Introduction

Natural thermal waters have been used for centuries as part of the healing process and the treatment of the diseases. Although there is evidence supporting that thermal mineral water treatment was clinically effective in a variety of dermatological conditions such as atopic dermatitis, psoriasis, and urticaria, there is still a lack of knowledge on the mechanism of effectiveness at the cellular and molecular level. Researchers have recently published an increasing number of in vitro studies providing some insight into the external application of thermal waters on the skin (Joly et al. 2014). Recent in vitro studies with the cell cultures and protein imaging revealed information regarding anti-inflammatory, antioxidant, UVB-protective, anti-allergic, and antiangiogenic properties of these waters (Jung et al. 2009; Varga et al. 2015). The French cosmetic industry has introduced dermocosmetic products formulated with thermal mineral waters (Beauvais et al. 1998; Ghersetich et al. 2001; Ferreira et al. 2010). Researchers reported a thermo-mineral water with known anti-allergic and anti-inflammatory properties which was claimed to be due to inhibition of the mast cell activation (Joly et al. 1998). The anti-inflammatory effect of another natural water on keratinocyte function of human Langerhans cells was also reported (Staquet et al. 2002). The treatment with a mineral water from Korea decreased the expression of pro-inflammatory cytokines associated with the Toll-like receptor stimulation to the HaCaT cells (Lee et al. 2012). A commercial thermal water from France was reported to protect the skin from dehydration based on TauT and SVCT1 expression (Verdy et al. 2012). Thermo-mineral waters were reported to suppress ROS in the keratinocytes after UVB exposure (Joly et al. 2014).

Although the above studies partially revealed the interaction between mineral waters and the skin at cellular level, there is still a lack of understanding of this interaction. Therefore, there is a need for further research on the mechanisms of beneficial effects of external application of thermo-mineral waters. The use of cell cultures is a fast, reliable, and precise method for determining the mechanism of these effects. Therefore, the purpose of the present study was to investigate the anti-inflammatory and anti-angiogenic effects of two different thermal mineral waters from Turkey in human keratinocytes (HaCaT).

Materials and methods

Chemical analysis of thermal waters

Thermal mineral waters were taken from the two traditional and historical thermal resorts of Turkey: Bursa and Bolu. Bursa is a well-known thermal town located in the north west of Turkey south coast of Marmara Sea. The thermal mineral sources of Bursa are being used for treating mostly rheumatic diseases for centuries. Bolu is located in the central Anatolia where one of the thermal water hospitals is being in service for rehabilitation of musculoskeletal. The physicochemical analyses of the thermal mineral waters (BJ1, Bursa, Turkey; BG, Bolu, Turkey) were carried out at the balneology laboratory of Istanbul University. All the analyses were performed based on the Standard Methods for the Examination of Water and Wastewater (Federation WE 2005).

Cell culture

The human keratinocyte cells (HaCaT) were maintained in high-glucose Dulbecco’s Modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM l-glutamine, and 100 U/mL gentamicin in an incubator at 37 °C in a humidified atmosphere at 5% CO2 (Supplements and media, Sigma Aldrich, USA).

Cytotoxicity assay

The Cell Proliferation Kit II (XTT) (Roche Diagnostics, Germany) was used for cell viability. 104 cells were seeded 96-well culture plates. After 24 h, the medium was replaced and cells were exposed to different concentrations of thermal waters (50, 25, and 10%) for 48 h. Fifty microliters XTT reagent and activator reagents were added and cells were incubated at same condition for 4 h in order that XTT reagent was reduced to formazan compound. The absorbance was read in microplate reader (BIO-RAD, Japan) at 450 nm with 620 nm reference wavelength. Cell survival was calculated from the equation:

$$ \mathrm{Cell}\ \mathrm{viability}\ \left(\%\right)=\left[\mathrm{Mean}\ \mathrm{OD}\ \mathrm{of}\ \mathrm{the}\ \mathrm{test}\ \mathrm{group}/\mathrm{Mean}\ \mathrm{OD}\ \mathrm{of}\ \mathrm{control}\ \mathrm{group}\right]\times 100 $$

Reverse transcription

HaCaT cells were incubated for 3 days with 10% concentration of thermal waters before total RNA isolation. TRI reagent (Sigma Aldrich) was used for RNA isolation. The concentration and purity of isolated RNA samples were determined using BioSpec-nano (Japan). Transcriptor First Strand cDNA Synthesis Kit was used for reverse transcriptase (Roche Molecular Diagnostics, Germany). Total volume of reaction mixture was 13 μl containing 1 μg total RNA, 2 μM sequence-specific primer, and water. The mixture was heated for 10 min at 65 °C. The mixture volume was completed to 20 μl by adding 10 U of transcriptor reverse transcriptase, 20 U of protector RNase inhibitor, 1× buffer, and 1 mM deoxy nucleotide, and the mixture was incubated at 50 °C for 60 min and then 85 °C for 5 min.

Real-time quantitative polymerase chain reaction

The experiments were conducted with Light Cycler 96 (Roche Diagnostics, USA) and Fast Start DNA Green Master Kit (Roche Molecular Diagnostics, Germany). Each 20 μL reaction contained 10 μL SYBR Green Master Mix (2×), 0.5 μM of reverse and forward primers (Table 1), 2.5 ng cDNA, and appropriate amount of nuclease-free water. All samples were run as triplicates in each run including a non-template control and four standards (1:10, 1:100, and 1:1000). All reactions subjected to initial denaturation step at 95 °C for 10 min and 45 cycle of 3-step amplification. Melting curve analysis was performed to confirm specificity of the amplified products. PCR reaction was carried out in triplicate. For quantitation of RT-qPCR results, ΔΔCt method was used.

Table 1 Nucleotide sequence of real-time PCR primers for genes
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Statistical analysis

All data are representative of three experiments and expressed as mean ± standard deviation. Statistical evaluation was performed by non-parametric Mann–Whitney test (GraphPad prism 6) and statistical significance was defined as p < 0.05.

Results

Chemical analysis of thermal waters

The chemical analysis of thermo-mineral waters is given in Table 2.

Table 2 Chemical analysis of thermal waters
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Cytotoxicity analysis (XTT cell proliferation assay)

The concentration of the tested thermal waters showed no toxic effect for HaCaT cells (Fig. 1).

Fig. 1
figure 1

Cytotoxicity analysis result of thermal waters (BJ1, Bursa, Turkey; BG, Bolu, Turkey) in HaCaT cells

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RT-PCR

The tested thermal waters decreased the expression of IL-1α (BJ1 0.07 ± 0.043 p = 0.0024 and BG 0.623 ± 0.125 p = 0.0303) when compared with the untreated control cells. However, a significant downregulation of TNF-α was found only with BJ1 and BG thermal waters (0.4123 ± 0.013 p = 0.0001 and 0.7745 ± 0.262 p = 0.0238 respectively). BJ1 and BG waters showed downregulation of the gene expression of VEGF significantly (0.018 ± 0.031 p = 0.043 and 0.8526 ± 0.115 p = 0.012 respectively) (Table 3).

Table 3 Fold changes of the genes, obtained by the treatment of HaCaT cells with thermal waters
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Discussion

The biological effects of thermal waters at cellular level have been reported since early 1990s (Beauvais et al. 1998; Joly et al. 1998; Celerier et al. 1995). The most significant effect of thermo-mineral waters was the anti-inflammatory activity. Among the studied anti-inflammatory factors were IL-1α, IL-2, IL-6, IL-8, TNFα, and other cytokines (Jung et al. 2009; Lee et al. 2012; Seite 2013), and also, the anti-angiogenic effect of VEGF suppression (Chiarini et al. 2006). The cells were incubated with the thermal waters at different concentrations to determine the cell viability. Based on our results, thermal waters were not cytotoxic at any concentration. Fluctuations in cell viability were not significant in this test. Therefore, cell viability results could not be correlated with factor levels such as VEGF at this stage. The only purpose of this stage was to determine the optimum concentration of thermal waters for further studies. One of the studied waters was BJ1 which significantly suppressed IL-1α, TNFα, and VEGF (Fig. 2). Whereas, the other thermo-mineral water BG suppressed TNFα and IL-1α, although significant but less pronounced effect on the VEGF expression. This observed difference might be attributed to their specific mineral content. The main difference of BG from BJ1 was sulfur content (1278 mg/L) (Table 2). Sulfur, which is found as H2S form in thermal mineral waters, has a well-known anti-inflammatory and anti-oxidative effect in HaCaT cells (Carbajo and Maraver 2017; Yang et al. 2011); therefore, the anti-inflammatory effect of BG water might be attributed to its sulfur content. On the other hand, silicium, zinc, sodium bicarbonate, and boron ingredients which are common elements in both thermal waters might be also responsible for the suppression of pro-inflammatory cytokines. In the previous studies, researchers attributed the suppression of interleukins to selenium and strontium (Chiarini et al. 2006). Therefore, a specific element in thermo-mineral water may induce a biological effect at least in the cell cultures representing the skin. High zinc content of both waters (BJ1 = 0.130 mg/L and BG = 0.1 mg/L) may be responsible for the anti-inflammatory effect. The anti-inflammatory effect of zinc was previously reported (Prasad 2014). Furthermore, researchers have also demonstrated boron as a component of a thermo-mineral water acted on keratinocytes as a wound-healing agent (Chebassier et al. 2004). Therefore, one of our thermal waters, BG has high boron content (51.5 mg/L); BG could be a good candidate on keratinocyte proliferation. The other common chemical components of BJ1 and BG were sodium, calcium, and bicarbonate. Calcium was found to be essential in wound healing and keratinocyte differentiation in the skin (Lansdown 2002; Elsholz et al. 2014). The BJ1 thermal water contains a higher level of calcium (88 mg/L) with respect to BG (0.9 mg/L). We have discussed the individual elements and their effects in HaCaT cells; however, it must be born in mind that the total chemical composition of the studied waters might be responsible for the observed overall effect. Finally, there are some limitations in this study. First of all, it is a cell culture study. The changes in the cells do not necessarily reflect themselves at the tissue or organ level. Secondly, our conclusions relating to the minerals of the thermal waters with the measured factor levels do not cover the entire range of elements dissolved in water. In addition, we did not use primary cells as control. We only used standardized HaCaT cells for the repeatability and reproducibility of the tests. We finally underline the fact that the findings of this study were based on selected gene levels not based on the protein synthesis in the cell.

Fig. 2
figure 2

The fold change results of gene expression analysis for TNFα, IL1α, and VEGF in HaCaT cell incubation 10% thermal waters of BJ1 and BG for 72 h

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Conclusion

In conclusion, BJ1 and BG thermo-mineral waters, when incubated with HaCaT cells, caused significant downregulation of TNFα, IL-1α, and VEGF gene expressions. These findings might give some insight on the underlying mechanisms of the therapeutic benefit of balneotherapy and topical applications of mineral waters. Clinical studies are needed for confirmation of the possible benefits of these waters in the management of some skin diseases such as rosacea and psoriasis where inflammation and angiogenesis are involved.