Tyndalized L. acidophilus, or ID-ACT3302, was obtained from Ildong Pharmaceutical Co., Ltd. (Seoul, South Korea). A total of 21 6-week-old HR-1 hairless male mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). UVB irradiation was administered using a UVM-225D Mineralight UV Display Lamp (UVP, Inc., Upland, CA, USA). Rabbit anti-phospho-stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK; cat. no. 9251), anti-SAPK/JNK (cat. no. 9252), anti-phospho-p44/42 mitogen-activated protein kinase (MAPK) extracellular signal-regulated protein kinases 1 and 2 (ERK1/2; cat. no. 9101), anti-p44/42 MAPK (ERK1/2; cat. no. 9102), anti-phospho-p38 MAPK (cat. no. 9211) and anti-p38 MAPK (cat. no. 9212) polyclonal antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Goat anti-matrix metalloproteinase (MMP)-1 polyclonal antibody (cat. no. sc12348), mouse anti-MMP-9 monoclonal antibody (cat. no. sc-21733), goat anti-β-actin polyclonal antibody (cat. no. sc-1616) and horseradish peroxidase (HRP)-conjugated anti-rabbit (cat. no. sc-2030) and anti-goat (cat. no. sc-2020) secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

After purchase, the HR-1 hairless male mice were stabilized for 1 week prior to the commencement of the study. The animals were housed in a climate-controlled facility at a temperature of 24°C, at 50% humidity with a 12-h dark:light cycle and ad libitum access to food and water. All experimental protocols were approved by the Korea Institute of Oriental Medicine Institutional Animal Care and Use Committee (Daejeon, South Korea). The mice were divided into 3 groups as follows: Control (n=5), UVB-treated vehicle (n=5), and UVB-treated tyndalized L. acidophilus (n=5) groups. Mice from the UVB-treated tyndalized L. acidophilus group were orally administered 0.1 ml water containing 100 mg of tyndalized L. acidophilus/kg body weight/day. The control group did not receive irradiation with UVB, or any other form of treatment. The vehicle group mice were orally administered 0.1 ml water and underwent irradiation with UVB.

UVB irradiation was administered using a UVM-225D Mineralight UV Display Lamp (UVP) that emitted at a wavelength of 302 nm. The strength of the UV radiation was measured using a HD2102-2 UV meter (Delta OHM Srl, Padova, Italy). UVB radiation was applied to the backs of the mice 3 times/week for 12 weeks. The amount of irradiation was progressively increased from 60 mJ/cm²/exposure during week 1 (1 minimal erythematous dose = 60 mJ/cm²) to 90 mJ/cm²/exposure during week 7.

A corneometer (Courage + Khazaka electronic GmbH, Cologne, Germany) was used to determine the hydration levels of the skin, and a Tewameter (Courage + Khazaka electronic GmbH) was used to measure TEWL, which is an indicator of the barrier function of the skin's epidermis.

The dorsal skin was removed from each hairless mouse and fixed in 10% neutral-buffered formalin. Using a conventional method, the fixed tissue samples were washed with distilled water, dehydrated using an ethanol gradient, cleared with xylene and embedded in paraffin wax, after which 5 µm sections were cut using a microtome. The tissue sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome stain for collagen fiber analysis. The thickness of the epidermis was measured under light microscopy using an eyepiece micrometer (Olympus Corporation, Tokyo, Japan).

Protein was extracted from the skin tissue samples using radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc., Rockford, IL, USA), following tissue homogenization (Precellys^® 24; Bertin Technologies, Montigny-le-Bretonneux, France). Total protein concentration was determined using the DC Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein lysates (20 µg) from each sample were electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel and then transferred to polyvinylidene fluoride membranes. The membranes were blocked with a 5% skimmed milk solution for 1 h at room temperature. Following blocking, the blots were incubated overnight at 4°C with a the primary antibodies at a dilution of 1:1,000. The blots were washed 3 times for 10 min each in Tris-buffered saline (Bio-Rad Laboratories, Inc.). The membranes were then incubated for 2 h with HRP-conjugated anti-rabbit and anti-goat secondary antibodies at a dilution of 1:5,000. The proteins were detected using an enhanced chemiluminescence solution. Images were captured by ImageQuant LAS 4000 digital imaging system (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) and were analyzed using Multi Gauge software, version 3.0 (Fujifilm, Tokyo, Japan).

All measurements were undertaken in triplicate, and all values are presented as the mean ± standard error. An analysis of variance and Tukey's test was used to determine differences in the results among the study groups. Statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

The hydration of the stratum corneum was significantly reduced in the UVB-treated vehicle group, as compared with the control group (P<0.0001; Fig. 1A). The hydration of the skin from the UVB-treated tyndalized L. acidophilus group mice was not reduced to the same extent as that observed in the UVB-treated vehicle group and it was significantly different, as compared with the UVB-treated vehicle group (P<0.001; Fig. 1A).

TEWL was significantly higher in the UVB-treated vehicle group (Fig. 1B; P<0.0001), as compared with the control group. However, TEWL was significantly reduced in the UVB-treated tyndalized L. acidophilus group, as compared with the UVB-treated vehicle group (P<0.001; Fig. 1B).

To determine the wrinkle-reducing effects of tyndalized L. acidophilus, skin was removed from the hairless mice and skin tissue sections were stained. H&E staining of the skin confirmed that the thickness of the stratum corneum and the epidermis had increased in the UVB-treated vehicle group compared with the skin from the control group (Fig. 2A and B; P<0.001). Masson's trichrome staining revealed that the collagen was uniformly distributed in the dermal layer (Fig. 2C). Collagen fibers were increased in the UVB-treated tyndalized L. acidophilus group compared with the UVB-treated vehicle group (Fig. 2C). These results suggest that tyndalized L. acidophilus reduced the level of skin-wrinkling resulting from UV irradiation.

To evaluate the anti-wrinkle effect of tyndalized L. acidophilus, changes in the thickness of the epidermis were investigated by measuring the distance from the keratin layer to the epidermal basement membrane in skin sections stained with H&E using a microscope. The skin epidermal thickness was significantly increased in the UVB-treated vehicle group, as compared with the control group (P<0.001; Fig. 2B). The skin epidermal thickness of the the UVB-treated tyndalized L. acidophilus group was decreased by 54.1%, as compared with the UVB-treated vehicle group, and this difference was statistically significant (P<0.01). These results suggest that tyndalized L. acidophilus is able to reduce the thickness of the epidermis and may be an effective anti-wrinkle agent.

UVB irradiation increased the expression of the MMPs, MMP-1 and MMP-9. Orally administered 100 mg tyndalized L. acidophilus reduced the expression levels of MMP-1 and MMP-9 (Fig. 3A). Furthermore, tyndalized L. acidophilus treatment suppressed the UVB-induced upregulation of MMP-1 and MMP-9. The effect of tyndalized L. acidophilus on UVB-induced MAPK phosphorylation was examined in mouse skin, as MMP-9 is primarily regulated by MAPK activation. As revealed in Fig. 3B, UVB irradiation induced the phosphorylation of p38, ERK 1/2, and JNK. Pretreatment with tyndalized L. acidophilus attenuated the phosphorylation of p38, ERK1/2, JNK, and MAPK in UVB-irradiated hairless mouse skin. In addition, tyndalized L. acidophilus inhibited the UVB-induced phosphorylation of MAPK/ERK kinase (MEK; Fig. 3C). The aforementioned data suggest that tyndalized L. acidophilus protects mouse skin from UVB-induced damage.