Abstract
BACKGROUND: Bone mass loss resulting from mechanical unloading in a microgravity environment constitutes a primary impediment to the advancement of space exploration for astronauts. However, the underlying mechanism remains unclear. In this study, we primarily investigated the impact of pyroptosis on osteoblasts under simulated microgravity and its influence on osteoblast functionality. METHODS: A rotary cell culture system was employed to establish a simulated microgravity environment. The proliferation of osteoblasts was assessed by cell counting kit-8 (CCK-8) assay. Lactate dehydrogenase (LDH) Release Assay Kit was used to measure cell necrosis. Osteoblast differentiation and mineralization were evaluated using an ALP kit and alizarin red staining. Fluorescence Hoechst/PI double staining and scanning electron microscopy (SEM) were used to detect pyroptosis, and a caspase-1 kit measured caspase-1 activity. The expression of NLRP3, caspase-1, GSDMD, IL-1β, IL-18, OCN, and COL-I was analyzed by qPCR and Western blot. Additionally, ELISA was used to quantify the release of IL-1β and IL-18. RESULTS: The PI fluorescence in osteoblasts exhibited significant enhancement under simulated microgravity conditions, accompanied by increased membrane pore formation, decreased cell proliferation, and elevated LDH release. Moreover, the expression levels of NLRP3, caspase-1, GSDMD, IL-1β, and IL-18 were upregulated while caspase-1 activity was increased. Treatment with MCC950 and VX-765 effectively attenuated pyroptosis levels as well as caspase-1 activity while reducing the expression of NLRP3, GSDMD, IL-1β, and IL-18. Notably, this treatment significantly enhanced the expression of OCN and COL-I. CONCLUSION: Under simulated microgravity conditions, pyroptosis occurs in osteoblasts and alters their osteogenic differentiation function. Pyroptosis modulates the functionality of osteoblasts and contributes to the mechanical response process, potentially serving as one of the mechanisms underlying mechanical-regulated osteoblast function in a microgravity environment. This finding may offer a novel approach for addressing bone tissue damage and repair under extreme mechanical conditions. CLINICAL TRIAL NUMBER: Not applicable.