Polyherbal formulation combining multiple herbs is suggested to achieve enhanced therapeutic effects and reduce toxicity. Harak herbal formula (HRF) extracts were proposed to regulate skin responses to UVR through their ability to suppress UVA-induced matrix metalloproteinase-1 (MMP-1) and pigmentation via promoting antioxidant defenses in in vitro models. Therefore, natural products targeting Nrf2 (nuclear factor erythroid 2-related factor 2)-regulated antioxidant response might represent promising anti-photoaging candidates. Hesperetin (HSP) was suggested as a putative bioactive compound of the HRF, as previously shown by its chemical profiling using the liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). In this study, we explored the anti-photoaging effects of HRF extracts and HSP on normal human dermal fibroblasts (NHDFs) and mouse skin exposed to UVA irradiation. Pretreatment of NHDFs with HRF extracts and HSP protected against UVA (8 J/cm2)-mediated cytotoxicity and reactive oxygen species (ROS) formation. The HRF and HSP pretreatment also attenuated the UVA-induced MMP-1 activity and collagen depletion concomitant with an upregulation of Nrf2 activity and its downstream genes (GST and NQO-1). Moreover, our findings provided the in vivo relevance to the in vitro anti-photoaging effects of HRF as topical application of the extracts (10, 30 and 100 mg/cm2) and HSP (0.3, 1, and 3 mg/cm2) 1 h before UVA exposure 3 times per week for 2 weeks (a total dose of 60 J/cm2) mitigated MMP-1 upregulation, collagen loss in correlation with enhanced Nrf2 nuclear accumulation and its target protein GST and NQO-1 as well as reduced 8-hydroxy-2′-deoxyguanosine (8-OHdG) in irradiated mouse skin. Thus, our findings revealed that HRF extracts and HSP attenuated UVA-induced photoaging via upregulating Nrf2, together with their abilities to reduce ROS formation and oxidative damage. Our study concluded that the HRF and its bioactive ingredient HSP may represent potential candidates for preventing UVA-induced photoaging via restoration of redox balance.

AimsOxidative stress and mitochondrial dysfunction play a role in the process of skin photoaging via activation of matrix metalloproteases (MMPs) and the subsequent degradation of collagen. The activation of nuclear factor E2-related factor 2 (Nrf2), a transcription factor controlling antioxidant and cytoprotective defense systems, might offer a pharmacological approach to prevent skin photoaging. We therefore investigated might offer a pharmacological approach to prevent skin photoaging. We therefore investigated protective effect of the novel mitochondria-targeted hydrogen sulfide (H2S) delivery molecules AP39 and AP123, and non-targeted control molecules on UVA-induced photoaging in normal human dermal fibroblasts (NHDFs) in vitro and the skin of BALB/c mice in vivo. ResultsIn NHDFs AP39 and AP123 (50-200 nM) but not non-targeted controls suppressed UVA (8 J/cm 2 )-mediated cytotoxicity and induction of MMP-1 activity, preserved cellular bioenergetics and increased the expression of collagen and nuclear levels of Nrf2. In in vivo experiments, topical application of AP39 or AP123 (0.3-1 µM/cm 2 ; but not non-targeted control molecules) to mouse skin prior to UVA (60 J/cm 2 ) irradiation prevented skin thickening, MMP induction, collagen loss oxidative stress markers 8-hydroxy-2'-deoxyguanosine (8-OHdG), increased Nrf2-dependent signaling as well as increased manganese superoxide dismutase (MnSOD) levels and levels of the mitochondrial biogenesis marker peroxisome proliferator-activated receptor-gamma coactivator (PGC-1α). Innovation and ConclusionTargeting H2S delivery to mitochondria may represent a novel approach for the prevention and treatment of skin photoaging, as well as being useful tools for determining the role of mitochondrial H2S in skin disorders and aging.

The skin microenvironment created by keratinocytes (KC) influences stress responses of melanocytes (MC) to UVB insult. Here, we investigated paracrine factors involved in the regulatory role of microenvironment created by KC in UVB-mediated MC responses using RNA sequencing analysis as well as in vitro and in vivo models. RNA-Seq showed that G-CSF and CCL20 genes were highly upregulated in UVB-irradiated KC and their levels best correlated with paracrine protective effects of KC on stress responses of MC to UVB. Recombinant G-CSF and CCL20 treatment revealed the strongest modulatory effects on UVB-induced MC responses by mitigating apoptosis and ROS formation and upregulating tyrosinase and tyrosinase-related protein-1 (TRP-1) involved in the melanogenic pathway. A similar correlation between G-CSF and CCL20 expression in KC and the tyrosinase level in MC was also observed in the UVB-irradiated mouse skin. Our study reports for the first time that G-CSF and CCL20 might play a regulatory role in the KC's paracrine effects on UVB-mediated MC damage and also provides translational insights for the development of biomarkers for predicting susceptibility to photodamage.