Investigating the Effects of Chelidonic Acid on Oxidative Stress-Induced Premature Cellular Senescence in Human Skin Fibroblast Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells Culture and Treatment
2.2. Developing a Stress-Induced Premature Senescence (SIPS) Model
2.2.1. Doubling Time
2.2.2. Staining Cells for SA-βgal
2.2.3. Determining the Sublethal Dose of H2O2 as an Oxidative Stress Agent
2.2.4. Determining the Sublethal Dose of CA
2.3. Free Radical Measurements
2.3.1. Staining Cells for SA-βgal
2.3.2. Cytotoxicity Testing: AO/DAPI Double Staining
4′,6-Diamino-2-Phenylindole (DAPI) Staining
Acridine-Orange Staining (AO)
2.3.3. Genotoxicity Testing: Comet Assay
2.3.4. H2O2 Measurements
2.3.5. Malondialdehyde Levels
2.4. Determination of Antioxidant Activity
2.4.1. Superoxide Dismutase Activity
2.4.2. Glutathione Peroxidase (GSH-Px) Activity
2.5. Molecular Docking Experiments
2.6. Gen Expression and Protein Analysis
2.6.1. Real-Time PCR Analysis
Total RNA Isolation and First-Strand cDNA Synthesis
Quantitative, Real-Time PCR
2.6.2. Western Blot Analysis
2.7. Statistical Analysis
3. Results
3.1. Developing a Stress-Induced Premature Senescence (SIPS) Model
3.1.1. Doubling Time
3.1.2. Staining Cells for SA-βgal
3.1.3. Determining the Sublethal Dose of H2O2 as an Oxidative Stress Agent
3.1.4. Determining the Sublethal Dose of CA
3.2. Free Radical Measurements
3.2.1. Staining Cells for SA-βgal
3.2.2. DAPI-Acridine Orange (AO) Staining
3.2.3. Comet Assay
3.2.4. H2O2 Measurements
3.2.5. MDA Levels
3.3. Antioxidant Activity
3.4. Molecular Docking
3.5. Gene Expression and Protein Analysis Results
3.5.1. RT PCR Results
Evaluation of Total RNA Isolation Results
Selection of Candidate Reference Genes and Evaluation of Amplification Specificity, PCR Efficiency, and Gene Expression Levels
3.5.2. Protein Analysis Results
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Antibodies | Source | Catalog |
---|---|---|
Anti-p16 polyclonal antibody | Proteintech | 10883-1-AP |
Anti-p21 polyclonal antibody | Proteintech | 10355-1-AP |
Anti-pRB1 monoclonal antibody | Proteintech | 67521-1-16 |
Anti- β-Actin Rabbit mAb | ABclonal | AC026 |
HRP-conjugated Goat anti-Rabbit IgG (H + L) | ABclonal | AS014 |
HRP-conjugated Goat anti- Mouse IgG (H + L) | ABclonal | AS071 |
Number | Complexes | PDB Code | Docking Scores (kcal/mol) | Types of Interactions | Interacting Residues |
---|---|---|---|---|---|
1 | P21-Chelidonic acid | 5XVG [63] | −12.68 | Conventional H-bonding | LEU 475. TYR 492, VAL 476, LYS 473 |
2 | P16-Chelidonic acid | 1GIH [64] | −11.99 | Conventional H-bonding, π Anion, π Sigma | ARG 126, VAL 164, THR 165, THR 14, ASP 127, ARG 169, LYS 129 |
3 | P53-Chelidonic acid | 4MZI [65] | −10.13 | Conventional H-bonding, π Alkyl | LYS 132, ARG 273, PRO 250, ARG 248, SER 240 |
4 | PRB1-Chelidonic acid | 1AD6 [66] | −6.81 | Van der waals, Conventional and Carbon H-bonding, π Alkyl | ASN 390, SER 499, ARG 445, ARG 500, |
5 | NRF2-Chelidonic acid | 3WN7 [67] | −7.71 | Conventional and Carbon H-bonding | Val 463, ILE 416, ARG 415, VAL 465, GLY 511, GLY 509 |
6 | NF-κB-Chelidonic acid | 1SVC [68] | −7.89 | Van der waals, Conventional and Carbon H-bonding, π Alkyl | ARG 57, SER 243, ASN 250, LYS 244 |
7 | JAK1-Chelidonic acid | 4L00 [69] | −11.31 | Van der waals, Conventional and Carbon H-bonding, π Alkyl | ARG 577, ARG 643 |
8 | JAK2-Chelidonic acid | 4GL9 [70] | −7.17 | Conventional and Carbon H-bonding, π Cation, π Sigma | LYS 1009, GLU 1006, LYS 1005, LYS 1030 |
9 | AMPK-Chelidonic acid | 7OPM [71] | −7.72 | Conventional and Carbon H-bonding | GLU 186, LEU 184, LYS 203 |
10 | STAT3-Chelidonic acid | 5AX3 [72] | −6.94 | Van der waals, Conventional H-bonding | LYS 221, THR 181, LYS 142 |
11 | HSP90-Chelidonic acid | 6KSQ [73] | −7.68 | Van der waals, Conventional and Carbon H-bonding, π Alkyl | THR 425, SER 476, GLU 461, THR 425, ALA 428, LYS 478, ARG 464 |
12 | FOXO4-Chelidonic acid | 3L2C [74] | −7.09 | Conventional H-bonding | Glu 125, ARG 429 |
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Turkoglu, B.; Mansuroglu, B. Investigating the Effects of Chelidonic Acid on Oxidative Stress-Induced Premature Cellular Senescence in Human Skin Fibroblast Cells. Life 2024, 14, 1070. https://doi.org/10.3390/life14091070
Turkoglu B, Mansuroglu B. Investigating the Effects of Chelidonic Acid on Oxidative Stress-Induced Premature Cellular Senescence in Human Skin Fibroblast Cells. Life. 2024; 14(9):1070. https://doi.org/10.3390/life14091070
Chicago/Turabian StyleTurkoglu, Burcu, and Banu Mansuroglu. 2024. "Investigating the Effects of Chelidonic Acid on Oxidative Stress-Induced Premature Cellular Senescence in Human Skin Fibroblast Cells" Life 14, no. 9: 1070. https://doi.org/10.3390/life14091070