Abstract: BACKGROUND: Periprosthetic osteolysis is the most common long-term complication of total joint arthroplasty. Many studies suggest that the inflammasome may play an important role during the osteolysis. Melatonin is a rhythm-regulated hormone secreted by the pineal gland with many functions including anti-inflammatory, anti-oxidation, and antitumor, but its effects on osteolysis and inflammasome have yet to be explored.
OBJECTIVE: To explore the effect of melatonin on the osteolysis induced by wear particles and the role of melatonin on the activation of NLRP3 inflammasome.
METHODS: (1) In vivo test: Fifteen C57BL/6 mice were randomly divided into sham operation group, osteolysis group and melatonin group by random number table method, with 5 mice in each group. The osteolysis model of the osteolysis group and the melatonin group was established by injecting cobalt-chromium-molybdenum (CoCrMo) particles into the sagittal suture of the skull. After injection, the melatonin group was intraperitoneally injected with 50 mg/(kg•d) of melatonin for 14 consecutive days. After drug intervention, the mouse calvarium was collected for micro-CT analysis to observe the micro-structural changes around the sagittal suture. (2) In vitro test: Mouse bone marrow-derived macrophages and THP-1 cells (which had been induced to differentiate into macrophages) were taken and divided into seven groups: normal group, lipopolysaccharide group, lipopolysaccharide+CoCrMo group and melatonin 0.5, 1, 1.5, 2 mmol/L groups (lipopolysaccharide and CoCrMo were added to the melatonin intervention groups). After the intervention for 6 hours, the expression of related proteins (NLRP3, Caspase-1, interleukin-1β, and gasdermin D, gasdermin D-N terminal) in the inflammasome of cell lysate or cell culture supernatant was detected by western blot assay. Cytotoxicity and cell death were observed through lactate dehydrogenase release and live-dead fluorescence staining. 
RESULTS AND CONCLUSION: (1) In vivo test: Micro-CT scanning 3D reconstruction images showed that the bone mass around the sagittal suture of the skull of mice in the osteolysis group was significantly reduced, and the bone tissue structure was severely damaged. Compared with the osteolysis group, the bone mass around the sagittal suture of the skull in the melatonin group was significantly increased, and the destruction of tissue structure was reduced. (2) In vitro test: For mouse bone marrow-derived macrophages, lipopolysaccharide significantly up-regulated NLRP3 protein expression in cell lysate, and melatonin intervention could reduce NLRP3 protein expression in a dose-dependent manner. CoCrMo particles significantly up-regulated the protein expressions of the gasdermin D-N terminal in cell lysate and Caspase-1 and interleukin-1β in the supernatant of cell culture, while melatonin intervention could reduce the expression of these proteins in a dose-dependent manner. For THP-1 cells, the protein expressions of Caspase-1 and interleukin-1β in the supernatant of cell culture were significantly up-regulated by CoCrMo particles, and the expression of these proteins was decreased dose-dependent by melatonin intervention. Lactate dehydrogenase release and live-dead fluorescence staining showed that CoCrMo particles significantly increased the release of lactate dehydrogenase and cell death in the supernatant of mouse bone marrow-derived macrophage culture, and melatonin intervention could reduce the release of lactate dehydrogenase and cell death. (3) The results show that melatonin can inhibit particle-induced inflammasome activation and pyroptosis to suppress periprosthetic osteolysis.

Key words: CoCrMo particle, osteolysis, macrophage, inflammasome, pyroptosis, melatonin