**4. Discussion**

Various skin-whitening agents are in use in the cosmetic industry. Many of these agents are from the chemical origin and are suffering from the limitations of causing various side effects including the cancers [24–26]. Therefore, the identification of safe and natural skin-whitening agents represent the need of the hour. Ectoine (Figure 1A) has been known to be used as an active ingredient in face creams and other cosmetic agents. This acts as a skin moisturizing agen<sup>t</sup> and also considered to delay the premature skin-aging as well [27]. Almost all known skin-whitening agents target the downregulation of tyrosinase enzyme activity in UV-irradiated cells that decreases the melanogenesis in skin cells. Yao et al. demonstrated the whitening properties of biosynthesized Ectoine and suggested that it is a putative whitening agent. In their study, they tested the high concentration (500 μM) of Ectoine for its

whitening e ffect on mouse melanoma (B16F0) and human melanoma (A2058) cell lines and concluded that Ectoine is a safe and potential agen<sup>t</sup> for the cosmetic and clinical application [20]. However, in this study, we further tested the beneficial e ffects of low concentrations of Ectoine (0.5–1.5 μM) on UVA-irradiated HaCaT cells and the underlying molecular mechanisms were deciphered. In our study, it was shown that Ectoine, through the Nrf2/ARE pathway, has not only induced the expression of antioxidant gene expression but also downregulated the α-MSH levels in UVA-irradiated HaCaT cells via the suppression of POMC. A decrease in the α-MSH levels was correlated with downregulation of tyrosinase enzyme activity leading to the decrease in the melanin production. From our knowledge, this is first the report that was evidenced by the mechanism elicited by Ectoine in UVA-irradiated HaCaT cells. This study delineated the molecular mechanisms exhibited by Ectoine in HaCaT cells as the cellular model system.

We first determined the sub-lethal concentrations of Ectoine as well as the e ffect of UVA radiation on the viability of HaCaT cells. Our MTT data indicated that low concentrations of Ectoine (0.5–1.5 μM) had no significant e ffect on the viability of HaCaT cells (Figure 1B). Ectoine pre-treatment increased the viability of 3 J/cm<sup>2</sup> UVA-irradiated HaCaT cells (Figure 1B). Based on these observations, we continued our further experiments using 1.5 μM of Ectoine pre-treatment and UVA irradiation at 3 J/cm<sup>2</sup> dosage.

UVA irradiation-induced ROS production in skin keratinocytes is a well-known fact [28]. Therefore, we also tested for any beneficial e ffects from Ectoine pre-treatment in the UVA-radiation induced ROS production in HaCaT cells. Our DCF-fluorescence intensity data indicated that pre-treatment with 1.5 μM of Ectoine significantly downregulated the UVA-radiation induced ROS production in keratinocytes. It was also observable that 1.5 μM of Ectoine could cause a basal level increase in the ROS levels in HaCaT cells that were shown to be statistically significant (Figure 1D,E).

Rousseau et al. reported that POMC, is secreted by human epidermal keratinocytes and melanocytes and has stimulated the melanogenesis [29]. By keeping this in view, we too tested the effect of UVA irradiation and Ectoine pre-treatments on the melanogenesis associated proteins in HaCaT cells. Our Western blot data indicated the dose-dependent downregulation of the expression of α-MSH and POMC proteins in UVA-irradiated HaCaT cells was caused by the pretreatment by Ectoine. Conversely, Ectoine pre-treatment has a di fferential e ffect on the expression pattern of melanogenesis associated proteins. Notably, almost all tested proteins (Tyrosinase, TRP-1, TRP-2, c-AMP protein kinase, CREB, and MITF) showed decreased expressions with increasing concentrations of Ectoine pre-treatment in UVA-irradiated HaCaT cells (Figure 2A,B). This data signifies the fact that Ectoine possesses anti-melanogenic properties in UVA-irradiated HaCaT cells.

The anti-melanogenic e fficacy of Ectoine was further tested in B16F10 cells, a well-known melanoma cell line used in melanogenesis studies [30]. One of the notable observations in our study was that, in contrast to the HaCaT cells, high concentrations of Ectoine (100–400 μM) were necessary to suppress the melanin synthesis in α-MSH-stimulated B16F10 cells (Figure 3B). Our Western blot data indicated that Ectoine dose-dependently downregulated the expression of tyrosinase and p-CREB proteins in α-MSH-stimulated B16F10 cells, leading to the aforesaid e ffect (Figure 3C). Therefore, we also tested if these high concentrations of Ectoine could a ffect the viability of B16F10 cells. Our MTT results indicated that high concentrations of Ectoine (100–400 μM) had no e ffect on the viability of B16F10 cells (Figure 3A). These results signify that keratinocytes play a key role in Ectoine mediated anti-melanogenesis and depigmenting e ffects.

The role of transcription factor Nrf2 in skin cells metabolism was well documented [31]. Therefore, we further tested the mechanisms played by Nrf2/Keap-1 pathway in Ectoine mediated e ffects in keratinocytes. Figure 4A shows that Ectoine dose-dependently and significantly increased the Nrf2/Keap-1 ratio with a maximum e ffect was observed at 1.5 μM Ectoine concentration. It was also observed that 1.5 μM Ectoine favored the nuclear translocation of Nrf2 protein with the maximum expression of Nrf2 from the nuclear protein fraction observed at the 2 h time point (Figure 4B). Data obtained from the immunofluorescence staining of HaCaT cells has also supported this e ffect (Figure 4D).

In human melanocytes and keratinocytes, Marrot et al. and others have explained the importance of Nrf2 defensive pathway in photo-oxidative stress responses [32]. We too studied the effect of Ectoine mediated antioxidant protein expression in HaCaT cells. Our time curve data indicated that Ectoine mediated expression of all three anti-oxidant proteins (HO-1, NQO-1, γ-GCLC), and Nrf2 were shown to express in a biphasic manner with the increasing time (0.5–12 h) with an observable effect was noted at 4 h time point (Figure 5A). From this, a concentration curve that measures the effect of Ectoine concentration on antioxidant protein expression was also determined at 4 h time point. Figure 5B shows that in comparison to the untreated cells, Ectoine treatment has dose-dependently increased the expression of HO-1, NQO-1, γ-GCLC proteins. We also measured how Ectoine concentration exhibited protective effects in HaCaT cells that were exposed to UVA radiation. Western blot data showed that Ectoine dose-dependently increased the expression of anti-oxidant proteins with a dramatic upregulation in the Nrf2/Keap-1 ratio as well (Figure 5C,D). These results indicated that Ectoine pretreatment (1.5 μM, 4 h) has the potential effect to induce antioxidant protein expression in HaCaT cells that could counteract the deleterious effects posed by UVA exposure.

Later, we determined the signaling pathways that mediated the activation of nuclear translocation of Nrf2 as well as the expression of anti-oxidant proteins. Our pharmacological inhibitor data revealed that p38, PI3/AKT, PKC, and CKII pathways were involved in the Ectoine mediated activation of nuclear NRf2. This data was consistent with the antioxidant expression data as well which showed that pharmacological inhibition of these four pathways down-regulated the expression of anti-oxidant proteins (HO-1, NQO-1, γ-GCLC). Except for the AKT activation (p-AKT), all three pathways (p38, PKC, and CKII) were activated at longer time points (after 30 min). The AKT pathway was demonstrated to be the first pathway activated (within 15 min) after exposure to Ectoine (Figure 6A–C).

Nrf2 knock-down technique has helped us to further demonstrate and confirm the key role played by Nrf2 in Ectoine mediated antioxidant and anti-melanogenic effects in HaCaT cells. Data showed that compared to the control siRNA cells, Nrf2 knockdown cells exposed to 1.5 μM Ectoine exhibited significant downregulation in the expression of HO-1, NQO-1, γ-GCLC antioxidant proteins (Figure 7A). On the other hand, these knockdown cells pre-treated with Ectoine and exposed to the UVA radiation did not affect the α-MSH expression, which confirmed that Nrf2 plays a key role in α-MSH expression in HaCaT cells (Figure 7B). In addition to the α-MSH expression, our DCF fluorescence data also revealed that Nrf2 knockdown is involved in the regulation of intracellular ROS production in UVA irradiated HaCaT cells that were pre-treated with 1.5 μM Ectoine (Figure 7C,D).
