Silibinin Downregulates Types I and III Collagen Expression via Suppression of the mTOR Signaling Pathway
<p>Chemical structure of silibinin. The chemical formula of silibinin is C<sub>25</sub>H<sub>22</sub>O<sub>10,</sub> and the molecular weight is 482.441 g/mol.</p> "> Figure 2
<p>Clinical photographs of the keloid scars included in the present study. (<b>A</b>,<b>B</b>) Keloid scars on the right ear helix that occurred after piercing. (<b>C</b>) A keloid scar on the abdomen that occurred after an orchiopexy surgery. (<b>D</b>) A keloid scar on the anterior chest that occurred after a laceration. (<b>E</b>) A keloid scar on the abdomen that occurred after a total abdominal hysterectomy and bilateral salpingo-oophorectomy.</p> "> Figure 3
<p>Expression of mTOR in keloid tissues. (<b>A</b>) Phosphorylation of mTOR is increased in active keloid scars compared to normal tissue specimens. The scale bar indicates 500 μm. (<b>B</b>) Semi-quantitative analysis showed that the p-mTOR signaling was increased by 21.3-fold in keloid tissue compared to normal tissue (** <span class="html-italic">p</span> < 0.01). (<b>C</b>) mTOR phosphorylation is increased in the cytoplasm of fibroblasts in keloid tissue.</p> "> Figure 4
<p>Effect of silibinin on cell viability of skin fibroblasts. (<b>A</b>) Human dermal fibroblast. (<b>B</b>) Keloid-derived fibroblast. Cells were seeded at a density of 1 × 10<sup>4</sup> cells/well in 96-well plates overnight and then treated with different concentrations of silibinin (0–200 µM) at 37 °C for 24 h. Cell viability was analyzed using the CCK-8 assay kit. The viability of the cells was not markedly suppressed by treatment with silibinin at a concentration of 200 μM. HDF, human dermal fibroblast; KF, keloid-derived fibroblast; CCK-8, Cell Counting Kit-8.</p> "> Figure 5
<p>Effect of silibinin on collagen types I and III expression. Relative mRNA expression levels were compared between control and silibinin treatments. Silibinin treatment resulted in a dose-dependent reduction in COL1A1 (<b>A</b>,<b>B</b>) and COL3A1 (<b>C</b>,<b>D</b>) mRNA transcripts in TGF-β1-treated human dermal and keloid-derived fibroblasts. Bars represent mean ± SD, <span class="html-italic">n</span> = 3 − 6 (* <span class="html-italic">p</span> < 0.05, *** <span class="html-italic">p</span> < 0.001, ns, non-significant).</p> "> Figure 6
<p>Effect of silibinin on the mTOR signaling pathway. (<b>A</b>) Silibinin effectively downregulated the mTOR pathway, including mTOR and its key components—p70S6K, S6, and 4E-BP1—in both HDFs and KFs. (<b>B</b>–<b>E</b>) Results of quantitative analysis. The data are presented as a ratio of the signal intensity of the phosphorylated form to the total form. <span class="html-italic">n</span> = 3 (* <span class="html-italic">p <</span> 0.05, ** <span class="html-italic">p</span> < 0.01, ns, non-significant).</p> "> Figure 6 Cont.
<p>Effect of silibinin on the mTOR signaling pathway. (<b>A</b>) Silibinin effectively downregulated the mTOR pathway, including mTOR and its key components—p70S6K, S6, and 4E-BP1—in both HDFs and KFs. (<b>B</b>–<b>E</b>) Results of quantitative analysis. The data are presented as a ratio of the signal intensity of the phosphorylated form to the total form. <span class="html-italic">n</span> = 3 (* <span class="html-italic">p <</span> 0.05, ** <span class="html-italic">p</span> < 0.01, ns, non-significant).</p> "> Figure 7
<p>Major pathways downstream of mTORC1 signaling in mRNA translation. mTORC1 promotes protein synthesis by phosphorylation of two key effectors, eIF4E binding protein 1 (4E-BP1) and p70S6 Kinase (p70S6K). 4E-BP1 inhibits translation by binding and sequestering eukaryotic translation initiation factor-4E (eIF4E) to prevent assembly of the eIF4F complex. eIF4F complex mediates the recruitment of ribosomes to mRNA, which is the rate-limiting step for translation. mTORC1 phosphorylates 4E-BP1 at multiple sites to trigger its dissociation from eIF4E, allowing 5′-cap-dependent mRNA translation to occur. Unrelated to 4E-BP1, mTORC1 phosphorylates p70S6K, stimulating its subsequent phosphorylation. p70S6K phosphorylates and activates several substrates that promote mRNA translation initiation, including S6.</p> "> Figure 8
<p>Summary and logic flowchart of the study. Silibinin treatment significantly reduced the expressions of collagen I and III in normal human dermal fibroblasts and keloid-derived fibroblasts, with suppression of the mTOR signaling pathway (highlighted in the yellow box). The relationship between mTOR, collagen synthesis, and keloid is described in the introduction and discussion sections of the manuscript.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Clinical Information of Investigated Tissue Samples
2.2. mTOR Phosphorylation Is Elevated in Active Keloid Scar Compared to the Normal Tissue Specimen
2.3. Effect of Silibinin on Cell Viability in HDFs and KFs
2.4. Silibinin Significantly Reduced Collagen I and III mRNA Transcripts in TGF-β1-Treated HDFs and KFs
2.5. Silibinin Effectively Suppressed the mTOR Signaling Pathway in HDFs and KFs
3. Discussion
4. Materials and Methods
4.1. Collection of Keloid and Normal Tissue Samples
4.2. Cell Lines, Cell Culture, and Isolation of Fibroblasts from Keloid Tissues
4.3. Natural Phytochemical
4.4. Cell Viability Assay
4.5. Histological Examination and Immunohistochemistry (IHC) Analyses
4.6. Quantitative Reverse Transcription–Polymerase Chain Reaction (qRT-PCR)
4.7. Immunoblotting Assay
4.8. Statistical Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Sex | Age (y) | Site | Duration of Scar (y) | Recurrence after Surgical Excision | Experiments Involving Tissue Sample | |
---|---|---|---|---|---|---|
Participants with keloid scars | ||||||
1 | F | 25 | Right ear | 1 | Yes | Cell isolation, IHC |
2 | F | 29 | Right ear | 2 | No | Cell isolation, IHC |
3 | M | 11 | Abdomen | 2.5 | No | IHC |
4 | F | 76 | Anterior chest | 1 | Yes | IHC |
5 | F | 51 | Abdomen | 2 | Yes | IHC |
Donors of normal skin | ||||||
1 | F | 40 | Abdomen | - | IHC | |
2 | F | 38 | Abdomen | - | IHC | |
3 | F | 55 | Abdomen | - | IHC |
Target Gene | Primer Sequences (5′ → 3′) |
---|---|
COL1A1 | Forward: 5′-TGTTCAGCTTTGTGGACCTCCG-3′ Reverse: 5′-CCGTTCTGTACGCAGGTGATTG-3′ |
COL3A1 | Forward: 5′-GAAGATGTCCTTGATGTGC-3′ Reverse: 5′-AGCCTTGCGTGTTCGATAT-3′ |
GAPDH | Forward: 5′-CATGAGAAGTATGACAACAGCCT-3′ Reverse: 5′-AGTCCTTCCACGATACCAAAGT-3′ |
Antibody | Host | Clonality | Isotype | Dilution | Product Code | Source |
---|---|---|---|---|---|---|
Primary antibodies | ||||||
Collagen I | Rabbit | polyclonal | IgG | 1:1000 | NB600-408 | Novus Biologicals |
Collagen III | Rabbit | polyclonal | IgG | 1:1000 | Ab7778 | Abcam |
mTOR | Rabbit | polyclonal | IgG | 1:500 | PA5-34663 | Invitrogen |
p-mTOR (Ser2448) | Rabbit | polyclonal | IgG | 1:1000 | 44-1125G | Invitrogen |
4E-BP1 | Rabbit | monoclonal | IgG | 1:1000 | 9644 | Cell Signaling Technology |
p-4E-BP1 (Thr37/46) | Rabbit | monoclonal | IgG | 1:1000 | 2855 | Cell Signaling Technology |
p70S6K | Rabbit | monoclonal | IgG | 1:1000 | 9202 | Cell Signaling Technology |
p-p70S6K (Thr421/Ser424) | Rabbit | monoclonal | IgG | 1:1000 | 9204 | Cell Signaling Technology |
S6 | Mouse | monoclonal | IgG1 | 1:1000 | 2317 | Cell Signaling Technology |
p-S6 (Ser235/236) | Rabbit | polyclonal | IgG | 1:1000 | 2211 | Cell Signaling Technology |
p-S6 (Ser240/244) | Rabbit | monoclonal | IgG | 1:1000 | 5364 | Cell Signaling Technology |
GAPDH | Rabbit | monoclonal | NA | 1:1000 | 2118 | Cell Signaling Technology |
Secondary antibodies | ||||||
anti-rabbit IgG | Goat | NA | NA | 1:2000 | 7074 | Cell Signaling Technology |
anti-mouse IgG | Goat | polyclonal | IgG | 1:2000 | SC-2005 | Santa Cruz Biotechnology |
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Choi, S.; Ham, S.; Lee, Y.I.; Kim, J.; Lee, W.J.; Lee, J.H. Silibinin Downregulates Types I and III Collagen Expression via Suppression of the mTOR Signaling Pathway. Int. J. Mol. Sci. 2023, 24, 14386. https://doi.org/10.3390/ijms241814386
Choi S, Ham S, Lee YI, Kim J, Lee WJ, Lee JH. Silibinin Downregulates Types I and III Collagen Expression via Suppression of the mTOR Signaling Pathway. International Journal of Molecular Sciences. 2023; 24(18):14386. https://doi.org/10.3390/ijms241814386
Chicago/Turabian StyleChoi, Sooyeon, Seoyoon Ham, Young In Lee, Jihee Kim, Won Jai Lee, and Ju Hee Lee. 2023. "Silibinin Downregulates Types I and III Collagen Expression via Suppression of the mTOR Signaling Pathway" International Journal of Molecular Sciences 24, no. 18: 14386. https://doi.org/10.3390/ijms241814386