<i>Enterococcus faecalis</i> induces necroptosis in human osteoblastic MG63 cells through the RIPK3 / MLKL signalling pathway
Xingzhu Dai, Zilong Deng, Yuqing Liang, Ling Chen, Weiyi Jiang, Wanghong Zhao
Abstract
AIM: To explore the activation of necroptosis triggered by Enterococcus faecalis in human osteoblastic MG63 cells and provide new insights into the pathogenesis of refractory apical periodontitis. METHODOLOGY: The viability of MG63 cells exposed to live E. faecalis was investigated using the cell counting kit-8 assay. The relative expressions of specific markers for necroptosis, namely p-RIPK3 and p-MLKL, were determined by western blotting. Cells pretreated with necrosulfonamide and GSK'872, which are specific inhibitors for MLKL and RIPK3, respectively, were then subjected to lactate dehydrogenase (LDH) cytotoxicity assay, flow cytometry analysis and Hoechst 33342/PI double fluorescence staining. Lentiviral-delivered short hairpin RNA (shRNA) targeting MLKL was employed to further confirm the activation of necroptosis in MG63 cells infected with E. faecalis. Transmission electron microscopy was additionally used to observe the morphological characteristics. Statistical analysis was conducted using Student's t-tests or one-way ANOVA followed by the Student-Newman-Keuls test. RESULTS: The infection with E. faecalis significantly inhibited the viability of MG63 cells in a multiplicity of infection- and infection time-dependent manners (P < 0.05). In line with this, the expression levels of necroptosis-related markers, p-RIPK3 and p-MLKL, were significantly increased postinfection (P < 0.05). Significant reductions in death rate were detected in the case of E. faecalis-infected MG63 cells following pretreatment with the inhibitors of RIPK3 and MLKL (P < 0.01). Furthermore, silencing of MLKL by shRNA significantly decreased LDH release (P < 0.01) and resulted in less mitochondrial swelling and vacuole-like changes, as well as reduced endoplasmic reticulum expansion. CONCLUSIONS: Enterococcus faecalis infection-induced necroptosis of MG63 cells via the RIPK3/MLKL signalling pathway, which may exert a negative influence on the healing process of refractory apical periodontitis. This study may offer novel insights into the pathogenesis and potential therapeutic targets of refractory apical periodontitis.