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.

中文翻译：

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粪肠球菌通过RIPK3 / MLKL信号通路诱导人成骨MG63细胞坏死。

目的探讨人粪成球菌MG63细胞中粪肠球菌触发的坏死性激活，并为难治性根尖周炎的发病机理提供新的见解。方法学使用细胞计数试剂盒8分析了暴露于活粪肠球菌的MG63细胞的生存能力。通过western blotting确定坏死性特异性标志物p-RIPK3和p-MLKL的相对表达。然后分别用坏死磺酰胺和GSK'872（分别是MLKL和RIPK3的特异性抑制剂）预处理的细胞进行乳酸脱氢酶（LDH）细胞毒性测定，流式细胞术分析和Hoechst 33342 / PI双重荧光染色。靶向MLKL的慢病毒传递的短发夹RNA（shRNA）被用于进一步证实感染屎肠球菌的MG63细胞中的坏死病激活。另外使用透射电子显微镜观察形态特征。使用Student's t检验或单向ANOVA进行统计分析，然后进行Student-Newman-Keuls检验。结果粪肠球菌感染以多种感染和感染时间依赖性方式显着抑制MG63细胞的活力（P <0.05）。与此相符，感染后坏死病相关标志物p-RIPK3和p-MLKL的表达水平显着增加（P <0.05）。在E病例中，死亡率显着降低。用RIPK3和MLKL的抑制剂预处理后，被粪便感染的MG63细胞感染（P <0.01）。此外，shRNA使MLKL沉默可显着降低LDH释放（P <0.01），并减少线粒体肿胀和液泡样变化，并减少内质网扩张。结论粪肠球菌感染通过RIPK3 / MLKL信号通路引起的MG63细胞坏死性坏死，可能对难治性根尖周炎的愈合过程产生负面影响。这项研究可能为难治性根尖周炎的发病机理和潜在的治疗靶点提供新的见解。以及减少内质网扩张。结论粪肠球菌感染通过RIPK3 / MLKL信号通路引起的MG63细胞坏死性坏死，可能对难治性根尖周炎的愈合过程产生负面影响。这项研究可能为难治性根尖周炎的发病机理和潜在的治疗靶点提供新颖的见解。以及减少内质网扩张。结论粪肠球菌感染通过RIPK3 / MLKL信号通路引起MG63细胞坏死性坏死，可能对难治性根尖周炎的愈合过程产生负面影响。这项研究可能为难治性根尖周炎的发病机理和潜在的治疗靶点提供新颖的见解。