**3. Results**

#### *3.1. Ectoine Inhibited UVA-Induced ROS Generation in HaCaT Cells*

First, we tested for the cytotoxic e ffects of Ectoine (Figure 1A) on UVA-irradiated HaCaT cells. Our MTT data indicated that when compared to the untreated control cells, Ectoine pre-treated (0.5–1.5 μM) and 3 J/cm<sup>2</sup> UVA exposed HaCaT cells were unable to show a significant decrease in cell viability (Figure 1B). Further, Ectoine pretreatment attenuated the UVA (3 J/cm2)-induced cell death in a dose-dependent manner (Figure 1B). In addition to our fluorescence data, which indicated that, when compared to the control cells, 3 J/cm<sup>2</sup> UVA irradiation and Ectoine alone treatments (1.5 μM) significantly upregulated ROS levels by 5- and 2-fold, respectively. However, in the case of Ectoine pretreatment ROS levels were significantly downregulated and we can infer that Ectoine has an antioxidant e ffect against UVA irradiation. This also induces basal levels of ROS in HaCaT cells (Figure 1C,D).

**Figure 1.** Ectoine inhibits UVA-induced ROS production in human keratinocyte (HaCaT) cells. (**A**) Ectoine's chemical structure. (**B**) To determine cell viability, an MTT assay was used. Cells were treated with Ectoine (0.5, 1, and 1.5 μM) for 24 h. Then, they were irradiated with 3 J/cm<sup>2</sup> or without UVA. (**C**,**D**) Cells were pre-treated with Ectoine (0 or 1.5 μM) for 24 h and then irradiated with 3 J/cm<sup>2</sup> or without UVA. For each condition, we used the percentage of the fluorescence intensity of the DCF-stained cells as determined by Olympus Softimage. Statistical significance was assigned as \*\*\* *p* < 0.001 compared to the untreated control cells and ### *p* < 0.001 compared to the UVA-exposed HaCaT cells.

#### *3.2. Ectoine Suppressed POMC and* α*-MSH Expressions in UVA-Irradiated HaCaT Cells*

UVA exposed keratinocytes were stimulated for their ROS-p53 mediated POMC and also a small peptide hormone α-MSH that is derived from POMC [23]. Therefore, we determined the alterations in expression patterns of α-MSH, POMC, and other associated proteins in Ectoine pre-treated HaCaT cells and then exposed them to UVA (3 J/cm2). Western blot data indicated that UVA-induced upregulation of α-MSH and POMC expressions were downregulated by Ectoine pretreatment; whereas, Ectoine treatment without UVA irradiation has completely inhibited the α-MSH and POMC expressions of non-irradiated HaCaT cells (Figure 2A). Later, we tested the effect of 'conditioned-medium' (10 mL/100 mm plate), obtained from the Ectoine pre-treated and UVA irradiated HaCaT cells, on the melanogenesis of B16F10 melanoma cells. Figure 2B shows this conditioned medium downregulated the tyrosinase, TRP-1, TRP-2, c-AMP protein kinase, p-CREB, CREB, and MITF levels in B16F10 cells.

**Figure 2.** Ectoine suppresses UVA-induced POMC and α-MSH expression in HaCaT cells. (**A**) Western blotting showed us protein levels of α-MSH and POMC. Cells were pre-treated with Ectoine (0–1.5 μM) for 24 h and then irradiated or not with 3 J/cm<sup>2</sup> UVA. (**B**) Effect of HaCaT conditioned medium on B16F10 cells melanin synthesis. HaCaT cells were pre-treated with vehicle (PBS) or Ectoine (0.5–1.5 μM) for 24 h. Subsequently, these cells were exposed or non-exposed to the 3 J/cm<sup>2</sup> UVA-irradiation. After 1–24 h, the conditioned medium (10 mL/100 mm dish) was collected and tested on B16F10 cells for the expression of various proteins through western blot method. Lanes 1 and 2 were indicating the experimental conditions of conditioned medium obtained from vehicle (PBS) pre-treated and UVA non exposed (lane #1) and exposed (lane #2). Whereas, lanes 3–5, and 6 were indicating the experimental conditions of conditioned medium obtained from Ectoine pre-treated (0.5–1.5 μM) and UVA exposed, and non-exposed, respectively. Western blot analysis measured the protein levels of tyrosinase, TRP-1, TRP-2 (for 24 h), c-AMP protein kinase (for 1 h), p-CREB, CREB (for 2 h), and MITF (for 4 h).

#### *3.3. Ectoine Downregulated Melanin and Tyrosinase Expression in* α*-MSH-Stimulated B16F10 Cells*

B16F10 melanoma cells were first subjected to the higher concentrations of Ectoine and the effect of cytotoxicity was determined using MTT assay. Figure 3A shows that Ectoine had no significant impact on the viability of B16F10 cells at higher concentrations (100–400 μM for 72 h). However, cell viability was not effected at 24 and 48 h of Ectoine treatment (data not shown). Therefore, these concentrations were used to determine the effect of Ectoine on α-MSH-stimulated melanogenesis in B16F10 cells. Melanin quantification data showed that, compared to the control cells, treatment with α-MSH (1 μM) alone significantly upregulated the melanin levels by more than 25%. However, compared to α-MSH alone treatment, cells exposed to increasing concentrations of Ectoine (100–400 μM at 72 h) dose-dependently and significantly downregulated the percentage of melanin content with maximum downregulation of only 85% (or −15% than untreated control) was observed at 400 μM of Ectoine pretreatment (Figure 3B). Moreover, our Western blot data also showed that α-MSH stimulated tyrosinase (24 h) and p-CREB (2 h) expressions were significantly downregulated with increasing concentrations of Ectoine pretreatments in these melanoma cells (Figure 3C).

146

**Figure 3.** Ectoine downregulated the melanogenesis in α-MSH-stimulated B16F10 cells. (**A**) High concentrations of Ectoine (100–400 μM, 72 h) affect the cell viability of B16F10 as determined by an MTT assay. (**B**) Cells were pre-treated with Ectoine (0–400 μM, 1 h) followed by stimulation without or with 1 μM α-MSH for 72 h. The percentage of melanin content was quantified from total cell lysates. (**C**) Cells were pre-treated with Ectoine (0–200 μM for 1 h) and then treated with or without α-MSH (1 μM) for 24 or 2 h. Western blot analysis measured the expressions of tyrosinase and p-CREB proteins. Statistical significance was assigned as \*\* *p* < 0.01; \*\*\* *p* < 0.001 compared to the untreated control cells and # *p* < 0.05, ### *p* < 0.001 compared to the α-MSH treated B16F10 cells.

#### *3.4. Ectoine Facilitated Nrf2 Nuclear Translocation in HaCaT Cells*

Nrf2-Keap-1 is an important cytoprotective pathway that protects the skin cells from ROS and electrophile insult caused by UVA exposure. Nrf2 and Keap1 maintain a stoichiometric ratio in the cytoplasm. In a cytoprotective pathway, Nrf2 dissociates from Keap-1 and, for the expression of various antioxidant genes, translocate to the cellular nucleus. Therefore, the ratio of Nrf2/Keap-1 is a key factor. Here, our Western blot data indicated a shift in the ratio of Nrf2/Keap-1 that was towards more Nrf2 expression with the increasing concentrations of Ectoine, signifying that Ectoine favors the nuclear translocation of Nrf2 in HaCaT cells (Figure 4A,B). Also, the expression of Nrf2 mRNA levels was shown to be significantly elevated in the 1.5 μM Ectoine pre-treated cells (Figure 4C). This was also consistent with our fluorescence image data (Figure 4D).

**Figure 4.** Ectoine upregulated the nuclear translocation of antioxidant marker Nrf2 in HaCaT cells. (**A**) Effect of Ectoine on protein expressions of Nrf2 and Keap-1 in HaCaT cells. Western blot method measured the expressions of Nrf2 and Keap-1 in cells treated with Ectoine (0–1.5 μM) for 2 h (**B**) Effect of time on the Ectoine mediated nuclear and cytosolic expressions of Nrf2. Cells were exposed to 1.5 μM Ectoine for different time points and Western blot method measured the expressions of nuclear and cytosolic Nrf2. (**C**) Cells were pre-treated with Ectoine (1.5 μM, 1 h) followed by the isolation of total mRNA from HaCaT cells. 1 μg of total mRNA was used to measure the expression of the Nrf2 gene through the RT-PCR method. As an internal control, GAPDH was used. (**D**) Immunofluorescence staining of HaCaT cells. Cells were treated with 1.5 μM of Ectoine for 2 h and the nuclear localization of Nrf2 were visualized by immunofluorescence method. Cells were stained with DAPI (1 μg/mL) for 5 min and examined by fluorescence microscopy (scale bar 100 μM). Statistical significance was assigned as \*\*\* *p* < 0.001 compared to the untreated control cells.

#### *3.5. Ectoine Upregulated the Expression of HO-1, NQO-1, and* γ*-GCLC Proteins in HaCaT Cells*

To determine the effect of time on Ectoine mediated nuclear translocation of Nrf2 and the subsequent downstream expression of HO-1, NQO-1, and γ-GCLC proteins, HaCaT cells were exposed to 1.5 μM Ectoine and the cellular proteins were harvested 0.5, 1, 2, 4, 8, or 12 h after the Ectoine treatment. Western blot data indicated that, except for the γ-GCLC protein, 1.5 μM Ectoine caused the maximum expression of HO-1, Nrf2, and NQO-1 proteins at the 4 h time point. γ-GCLC was shown at 8 h time point (Figure 5A). Data obtained from the time-curve lead us to test the effect of Ectoine concentration on the expression of antioxidant proteins at 4 h time point. Figure 5B shows that all three antioxidant proteins exhibited maximum expression at 1.5 μM of Ectoine concentration. Later, the effects of Ectoine pre-treatment were tested on the expression of Nrf2 and Keap-1 ratio in the UVA-irradiated HaCaT cells. Western data analysis indicated that pre-treatment with 1.5 μM of

Ectoine exhibited an increase in the ratio of Nrf2/Keap-1 in UVA-irradiated HaCaT cells (Figure 5C). We also saw consistent data with the increased expression of NQO-1, HO-1, and γ-GCLC proteins in Ectoine pre-treated HaCaT cells that were irradiated with 3 J/cm<sup>2</sup> UVA (Figure 5D). This data infers that Ectoine pre-treatment plays a protective role in UVA-irradiated HaCaT cells.

**Figure 5.** Ectoine mediated differential expressions of antioxidant genes in UVA irradiated HaCaT cells. (**A**,**B**) Western blot method was used to determine the effects of time (0–12 h) and Ectoine concentrations (0–1.5 μM) on the expressions of Nrf2 and antioxidant genes (HO-1, NQO-1, and γ-GCLC) in HaCaT cells. (**C**,**D**) Effect of Ectoine pretreatment effect on the Nrf2/Keap-1 ratio of HaCaT cells irradiated with UVA. Cells were pre-treated with Ectoine (0–1.5 μM for 24 h), and then irradiated in the absence or presence of 3 J/cm<sup>2</sup> UVA. The ratio of Nrf2/Keap-1 (**C**) and antioxidant gene (HO-1, NQO-1, and γ-GCLC) expressions (**D**) were determined by Western blotting. Statistical significance was assigned as \*\*\* *p* < 0.001 compared to the untreated cells and ### *p* < 0.001 compared to the UVA exposed HaCaT cells.

#### *3.6. Various Signaling Pathways Were Involved in the Activation of Nrf2 in Ectoine Treated HaCaT Cells*

We determined the signaling pathways involved in the Ectoine mediated nuclear translocation of Nrf2. HaCaT cells were pre-treated with pharmacological inhibitors of PI3K/AKT, ERK, p38, JNK, PKC, ROS, and CKII signaling pathways, followed by 1.5 μM Ectoine. Western blot data of nuclear Nrf2 showed that p38 MAPK, PI3K/AKT, PKC, and CKII pathways were involved in this mechanism (Figure 6A). From the obtained information, we also determined the effect of Ectoine pre-treatment on the role played by these pathways in the expression of antioxidant proteins. Figure 6B shows that pharmacological inhibition of MAPK, p38, PI3K/AKT, CKII, and PKC pathways down-regulated the expression of NQO-1, HO-1, and γ-GCLC antioxidant proteins in HaCaT cells. Moreover, the time taken for the phosphorylation of AKT, p38, and the expression of PKC and CKII while exposed to Ectoine indicates that, except for the p-AKT, the phosphorylation of p38 and the expressions of PKC and CKII took place at the later time points only (after 30 min) (Figure 6C). In the case of AKT, phosphorylation was observed from the 15 min time point that has reached a peak at 30 min (Figure 6C). These cumulative results suggested that p38, AKT, PKC, and CKII signaling pathways activated the Ectoine mediated nuclear translocation of Nrf2 leading to the expression of antioxidant proteins.

**Figure 6.** Ectoine mediated the activation of nuclear Nrf2 through p38, AKT, PKC, and CKII signaling pathways in HaCaT cells. (**A**) Cells were pre-treated with p38 inhibitor (SB203580, 20 μM), ERK inhibitor (PD98059, 30 μM), JNK inhibitor (SP600125, 25 μM), PI3K/AKT inhibitor (LY294002, 30 μM), PKC inhibitor (GF109203X, 2.5 μM), AMPK inhibitor (compound C, 10 μM), Casein kinase II inhibitor (CKII, 20 μM), or antioxidant NAC (1 mM) for 30 min, followed by exposure to Ectoine (1.5 μM) for 2 h. Western blot was performed to analyze the nuclear Nrf2 expression against histone proteins as internal control (**B**) HO-1, NQO-1, and γ-GCLC protein levels were evaluated using immunoblot analysis. Cells were pre-treated with inhibitors for p38 (SB203580, 20 μM), PI3K/AKT (LY294002, 30 μM), PKC (GF109203X, 2.5 μM), and CKII (20 μM) for 30 min followed by exposure to Ectoine (1.5 μM) for 4–8 h. (**C**) Ectoine activated the p38, AKT, PKC, and CKII signaling pathways. Cells were pre-treated with Ectoine (1.5 μM) for 0–120 min, and the protein expressions of p-p38, p38, p-AKT, AKT, PKC, and CKII were measured by immunoblot analysis.

#### *3.7. Ectoine Mediated Anti-Melanogenic E*ff*ect was Suppressed due to the Knockdown of Nrf2*

The role of Nrf2 in Ectoine mediated anti-melanogenesis was determined by silencing the Nrf2 in HaCaT cells. Data from the Western blot indicated that Nrf2 knockdown cells exposed to 1.5 μM Ectoine showed minimum expression of NQO-1, HO-1, and γ-GCLC antioxidant proteins (Figure 7A). Later, we tested the effect of Nrf2 knockdown on the expression of α-MSH levels in UVA irradiated (3 J/cm2) HaCaT cells. Western blot results indicated that to control the siRNA transfected cells, UVA-irradiation was significant in the upregulation of the expression of α-MSH levels in cells unexposed to Ectoine (Figure 7B). However, 1.5 μM Ectoine has suppressed this effect. For the other case, cells transfected with siNrf2 showed a decrease in the expression of α-MSH levels in both untreated and treated

cells (Figure 7B). Similar to α-MSH data, our fluorescence data also indicated that UVA irradiation significantly upregulated the ROS production in Ectoine untreated control siRNA cells (Figure 7C,D). However, this effect was significantly suppressed when the cells exposed to 1.5 μM Ectoine. On the other hand, Nrf2 transfected and UVA-irradiated HaCaT cells showed an approximately 8-fold increase in ROS levels compared to the Nrf2 transfected cells that were not irradiated with UVA but exposed to Ectoine treatment (Figure 7C,D). All this data signifies the Ectoine mediated protective role played by Nrf2 in the minimization of melanin production in UVA-irradiated HaCaT cells.

**Figure 7.** Nrf2 knockdown attenuated the protective effects of Ectoine in UVA-irradiated HaCaT cells. Cells were transfected with siRNA that is specific to either Nrf2 or a non-silencing control. (**A**) Transfected cells were pre-treated with Ectoine (0 or 1.5 μM for 2 or 12 h) and the expression of Nrf2 (for 2 h), or HO-1, NQO-1, and γ-GCLC (for 12 h) proteins in both control and siNrf2 were measured by Western blot analysis. (**B**) The effect of Nrf2 knockdown on the expression of α-MSH levels in UVA irradiated HaCaT cells were determined. Transfected cells were pre-treated with or without Ectoine (1.5 μM for 24 h) and then irradiated with 3 J/cm<sup>2</sup> UVA. The Western blot method measured the percentage of α-MSH levels (**C**,**D**) The effect of Nrf2 knockdown on the UVA radiation-induced ROS levels in transfected cells were measured by DCF fluorescence microscopy. Statistical significance was assigned as \*\*\* *p* < 0.001 compared to the untreated cells.
