Hesperetin activates CISD2 to attenuate senescence in human keratinocytes from an older person and rejuvenates naturally aged skin in mice

Background CDGSH iron-sulfur domain-containing protein 2 (CISD2), a pro-longevity gene, mediates healthspan in mammals. CISD2 is down-regulated during aging. Furthermore, a persistently high level of CISD2 promotes longevity and ameliorates an age-related skin phenotype in transgenic mice. Here we translate the genetic evidence into a pharmaceutical application using a potent CISD2 activator, hesperetin, which enhances CISD2 expression in HEK001 human keratinocytes from an older person. We also treated naturally aged mice in order to study the activator’s anti-aging efficacy. Methods We studied the biological effects of hesperetin on aging skin using, firstly, a cell-based platform, namely a HEK001 human keratinocyte cell line established from an older person. Secondly, we used a mouse model, namely old mice at 21-month old. In the latter case, we investigate the anti-aging efficacy of hesperetin on ultraviolet B (UVB)-induced photoaging and naturally aged skin. Furthermore, to identify the underlying mechanisms and potential biological pathways involved in this process we carried out transcriptomic analysis. Finally, CISD2 knockdown HEK001 keratinocytes and Cisd2 knockout mice were used to study the Cisd2-dependent effects of hesperetin on skin aging. Results Four findings are pinpointed. Firstly, in human skin, CISD2 is mainly expressed in proliferating keratinocytes from the epidermal basal layer and, furthermore, CISD2 is down-regulated in the sun-exposed epidermis. Secondly, in HEK001 human keratinocytes from an older person, hesperetin enhances mitochondrial function and protects against reactive oxygen species-induced oxidative stress via increased CISD2 expression; this enhancement is CISD2-dependent. Additionally, hesperetin alleviates UVB-induced damage and suppresses matrix metalloproteinase-1 expression, the latter being a major indicator of UVB-induced damage in keratinocytes. Thirdly, transcriptomic analysis revealed that hesperetin modulates a panel of differentially expressed genes that are associated with mitochondrial function, redox homeostasis, keratinocyte function, and inflammation in order to attenuate senescence. Intriguingly, hesperetin activates two known longevity-associated regulators, namely FOXO3a and FOXM1, in order to suppress the senescence-associated secretory phenotype. Finally, in mouse skin, hesperetin enhances CISD2 expression to ameliorate UVB-induced photoaging and this occurs via a mechanism involving CISD2. Most strikingly, late-life treatment with hesperetin started at 21-month old and lasting for 5 months, is able to retard skin aging and rejuvenate naturally aged skin in mice. Conclusions Our results reveal that a pharmacological elevation of CISD2 expression at a late-life stage using hesperetin treatment is a feasible approach to effectively mitigating both intrinsic and extrinsic skin aging and that hesperetin could act as a functional food or as a skincare product for fighting skin aging. Supplementary Information The online version contains supplementary material available at 10.1186/s12929-024-01005-w.


Additional file 1
Figure S1 CISD2 is mainly expressed in the proliferating keratinocytes of the epidermis in normal human skin from an older person.Related to Figure 1.

Figure S5
Hesperetin modulated gene expression profiles using HEK001 human keratinocytes from an older person, These are related to proteostasis, cellular senescence, stress response and redox homeostasis.
Related to Figure 5.

Figure S6
Hesperetin modulates the activity of FOXM1 and IL-1α, as well as the expression of FOXO3a downstream target genes in HEK001 keratinocytes.Related to Figure 6.

Table S1
Hesperetin-modulated changes in the upstream regulators and their downstream target genes in HEK001 keratinocytes    revealed an age-dependent decline in Cisd2 protein expression in the skin from the young (3-month), mid-age (14-month) and old (24-month) wild-type C57BL/6 male mice (n = 4-5).(C) Hesperetin (10 mg/kg/day i.p. for 30 days) enhances by 2.5-fold the Cisd2 protein levels in the skin of middle-aged mice at 14-month old (n=3).
The Cisd2 protein levels were determined by Western blot analysis and normalized using Gapdh.Data are presented as mean ± SD. *p < 0.05; **p < 0.005.In (B) the statistical analysis was performed by one-way ANOVA with Bonferroni multiple comparison test.In (C) the statistical analysis was performed by Student's t test.

Figure S2
Figure S2Toxicity testing of different dosages of hesperetin against HEK001 human keratinocytes from an

Figure S3
Figure S3Oral administration of hesperetin is able to ameliorate UVB-induced skin photoaging in WT mice.

Figure S4
Figure S4 Hesperetin enhances Cisd2 expression in the skin of WT mice.Related to Figure 4.

Figure S1 .
Figure S1.CISD2 is mainly expressed in the proliferating keratinocytes of the epidermis in normal human skin from an older person.Related to Figure 1.(A) The age, sex and collection sites of the skin samples of the human tissues array SKN1001.(B and C) Fluorescent immunohistochemistry (IHC) staining of CISD2 (B) and KRT14 (a marker of proliferating keratinocytes in the epidermis) (C) in the normal human skin of the SKN1001 tissue array.(D) The merged IHC image of CISD2, KRT14 and DAPI in the SKN1001 tissue array.Blue boxes indicate the representative samples in panel E (#S1 to #S4).Red boxes indicate the representative samples in panel F (#N1 to #N4).Scale bars in (B-D), 2 mm.(E) Representative images of IHC staining of CISD2 and KRT14 from the sun-exposed sites (Face and Neck) of human skin.(F) Representative images of IHC staining of CISD2 and KRT14 from the non-sunexposed sites, including chest, humeral back, thigh and anus of human skin.Scale bars in (E) and (F), 100 μm.

Figure S2 .
Figure S2.Toxicity testing of different dosages of hesperetin against HEK001 human keratinocytes from an older person.Related to Figure 2. (A) Toxicity testing of different dosages of hesperetin against HEK001 keratinocytes.Cell morphology of the HEK001 keratinocytes after treatment with hesperetin at different dosages (1 μM, 10 μM and 100 μM) for 4 days.The cytotoxic effects, including a reduced cell density and a smaller cell size are present in the HEK001 keratinocytes at a high concentration (100 μM) of hesperetin treatment.(B) Analysis of cell proliferation rates after treatment of HEK001 keratinocytes with different concentrations of hesperetin (1-100 μM).The HEK001 keratinocytes were treated with different doses of hesperetin as indicated.Vehicle (V), 0.1% DMSO.(C)Representative images of JC-1 staining of the different groups of HEK001 keratinocytes.Hesperetin (10 μM) protects against oxidative stress-induced mitochondrial dysfunction.All experiments were performed and repeated three independent times as biological replicates using HEK001 keratinocytes.Data are presented as mean ± SD.The statistical analysis was performed by one-way ANOVA with Bonferroni multiple comparison test.

Figure S3 .
Figure S3.Oral administration of hesperetin is able to ameliorate UVB-induced skin photoaging in WT mice.Related to Figure 3. (A) A gross view of the dorsal skin of Vehicle or Hesperetin treated WT and Cisd2KO mice before and after UVB exposure.(B) The protocol for oral treatment with hesperetin and its effect on UVB-induced skin damage in WT mice at 3-month old.The mice were pre-treated with hesperetin (30 mg/kg/day, oral administration) for 7 days, and then exposed to UVB (312 nm, 349 mJ/cm 2 /75 seconds) light once a day for 5 days in a UVB box.The mice were sacrificed 6 days after the first UVB exposure.(C) A gross view of the dorsal skin of Vehicle or Hesperetin treated WT mice before and after UVB exposure.(D) Masson's trichrome staining of skin sections from the different groups of mice.UVB exposure significantly induces skin damage and increases skin thickness, which are the major characteristics of photoaging, while hesperetin treatment ameliorates UVBinduced skin damage.(E) Quantitation of the thickness of total skin layer, epidermal layer and dermal layer, in the skin of WT mice.Data are presented as mean ± SD. *p < 0.05; **p < 0.005 by one-way ANOVA with Bonferroni multiple comparison test; not significant (n.s.).UT, untreated.

Figure S4 .
Figure S4.Hesperetin enhances Cisd2 expression in the skin of WT mice.Related to Figure 4. (A) In vivo imaging system (IVIS) analysis of luciferase activity was examined in transgenic mice carrying the CISD2 BAC Luc reporter.Hesperetin stimulated luciferase reporter activity in the right ear of the CISD2 BAC reporter mice.Hesperetin (10 μM) and vehicle (10% glycerol with 5% menthol) were topically applied twice a day for 7 days to the right (R) and left (L) ears of mice at 3-month old, respectively.Luciferase activity was monitored for 7 days after hesperetin or Vehicle treatment.(B) Western blot analysis of Cisd2 protein levels

Figure S5 .
Figure S5.Hesperetin modulated gene expression profiles using HEK001 human keratinocytes from an older person, these are related to proteostasis, cellular senescence, stress response and redox homeostasis.Related to Figure 5. (A) The heatmap pinpoints the proteostasis-related DEGs, including unfolded protein response (UPR), ubiquitination and proteasomal protein degradation, and chaperone-mediated protein folding, in HEK001 keratinocytes.(B) The heatmap pinpoints the cellular senescence-related DEGs, including aging, cell cycle arrest, and telomere maintenance in HEK001 keratinocytes.(C) The heatmap pinpoints the stress responserelated DEGs, including response to UV, hypoxia, and wounding, in HEK001 keratinocytes.(D) The heatmap pinpoints the redox homeostasis-related DEGs, including oxidative stress response, oxidation-reduction, and regulation of reactive oxygen species (ROS) metabolism, in HEK001 keratinocytes.(E and F) The heatmap pinpoints the immune response and inflammation-related DEGs in HEK001 keratinocytes, including TNF signaling, IKK/NF-κB signaling, cellular response to TGF-β stimulus and IL-17 signaling.

Figure S6 .
Figure S6.Hesperetin modulates the activity of FOXM1 and IL-1α, as well as the expression of FOXO3a downstream target genes in HEK001 keratinocytes.Related to Figure 6.(A) Significant activation of FOXM1 transcriptional signaling is based on the activation z-score (z-score > +2.0 and p-value of overlap < 0.01) from IPA upstream regulator analysis of the Hesperetin modulated DEGs of HEK001 keratinocytes.The mRNA expression levels of the Hesperetin modulated DEGs are associated with