Arzanol, a natural phloroglucinol α‐pyrone, protects HaCaT keratinocytes against H2O2‐induced oxidative stress, counteracting cytotoxicity, reactive oxygen species generation, apoptosis, and mitochondrial depolarization

Skin oxidative stress results in structural damage, leading to premature senescence, and pathological conditions such as inflammation and cancer. The plant‐derived prenylated pyrone–phloroglucinol heterodimer arzanol, isolated from Helichrysum italicum ssp. microphyllum (Willd.) Nyman aerial parts, exhibits anti‐inflammatory, anticancer, antimicrobial, and antioxidant activities. This study explored the arzanol protection against hydrogen peroxide (H2O2) induced oxidative damage in HaCaT human keratinocytes in terms of its ability to counteract cytotoxicity, reactive oxygen species (ROS) generation, apoptosis, and mitochondrial membrane depolarization. Arzanol safety on HaCaT cells was preliminarily examined by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and microscopic observation. The arzanol pre‐incubation (5–100 μM, for 24 h) did not induce cytotoxicity and morphological alterations. The phloroglucinol, at 50 μM, significantly protected keratinocytes against cytotoxicity induced by 2 h‐incubation with 2.5 and 5 mM H2O2, decreased cell ROS production induced by 1 h‐exposure to all tested H2O2 concentrations (0.5–5 mM), as determined by the 2′,7′‐dichlorodihydrofluorescein diacetate (H2DCFDA) assay, and lipid peroxidation (thiobarbituric acid reactive substances [TBARS] method). The 2‐h incubation of keratinocytes with H2O2 determined a significant increase of apoptotic cells versus control cells, evaluated by NucView® 488 assay, from the dose of 2.5 mM. Moreover, an evident mitochondrial membrane potential depolarization, monitored by fluorescent mitochondrial dye MitoView™ 633, was assessed at 5 mM H2O2. Arzanol pre‐treatment (50 μM) exerted a strong significant protective effect against apoptosis, preserving the mitochondrial membrane potential of HaCaT cells at the highest H2O2 concentrations. Our results validate arzanol as an antioxidant agent for the prevention/treatment of skin oxidative‐related disorders, qualifying its potential use for cosmeceutical and pharmaceutical applications.

H 2 O 2 concentrations (0.5-5 mM), as determined by the 2 0 ,7 0dichlorodihydrofluorescein diacetate (H 2 DCFDA) assay, and lipid peroxidation (thiobarbituric acid reactive substances [TBARS] method).The 2-h incubation of keratinocytes with H 2 O 2 determined a significant increase of apoptotic cells versus control cells, evaluated by NucView ® 488 assay, from the dose of 2.5 mM.Moreover, an evident mitochondrial membrane potential depolarization, monitored by fluorescent mitochondrial dye MitoView™ 633, was assessed at 5 mM H 2 O 2 .Arzanol pretreatment (50 μM) exerted a strong significant protective effect against apoptosis, preserving the mitochondrial membrane potential of HaCaT cells at the highest H 2 O 2 concentrations.Our results validate arzanol as an antioxidant agent for the prevention/treatment of skin oxidative-related disorders, qualifying its potential use for cosmeceutical and pharmaceutical applications.
Arzanol was efficient in protecting different cell lines and animals from oxidative stress (Rosa et al., 2011(Rosa et al., , 2017) ) and also exhibited radical scavenging activity in various in vitro systems of lipid peroxidation, surpassing or competing with standard lipophilic antioxidants, like vitamin E, and hydrophilic ones, like vanillyl alcohol (Rosa et al., 2007(Rosa et al., , 2011)).
The HaCaT cell line is a spontaneously immortalized human keratinocyte cell line from adult skin.These cells are physiologically similar to normal human keratinocytes and retain epidermal differentiation ability, reconstituting a stratified and differentiated epidermal structure after transplantation onto nude mice (Boukamp et al., 1988).
HaCaT cells are extensively used as a model to study the oxidative stress in human epidermal cells, and the H 2 O 2 -induced HaCaT cell damage model has been extensively applied to find antioxidant effects of pharmacological treatment of skin pathologies (Bae et al., 2014;Liu, Tang, et al., 2023;Zhang et al., 2020).
Hydrogen peroxide is a small electroneutral molecule, generated in numerous biological processes, that reacts with transition metal centers, selenoproteins, and with thiol-based proteins, such as catalase, glutathione peroxidases, and peroxiredoxins (Winterbourn, 2013(Winterbourn, , 2018)).H 2 O 2 is widely used to induce oxidative damage in cellular models, and a strong production of ROS has been observed in H 2 O 2treated cells (Park et al., 2020;Zhang et al., 2020).
In light of these findings, the current study, for the first time, evaluated the protective effect of arzanol in HaCaT cells against

| Arzanol extraction and isolation
Arzanol (Figure 1) was isolated from an acetone extract of H. italicum ssp.Microphyllum, collected in 2021 (Sardinia, Italy), according to literature (Appendino et al., 2007).A batch of the plant is stored in the phytochemistry laboratory of Novara (Italy) with the code HM-CA2021.Approximately 500 g of the dried aerial parts (leaves and flowerheads) were extracted two times with 2.5 L of acetone in a vertical percolator.After that, the extract was filtered under vacuum in a sintered filter to remove the vegetable material and the solvent was then evaporated with rotary evaporator to obtain a gummish residue.
This latter residue was dissolved in EtOAc-petroleum ether solution 1:1 to promote the precipitation of arzanol.Characterization was made by spectroscopic methods ( 1 H NMR, 13 C NMR, UV, IR, and HR-MS), and the arzanol structure was confirmed by comparison of its physical properties with scientific data reported by Appendino et al. (2007).The final purity of arzanol was 98%.

| Cell culture
Human keratinocytes HaCaT cell line was obtained from CLS-Cell Line Services (Eppelheim, Germany).The cells were cultured in high glucose DMEM supplemented with 10% FBS, 100 units/mL of penicillin, and 100 μg/mL of streptomycin and kept at 37 C in a water-saturated humidified incubator at 5% CO 2 .The cells were split once a week when the cells were confluent using Trypsin 0.25%-EDTA and were plated at a density of 1 Â 10 5 /mL and grown until reaching about 80% confluence in experimental studies.

| Arzanol protection against H 2 O 2 inducted cytotoxicity
MTT viability assay was also applied to determine the rate of protection of arzanol (24 h of pre-incubation) against the cytotoxic effect induced in HaCaT cells by H 2 O 2 exposure.Cells were seeded at a density of 10 5 cells/mL in 96-well plates in a complete culture medium.After 48 h of incubation, the cells were treated with 50 μM arzanol for 24 h.After the medium removal, keratinocytes were exposed for 2 h to different concentrations of H 2 O 2 (from 0.5 to 5 mM, in DMEM solution) in the absence (oxidized controls) and in the presence of arzanol.Cells were then subjected to the MTT assay as described above.Viability data were expressed as a percentage of respective control cells (untreated cells).The effect of the maximal amount of DMSO (0.5%) was also evaluated in this system.

| Intracellular ROS assay
The redox status of HaCaT keratinocytes in response to H 2 O 2 in the absence and in the presence of arzanol was determined.The intracellular ROS production was determined by the H 2 DCFDA assay (Sogos et al., 2021), a method based on the structure of 2 0 -7 0 dichlorofluorescein (DCF) that interacts with reactive species to lead to a fluorescence (Pavelescu, 2015).The acetyl groups of H 2 DCFDA enable penetration through the cell membrane and, into the cell, cytoplasmic esterases cleave it, releasing the reduced state of DCF (DCF-H 2 ) which reacts with intracellular reactive species and fluoresces (Pavelescu, 2015;Petretto et al., 2023).HaCaT cells were seeded in 96-well plates at 10 5 cells/mL density in 100 μL/well of complete culture medium and cultured for 48 h.Then, fresh medium without (control cells) and with 50 μM arzanol was added, and cells were incubated for a further 24 h.Next, the cells, after washing with phosphate-buffered saline (PBS) solution (pH 7.4), were incubated with 10 μM H 2 DCFDA in PBS, at 37 C for 30 min.After incubation, the H 2 DCFDA solution was discharged, and cells were washed before the addition of PBS alone in control cells or different H 2 O 2 concentrations (0.5, 1, 2.5, and 5 mM) in cells without (oxidized controls) and with arzanol pre-treatment.The effect on ROS generation was also evaluated in HaCaT cells after 24 h of incubation with only 50 μM arzanol.ROS production was detected and monitored (every 5 min) for 1 h by using an Infinite 200 Tecan microplate reader at a controlled temperature of 37 C.The reading was performed using an excitation wavelength of 490 nm and an emission wavelength of 520 nm.Data were collected and analysed using the Tecan I-control 1.5 V software.Fluorescence data were normalized to control cells.

| Thiobarbituric acid reactive substances (TBARS) assay
The antioxidant activity of arzanol against the H 2 O 2 oxidation in HaCaT cells was also monitored by the evaluation of its effect on the level of low-molecular-weight end products (mainly malondialdehyde, MDA) (TBARS method), formed during the decomposition of primary lipid peroxidation products (Aguilar Diaz De Leon & Borges, 2020;Rosa et al., 2007).HaCaT cells were cultured in a complete culture medium in 60 mm plates and a density of 1.5 Â 10 5 cells/mL in 4 mL/ plate for 48 h.Afterwards, the cells were incubated in the absence (control cells) and in the presence of 50 μM arzanol in a fresh medium for another 24 h.After the medium removal and washing with PBS solution (pH 7.4), keratinocytes, without (oxidized controls) and with arzanol pre-treatment, were exposed for 1 h at 37 C to different concentrations of H 2 O 2 (0.5, 1, 2.5, and 5 mM) in PBS solution.After PBS removal, 10% TCA (250 μL) was added and then cell pellets were transferred to a tube.Aliquots (500 μL) of 0.6% TBA were added, and cell samples were then incubated at 95 C for 45 min.After cooling on ice, the samples were centrifuged at 5000 rpm for 10 min, and the resulting pellets were dissolved in DMSO.TBARS production (Rosa et al., 2007) was detected using an Infinite 200 Tecan microplate reader.The reading was performed at 540 wavelengths.The collected data were analysed using the Tecan I-control 1.5 V software and normalized to control cells.

| Apoptosis and mitochondrial activity assay
The protective effect of arzanol against the changes in the mitochondrial membrane potential and apoptosis, inducted by H 2 O 2 oxidation in HaCaT cells, was evaluated through the NucView ® 488 and Mito-View™ 633 Apoptosis Assay Kit, as previously reported (Sogos et al., 2021).NucView ® 488 consists of a fluorogenic DNA dye and a DEVD substrate moiety.This complex, both non-fluorescent and nonfunctional, crosses the cellular membrane, and it can be cleaved by caspase-3/7 in the cytoplasm to release a high-affinity DNA dye that, after migration to the nucleus, dyes DNA with green fluorescence.
MitoView™ 633, a far-red fluorescent mitochondrial dye, enters the cell and accumulates in mitochondria, becoming fluorescent with staining dependently on the potential of the mitochondrial membrane (higher in healthy cells and lower in apoptotic cells) (Sogos et al., 2021).
HaCaT cells were seeded onto 96-well plates at 10 5 cells/mL and incubated for 48 h.Then, cells were treated for 24 h with 50 μM arzanol in a fresh medium.After incubation, the medium was carefully removed

| Effect on HaCaT cell viability (MTT assay)
The cytotoxic potential of arzanol was evaluated by using the colorimetric MTT assay, a method frequently used as an indicator of cell viability, proliferation, and cytotoxicity (Petretto et al., 2023).Figure 2 shows the viability, expressed as percent of the control, induced by incubation for 24 h with different amounts of arzanol (5-100 μM) (Figure 2A) and DMSO (0.05-1%) (Figure 2B) in HaCaT cells.
The results of this test (Figure 2A) showed that arzanol, after 24 h of incubation, did not decrease HaCaT cell viability at concentrations from 5 to 100 μM with regard to control cells.DMSO, used to dissolve arzanol, did not affect cell viability from 0.05% to 0.5%, whereas a 21% significant reduction in viability ( p < 0.01 versus control cells) was observed at 1% (Figure 2B).
Based on these data, a 50-μM concentration of arzanol, containing 0.5% of DMSO, was used as the tested maximal concentration of the phloroglucinol in successive experiments.

| Arzanol protection against H 2 O 2 inducted cytotoxicity
The arzanol protection against H 2 O 2 -inducted cytotoxicity was then assessed by MTT assay.

| Intracellular ROS
The effect of arzanol against the intracellular ROS generation induced in HaCaT keratinocytes by the H 2 O 2 treatment was determined by the H 2 DCFDA assay.

| TBARS assay
The arzanol antioxidant activity against the formation of secondary products of lipid peroxidation induced by H 2 O 2 in HaCaT cells was monitored by TBARS method.Figure 6 shows TBARS formation, expressed as percent of the control, measured in control cells, cells pre-incubated with arzanol 50 μM, and cells exposed for 1 h to different amounts of H 2 O 2 (0.5, 1, 2.5, and 5 mM) in the absence and in the presence (after 24 h-pre-incubation) of arzanol 50 μM.The exposure to H 2 O 2 induced a concentration-dependent rise in the secondary lipid oxidative products, and a marked significant ( p < 0.001) increase (188% with respect to control cells) was observed at the highest H 2 O 2 tested concentrations (5 mM).
The cells treated with arzanol showed TBARS values similar to control cells at all the H 2 O 2 tested concentrations, exhibiting significant protection versus the corresponding H 2 O 2 -oxidized cells at 2.5 ( p < 0.05) and 5 mM ( p < 0.001).
For the first time, in this study, the antioxidant effect of arzanol was natural extracts and compounds against oxidative stress in human epidermal cells and skin pathologies (Jiang et al., 2020;Mirata et al., 2023;Wang et al., 2022).In our experimental conditions, HaCaT cells were cultured in DMEM medium containing high calcium (Ca 2+ ) amount, a condition favorable for keratinocyte differentiation (Colombo et al., 2017).Calcium represents one the most important inductors of keratinocyte differentiation in the epidermis, increasing from the basal to the granular layer (Colombo et al., 2017).
At first, the cytotoxicity of arzanol on HaCaT cells was examined after 24 h of incubation.The results of the MTT assay showed that, at all tested concentrations, arzanol was not toxic for HaCaT cells.Moreover, confirming this, no difference in morphology was observed between arzanol-treated and control cells.According to our results, previous studies showed no cytotoxic effects of arzanol in peripheral blood mononuclear cells, obtained from healthy adult donors (Bauer et al., 2011), immortalized Vero cell line, and differentiated CaCo-2 cells (Rosa et al., 2011), as a model of the intestinal epithelium.Conversely, the phloroglucinol showed the ability to decrease viability in cancer cells as epithelial cancer cell lines (A549, RT-112, HeLa, CaCo-2) and B16F10 melanoma cells (Bauer et al., 2011;Deitersen et al., 2021;Rosa et al., 2017), indicating its selective cytotoxicity versus tumoral cells, maybe interacting with targets of the cell proliferation and survival signaling pathways (Rosa et al., 2017).
Then, the protective effect of 50 μM arzanol (after 24 h of preincubation) was assessed by MTT assay against the rate of cytotoxicity induced in HaCaT cells by 2 h oxidation with the peroxide H 2 O 2 .In our experimental conditions, the exposure of keratinocytes to H 2 O 2 markedly reduced viability in a concentration-dependent manner from the dose of 0.75 to 5 mM, with a range of 30-50% cell viability observed at 1 mM.A dose-dependent decrease in HaCaT cell viability was previously reported in HaCaT cells exposed to H 2 O 2 (100-500 μM) for 12 h, by MTT assay, with a 50% cell mortality at the concentration of 350 μM H 2 O 2 (Zhang et al., 2020), whereas a survival rate of about 60% was reported by Park et al. (2020) for HaCaT cells exposed to 500 μM H 2 O 2 for 24 h.In our study, the H 2 O 2 cytotoxic effect was also confirmed by microscopic observation, which allowed to assess the presence of apoptotic features and decreased cell density.Similar  (Bayati et al., 2011;Farshori et al., 2021;Liao et al., 2022 (Park et al., 2020).
H 2 O 2 is widely used to induce oxidative damage in cellular models (Park et al., 2020;Zhang et al., 2020).Because of its small size and lack of charge, H 2 O 2 crosses through the membrane barrier, so exposed cells cannot block its entry, and, in vivo, can diffuse over long distances into different organs (Lenzen et al., 2022;Mahaseth & Kuzminov, 2017).In the cells, H 2 O 2 can interact with free Fe 2+ ions and hydroxyl radical (HO • ) are produced by Fenton's reaction, which is thought to be the main mechanism for oxidative damage (Bae et al., 2014;Bayati et al., 2011;Ransy et al., 2020;Yan et al., 2022).
The HO • radicals are extremely toxic, highly reactive with an ultrashort half-life (10 À9 s), and capable of destroying any biological structure; however, their action is limited to the site of generation (Lenzen et al., 2022;Mahaseth & Kuzminov, 2017).Hydroxyl radicals damage DNA, acting on aromatic structures of RNA/DNA purines and pyrimidines by addition reactions at double bonds, attack sugars such as deoxyribose of DNA, and can act on biological membranes by the oxidative attack on polyunsaturated lipids by hydrogen extraction, triggering the autocatalytic process of lipid peroxidation (Bayati et al., 2011;Lenzen et al., 2022;Mahaseth & Kuzminov, 2017).
In this study, the H 2 O 2 oxidation significantly induced a dosedependent ROS increase (H 2 DCFDA assay) in keratinocytes during 1 h of exposure, compared to the basal level of control cells, in line with previous studies (Park et al., 2020;Zhang et al., 2020).Preincubation for 24 h with arzanol 50 μM significantly reduced ROS because of hydrogen atom donor properties (Rosa et al., 2007(Rosa et al., , 2011)).
Moreover, arzanol, at non-cytotoxic concentrations, exerted noteworthy protection on tert-butyl hydroperoxide (TBH)-induced oxidative damage in a line of fibroblasts derived from monkey kidney (Vero cells) and in human intestinal epithelial cells (Caco-2) (Rosa et al., 2007(Rosa et al., , 2011)).The antioxidant activity of arzanol was demon- lipids induced by Fe-NTA at 1 h of oxidation (Rosa et al., 2017).The observed protective effect of arzanol in HaCaT cells was probably attributable to its scavenging ability against the H 2 O 2 -induced production of HO • and ROO • radicals.
Previous studies evidenced that the ROS production by the H 2 O 2 oxidation induces mitochondria-mediated apoptosis (Park et al., 2020;Zhang et al., 2020).An increase in apoptotic cells (approximately ninefold higher than control cells) was previously reported for HaCaT cells exposed to 500 μM H 2 O 2 for 24 h (Park et al., 2020).Moreover, an increase (2.4-fold) in the caspase-3 expression was reported in HaCaT cells after 4 h of exposure to 350 μM H 2 O 2 (Zhang et al., 2020), coupled with a marked decrease in the mitochondrial membrane potential.Mitochondrial membrane potential drop is a landmark event of early apoptosis, and the expression of caspase-3 protein promoted the acceleration of the mitochondrial apoptotic pathway (Sogos et al., 2021;Zhang et al., 2020).Therefore, the protective effect of H 2 O 2 -induced oxidative damage.To this goal, experiments were designed to evidence the ability of the phloroglucinol to reduce cytotoxicity and ROS generation because of the peroxide.In addition, the arzanol protection on apoptosis and mitochondrial membrane potential depolarization observed in oxidated HaCaT cells were also explored.The results of this work were expected to provide indications on the nature of the molecular mechanism(s) underlying the antioxidative activity of arzanol, for future potential dermatological, cosmetic, and pharmaceutical applications in skin oxidative-related diseases and other related conditions.2 | MATERIALS AND METHODS2.1 | MaterialsCell culture materials, such as Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, and trypsin 0.25%-EDTA were all purchased from EuroClone (Pero, MI, Italy).Dimethyl sulfoxide (DMSO), 3thiobarbituric acid (TBA), and hydrogen peroxide (H 2 O 2 ) solution 30% (w/w), were purchased from Merck Life Science (Milan, Italy).NucView ® 488 and MitoView ™ 633 apoptosis assay kits were obtained from Biotium (Fremont, CA, USA).
and cells were treated with NucView ® 488 and MitoView™ 633 (mitochondrial dye) probes, according to the manufacturer's instructions, and different concentrations of H 2 O 2 (0.25, 0.5, 0.75, 1, 2.5, and 5 mM) in fresh medium and then incubated at 37 C.The microscopic observations were finally made after 2 and 24 h of incubation using a ZOE™ Fluorescent Cell Imager.Instrument gain and offset values were adjusted using control (untreated cells) and remained constant for all subsequent experiments.Image analysis of NucView ® 488 and Mito-View™ 633 images was performed with ImageJ software (version 1.53e).Briefly, background fluorescence was subtracted from images, and histogram values of fluorescence intensity were expressed as percent of control cell fluorescence.For each sample, eight images (from two different experiments) were processed for image analysis.

Figure
Figure2Cshows representative images of phase contrast microscopy of control HaCaT cells and cells treated for 24 h with arzanol 50 μM or DMSO 0.5%.Control (untreated) keratinocytes, at a non-complete confluency, appeared enlarged, elongated, and linked to each other.The arzanol treatment did not induce, from 5 to 50 μM, a change in cell morphology and a reduction in the cell number, and treated cells appeared very similar to control cells.The vehicle molecule DMSO did not affect HaCaT cell morphology from 0.05% to 0.5%, and DMSO-treated cells showed the same microscopic features as control cells.MTT assay was also applied to evaluate the H 2 O 2 effect on HaCaT cell viability.Figure3Ashows the viability (as percent of the control) induced by 2 h-incubation with different amounts of H 2 O 2 (0.05-5 mM) in HaCaT cells, whereas the corresponding morphological images of phase contrast microscopy are reported in Figure3B.The exposure to H 2 O 2 did not markedly change the viability rate with respect to control cells from 0.05 to 0.25 mM of oxidant.At higher tested concentrations (from 0.5 to 5 mM), H 2 O 2 induced a significant cytotoxic effect in a concentration-dependent manner, with cell viability reduction values in the range of 21-83%.Microscopic observations (Figure3B) did not show an evident cell morphology and density alteration in the H 2 O 2 dose range of 0.05-0.75mM, whereas, at the high concentrations, areas with a decreased cell density and cell-to-cell packing were noted.Moreover, some cells displayed an ill-defined cytoplasm, changes in size, pyknotic nuclei, and fragmentation.

Figure 4
Figure 4 shows the viability, expressed as percent of control cells, induced by 2-h incubation with different amounts of H 2 O 2 (0.5-5 mM) in HaCaT cells measured in control oxidized samples (OX) and in samples oxidized after a 24 h-pre-incubation with 50 μM arzanol.A slight viability reduction was observed in the range of 0.5-1 mM H 2 O 2 concentration both in the absence and in the presence of arzanol.A significant protection against H 2 O 2 -induced cytotoxicity was evident at the H 2 O 2 concentrations of 2.5 mM ( p < 0.01 versus the respective oxidized control) and 5 mM ( p < 0.05), with arzanoltreated cells showing viability, with to control cells, of about 55-58%.

Figure 5
Figure 5 shows the ROS-induced fluorescence, expressed as percent of the control, measured during 1 h in HaCaT control cells, cells pre-incubated with arzanol 50 μM, and cells exposed to different amounts of H 2 O 2 (0.5, 1, 2.5, and 5 mM) in the absence (oxidized samples) and in the presence of arzanol 50 μM (after 24 h-preincubation).The H 2 O 2 -treatment induced a significant ( p < 0.001) increase in cell fluorescence during 60 min time of exposure compared to the ROS basal level of control cells, and the highest relative intensity of fluorescence was observed in HaCaT keratinocytes at 5-mM concentration.

Finally
, the effect of arzanol against apoptosis and changes in the mitochondrial membrane potential induced by H 2 O 2 oxidation in HaCaT cells was evaluated.

Figure
Figure 7A shows images of phase contrast, red emission (MitoView 633), and green emission (NucView 488) obtained for HaCaT control cells, cells pre-incubated with arzanol 50 μM, and H 2 O 2 -oxidized cells in the absence and in the presence of arzanol 50 μM (24 h-pre-incubation).Quantitative data of fluorescence intensity (expressed as a percentage of control cells) are depicted in Figure 7B.Observations made after 2 h of incubation with the oxidant ascertained a slight increase, with respect to control cells, of the apoptotic signal (green staining) in cells incubated with H 2 O 2 from 0.5 to 1 mM, whereas the treatment with 2.5 and 5 mM H 2 O 2 determined a significant (p < 0.001) increase in apoptotic cell number compared to the control, with values of 260% and 1031% of control, respectively.Moreover, associated altered cell morphology (brightfield) was observed at the highest tested H 2 O 2 concentrations.H 2 O 2 oxidation did not induce an evident change in the red fluorescence signal in the range of 0.5-2.5 mM, whereas the dose of 5 mM markedly reduced the red emission, indicating a depolarization of mitochondrial membrane potential.Interestingly, a strong significant ( p < 0.001) protective effect of arzanol 50 μM was evident against apoptosis induced in HaCaT cells by 5 mM H 2 O 2 , and arzanol-treated cells showed a fluorescent intensity of 280%, about three times lower than the corresponding oxidized sample.However, the arzanol treatment reduced, unless not significantly, the green signal also at the other oxidant doses.Moreover, the phloroglucinol preserved HaCaT cells against mitochondrial membrane depolarization induced by 5 mM H 2 O 2 addition, with arzanol-treated cells showing red fluorescence intensity similar to that of control cells.HaCaT cells treated with only 50 μM arzanol for 24 h showed a lower apoptotic profile than control cells.These results demonstrated a protective role of arzanol in HaCaT cells against apoptosis and mitochondrial membrane depolarization induced by H 2 O 2 oxidation.
evaluated in human HaCaT keratinocytes against the oxidative stress induced by H 2 O 2 with the aim of exploring its beneficial potential effects on human skin oxidative conditions.Cultured human HaCaT cells, being physiologically similar to normal human keratinocytes, represent a cell model amply used to assess the protective effect of F I G U R E 4 Viability, expressed as percent of control cells (0), induced by incubation for 2 h with different amounts of H 2 O 2 (0.5-5 mM) in HaCaT cells measured in control oxidized samples (OX) and in samples oxidized after a 24 h-pre-incubation with 50 μM arzanol (MTT assay).Three independent experiments are performed, and data are presented as mean and SD (n = 21).*** = p < 0.001 versus control (0) (one-way ANOVA and Bonferroni post test); = p < 0.01, = p < 0.05 between oxidized cells in the absence and in the presence of 50 μM arzanol (Student's unpaired t-test with Welch's correction).ANOVA, analysis of variance; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

F
I G U R E 5 ROS-induced fluorescence, expressed as percent of the control (Ctrl), measured for 1 h, at different time points, in HaCaT control cells (Ctrl), cells pre-incubated with arzanol 50 μM (ARZ), and cells exposed to different amounts of H 2 O 2 (0.5, 1, 2.5, and 5 mM) in the absence (oxidized samples, OX) and in the presence (after 24 h-pre-incubation) of arzanol 50 μM (OX + ARZ).Three independent experiments are performed, and data are presented as mean and SD (n = 9).Evaluation of the statistical significance of differences between groups was performed by one-way ANOVA followed by the Bonferroni multiple comparisons test.At each time point: *** = p < 0.001, ** = p < 0.01, * = p < 0.05 versus the respective Ctrl; = p < 0.001 versus the respective OX samples.ANOVA, analysis of variance; ROS, reactive oxygen species.F I G U R E 6 TBARS formation, expressed as percent of the control (Ctrl), measured in HaCaT control cells (Ctrl), cells pre-incubated with arzanol 50 μM (ARZ), and cells exposed for 1 h to different amounts of H 2 O 2 (0.5, 1, 2.5, and 5 mM) in the absence (oxidized samples, OX) and in the presence (after 24 h-pre-incubation) of arzanol 50 μM (OX + ARZ).Three independent experiments are performed, and data are presented as mean and SD (n = 12).*** = p < 0.001 versus the Ctrl (one-way ANOVA and Bonferroni post test); = p < 0.001, = p < 0.05 between oxidized cells in the absence and in the presence of 50 μM arzanol (Student's unpaired t-test with Welch's correction).TBARS, thiobarbituric acid reactive substances.morphological alterations were observed in other cell types exposed to H 2 O 2 -damage, such as human umbilical vein endothelial cells, neuroblastoma cell line SK-N-MC, and HepG2 cells
production with respect to H 2 O 2 -oxidized HaCaT cells at all oxidation times and tested oxidant concentrations, confirming the antioxidant properties of this phloroglucinol.Interestingly, the treatment with arzanol 50 μM significantly reduced the level of internal physiologically ROS generation in HaCaT cells with respect to control cells.Then, the protective effect of arzanol against the lipid peroxidation induced by H 2 O 2 in HaCaT cells was monitored by TBARS assay.TBARS analysis is amply used to measure the formation of the secondary byproduct of lipid peroxidation, primarily composed of MDA (Aguilar Diaz De Leon & Borges, 2020).Our data showed a significant antioxidant activity of arzanol against lipid peroxidation, with a remarkable reduction in TBARS generation, evident at the highest H 2 O 2 concentrations.According to our findings, previous studies evidenced the antioxidant properties of the natural compound arzanol in different in vitro chemical systems of lipid peroxidation and in cultured cell models of oxidative stress.The phenolic compound preserved the oxidative modification of pure lipids (linoleic acid and cholesterol), liposomes, and human low-density lipoprotein (LDL), reducing the formation of their oxidative products (hydroperoxides, oxysterols, and MDA), due to its noteworthy efficacy in scavenging lipid peroxyl radicals (ROO • ) strated also in the ferric-nitrilotriacetate (Fe-NTA) model of in vivo oxidative stress.Fe-NTA administration induces in animals a state of sustained oxidative stress in association with iron excess that leads to high production of free radicals, especially HO • radicals (Rosa et al., 2005, 2017).The intraperitoneal administration of arzanol exerted a protective effect on the oxidative degradation of plasmatic S C H E M E 1 Schematic representation of the protective effect of arzanol against H 2 O 2 -induced oxidative stress in in HaCaT cells.[Colour figure can be viewed at wileyonlinelibrary.com] Scheme 1.