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. 2021 Dec 16;16(12):e0260545.
doi: 10.1371/journal.pone.0260545. eCollection 2021.

Senotherapeutic-like effect of Silybum marianum flower extract revealed on human skin cells

Jieun Woo  1 , Seoungwoo Shin  1 , Eunae Cho  1 , Dehun Ryu  1 , David Garandeau  2 , Hanane Chajra  2 , Mathilde Fréchet  2 , Deokhoon Park  1 , Eunsun Jung  1
Affiliations

Senotherapeutic-like effect of Silybum marianum flower extract revealed on human skin cells

Jieun Woo et al. PLoS One. .

Abstract

Cellular senescence causes irreversible growth arrest of cells. Prolonged accumulation of senescent cells in tissues leads to increased detrimental effects due to senescence associated secretory phenotype (SASP). Recent findings suggest that elimination of senescent cells has a beneficial effect on organismal aging and lifespan. In this study, using a validated replicative senescent human dermal fibroblasts (HDFs) model, we showed that elimination of senescent cells is possible through the activation of an apoptotic mechanism. We have shown in this replicative senescence model, that cell senescence is associated with DNA damage and cell cycle arrest (p21, p53 markers). We have shown that Silybum marianum flower extract (SMFE) is a safe and selective senolytic agent targeting only senescent cells. The elimination of the cells is induced through the activation of apoptotic pathway confirmed by annexin V/propidium iodide and caspase-3/PARP staining. Moreover, SMFE suppresses the expression of SASP factors such as IL-6 and MMP-1 in senescent HDFs. In a co-culture model of senescent and young fibroblasts, we demonstrated that senescent cells impaired the proliferative capacities of young cells. Interestingly, when the co-culture is treated with SMFE, the cell proliferation rate of young cells is increased due to the decrease of the senescent burden. Moreover, we demonstrated in vitro that senescent fibroblasts trigger senescent process in normal keratinocytes through a paracrine effect. Indeed, the conditioned medium of senescent HDFs treated with SMFE reduced the level of senescence-associated beta-galactosidase (SA-β-Gal), p16INK4A and SASP factors in keratinocytes compared with CM of senescent HDFs. These results indicate that SMFE can prevent premature aging due to senescence and even reprograms aged skin. Indeed, thanks to its senolytic and senomorphic properties SMFE is a candidate for anti-senescence strategies.

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Conflict of interest statement

No authors have competing interests.

Figures

Fig 1
Fig 1. Establishment of senescence in fibroblast by successive passages in culture (replicative senescence model).
(A) Calculation of the population doubling time and (B) Measurement of SA-β-gal activity according to the number of subcultures. Representative microphotographs are shown. Data are represented as mean ± SEM of three independent assays. **p < 0.01 compared to passage 9.
Fig 2
Fig 2. Inhibitory effect of SMFE against DNA damage marker and cell cycle markers.
Senescent HDFs were treated with the indicated concentrations of extracts for 3 days. (A) Quantification of γ-H2AX staining intensity. (B, C) qRT–PCR for p16INK4A and p53 mRNA expression. (D) Quantification of p21CIP1-staining intensity. Data are represented as mean ± SEM of three independent assays. *p < 0.05; **p < 0.01 compared to young cell control (YC), #p < 0.05; ##p < 0.01 compared to senescent cell control.
Fig 3
Fig 3. Senolytic effect of SMFE in a replicative senescence model.
(A) Effect of SMFE on the viability of young cells (p8) and senescent cells (p40) after the cells were treated with the indicated concentrations of extracts for 3 days. (B) SA-β-gal activity in senescent cells. Representative flow cytometry plots are shown. Quantification of the percentage of positively stained cells (SA-β-gal FITC-positive) as in flow cytometry plots. Data are represented as mean ± SEM of three independent assays. *p < 0.05; **p < 0.01 compared to young cell control, #p < 0.05; ##p < 0.01 compared to senescent cell control. YC, young cell control; SC, senescent cell control.
Fig 4
Fig 4. Induction of apoptosis by SMFE through caspase-3/PARP pathway.
(A) Representative flow cytometry plots of apoptosis assay. Young and senescent cells were treated with vehicle or SMFE (100, 200 μg/mL) for 3 days. Cell apoptosis was assayed by flow cytometer after annexin V and PI staining. Quantification of the percentage of apoptotic cells (annexin V-FITC positive) 3 days after treatment as in (A) (right). (B-C) Representative immunofluorescence image and fluorescence intensity analysis of cleaved PARP (B) and cleaved caspase-3 (C) in young and senescent cells 3 days after incubation with vehicle or SMFE (100, 200 μg/mL). ABT-737 treated cells were used as a positive control. Data are represented as mean ± SEM of three independent assays. *p < 0.05; **p < 0.01 compared to young cell control, #p < 0.05; ##p < 0.01 compared to senescent cell control. YC, young cell control.
Fig 5
Fig 5. Effect of SMFE on removal of senescent cells and cell proliferation.
(A) Schematic representation of co-culture process (B) Rate of young and senescent cells after SMFE treatment for 3 days. PKH26 (senescent, p40, red)-positive cells and PKH67 (young, p8, green) -positive cells were calculated the percentage compared to the total cell number. (C-D) The young and senescent cells were mixed 1:9, and treated with the SMFE for 3 days, then the cells harvested and re-seeded. After 3 days of incubation, cell growth rate and population doubling time measured by cell counting (C) Cell proliferation analyzed after 6 days of incubation of re-seeding cells. (D) The population doubling time analysis. Data are represented as mean ± SEM of three independent assays. *p < 0.05; **p < 0.01 compared to the vehicle.
Fig 6
Fig 6. Effect of SMFE on SASPs and type-1 procollagen expression in senescent cells.
Senescent HDFs were treated with the indicated concentrations of extracts for 3 days. The secretion of MMP-1 (A), IL-6 (B) and collagen production (C) in the culture supernatant was measured using an ELISA kit. Data are represented as mean ± SEM of three independent assays. **p < 0.01 compared to young cell control, #p < 0.05; ##p < 0.01 compared to senescent cell control.
Fig 7
Fig 7. Effect of F-CM with or without SMFE on adult keratinocytes senescence.
(A) Process for the preparation of concentrated F-CM. (B) SA-β-gal activity in senescent cells. Representative flow cytometry plots are shown. Quantification of the percentage of positively stained cells (SA-β-gal FITC-positive) as in flow cytometry plots. (C) qRT–PCR for p16INK4A mRNA expression. (D-F) qRT–PCR for MMP-1, IL-6, and IL-1α mRNA expression. Data are represented as mean ± SEM of three independent assays. *p < 0.05; **p < 0.01 compared to young cell control, #p < 0.05; ##p < 0.01 compared to senescent cell control. YC, young cell control; SC, senescent cell control; CM, conditioned medium.
Fig 8
Fig 8. HPLC chromatogram of SMFE at 290 nm.
(A) Silymarin analysis of SMFE. (B) Phenolic compound analysis of SMFE. 1, Taxifolin; 2,Silychristin; 3, Silydianin; 4,Silybinin A,B; 5, Isosilybin; 6, Scopolin; 7, chlorogenic acid; 8, Scopoletin; 9, Myricetin; 10, Quercetin; 11, Luteolin; 12, Apigenin; 13, Kaempferol.
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Grants and funding

This work was funded by the Ministry of Trade, Industry and Energy, http://www.motie.go.kr/ (Grant number P-001-7747, to EJ). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.