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Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis
Published on: August 7, 2018
Enterococcus faecalis induces necroptosis in human osteoblastic MG63 cells through the RIPK3 / MLKL signalling
1Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou, China.
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.
Insights
Enterococcus faecalis infection triggers necroptosis in human osteoblastic cells via the RIPK3/MLKL pathway. This finding offers new insights into refractory apical periodontitis and potential therapeutic targets.
Area of Science:
- Cell Biology
- Immunology
- Oral Pathology
Background:
- Refractory apical periodontitis is a complex dental infection.
- Enterococcus faecalis is a common pathogen implicated in persistent root canal infections.
- The cellular mechanisms underlying E. faecalis-induced damage in osteoblasts require further elucidation.
Purpose of the Study:
- To investigate the role of necroptosis in human osteoblastic MG63 cells upon infection with Enterococcus faecalis.
- To elucidate the specific signaling pathway involved in this process.
- To provide insights into the pathogenesis of refractory apical periodontitis.
Main Methods:
- MG63 cell viability was assessed using the cell counting kit-8 assay.
- Western blotting was used to measure the expression of necroptosis markers (p-RIPK3, p-MLKL).
- Inhibitors (necrosulfonamide, GSK’872) and MLKL-targeting shRNA were used to confirm necroptosis activation, alongside LDH assay, flow cytometry, and electron microscopy.
Main Results:
- E. faecalis infection significantly reduced MG63 cell viability in a dose- and time-dependent manner.
- Expression of p-RIPK3 and p-MLKL was significantly upregulated following E. faecalis infection.
- Inhibitor treatment and MLKL silencing markedly reduced cell death and specific indicators of necroptosis.
Conclusions:
- Enterococcus faecalis infection induces necroptosis in MG63 cells through the RIPK3/MLKL signaling pathway.
- This necroptotic process may negatively impact the healing of refractory apical periodontitis.
- The study highlights potential therapeutic targets for refractory apical periodontitis.
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