Eldecalcitol Inhibits LPS-Induced NLRP3 Inflammasome-Dependent Pyroptosis in Human Gingival Fibroblasts by Activating the Nrf2/HO-1 Signaling Pathway

Purpose: Periodontitis is a major chronic oral disease that is accelerated by activation of the NLRP3 inflammasome and the resulting Pyroptosis. According to recent studies, active vitamin D and its analogs have been reported to have great anti-inflammatory effects. However, the anti-inflammatory mechanism of a newly found vitamin D analog, eldecalcitol (ED-71), is still unclear. This study investigates whether ED-71 could protect human gingival fibroblasts (HGFs) from LPS-induced Pyroptosis and, if so, determine its underlying mechanism.

Methods: After HGFs were treated with LPS alone or with LPS and ED-71, their viability was measured by CCK8 assay. The degrees of inflammation and Pyroptosis were measured via LDH assay, H₂O₂ assay, fluorescent staining, flow cytometry, and Western blots. Intracellular ROS, Hoechst 33,342, and PI stains were assessed with a fluorescence microscope. ROS inhibitor NAC, NLRP3 Inhibitor MCC950, and Nrf2 inhibitor ML385 were added to further clarify the mechanism.

Results: LPS induced cytotoxicity in HGFs, as shown by CCK8 assay. LPS also increased intracellular ROS, H₂O₂ levels, release of LDH, and expression of the pyroptosis-related proteins NLRP3, Caspase-1, and IL-1β. NAC and MCC950 reduced LPS-induced NLRP3, Caspase-1, and IL-1β. Pretreatment with ED-71 effectively inhibited the LPS-induced Pyroptosis and was associated with activation of the Nrf2/HO-1 signaling pathway. This beneficial effect of ED-71 was suppressed by ML385.

Conclusion: This study demonstrates the therapeutic effect of ED-71 on LPS-induced NLRP3 inflammasome-dependent Pyroptosis in HGFs and further reveals that ED-71 can inhibit Pyroptosis by activating the Nrf2/HO-1 pathway. Our results thus suggest that ED-71 is a potential candidate for the treatment of periodontitis.