α-Lipoic Acid Inhibits Apoptosis by Suppressing the Loss of Ku Proteins in Helicobacter pylori-Infected Human Gastric Epithelial Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell line and Culture Conditions
2.2. Bacterial Strain and Growth Conditions
2.3. Reagents
2.4. Infection of AGS Cells with H. pylori
2.5. Experimental Protocols
2.6. Preparation of Whole-Cell Extracts and Nuclear Extracts
2.7. Measurement of Cell Viability and DNA Fragmentation
2.8. Measurement of Intracellular ROS Levels
2.9. Electrophoretic Mobility Shift Assay (EMSA)
2.10. Western Blotting
2.11. Immunofluorescence Staining
2.12. Co-Immunoprecipitation Assay
2.13. Statistical Analysis
3. Results
3.1. H. pylori Induces DNA Fragmentation and Apoptosis in AGS Cells
3.2. α-LA Inhibits H. pylori-Induced DNA Fragmentation and Apoptosis in AGS Cells
3.3. α-LA Inhibits H. pylori-Induced Increases in ROS and γ-H2AX in AGS Cells
3.4. α-LA Inhibits H. pylori-Induced Decreases in Nuclear Ku70/80 in AGS Cells
3.5. Both NAC and MG-132 Inhibited Cell Death and Loss of Nuclear Ku Induced by H. pylori in AGS Cells
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
AGS | gastric epithelial adenocarcinoma cell line |
α-LA | α lipoic acid |
AIF | apoptosis-inducing factor |
H. pylori | Helicobacter pylori |
DAPI | 4′,6-diamidino-2-phenylindole |
DCF | dichlorofluorescein |
DCFH-DA | dichlorofluorescein diacetate |
DDRs | DNA damage responses |
DMSO | dimethyl sulfoxide |
DNA-PKcs | DNA-dependent protein kinase catalytic subunit |
DSBs | double-strand breaks |
ECL | enhanced chemiluminescence |
EDTA | ethylene diaminetetraacetic acid |
EGTA | ethylene glycol tetraacetic acid |
EMSA | electrophoretic mobility shift assay |
FBS | fetal bovine serum |
HR | homologous recombination |
H2AX | H2A histone family member X |
MOI | multiplicity-of-infection |
NAC | N-acetycysteine |
NHEJ | non-homologous end joining |
NP-40 | nonidet P-40 |
8-OH-dG | 8-hydroxy-2′-deoxyguanosine |
PMSF | phenylmethylsulfonylfluoride |
ROS | reactive oxygen species |
RPMI | Roswell Park Memorial Institute |
SDS | sodium dodecylsulfate |
TBST | Tris-buffered saline and 0.2% Tween-20 |
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Park, D.; Lim, J.W.; Kim, H. α-Lipoic Acid Inhibits Apoptosis by Suppressing the Loss of Ku Proteins in Helicobacter pylori-Infected Human Gastric Epithelial Cells. Nutrients 2022, 14, 3206. https://doi.org/10.3390/nu14153206
Park D, Lim JW, Kim H. α-Lipoic Acid Inhibits Apoptosis by Suppressing the Loss of Ku Proteins in Helicobacter pylori-Infected Human Gastric Epithelial Cells. Nutrients. 2022; 14(15):3206. https://doi.org/10.3390/nu14153206
Chicago/Turabian StylePark, Dayong, Joo Weon Lim, and Hyeyoung Kim. 2022. "α-Lipoic Acid Inhibits Apoptosis by Suppressing the Loss of Ku Proteins in Helicobacter pylori-Infected Human Gastric Epithelial Cells" Nutrients 14, no. 15: 3206. https://doi.org/10.3390/nu14153206