osteogenic differentiation caused by TNF-alpha. Further, immunofluorescence and coimmunoprecipitation of several closely related proteins were performed to verify the possible presence of PANoptosome.ConclusionIn summary, inhibition PANoptosis may serve as a new therapeutic target for bone infection and may plays an important role in osteogenic differentiation and inflammatory cell death in bone infection.