Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou, China.
Int Endod J. 2020 Sep;53(9):1204-1215. doi: 10.1111/iej.13323. Epub 2020 Jun 7.
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.
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.
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.
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.
探讨粪肠球菌诱导人成骨肉瘤 MG63 细胞发生坏死性凋亡的作用，并为难治性根尖周炎的发病机制提供新的见解。
用细胞计数试剂盒-8 法检测暴露于活粪肠球菌的 MG63 细胞活力。用 Western blot 法测定坏死性凋亡特异性标志物 p-RIPK3 和 p-MLKL 的相对表达。然后用 MLKL 和 RIPK3 的特异性抑制剂 necrosulfonamide 和 GSK'872 预处理细胞，进行乳酸脱氢酶（LDH）细胞毒性试验、流式细胞术分析和 Hoechst 33342/PI 双荧光染色。用靶向 MLKL 的慢病毒短发夹 RNA（shRNA）进一步确认感染粪肠球菌后 MG63 细胞中坏死性凋亡的激活。此外，还使用透射电子显微镜观察形态学特征。采用 Student's t 检验或单因素方差分析加 Student-Newman-Keuls 检验进行统计学分析。
粪肠球菌感染以感染复数和感染时间依赖的方式显著抑制 MG63 细胞的活力（P<0.05）。与此一致的是，感染后坏死性凋亡相关标志物 p-RIPK3 和 p-MLKL 的表达水平显著增加（P<0.05）。用 RIPK3 和 MLKL 的抑制剂预处理后，粪肠球菌感染的 MG63 细胞的死亡率显著降低（P<0.01）。用 shRNA 沉默 MLKL 后，LDH 释放明显减少（P<0.01），线粒体肿胀和空泡样变化减少，内质网扩张减少。
粪肠球菌感染通过 RIPK3/MLKL 信号通路诱导 MG63 细胞发生坏死性凋亡，这可能对难治性根尖周炎的愈合过程产生负面影响。本研究可为难治性根尖周炎的发病机制和潜在治疗靶点提供新的见解。
