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

**Background:** Skin aging involves intrinsic (chronological) and extrinsic (e.g., UV) factors, with keratinocyte senescence playing a key role. CISD2 is a pro-longevity gene that declines with age; its overexpression in transgenic mice delays skin aging. Hesperetin, a citrus flavanone, was previously identified as a CISD2 activator. This study aimed to translate genetic evidence into a pharmaceutical application by testing hesperetin's anti-aging effects on human keratinocytes from an older person and on naturally aged mouse skin.

**Methods:** The study used HEK001 human keratinocytes (from a 65-year-old male) and Ker-CT neonatal keratinocytes. CISD2 expression was assessed by Western blot and qPCR. Mitochondrial function was measured via Seahorse OCR and JC-1 membrane potential. Oxidative stress was induced with H2O2 (5 μM or 98 μM). UVB damage was induced at 20 J/m². CISD2 knockdown was achieved with lentiviral shRNA. In vivo, 3-month-old WT and Cisd2 KO mice received hesperetin (10 mg/kg/day i.p. or oral) for 7 days before UVB (349 mJ/cm²/day for 5 days). For natural aging, 21-month-old WT mice received dietary hesperetin (100 mg/kg/day) for 5 months. Skin histology (Masson's trichrome), ROS/RNS levels, and MMP-1 were analyzed. RNA-seq was performed on HEK001 cells treated with 10 μM hesperetin for 48 h.

**Key Results:** In human skin tissue arrays, CISD2 was mainly expressed in proliferating basal keratinocytes and was significantly lower in sun-exposed sites (face, neck) versus protected sites (chest, thigh). In HEK001 cells, hesperetin (10 μM, 48 h) increased CISD2 protein ~2-fold, enhanced basal and ATP-linked OCR, and reduced H2O2-induced ROS and mitochondrial depolarization; these effects were lost in CISD2 knockdown cells. Hesperetin also suppressed UVB-induced MMP-1 expression (intracellular and secreted pro-MMP-1). In WT mice, UVB reduced skin CISD2 by 50%, while hesperetin increased it 2.6-fold and prevented UVB-induced skin thickening, ROS/RNS elevation, and MMP-1 increase. In Cisd2 KO mice, hesperetin had no protective effect. In naturally aged mice (21→26 months), dietary hesperetin restored skin CISD2 to young (3-month) levels, increased hair follicle density, sebaceous gland size and nuclear number, and reduced ROS/RNS. RNA-seq identified 1723 DEGs (821 up, 902 down) modulated by hesperetin, with enrichment in pathways related to mitochondrial function, redox homeostasis, keratinocyte function, and inflammation. IPA upstream analysis revealed activation of FOXO3a and FOXM1, and suppression of SASP factors (e.g., IL-1α, MMP-1). Hesperetin also upregulated OXPHOS genes (Complex I-V) and mitochondrial transcription factors TFAM and TFB1M.

**Clinical Implications:** This study provides proof-of-concept that pharmacological activation of CISD2 with hesperetin can attenuate both intrinsic and extrinsic skin aging. The findings are supported by a prior clinical trial (NCT04015063) showing that topical 0.1% hesperetin improved wrinkles, hydration, and elasticity in photoaged skin. Hesperetin, as a natural citrus compound with no observed toxicity in long-term mouse studies, represents a promising candidate for functional foods or skincare products to combat skin aging. The mechanism involves CISD2-dependent mitochondrial protection, FOXO3a/FOXM1 activation, and SASP suppression.