Further assessment of Salvia haenkei as an innovative strategy to counteract skin photo-aging and restore the barrier integrity

Safe profile of SH extract and antiaging properties

Oxidative stress in skin plays a major role in the aging process and in the pathogenesis of several skin disorders [22]. Matic et al. already demonstrated the anti-senescence activity of Salvia haenkei also correlated to its scavenging properties [18]. These protective effects might be exploited to develop a novel skin-repairing treatment able to preserve the integrity of the barrier. At the aim of deeply investigate SH properties, the extract activity on cell viability and ROS generation in HaCaT cell model was firstly verified.

The cell viability assay was performed to address possible cytotoxic effects of the Salvia haenkei extract on keratinocytes. HaCaT cells were treated with SH extract (0.01–10 μg/ml) for 24 and 48 hours. Cell viability was assessed by Trypan blue Exclusion Assay. Figure 1A, 1B shows that treatment with SH extract does not affect cell viability confirming the safe profile of the extract on HaCaT cells at the used experimental conditions.

The activity of Salvia haenkei extract on ROS generation was assayed in HaCaT cells after 3-24 hours of treatment and before and after H₂O₂ exposure. Results in Figure 2A show that SH is able to reduce basal ROS levels within the cells, particularly for the 3 hour-treatment time. Besides a significant increase in ROS levels after H₂O₂ stimulation (Figure 2C), SH is able to prevent the production of ROS. SH extract (0.01 μg/ml) significantly reduced (-25%) ROS generation induced by the oxidative stimulus (Figure 2B), showing an effect comparable with NAC after 3 hours of treatment. 24 hours of SH treatment appear to be more effective in counteracting the ROS increment, significantly reducing ROS levels at 1 and 10 μg/ml (Figure 2B). These results confirm Salvia haenkei antioxidant effects, as previously observed [18], showing an antioxidant basal effect already starting from short treatments; the effect in preventing oxidative stimulus-induced increase is most relevant for longer treatment times.

It is reported that ultraviolet radiations induce increase in intracellular level of ROS in human skin causing damages and premature aging and other pathologies [23–25]. Therefore, the effect of SH extract on UVB-mediated ROS production has been evaluated. As expected, results in Figure 3C show a significant increase in intracellular ROS level following UVB stimulation. Treatment with SH, which induces a tendency to reduce basal ROS levels within the cells (Figure 3A), is able to prevent the increase induced by UVB stimulation (Figure 3B). 3 hours of SH treatment induce a tendency to reduce ROS levels; a longer SH treatment is more effective in counteracting the oxidative stress, showing significant decrease as reported in Figure 3A. These data underline the antioxidant activity of Salvia haenkei also in UVB mediated oxidative stress.

As mentioned, increasing evidence suggests that senescent cells accumulate in chronologically and photo-aged skin, contributing to age related-skin alterations and pathologies [26]. The effect of Salvia haenkei was therefore evaluated on p21 and p27 senescence markers (which prevent phosphorylation of the retinoblastoma protein (Rb) and with inhibitory activity on CDK2-cyclin E, respectively [27]) after photo-stress induced by ultraviolet radiation. Results in Figure 4 show that UVB irradiation induces a significant increase in p21 and p27 mRNA levels as compared to the unstimulated control. A similar trend in prevention of increase is observed in cells treated with SH. SH 1 μg/ml induces a significant reduction of p21 levels compared to UVB stimulated control, while the other concentrations induce a tendency to reduce (Figure 4A). P27 levels are significantly reduced by pretreatment with SH 0.01, 0.1 and 10 μg/ml (Figure 4B).

It is also established that senescent cells negatively affect the microenvironment by secreting a pro-inflammatory mixture of chemokines, cytokines, growth hormones and proteases, known as senescence-associated secretory phenotype (SASP) [28]. Results in Figure 5 show that UVB stimulation induces a significant increase in IL1α (A), IL6 (B), and IL18 (C) mRNA levels with respect to the unstimulated control. Pretreatment with SH presents a strong activity in counteracting the increase in cytokine levels. As shown in Figure 5A, SH 10 μg/ml significantly decreased IL1α levels; the increase in IL6 is significantly counteracted by SH 0.01,0.1 and 10 μg/ml (Figure 5B) while all the tested concentrations are able to significantly reduce IL18 levels (Figure 5C).

Skin barrier maintenance

Given the above reported results on an epithelial cell line, the effect of SH extract on several proteins and processes involved in skin barrier maintenance and tissue damage repair was evaluated.

Sirtuin-1 expression

Several in vitro studies reported that UVB radiations induce a decrease in Sirtuin1 (SIRT1) protein levels of human fibroblasts, indicating an involvement of SIRT1 in the UVB-mediated damage [29]. So, the effect of Salvia haenkei extract on Sirtuin1 protein expression was evaluated by western blot assay (Figure 6). As expected, UVB stimulation induced a significant decrease in SIRT1 protein expression in HaCaT cells. The activity of SH extract on SIRT1 levels was therefore investigated. Interestingly, results show that 1 μg/ml SH extract, as compared to control, induced an increase of SIRT1 expression (+40%) under basal conditions, indicating a potential stimulating-effect on SIRT1 levels. As expected, UVB stimulation induces a significant reduction of SIRT1 levels. Results show that the pretreatment with 0.01 μg/ml SH extract exhibits ameliorative effect in Sirtuin 1 levels, as compared to the UVB-treated control.

Wound healing assay

Keratinocytes are the main constituents of epidermis, providing a barrier between the organism and the environment. Therefore, their proliferation and migration play a key role in the maintenance of an effective barrier and in wound healing following injuries [30, 31]. Thus, the effect of SH on HaCaT cell migration was evaluated. Images in Figure 7A show that treatment with SH leads to a reduction in the thickness of the scratch indicating an effect on HaCaT cell migration. Figure 7B reports kymograms construction of the migration velocity of HaCaT cells; images show a concentration-dependent increase in cells migration. Graph in Figure 7C indicates a significant increase in cells migration speed after 4-8 hours of treatment with 0.01 μg/ml SH extract. The maximum increase is reached in the 8-12 hours interval, where the rate in cell migration speed of cells is about 165% with respect to the control. These data, as the results obtained on cell proliferation, indicate that the lowest concentration of SH extract is able to induce an increase in cell migration speed not related to a hyperproliferative phenotype.

Tight junctions’ maintenance

As well as stratum corneum, tight junctions (TJs) play an important role in skin integrity, acting as a barrier for water and solutes, and being involved in differentiation, proliferation, cell polarity, and signal transduction processes of cells. Recent studies demonstrated that UV-light alters expression and localization of single proteins of TJ structure, which in turn disrupts the barrier function of skin [32].

In order to evaluate Salvia haenkei extract’s effect as possible protective agent against TJs disruption, expression of occludin was measured by immunocytochemistry following exposure to UVB radiation. Images in Figure 8A, 8B show that treatment with both concentrations of SH induces an increase in occludin protein expression, promoting junction’s formation useful to the maintenance of the epidermal barrier integrity. As expected, following exposure to UVB radiation, cells show an irregular and not defined membrane ring (Figure 8A). SH treatment, especially at the higher concentration, preserves the morphology of the membrane maintaining the structure comparable to the unstimulated control. SH extract promotes junction’s reinforcement and prevents the damage induced by UVB exposure, maintaining the functionality of the skin barrier.

Filaggrin expression

The effect of Salvia haenkei extract on filaggrin (FLG) protein expression was evaluated by immunocytochemistry. Filaggrin is a structural protein fundamental in the development and maintenance of the skin barrier. Results in Figure 9A, 9B show a concentration-dependent increase in filaggrin protein expression following treatment for 24h with SH extract. Furthermore, the expression of F-actin protein indicates a regular distribution of cells, following the treatment with SH, which leads to a well-organized structural continuity of the epithelial barrier. These data indicate that SH has a beneficial impact on skin barrier, improving filaggrin expression, which is essential for the correct formation and function of the skin.

Metalloproteinase expression

Skin exposure to ultraviolet radiations, induces impairments in the extracellular matrix (ECM) causing skin wrinkling, sagging, and laxity, the major characteristics of skin aging. Matrix metalloproteinases (MMPs) are the principal responsible for degrading the ECM components such as collagen, elastin, fibronectin, and proteoglycans [33]. A large amount of studies underlines that skin exposure to UV irradiation induces upregulation of different MMPs, impacting skin appearance and health [34–37].

Results in Figure 10 show that UVB radiation increases the mRNA expression of the gelatinase MMP-2 which degrades basement membrane collagens and denatured structural collagens [38]. Treatment with SH is able to significantly reduce MMP-2 mRNA levels (SH 0.01-0.1-10 μg/ml) indicating activity of SH extract in the maintenance of the structural integrity of the skin, damaged by photo-stress.

Cell culture and UVB irradiation

HaCaT cells (human spontaneously immortalized keratinocytes) were cultured in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum Life-Technologies, Waltham, MA, USA), 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Lonza, Basilea, Switzerland). Cells were maintained under a humidified atmosphere of 5% CO₂ in air and incubated at 37° C.

When requested by the experimental protocol, cells were exposed to 30 KJ/m² UVB radiation, supplied by a Philips Medical Narrowband UVB PL-S lamp. To prevent light absorption by tissue-culture medium, this was removed prior to irradiation, and replaced with DMEM FBS and red phenol free, following the irradiation protocol [47]. After UVB irradiation, cells were fed with fresh growth medium or used for experiment following the protocol.

Cell viability - Trypan blue exclusion assay

35 x10³ cells were plated on 12 well plates and, following overnight incubation, were exposed to different concentrations of Salvia haenkei hydroethanolic extract according to experimental protocols. The extract has been kindly provided by IBSA Farmaceutici Italia (Lodi, Italy) and presents a content of the marker rosmarinic acid of 0.6% w/w by HPLC. After treatment, cells were washed, detached with 0.25% trypsin-0.2% EDTA and suspended in trypan blue (Sigma-Aldrich, St Louis, MO, USA) at 1:1 ratio in medium solution. Cells were counted using a chamber Burker hemocytometer.

ROS fluorescence assay

ROS were quantified using 2′,7′-dichlorofluorescin-diacetate (H₂-DCF-DA, Sigma-Aldrich, St Louis, MO, USA), as previously described [48]. Briefly, cells (5x10³) were seeded into 96-well plates, allowed to adhere overnight and then exposed to SH extract for 3 or 24 hours. N-acetylcysteine 5mM (Sigma-Aldrich, St Louis, MO, USA) was used as positive control.

For the oxidative stress condition, the ROS level was measured after addition of 50 μM H₂-DCF-DA in absence or presence of H₂O₂ [48]. DCF fluorescence intensity was measured at excitation 485 nm—emission 535 nm, using a Multilabel Plate Reader VICTOR X3 (PerkinElmer, Waltham, MA, USA).

For the evaluation of ROS levels in UVB stress conditions, the protocol was slightly modified, by incubating cells in SH treatments - 50 μM H₂-DCF-DA solution in PBS. Following 25 minutes of incubation at 37° C, the plate was exposed to UVB radiations and the DCF fluorescence intensity was immediately measured at excitation 485 nm—emission 535 nm, using a Multilabel Plate Reader VICTOR X3 (PerkinElmer, Waltham, MA, USA).

Fold increase in ROS production was calculated using the equation: $\left(F_{treatment}-F_{blank}|F_{control}-F_{blank}\right)$, where F is the fluorescence reading.

RNA expression/quantitative real-time PCR

10x10⁴cells were seeded into 6-well plates, and, following overnight incubation, were exposed to SH extract for 24 hours. UVB stimulation was performed as already described, and SH treatment in complete medium was applied for another 16 hours.

RNA was isolated with the TRIzol method (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and retro-transcription was performed with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA USA) according to the manufacturer’s instructions. Quantitative PCR (qPCR) reactions were performed with a QuantStudio™ 5 (Invitrogen) using Power SYBR™ Green PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA USA) and the specific primers reported below.

Primer sequences were obtained from PrimerBank (http://pga.mgh.harvard.edu/primerbank/index.html).

GAPDH expression level was used as a reference for the normalization of each value level. The primer sequences used were as follows: p21 forward, 5’-TGTCCGTCAGAACCCATGC-3’; reverse, 5’-AAAGTCGAAGTTCCATCGCTC-3’, p27 forward, 5’-TAATTGGGGCTCCGGCTAACT-3’; reverse, 5’-TGCAGGTCGCTTCCTTATTCC-3’; IL-1α forward, 5’-GACGGTTGAGTTTAAGCCAATCC-3’; reverse 5’-CAGGAAGCTAAAAGGTGCTGAC-3’; IL-6 forward, 5’-TACATCCTCGACGGCATCTC-3’; reverse, 5’-TGCCTCTTTGCTGCTTTCAC-3’. IL-18 forward, 5’-CCAAGGAAATCGGCCTCTATTTG-3’; reverse, 5’-ATATGGTCCGGGGTGCATTATC-3’; MMP2 forward, 5’-TACAGGATCATTGGCTACACACC-3’; reverse, 5’-GGTCACATCGCTCCAGACT-3’; GAPDH forward 5’-AATCCCATCACCATCTTCCA-3’; reverse, 5’- TGGACTCCACGACGTACTCA-3’.

Immunoblot assay

6-multiwell plates were seeded with 75x10³ cells and incubated overnight. Cells were then treated according to the protocol and then lysed with ice-cold lysis buffer, supplemented with the protease inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany). Cell lysates were then centrifuged at 14000 rpm for 15 minutes at 4° C and the supernatant protein content was determined by the Lowry procedure (Bio-Rad DC Protein Assay, MA, USA).

Equal amount of protein was loaded on a polyacrylamide gel and electrophoretically separated in running buffer. After electrophoresis, the proteins were blotted onto a Hybond-P PVDF membrane (Amersham Biosciences, Buckinghamshire, UK). After blocking with a 10% skim milk solution, the membrane was exposed to the primary antibody anti-SIRT1 (1:250; Sigma-Aldrich, St Louis, MO, USA), and, following overnight incubation, was washed and exposed to HRP-conjugated anti-rabbit secondary antibody (1:3500; PerkinElmer, MA, USA). The signal was visualized with an enhanced chemoluminescent kit (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer's instructions and analyzed by Molecular Imager VersaDoc MP 4000 (Bio-Rad, Hercules, CA, USA). All the proteins were normalized to calnexin (1:1000; Santa Cruz Biotechnology INC, Santa Cruz, CA, USA).

Wound healing assay

10x10⁴ cells were seeded in 4-well culture chambers with glass bottom (Sarstedt AG & Co, Nümbrecht, Germany) pre-coated with collagen. A two-wells silicone insert with a defined cell-free gap was used to mechanically form a cell-free area. Once reached the surface confluence, the silicon insert was removed, and cells were treated with SH extract 0.01 and 0.1 μg/ml. The HaCaT migration in the cell free gap was recorded for 24 hours using an in live cells time lapse confocal microscope LSM 800 software ZEN 2.1 blue edition (Carl Zeiss, Jenza, Germany), magnification 20X. Data were analyzed with the dedicated software.

Immunofluorescence microscopy

10x10⁴ cells were seeded on glass coverslips precoated with collagen in 24-well plates, allowed to attach overnight, and treated with SH extract according to protocol. For detection of β-catenin and occludin proteins, cells were exposed to UVB radiation, as described in the protocol above. After irradiation, cells were fed with fresh growth medium or used for experiment following the protocol. For immunofluorescence assay, 4 hours after the UVB irradiation, cells were washed, fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100 in PBS and stained for 1 hour at 37° C with different protein-specific antibody: mouse monoclonal anti-occludin Invitrogen Life Technologies, Waltham, MA, USA mouse anti-filaggrin (Sigma-Aldrich, St Louis, MO, USA). After PBS wash, cells were incubated with secondary antibodies/fluorescein isothiocyanate (Alexa Fluor 488 anti-mouse or Alexa Fluor 536 anti-rabbit immunoglobulin G, Molecular Probes, Invitrogen Life Technologies, Waltham, MA, USA) and with phalloidin for 1 hour at room temperature. Following 10 minutes of treatment with RNase, the coverslips were mounted on glass slides by using Mowiol 40–88 (Sigma-Aldrich, St Louis, MO, USA) added of propidium iodide. Images were acquired through confocal microscope LSM 800, magnification 60X, software ZN 2.1 blue Edition (Carl Zeiss, Jenza, Germany) and quantified using ImageJ software.

Statistical analysis

The statistical analysis was performed using GraphPad Prism version 7.02 for Windows (GraphPad Software, San Diego, CA, USA). Unless stated otherwise, results are presented as mean ± SEM. The Student’s t-test was used and p values < 0.05 were considered as statistically significant.