Nuciferine inhibits osteoclast formation through suppressing glycolysis metabolic programming and ROS production.

Abstract

Nuciferine (NCF) is a bioactive compound from lotus leaves and has been proven to prevent osteoclastogenesis and ovariectomy-induced osteoporosis by our previous research. However, the underlying mechanism is still unclear. In this research, Raw264.7 cells were induced into osteoclast with or without NCF. CCK-8 and Edu assays were performed to detect the effects of 30 μM NCF on cell viability and proliferation. TRAP staining and bone resorption assays were performed to observe the role of NCF in osteoclastogenesis and bone resorption. RT-PCR and Western blot were performed to detect the effects of NCF on osteoclast-related genes, glycolysis-related genes, and reactive oxygen species (ROS)-related genes. Seahorse assays, lactate concentration and glucose consumption were performed to observe cell metabolism change. DCFH-DA fluorescent probe was used to detect ROS level. In this work, 30 μM NCF could not influence cell viability and cell proliferation. Osteoclast differentiation could be inhibited by 30 μM NCF. Bone resorption assay could also observe that bone resorption ability was successfully inhibited by 30 μM NCF. In seahorse assay, we discovered that NCF could decrease extracellular acid rate and increase oxygen consumption. RT-PCR and Western blot results showed that NCF could decrease the expression of hexokinase2, pyruvate kinase muscle 2, and lactate dehydrogenase A and that NCF could also weaken the concentration of lactate. However, pyruvate kinase muscle 2 activator (GC69716) and lactate addition could promote osteoclastogenesis and bone resorption and promote the expression of c-Fos and nuclear factor of activated T cells c1. Besides, NCF could also inhibit the production of ROS. In conclusion, NCF might inhibit osteoclast formation through inhibiting glycolysis metabolism and ROS production.