Antiwrinkle and Antimelanogenesis Effects of Tyndallized Lactobacillus acidophilus KCCM12625P

UVB irradiation can induce generation of reactive oxygen species (ROS) that cause skin aging or pigmentation. Lactobacillus acidophilus is a well-known probiotic strain that regulates skin health through antimicrobial peptides and organic products produced by metabolism and through immune responses. In this study, we investigated the antioxidative, antiwrinkle, and antimelanogenesis effects of tyndallized Lactobacillus acidophilus KCCM12625P (AL). To analyze the effects of AL on UV irradiation-induced skin wrinkle formation in vitro, human keratinocytes and human dermal fibroblasts were exposed to UVB. Subsequent treatment with AL induced antiwrinkle effects by regulating wrinkle-related genes such as matrix metalloproteinases (MMPs), SIRT-1, and type 1 procollagen (COL1AL). In addition, Western blotting assays confirmed that regulation of MMPs by AL in keratinocytes was due to regulation of the AP-1 signaling pathway. Furthermore, we confirmed the ability of AL to regulate melanogenesis in B16F10 murine melanoma cells treated with α-melanocyte-stimulating hormone (α-MSH). In particular, AL reduced the mRNA expression of melanogenesis-related genes such as tyrosinase, TYRP-1, and TYRP-2. Finally, we used Western blotting assays to confirm that the antimelanogenesis role of AL was due to its regulation of the cyclic adenosine monophosphate (cAMP) signaling pathway. Collectively, these results indicate that AL has an antiwrinkle activity in damaged skin and can inhibit melanogenesis. Thus, AL should be considered an important substance for potential use in anti-aging drugs or cosmetics.


Introduction
Skin is consistently in contact with the external environment, protecting the body from harmful factors. The skin not only protects the body from viruses and bacteria, but also prevents moisture loss [1]. The skin aging system is composed of several processes including those of hereditary factors and environmental factors that are normally associated with sun exposure. Repeated exposure to UVB irradiation can induce generation of reactive oxygen species (ROS) to cause skin aging and pigmentation [2,3]. Moreover, the increase in ROS generated by UV irradiation not only induces cell death, but also increases expression levels of matrix metalloproteinases (MMPs) [4][5][6]. This process is characterized by formation of coarse

In Vitro Antioxidant Effects of AL in Skin Cells
To investigate whether AL reduces ROS generation, the H2DCFDA-staining assay was used. ROS generation was induced by UVB irradiation (30 mJ/cm 2 ) of HaCaT cells, and AL reduced the ROS levels in a dose-dependent manner (Figure 1a). In the MTT assay, AL did not show cell cytotoxicity in the concentration range of 25-400 µg/mL AL (Figure 1b). The cell viability of HaCaT cells was decreased by UVB irradiation (30 mJ/cm 2 ) and recovered by AL, implying a cytoprotective effect against cell death caused by oxidative stress (Figure 1c). The antioxidant effect of AL was further investigated in vitro using a radical-scavenging activity assay. 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was incubated with either AL at a concentration of 25-400 µg/mL or ascorbic acid (500 µM) as a positive control for 20 min. AL reduced the ABTS radical level in a density-dependent manner (Figure 1d). Taken together, these data strongly suggest that AL has antioxidant effects. HaCaT cells were subjected to UVB irradiation (30 mJ/cm 2 ) and treated with the indicated dose of AL (50-200 µg/mL) for 24 h. The cytoprotective effects of AL were measured using the MTT assay. (d) The ABTS radical scavenging activity of AL at the indicated concentration (25-400 µg/mL) was measured. +: indicate treatment, −: indicate non-treatment. For all applicable experiments, statistical significance was evaluated using the Mann-Whitney U test. ## p < 0.01 compared with the normal group, ** p < 0.01 compared with the control group.

Antiwrinkle Effects of AL through Activation of the AP-1 Signaling Pathway in HaCaT Cells
ROS induced by UVB irradiation contributes to intrinsic aging such as photoaging. In particular, ROS induce wrinkles by inducing degradation of the extracellular matrix (ECM) through induction of MMPs and elastase enzymes in keratinocytes and fibroblasts [32][33][34]. To confirm the antiwrinkle effect of AL, we measured its elastase inhibition activity [35]. AL inhibited elastase activity in a dose-dependent manner (Figure 2a). MMP-1 expression was determined using ELISA, and the amount of MMP-1 induced by UVB was inhibited by AL ( Figure 2b). These data indicate that AL exerts antiwrinkle effects through inhibition of MMP-1 and elastase in keratinocytes with UVB-induced oxidative stress. UVB irradiation in human skin cells induces the expression level of other MMPs in addition to MMP-1 [36]. To clarify the antiwrinkle effects of AL regarding UVB-induced MMP expression, we determined the mRNA Fseveral MMPs, as well as sirtuin 1 (SIRT-1) under UVB-induced oxidative stress conditions using RT-PCR. AL suppressed the expression of MMP-1, -2, -3, and -9 in UVB-induced HaCaT cells in a dose-dependent manner. Additionally, the expression of SIRT-1, which has notable functions in cell survival and inflammatory processes [37][38][39], was increased to approximately its baseline level by AL (Figure 2c). ROS, which are secondary messengers to activate mitogen-activated protein kinase (MPAK), promote the expression of MMPs due to induction of the AP-1 signaling pathway [36]. Therefore, we conducted Western blotting to determine whether reduction of ROS due to the antioxidant effect of AL contributes to inhibition of AP-1 signal activity. AL significantly inhibited the AP-1 pathway by inhibiting phosphorylation of ERK and c-Fos in UVB-irradiated keratinocytes ( Figure 2d). These data show that the ROS-scavenging effect of AL on the UVB-induced oxidative stress response leads to anti-aging effects in keratinocytes.

Antiwrinkle Effects of AL in Human Dermal Fibroblast Cells
Though UVB is absorbed in the epidermis and has little effect on the dermis [40], such irradiation induces ROS generation of HDF cells in vitro [41,42]. Since dermal fibroblasts are the primary cells secreting MMPs and elastase [32,36], we evaluated the biological effect of UVB-induced oxidative response in HDF cells. First, the cytotoxicity of AL in HDF cells was examined by treating cells with increasing doses of AL for 24 or 48 h. Cell viability was then determined using the MTT assay, and AL had no appreciable cytotoxicity in HDF cells (Figure 3a,b). Since AL modulates elastase activity (Figure 2a), we performed an in vitro evaluation of the effects of AL in HDF following UVB irradiation. AL inhibited both elastase activity ( Figure 3c) and MMP-1 expression (Figure 3d) in HDF cells after UVB-induced oxidative stress. Taken together, these data indicate that AL has antiwrinkle effects in HDFs and keratinocytes under UVB-induced oxidative stress.
Type 1 procollagen, which modulates the structure of skin tissue, is one of the major markers of wrinkle development in the skin and is degraded by MMP-1 [43]. Interestingly, the type 1 pro-collagen alpha level in HDF was significantly increased by AL with or without UVB irradiation (Figure 3e,f). This indicates that AL regulates baseline MMP expression and induces collagen production in HDF cells ( Figure 3e). Finally, we clarified the effects of AL on expression of antiwrinkle-related genes using PCR analysis. The mRNA expression of MMP-1 and MMP-9 were inhibited by AL under UVB-irradiation conditions. Conversely, COL1A1 expression was increased by AL in HDF cells ( Figure 3g). Collectively, these data indicate that AL inhibits elastase and MMP-1 and induces type 1 pro-collagen to exert antiwrinkle effects.  For all applicable experiments, statistical significance was evaluated using the Mann-Whitney U test. # p < 0.05 and ## p < 0.01 compared with the normal group, and * p < 0.05 and ** p < 0.01 compared with the control group.

UVB irradiation induces melanogenesis through physiological responses in keratinocytes.
We explored the effects of AL on the melanogenesis response induced by UVB-irradiation in B16F10 cells. MTT assay confirmed that AL had no cytotoxicity in B16F10 cells at doses of 50-400 µg/mL for 48 h (Figure 4a). To investigate the effects of AL on melanogenesis, B16F10 cells were treated with AL and α-MSH, a melanocyte-stimulate hormone. The level of melanin secreted from the cells, as well as in the cells, was then determined. AL significantly inhibited melanin secretion ( Figure 4b) and intracellular content (Figure 4c) in a dose-dependent manner. Tyrosinase is an important molecule in the melanogenesis process; therefore, we investigated the tyrosinase-inhibition activity of AL using a mushroom tyrosinase and L-3,4-dihydroxyphenylalanine (L-DOPA) assay, as well as PCR analysis. Mushroom tyrosinase activity was not inhibited by AL (Figure 4d), and AL reduced the mRNA expression of tyrosinase and TYRP-1 but not TYRP-2 ( Figure 4e). These results indicate that AL does not directly affect tyrosinase activity. To clarify the molecular mechanism of AL, we examined whether it regulated the cAMP signaling pathway. In B16F10 murine melanoma cells stimulated with the melanocyte hormone α-MSH, AL inhibited phosphorylation of PKA and CREB and downregulated protein expression of tyrosinase and MITF (Figure 4f). These results strongly indicate that AL has antimelanogenesis effects through regulation of the cAMP signaling pathway.

Discussion
In this study, we explored the skin health benefits of tyndallized L. acidophilus (KCCM 12625P) in skin cells exposed to UVB irradiation to induce cell death and stimulate the photoaging process [44,45].
HaCaT cells treated with UVB irradiation showed an increase in ROS. Administration of AL inhibited ROS production (Figure 1a), and MTT assay confirmed that AL was not cytotoxic at the concentrations used (Figure 1c). In addition, treatment with AL prevented cell death by UVB, indicating that AL protects the skin and body from UVB irradiation (Figure 1b). The antioxidant effects of AL were found through ABTS radical-scavenging assays [46][47][48], which again demonstrates that AL exerts antioxidant effects (Figure 1d).
ROS induced by UV irradiation not only triggers cell death, but also increases matrix metalloproteinase (MMP) expression [4][5][6]. In addition, degradation of elastin induces wrinkles, and inhibitors of elastase activity can be used to prevent signs of aging [49,50]. We further evaluated the antiwrinkle effects of AL using keratinocytes and human dermal fibroblasts. Interestingly, AL increased the mRNA expression of SIRT-1 (Figure 2c), which has a notable function in cell survival and inflammatory processes [37][38][39]. Based on our results, it is assumed that AL protects cells through antioxidative effects, inhibits MMPs by regulating AP-1 signaling, and suppresses wrinkles by inhibiting elastase activity in keratinocytes.
Formation of skin wrinkles is a phenotype of the aging process [51,52], which is affected by several factors including genetic changes, hormonal alteration, UVB irradiation, and exposure to inflammationor oxidation-inducing agents [1,12,38]. In terms of cellular and molecular causes of wrinkle formation, a decrease of extracellular matrix proteins such as collagen in dermal fibroblasts is known as a major cause [53]. Moreover, upregulation of the activity of matrix metalloproteinases to degrade collagen is associated with loss of extracellular matrix proteins in the skin [53]. Thus, we determined the effects of AL on extracellular matrix protein levels in HDF cells. AL reduced the mRNA expression of MMP-1 and MMP-9, important skin-related genes regulated during aging [24,54], triggered by UVB irradiation and also increased the expression of procollagen (eg., COL1A1) ( Figure 3e). Therefore, these data show that AL exhibits an antiwrinkle effect by regulating MMPs and elastase in HDF cells, as well as keratinocytes. In addition, AL protects against skin barrier damage caused by UVB by inducing type I pro collagen alpha.
Excessive UVB irradiation causes melanin formation, which presents as pigmentation of the skin. In B16F10 cells stimulated with α-MSH, which activates various transcription factors and enzymes to induce melanin production, the mRNA expression of tyrosinase and TYRP-1 was decreased by AL (Figure 4e). To clarify the mode of action of AL in melanogenesis, we confirmed its effects on cell signaling mechanisms using Western blotting. Interestingly, AL regulated the protein levels of cAMP signaling pathway members such as tyrosinase, MITF, CREB, and PKA ( Figure 4f). Collectively, these results demonstrate that AL can modulate melanogenesis though the cAMP pathway.
In conclusion, we demonstrated that heat-killed (tyndallized) L. acidophilus KCCM12625P exhibits skin-protective activities. Our results show that the skin-protective characteristics of AL are mediated through various activities. First, AL exhibited antioxidant and cell-protective activity in keratinocytes through the AP-1 signaling pathway. Second, AL inhibited wrinkle-related factors and induced cell growth in keratinocytes. Finally, AL suppressed melanin secretion and intracellular melanin content in B16F10 cells by inhibiting cAMP pathway. The skin-protective activities of AL in human keratinocytes, human dermal fibroblast cells, and B16F10 murine melanoma cells are summarized in Figure 5. Therefore, this study strongly suggests that L. acidophilus KCCM12625P has potential for use in anti-aging drugs or cosmetics.

ROS Generation Assay
H2DCFDA staining assays were used to confirm reactive oxygen species (ROS) generation. HaCaT cells were treated with UVB irradiation (30 mJ/cm 2 ) and further treated with AL (50-200 µg/mL) for 24 h. The cells were then incubated with H2DCFDA (10 µM) at 37 • C for 20 min and washed with PBS three times. Fluorescence was measured using a flow cytometer (EMD Millipore Co., Billerica, MA, USA)

MTT Assay
Cell viability assays were used to determine the cytotoxicity activity of AL. HaCaT cells, human dermal fibroblast cells, or B16F10 cells were treated with AL (25-400 µg/mL) for 24 or 48 h. To confirm the cytoprotective activity of AL, oxidative stress was stimulated using UVB irradiation (30 mJ/cm 2 ), and cells were treated with AL (50-200 µg/mL) for 24 h. Absorbance was measured at 570 nM.

Radical-Scavenging Activity Assay
The radical-scavenging activity of AL was measured using the ABTS assay as previously reported [55]. AL (25-400 µg/mL) or ascorbic acid (100 µM) was mixed with ABTS solution (7.4 mM) in a 96-well plate. The plate was incubated for 20 min at room temperature, and the absorbance was measured at 730 nM using a SpectraMax 250 microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Elastase Inhibition Assay
Elastase (0.3 units/mL) was incubated with either AL (50-200 µg/mL) or ursolic acid (500 µM) for 30 min in a humidified 5% CO 2 incubator. The substrate STANA (400 µM) was then added, and the reaction was incubated for 15 min in a humidified 5% CO 2 incubator. The absorbance of the mixtures was measured at 410 nM using a SpectraMax 250 microplate reader.

RT-PCR
HaCaT or HDF cells were induced with UVB irradiation (30 mJ/cm 2 ) and then treated with AL (50-200 µg/mL) for 24 h. In B16F10 cells, oxidative stress was stimulated by α-MSH for 30 min, and the cells were treated with AL (50-200 µg/mL) for 12 h. For all cell types, RNA was extracted using TRI reagent as reported previously [56,57]. cDNA was synthesized from total RNA (1 µg) using MuLV reverse transcriptase according to the manufacturer's instructions. Primers for RT-PCR were listed in Table 1.

Western Blotting
HaCaT cells were treated with UVB irradiation (30 mJ/cm 2 ) and AL (50-200 µg/mL) for 24 h. Western blotting was conducted as previously described [56,57]. Briefly, total cell lysates were separated using SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Specific antibodies were used to detect the total and phosphorylated forms of the target proteins, and protein bands were visualized using enhanced chemiluminescence reagents.

ELISA
HDF cells (1 × 10 5 cells/well) were seeded into 6-well plates and incubated for 24 h. The culture media was changed to fresh media supplemented with AL (50-200 µg/mL) and incubated for 24 h. The media was collected from each well, and the levels of MMP-1 and type 1 procollagen alpha were measured using ELISA assay kits (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's instructions.

Tyrosinase Activity Assay
Mushroom tyrosinase (100 unit/mL) was incubated with AL (50-200 µg/mL) or kojic acid (300 µM) for 30 min in a humidified 5% CO 2 incubator. The reactions were then treated with L-DOPA (40 g/mL) for 5 min. Tyrosinase activity was determined from the absorbance of the mixture at 475 nm, which was measured with a SpectraMax 250 microplate reader.

Melanin Generation Assay
B16F10 cells (0.5 × 10 5 cells/well) were seeded into 12-well plates and incubated for 24 h. The culture media was changed to fresh media supplemented with either AL (50-200 µg/mL) or arbutin (1 mM). Melanin production was then stimulated with α-MSH (100 nM) for 48 h. To determine the amount of melanin secreted into the culture media, the absorbance of the culture media was measured at 475 nm using a SpectraMax 250 microplate reader. To determine the intracellular melanin content, harvested cells were lysed using a lysis buffer (50 mM Tris HCl pH 7.5, 20 mM glycerophosphate pH 7.5, 120 mM NaCl, and 2 % NP-40) and centrifuged at 10,000× g for 3 min. The concentrated cell pellets were resuspended in 10% DMSO in 1 N NaOH and incubated at 60 • C for 10 min. The absorbance at 405 nm was measured using a SpectraMax 250 microplate reader.

Statistical Analysis
All data are presented as mean ± standard deviation (SD) of at least three independent experiments. The experimental and control groups were compared using the Mann-Whitney U test, and a p-value < 0.05 was considered statistically significant. SPSS software (SPSS Inc., Chicago, IL, USA) was used for all statistical analyses.

Conflicts of Interest:
The authors declare no conflict of interest.