WITHDRAWN: ELP2 -NLRP3-GSDMD/GSDME mediated pyroptosis induced by TNF-α during osteogenic differentiation

Abstract

Elongated protein 2 (ELP2) affects inflammatory responses through the JAK-STAT3 cascade and downstream pathways which regulates ontogenesis. Our recent experiment suggests that TNF-a induced inflammation during ontogenesis is inextricably linked to pyroptosis and is critical for inhibiting osteogenic differentiation. However, we do not know the effect of ELP2 on pyroptosis under inflammatory microenvironment. Thus, this study aims to investigate the role of ELP2 in molecular mechanism for MC3T3-E1 cell pyroptosis. Firstly, using scanning electron microscope, we observed MC3T3-E1 cells underwent obvious cell membrane rupture and inflammation after 48h of TNF-α treatment under ontogenesis induction. Then, we targeted ELP2 associated with the NLRP3 inflammasome identified by bioinformatics analysis. Besides, the effect of ELP2 on the inflammasome was determined by the detection of STAT3/NLRP3/ASC protein in ELP2 knockdown and overexpression cells. The interaction between ELP2 and NLRP3 was confirm by co-immunoprecipitation. Pyroptosis downstream was demonstrated by activation of Caspase-1 and Caspase-3, cleavage of downstream gas protein D (GSDMD) and gas protein E (GSDME/DFNA5). Clarifying the effect of ELP2 on the degree of apoptosis by Fluorescent Staining and Flow Quantitative Analysis of Cell Death Staining. Finally, the effects of ELP2 on ontogenesis was evaluated by detecting the osteogenic associated protein OPN/OCN/RUNX2/ALP expression using western blot and qPCR, also we conducted the ALP staining after the cell was stimulated with TNF-α. From the data in this study, we get ELP2-NLRP3-GSDMD/GSDME mediated pyroptosis induced by TNF-α during osteogenic differentiation. Regulation of ELP2 may be a potential target for the treatment of bone infections.