| Objective To explore the effect and mechanism of rhizoma drynariae (Rhd) on lipotoxicity-mediated osteoblast pyroptosis caused by abnormal fat accumulation in the bone marrow cavity. Methods An ovariectomy (OVX) osteoporosis mouse model was constructed in vivo. Rhd was administered with gavage. After 8 weeks of administration, mice were weighed and the femoral tissue and serum were collected. HE staining was used to observe the fat accumulation status in the medullary cavity. An adipogenic-osteoblast co-culture system was constructed in vitro. Alizarin red S (ARS) staining was used to observe the number of calcified nodules. Alkaline phosphatase (ALP) staining was used to detect the alkaline phosphatase activity level. Lactate dehydrogenase (LDH) detection and Hoechst33342/PI staining were used to detect the pyroptosis level of osteoblasts (OBs) in co-culture. Enzyme-linked immunosorbent assay (ELISA) was used to detect the release amounts of interleukin-1β (IL-1β) and interleukin-18 (IL-18) in cell culture supernatant. Western blotting analysis was employed to quantify the expression levels of osteogenic capacity-related proteins, including runt-related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), and collagen-1 (COL-1). The NLRP3 specific inhibitor MCC950 was selected as a positive control to detect the pyroptosis-related proteins NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and cysteinyl aspartate specific proteinase-1 (CASP-1), Gasdermin D (GSDMD), IL-1β, and IL-18. Results Bone loss and massive fat accumulation occurred in the medullary cavity of OVX mice. Intragastric administration of Rhd could effectively alleviate this trend. In the adipogenic-osteogenic co-culture system, the co-culture environment inhibited the activity of OBs, ALP activity, and mineralized nodules in OBs, inhibited the expressions of RUNX2, BMP2, and COL-1 proteins, induced pyroptosis, and increased LDH. Release amount of LDH and PI positive cell staining rate increased. NLRP3, CASP-1, GSDMD, IL-1β and IL-18 protein expression levels were up-regulated. This trend was reversed after the Rhd intervention. Conclusion Lipotoxicity resulting from excessive fat accumulation impedes the activity of OBs and induces pyroptosis in OBs through the activation of the NLRP3 inflammasome. Rhd may exert an anti-osteoporotic effect by suppressing OB pyroptosis and inflammatory responses through the inhibition of NLRP3 inflammasome activation. |