Beneficial Effects of Lacticaseibacillus paracasei subsp. paracasei ABF21013 on Skin Health by Regulating MMPs Production and EGFR Signal Pathway

Abstract

Increased ultraviolet (UV) exposure due to ozone depletion significantly affects human skin health. Recent skin research has been exploring various interventions to counteract the effects of UV-induced photoaging. UV directly affects skin components such as collagen and natural moisturizing factors (NMF), which are essential for the skin's structural integrity and hydration. Recent studies on the gut-skin axis have shown that certain probiotic strains could significantly improve skin aging. So, we examined the effect of ABF21013 on pro-collagen production and hyaluronic acid secretion using skin tissue from UVB-induced mice treated with ABF21013. Our findings reveal that ABF21013 reduced the total number of wrinkles and skin thickness by up-regulating MMP degradation and pro-collagen biosynthesis. These results indicate that ABF21013 can regulate wrinkle formation through the enhancement of collagen. Additionally, we observed downregulated transepidermal water loss (TEWL) regarding skin moisturizing. We also detected increased expression of EGFR ligands, AMPKα, hyaluronan synthase (HAS) -1/-2, and filaggrin. We have proved that ABF21013 can protect the skin barrier by activating the EGFR signaling pathway. Taken together, these results demonstrated that Lacticaseibacillus paracasei subsp. paracasei ABF21013 can enhance skin function by maintaining hydration and elasticity in a UVB-irradiated mouse model.

Introduction

Ultraviolet (UV) radiation is an environmental factor encountered in sunlight, that exhibits intense harmful effects on the human skin. The depletion of the earth’s stratospheric ozone layer has precipitated a notable increase in UV exposure, especially UV-B radiation (280–315 nm), which can directly cause severe damage via DNA damage, producing cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone (6-4 photoproducts, 6-4 PPs) [1]. Therefore, the specific wavelengths of UV radiation cause DNA photodamage and induce the excess generation of reactive oxygen species (ROS) such as hydroxyl groups (OH.) and superoxides (O2^.-), reaching a peak oxidative stress and a surge in ectopic inflammatory reactions leading to mutations of the cell, senescence, apoptosis or causing structural and physical defects of tissues [2]. With continuous exposure, such extreme molecular agitation contributes to the deterioration of skin architecture, a phenomenon clinically manifested as photoaging, which can display as skin aging, characterized by wrinkle formation, loss of elasticity, and dehydration or dryness. Photoaging is identified as a factor, which accelerates the natural aging process. Moreover, past and recent studies have provided profound evidence of the damage to mitochondria, which play a key role in cellular energy metabolism, which had been revised through strong free radical scavenging ability and antioxidant properties in mouse models [3]. Photoaging represents an intricate interplay of biochemical and structural alterations, prominently involving the extracellular matrix (ECM). Collagen, a typical part of ECM protein, is indispensable for maintaining the skin’s tensile strength and elasticity. However, chronic UVB exposure disrupts this balance by simultaneously enhancing the expression of matrix metalloproteinases (MMPs) such as MMP-1, which catalyze collagen fiber bundle degradation, and decreasing the pro-collagen biosynthesis through inhibiting transcriptional regulators such as Smad3, finally affects the skin showing lose elasticity, ultimately leading to ECM decomposition. These merged processes create the background for dermal thinning, wrinkle formation, and the progressive loss of skin resilience [4].

Besides collagen degradation, UVB radiation profoundly impacts skin hydration, a key causation of barrier function and comprehensive skin health. Hyaluronic acid is central to maintaining hydration, a glycosaminoglycan synthesized by hyaluronan synthases (HAS) such as HAS1 and HAS2, facilitating water retention within the dermis and epidermis, while maintaining anti-inflammatory and antioxidative effects [5]. UVB-induced oxidative stress impairs hyaluronic acid synthesis and compromises the stratum corneum's integrity, leading to elevated transepidermal water loss (TEWL) [6]. The resulting desiccation and barrier dysfunction underscore the multidimensional nature of photoaging, extending its consequences beyond superficial aesthetic concerns to increased susceptibility to environmental insults and microbial invasion. While plenty of interventions, ranging from topical antioxidants and sunscreens to systemic retinoids have been developed to mitigate UV-induced damage, these modalities often fall short of reversing established photoaging or addressing the systemic consequences such as distal organ inflammation of prolonged UV exposure that may appear as type II lung inflammation proven in human and mice[7]. This therapeutic insufficiency has inspired interest in more holistic approaches, particularly those targeting the gut-skin axis, an emerging paradigm in skin health research as the intestinal microbiota significantly impacts distant organs, including the brain, lungs, and skin, with gut microbes influencing health and disease. While traditionally studied for their role in linking intestinal and skin conditions, recent evidence highlights the reverse relationship, where skin disruptions, such as wounding or hyaluronan digestion, directly influence gut microbiome composition and host defense. Skin injuries upregulate intestinal defense genes such as Reg3 and Muc2 and alter bacterial behavior, promoting intestinal permeability and exacerbating conditions like colitis. This bidirectional skin-gut axis underscores the interconnectedness of these systems, with implications for understanding and treating diseases involving both tissues [8].

The skin-gut intercommunication describes the bidirectional interplay between the integumentary system and the gut microbiota the integumentary system, mediated through immunological, metabolic, and endocrine pathways. Recent studies have demonstrated that probiotics, particularly strains of LAB (lactic acid bacteria), can modulate proinflammatory responses systemically, enhance extracellular matrix remodeling, modulate MMPs, and hyaluronic acid, and fortify skin barrier function [9]. These microorganisms exert their effects by influencing key molecular pathways in skin repair and hydration. LABs are renowned for encouraging fibroblast migration, epithelial function, and macrophage activation to increase cytokine production and aid in tissue repair. Therefore, certain probiotic strains, such as Lacticaseibacillus paracasei and Lactobacillus coryniformis, improve nutrient absorption, particularly vitamins D, E, and B12, which are essential for wound healing, and improve the skin barrier. Probiotics used topically or orally support enhanced immune cell activity and the strengthening of the epithelial barrier [10]. Among LAB, L. paracasei subsp. paracasei has garnered significant attention for its immunomodulatory and reparative properties [11]. Previous studies have revealed its capacity to attenuate inflammation, bolster ECM integrity, and improve hydration in various dermatological contexts. LP-PBL, a L. paracasei-derived postbiotic, had been proven to exhibit anti-inflammatory effects by inhibiting cytokines IL-23A, IL-33, and TSLP, while enhancing filaggrin and ZO-1 expression in keratinocytes. Nevertheless, despite these promising findings, the specific mechanistic underpinnings of L. paracasei subsp. paracasei in ameliorating UVB-induced photoaging remain incompletely elucidated.

To address these gaps, the current study investigates the therapeutic potential of L. paracasei subsp. paracasei ABF21013 (ABF21013, Deposit Number: KCTC 16093BP) in a UVB-induced photoaging model. Specifically, we evaluate its efficacy in regulating the key molecular processes associated with ECM integrity, hydration, and barrier function. The current study investigates the strain’s influence on pro-collagen biosynthesis, hyaluronic acid production, and TEWL regulation. Furthermore, we explore its impact on epidermal growth factor receptor (EGFR) signaling and related pathways, including keratinocyte migration (HB-EGF) and strengthens the skin barrier (Filaggrin) to reduce moisture loss, while promoting dermal regeneration to inhibit wrinkle formation (pro-collagen), which is critical in maintaining skin health [12-14]. By elucidating these mechanisms, we aim to contribute substantively to the burgeoning field of probiotics in dermatology. Our findings provide insights into the role of ABF21013 in mitigating the structural and functional hallmarks of photoaging and underscore its potential as a nutraceutical intervention for promoting skin health. Such an approach aligns with the broader trend toward integrative strategies that address environmental skin damage's systemic and multifactorial nature.

Materials and Methods

Mouse Handling and Ethical Approval

The mice were cared for and used following the guidelines established by the Animal Ethics Committee of Corestemchemon Inc. Specific approval for the mouse experiments was obtained under protocol CHEM-2023-IA0766-00 from the Institutional Official and the Institutional Animal Care and Use Committee (IACUC) at Corestemchemon Inc. (Republic of Korea). A total of 32 female HR-1 hairless mice, aged 5 weeks, were purchased from Narabiotech ((Republic of Korea). The mice were housed in the Gyeonggi Bio Center Animal Breeding Area, which was maintained at a temperature of 23 ± 3°C, with 55 ± 15% humidity, a ventilation rate of 10-20 air changes per hour, and a lighting schedule of 12 h (lights on at 8:00 AM and off at 8:00 PM). The illuminance was maintained at 150-300 Lux, and the mice were provided with nutritionally balanced rodent food (Dooyeolbiotech, Republic of Korea) and sterilized water. All reasonable efforts were made to alleviate suffering, including the use of anesthesia for painful procedures.

Composition of the Test Group, UVB Irradiation, and Sample Intake

To classify the experimental groups, the weights of the mice were measured immediately after their acquisition, and a sufficient number of mice with weights close to the average were selected. The weights of the mice were measured, ranked, and randomly assigned to ensure that the average weight of each group was evenly distributed. The test groups were designated as Untreated, UVB only, UVB+ABF21013LD, UVB+ABF21013HD with eight mice assigned to each group. The test substances, lactic acid bacteria ABF21013 (ABF21013, Deposit Number: KCTC 16093BP), were administered orally at a low dose (LD) of 1 × 10⁹ CFU/mouse and a high dose (HD) of 1 × 10¹⁰ CFU/mouse, once per day, five times per week for 12 weeks, resulting in a total of 60 administrations. UVB irradiation occurred simultaneously with the intake of probiotics at an intensity sufficient to induce the minimal erythema dose (MED). In the first week, 25 mJ/cm² (1 MED) was administered three times; in the second week, 50 mJ/cm² (2 MED) was administered three times; in the third week, 75 mJ/cm² (3 MED) was administered three times; and from the fourth to the twelfth week, 100 mJ/cm² (4 MED) was administered three times per week Transepidermal water loss (TEWL) and skin moisture content were measured, and replica samples were collected at weeks 0, 4, and 12. At week 12, the mice were sacrificed, and an analysis of wrinkles and moisturizing factors was conducted using skin tissue samples. (Fig. S5).

General Symptoms, Weight Changes, Food Consumption

During the administration and observation period, mortality and general symptoms were assessed daily. The onset date and severity of symptoms were recorded for each individual when abnormalities occurred. The administration began on Day 1, and observation continued for 12 weeks. Weight changes were measured weekly throughout the testing period. Food consumption was also measured weekly during the administration of the test substance. The measurement method involved providing a specific amount of food, measuring the remaining food in each breeding box after one week, and calculating the difference. Food consumption was expressed as the average intake per group (g/group/week).

Skin Elasticity, Skin Moisture Content, and Transepidermal Water Loss

Skin elasticity, moisture content, and TEWL were measured using a Cutometer (MPA580, Courage+Khazaka Electronic GmbH, Germany), a Corneometer (CM820, Courage+Khazaka Electronic GmbH), and a Tewameter (TM300, Courage+Khazaka Electronic GmbH), respectively. Measurements were conducted on the dorsal area of UVB-irradiated mice at weeks 0, 4, and 12, following the manufacturer's instructions.

Wrinkle Measurement

The measurement of wrinkle-related factors was conducted through image analysis following the creation of a skin replica. Three drops of catalyst were added to approximately 5 grams of silicone polymer and mixed thoroughly. The resulting solution was applied to the back of the mouse, and after about 10 minutes, once the polymer material had hardened, the replica was carefully removed from the skin. The manufactured replica was then analyzed using the Visioline VL650 (Courage+Khazaka Electronic GmbH) to assess the depth, number, length, and thickness of wrinkles, in accordance with the guidelines provided by the manufacturer.

Histological Analysis

On the day of necropsy 12 weeks after the experiment, the animals were anesthetized by intraperitoneal administration of 1 ml/kg of a mixture of Zoletil 1 mg/kg and Lumpun in a 4:1 (v/v) ratio. Skin tissues were collected from the anesthetized animals and histopathological analysis, ELISA, WB, and RT-qPCR analyses were performed.

Histomorphometry Analysis

After sacrificing the experimental animals, the dorsal skin tissue was extracted, sectioned longitudinally, and rapidly frozen in liquid nitrogen. For hematoxylin and eosin (H&E) staining and immunohistochemical analysis, the skin tissue was fixed in a 10% neutral buffered formalin solution for 24 h. The fixed skin tissue was then embedded in paraffin to create a paraffin block, and slides were prepared by trimming and sectioning to a thickness of 3-4 μm using a microtome (RM2255, Leica Biosystems, Germany). The prepared slides were stained with H&E to assess the thickness of the epidermis and the stratum corneum, while the expression levels of matrix metalloproteinase-1 (MMP-1) and MMP-13 in the skin tissue were evaluated using anti-MMP-1 and anti-MMP-13 antibodies. In this study, the following antibodies were utilized: anti-MMP1 (AB137332, Abcam, USA), anti-MMP13 (AB39012, Abcam).

ELISA Assay

Mouse skin tissues were rapidly frozen in liquid nitrogen and subsequently thawed by overnight rotation at 4°C in RIPA buffer containing a proteinase inhibitor (25 μl of RIPA buffer per 1 mg of tissue; Atto, Japan). The samples were then centrifuged at 13,000 rpm for 10 min) to obtain the supernatant extract. The hyaluronic acid (HA) content in the tissue was measured using the Quantikine ELISA Hyaluronan Immunoassay (#DHYAL0, R&D Systems, USA), following the manufacturer's instructions. A standardization curve for HA was generated using the Hyaluronan standard included in the kit. The total protein content of the mouse skin tissues was quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific, USA) on the supernatant obtained from the tissue extract. A standardization curve for total tissue protein was created using BSA (MP Biomedicals, #160069, Republic of Korea). The quantity of HA expressed in the dorsal tissues of mice was determined by dividing the amount of HA measured using the kit by the total protein content.

Western Blot Analysis

After dissecting the back skin tissue of the mouse, 25 μl of RIPA buffer (containing a protease and phosphate inhibitor cocktail) was added per 1 mg of tissue, followed by overnight rotation at 4°C. The supernatant was obtained through centrifugation at 3,000 rpm for 5 min. Subsequently, 150 μl of 2X reducing buffer was mixed with 150 μl of the supernatant, and the mixture was subjected to heat treatment at 100°C for 5 min. From this preparation, 20 μl was separated using a 10–12% SDS-PAGE system (25 mM Tris, 250 mM glycine, 0.1% SDS) and transferred onto a polyvinylidene fluoride (PVDF) membrane using a power supply set to 100 V for 2 h). The membrane was blocked with TBS-T buffer (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, 5% skim milk) for 1 h at room temperature (RT). Primary antibodies and HRP-conjugated secondary antibodies, both diluted in TBS-T buffer, were sequentially applied to the membrane. The target proteins were visualized using enhanced chemiluminescence (ECL) reagent (EZ-Western Lumi Pico, DG-WP500, DoGenBio, Republic of Korea) and a chemiluminescence imaging device (Chemidoc, iBright 750, Invitrogen, USA). In this study, the following antibodies were utilized: anti-procollagen (ABT257, Sigma-Aldrich, USA), anti-β-actin (sc-47778, Santa Cruz Biotechnology, USA), and mouse anti-rabbit IgG-HRP (sc-2357, Santa Cruz Biotechnology).

RT-qPCR

To measure mRNA expression levels, 50 μl of RNA-Bee reagent (CS-104B, AMSBIO, USA) was added per 1 mg of mouse skin tissue, and total RNA was isolated by overnight rotation at 4°C. cDNA was synthesized using the isolated total RNA and the iScript cDNA synthesis kit (Bio-Rad, USA). Target gene expression in mouse skin tissue was quantified using real-time PCR (AriaMx Real-Time PCR system, IG8830A-1FIL, Agilent Technologies, USA). The sequences presented in Table 1 were used for the forward and reverse primer pairs. mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Table 1.

Primers sequences.

Gene	Direction	Sequences
MMP-1	Forward	5’-GCAGCAAAGTATGGGCTGTTC-3’
	Reverse	5’-TGTCACATTGCTAGGGAAGCC-3’
MMP-13	Forward	5’-GACCCCAACCCTAAGCATCC-3’
	Reverse	5’-CCTCGGAGACTGGTAATGGC-3’
Filaggrin	Forward	5’-GTCATCCCATGGTCAGGCAG-3’
	Reverse	5’-ACTGGAGATGGTTTGGAGTGG-3’
Amphiregulin	Forward	5’-ACAGCGAGGATGACAAGGAC-3’
	Reverse	5’-GCCAATAGCTGCGAGGATGA-3’
EGFR	Forward	5’-GTCAGAGATGCGACCCTCAG-3’
	Reverse	5’-CCTTGGCAGACTTTCTTTTCCTC-3’
AMPKα	Forward	5’-AAAGTGAAGGTGGGCAAGCA-3’
	Reverse	5’-CTCCGAATCTTCTGCCGGTT-3’
HB-EGF	Forward	5’-CAGAGTGCAGGGATCGGTTT-3’
	Reverse	5’-CGGAACACGAACGGTAGACA-3’
HAS1	Forward	5’-GCTGTGTAGGGCTGTGAGAC-3’
	Reverse	5’-TGCTCTTTTTGCTTCGCCAC-3’
HAS2	Forward	5’-CAGAGTGCAGGGATCGGTTT-3’
	Reverse	5’-CGGAACACGAACGGTAGACA-3’
COL1A1	Forward	5’-AGTCGCTTCACCTACAGCAC-3’
	Revere	5’-TTCGATGACTGTCTTGCCCC-3’
GAPDH	Forward	5’-GCCTCGTCCCGTAGACAAAA-3’
	Revere	5’-GAAGGGGTCGTTGATGGCAA-3’

Statistical Analysis

All experiment results were analyzed using GraphPad Prism software version 10.2 (USA) Data are presented as mean values ± standard deviation (SD) or standard error of the mean (SEM). Statistical analyses were performed using were one-way ANOVA and two-way ANOVA with Tukey’s multiple comparisons test for statistically significant differences at p < 0.05. ‘*’ indicates significance compared to the untreated group, while ‘#’ indicates significance compared to the UVB only group.

Results

ABF21013 Attenuates UVB-Induced Skin Wrinkle Formation in the Mouse Model

To investigate the impact of ABF21013 on UVB-induced photoaging, wrinkle formation was evaluated at 4 and 12 weeks post-UVB exposure. UVB exposure significantly induced wrinkle formation in the skin, with visible changes in wrinkle area, length, number of wrinkles, and depth. The photographs of skin replicas showed that oral administration of ABF21013 before UVB irradiation alleviated wrinkle formation (Fig. 1A). At 4 weeks, the percentage of the wrinkle area was markedly increased in the UVB-only group (7.2 ± 2.0%), compared to the non-UVB exposed control group (2.5 ± 0.7%, p < 0.0001) (Fig. 1B). This increase in wrinkle formation was further aggravated at 12 weeks, with the wrinkle area reaching 9.9 ± 2.4%. In contrast, treatment with ABF21013 significantly reduced wrinkle formation. The low-dose group (L) exhibited a wrinkled area of 5.5 ± 2.0%, while the high-dose group (H) showed a marked reduction in wrinkle area to < 5.0 % (p < 0.0001 vs. UVB-only group) at 12 weeks (Fig. 1B). The total wrinkle length followed a similar pattern, with UVB exposure increasing the wrinkle length to 116.7 ± 26.6 mm at 12 weeks, compared to 45.7 ± 12.5 mm in the control group (p < 0.0001) (Fig. 1C). ABF21013 treatment significantly reduced the total wrinkle length, with the high-dose group showing a length of 35.3 ± 10.4 mm (p < 0.0001 vs. UVB-only group) (Fig. 1C). Similarly, the number of wrinkles and wrinkle depth were significantly increased in the UVB-only group (Fig. 1D and 1E). The high-dose ABF21013 group substantially reduced both parameters (p < 0.0001). In detail, the number of wrinkles, UVB-only group showed 347.6 ± 62.5, while the high-dose ABF21013 treatment group reduced this to 139.9 ± 37.5 at 12 weeks (Fig. 1D). In the measurement of wrinkle depth, the UVB-only group showed 12,382 ± 2,180 μm, while the high-dose treatment group reduced this to 4,036 ± 1,080 μm at 12 weeks, while high-dose showed an intermediate suppression by ABF21013 (Fig. 1E). These results indicate that ABF21013 significantly attenuates the UVB-induced increases in wrinkle area, total wrinkle length, number of wrinkles, and wrinkle depth.

Fig. 1. ABF21013 attenuates UVB-induced wrinkle formation in a dose-dependent manner.

(A) Representative images of mouse dorsal skin illustrate wrinkle formation across treatment groups at 4 and 12 weeks. All mice were selected as HR-1 hairless, five-week-old mice. UVB irradiation was exposed to mice 3 times per week. ABF21013 was orally administrated 5 times a week. One time per every four weeks, transepidermal water loss, skin hydration, and skin elasticity replica were measured (B-E). Quantitative analysis was performed to assess wrinkle area, total wrinkle length, wrinkle number, and wrinkle depth. UVB exposure induced significant wrinkle formation, which was visibly mitigated by ABF21013 treatment, particularly at the high dose. Images and data reflect the antiphotoaging effects of ABF21013 in the UVB-induced mouse model. Data were expressed as the mean ± SD and statically analyzed by Two-way ANOVA (n = 8).

ABF21013 Minimizes UVB-Induced Epidermal Thickness

To access how UVB-induced photoaging affects the epidermal thickness of the skin and how ABF21013 recovered the effects, the histological evaluation was conducted and H&E staining of skin sections revealed significant differences in epidermal thickness between the UVB-exposed and control groups. UVB exposure resulted in a marked increase in epidermal thickness, with the UVB-only group showing 78.8 ± 11.4 μm at 12 weeks compared to 29.4 ± 3.8 μm in the control group (p < 0.0001, Fig. 2A). The thickening of the epidermis was consistent with UVB-induced hyperplasia and skin damage. Treatment with ABF21013 significantly reduced epidermal thickness. The low-dose ABF21013 group exhibited 43.4 ± 4.1 μm and high-dose ABF21013 group showed 47.2 ± 7.7 μm which were also significantly different from the UVB-only group (p < 0.0001). Histological images (Fig. 2A) showed reduced hyperplastic cells in the epidermis after ABF21013 treatment, with reduced keratinocyte proliferation in the treatment groups. The reduction in epidermal thickness made us hypothesize whether ABF21013 inhibits UVB-induced cellular proliferation and promotes skin repair mechanisms. Then, we focused on how UVB-induced photoaging affects the integrity of skin and the barrier function physically. skin barrier function, evaluated by skin moisture content, transepidermal water loss (TEWL), and elasticity, was significantly impaired by UVB exposure. At 12 weeks, UVB exposure significantly reduced skin moisture content to 26.5 ± 6.8 AU, compared to 50.7 ± 4.9 AU in the untreated control group (p < 0.0001, Fig. 2B). This reduction in skin moisture content is consistent with the impairment of the skin barrier function caused by UVB irradiation. Nevertheless, the treatment with ABF21013 restored skin moisture content. The high-dose group exhibited a significant improvement in moisture content (54.5 ± 9.3 AU, p < 0.0001 vs. UVB-only group), while the low-dose group showed an intermediate value (57.4 ± 5.2 AU, p < 0.0001 vs. UVB-only group). Further, at 12 weeks, UVB exposure increased TEWL to 14.6 g/m²/h, whereas the dose-dependent treatment with ABF21013 significantly reduced it to 10.2 ± 0.9 g/m²/h and 6.8 ± 1.9 g/m²/h, respectively (p < 0.0001, Fig. 2C). The treatment of ABF21013 improved skin elasticity. In detail, at 12 weeks, a significant increase was observed in the ABF21013-treated group (low-dose; 0.56 ± 0.06, high-dose; 0.64 ± 0.11, R2 value) compared to the UVB-exposed group (0.33 ± 0.06, R2 value; p < 0.0001, Fig. 2D). These results suggest that ABF21013 helps to restore the skin's moisture balance, preventing the drying effect associated with UVB exposure.

Fig. 2. ABF21013 reduces UVB-induced epidermal thickening and restores skin barrier function.

(A) Histological analysis of mouse dorsal skin stained with H&E to evaluate epidermal thickness after 12 weeks of UVB exposure. UVB-induced hyperplasia and epidermal thickening were visibly reduced in groups treated with ABF21013, particularly at the low and high doses. (B-D) Skin elasticity (R2), moisture content, and transepidermal water loss (TEWL, g/m²/h) were measured at 0, 4, and 12 weeks. Red arrow; Stratum corneum, White arrow; Epidermis. Data were expressed as the mean ± SD and statically analyzed by Oneway and Two-way ANOVA (n = 8).

ABF21013 Attenuates Matrix Metalloproteinases (MMPs) Hyperexpression Aggravated by UVB-Induced Photoaging

Based on the observations of Figs. 1 and 2, we hypothesized whether wrinkle development followed by UVB exposure caused degradation of extracellular matrix, leading to collagen degradation, which ultimately induce wrinkle formation. As shown in Fig. 3, the relative mRNA expression demonstrated increased metalloproteinases 1 and 13 (MMP-1 and MMP-13, p < 0.01 and p < 0.001) in the UVB only treatment, while the UVB-only group demonstrated suppressed COL1A1 (p < 0.01). However, both low and high doses of ABF21013 suppressed the UVB-indued MMPs’ expression (MMP-1 and MMP-13), while MMP-13 expressions at low and high doses were suppressed with high significance (p < 0.001 vs UVB-only group) compared to the mRNA expression of MMP-1 (p < 0.05 and p < 0.01 vs UVB-only group). The mRNA expression of MMP-1 revealed that the dose-dependent treatment of ABF21013 is important in the reduction of extracellular matrix (ECM) degradation, MMP-13 indicated recovery of UVB-induced ECM degradation regardless of low or high doses of ABF21013 (p < 0.001). Meanwhile, mRNA relative expression of COL1A1 supported the hypothesis of the improvement of ECM by ABF21013 through the dose-dependent increase only at the high dose in UVB-induced condition (p < 0.01 vs UVB-only group) compared to the UVB-only group which showed a significant drop in COL1A1 (p < 0.01 vs untreated control). Furthermore, we confirmed these molecular level expressions followed by immunohistochemistry analysis of MMP-1 and MMP-13, respectively by Fig. 3B and 3C, demonstrating that both low and high doses are effective in suppressing the UVB-induced damage with the same statistical significance (p < 0.01). At the same time, UVB-only group induced remarkable damage compared to the untreated control with an invasive morphological malformation in the tissue structure (p < 0.01 vs untreated control). This suggests that UVB exposure induces extracellular matrix (ECM) degradation by upregulating MMP-1 and MMP-13 while suppressing COL1A1 expression, leading to wrinkle formation. ABF21013 treatment, particularly at higher doses, significantly reduces MMP-1 and MMP-13 expression, mitigates ECM degradation, and restores COL1A1 levels, indicating its protective role against UVB-induced damage. Immunohistochemistry confirmed these effects, showing that both low and high doses effectively suppress UVB-induced tissue damage and morphological alterations.

Fig. 3. ABF21013 reduces UVB-induced extracellular matrix (ECM) degradation and modulates MMP and COL1A1 expression.

(A) Relative mRNA expression levels of MMP-1, MMP-13, and COL1A1 were accessed across experimental groups, illustrating the impact of ABF21013 on ECM regulation after UVB exposure. (B, C) Immunohistochemistry was conducted and quantified for MMP-1 and MMP-13 in dorsal skin sections that shows the differential expression patterns in response to ABF21013 treatment, highlighting its protective effects on tissue integrity. Data were expressed as the mean ± SD and statically analyzed by One-way ANOVA (n = 7).

ABF21013 Recovers Skin Damage after UVB-Induced Photoaging Damage by Improving Essential Factors, Responsible for Skin Tissue Integrity and Hydration

Since ABF21013 had already proven that both low and high doses can attenuate and improve the UVB-induced wrinkle formation and their essential molecular marker expression. Nevertheless, we further demonstrated the role of upstream molecular signaling and their associated marker expression level followed by a UVB-induced by engaging molecular signals, including heparin-binding EGF-like growth factor (HB-EGF), epidermal growth factor receptor (EGFR), AMP-activated protein kinase α (AMPKα) and Amphiregulin (AREG), which is a member of EGF family. Figure 4 A showed that UVB-induced photoaging markedly downregulated the mRNA expressions of AREG (p < 0.01 vs untreated control), EGFR (p < 0.001), and AMPKα (p < 0.01). Surprisingly, UVB-induced photoaging showed no significant alteration in HB-EGF mRNA expression compared to its untreated control. However, the higher dose of ABF21013 showed an elevated expression followed by UVB-induced photoaging (p < 0.05). Anyhow, in this signal molecule marker expression evaluation, the top two upstream molecules, including EGFR and AMPKα mRNA expression, were markedly upregulated by ABF21013 after a UVB exposure (EGFR; p < 0.05, p < 0.0001 and AMPKα; p < 0.001). Next, we designed the study to check whether the marker for epidermal hydration and epidermal barrier function are regulated in order to discover the ability of ABF21013 to recover the damage after UVB-induced photoaging in a mouse model. Fig 4B demonstrated that UVB-induced photoaging downregulated the Hyaluronan synthase 1 and 2 (HAS1 and HAS2) compared to their untreated control, showing a substantial reduction in both HAS1 (p < 0.05 vs control) and HAS2 (p < 0.001 vs control). However, in the ABF21013 treatment groups (UVB+ABF21013_L, UVB+ABF21013_H), the increase in HAS1 and HAS2 mRNA expression showed a trend but was not statistically significant (compared to the UVB-only treatment group). Nevertheless, the mRNA expression of filaggrin indicated by a high dose of ABF21013 showed a further increase (p < 0.0001) compared to the UVB-only exposure group, which showed a significantly upregulated mRNA expression compared to its untreated group (p < 0.01). These findings suggest that ABF21013 effectively alleviates UVB-induced photoaging on skin wrinkle formation, through the modulation of key molecular pathways and markers. It restores EGFR and AMPKα mRNA expression, which are crucial upstream cellular repair regulators, while enhancing filaggrin expression to improve epidermal barrier function. Although HB-EGF showed no significant changes after UVB exposure, the high dose of ABF21013 increased its expression, indicating potential involvement in repair processes. While the recovery of HAS1 and HAS2 expressions was limited, the overall ability of ABF21013 to counteract UVB-induced damage highlights its potential as a therapeutic agent for photoaging.

Fig. 4. ABF21013 modulates key molecular markers and enhances skin tissue integrity and hydration in UVB-induced photoaging.

(A) Graphical representation of key molecular pathways influenced by ABF21013, illustrating its role in mitigating UVB-induced damage. mRNA expression levels of upstream signaling molecules (EGFR, AMPKα, AREG, and HB-EGF) were evaluated across experimental groups, in UVB irradiation (B) mRNA expression levels of epidermal hydration and barrier function markers (HAS1, HAS2 and FLG) were accessed to show ABF21013's effects on restoring skin integrity and hydration post-UVB exposure. Data were expressed as the mean ± SEM and statically analyzed by One-way ANOVA (n = 7).

ABF21013 Repairs Pro-Collagen Degradation after a UVB-Induced Photoaging

Since ABF21013 enhanced the extracellular matrix, the skin barrier function, and water retention by upregulating of key epidermal structural marker expression (filaggrin and MMPs) and through regulating structural parameters such as skin thickness and skin elasticity, we next made a hypothesis whether pro-collagen expression in skin tissue is improved or not. Before accessing the pro-collagen degradation by UVB exposure and effects of ABF21013 on pro-collagen expression after a UVB exposure, we accessed the hyaluronic acid (HA) content (ng/ml) of the mouse skin (from the dorsal tissues). UVB exposure markedly reduced the HA content (p < 0.01 vs untreated control), however, ABF21013 enhanced HA content dose-dependently after the UVB exposure with two different significant levels by low (p <0.01) and high doses (p < 0.0001) of ABF21013 compared to the UVB-only group (Fig. 5A). After recovery of HA followed by UVB-induced photodamaging, we accessed the pro-collagen protein expression level normalized to the loading control β-actin. However, Fig. 5B demonstrated that only a high dose upregulated the pro-collagen expression after UVB exposure compared to the UVB-only group (p < 0.01). In contrast, even UVB exposure was insignificant compared to the untreated control. These findings suggest that AB21013 can recover the skin barrier function and protect against UVB-induced photoaging while improving ECM structure by enhancing the pro-collagen expression.

Fig. 5. ABF21013 enhances hyaluronic acid (HA) levels and pro-collagen expression in UVB-induced photoaging.

(A) Hyaluronic acid (HA) content (ng/mL) were evaluated by ELISA assay in dorsal skin tissues between four experimental groups, showing ABF21013's dose-dependent restoration of HA levels after UVB-induced reduction. (B) Western blot analysis was performed to access the pro-collagen expression of the dorsal skin of the mice, normalized to β-actin, to demonstrate significant upregulation with high dose ABF21013 treatment, highlighting its role in improving extracellular matrix integrity and skin barrier repair following UVB exposure. Each lane represents an individual mouse. Normal group (lanes N1–N8) and UVB induced groups are denoted as (U1–U8). Low dose of ABF21013 treatment groups (L1–L8) and high dose of ABF21013 treatment groups are denoted as (H1-H8). Data were expressed as the mean ± SD or SEM and statically analyzed by One-way ANOVA (n = 8).

Discussion

The current study discovered the protective effects of ABF21013 against skin health in human dermal fibroblasts (Hs68), keratinocytes (HaCaT), and UVB-induced photoaging in the dorsal skin of a mouse model. Degradation of the extracellular matrix (ECM) is a major cause of wrinkles and dehydration, and its main components are pro-collagen and hyaluronic acid (HA), which is presumed to enhance extracellular matrix integrity by activating the EGFR signaling pathway (Fig. S1). In addition, CFS was shown to have photoprotective effects and reduce IL-6 inflammatory cytokines in UVB-exposed HaCaT keratinocytes which is end-point of EGFR signaling pathway (Fig. S2). Consistently, in vivo supplementation mitigated UVB-induced photoaging in a mouse model, suggesting that stimulation of EGFR-mediated collagen and hyaluronic acid synthesis at the cellular level directly contributes to physiological skin protection. Morever, we determined whether ABF21013 does not produce intestinal toxins and has the adhesion ability to intestinal cells. As a result, it is expected that ABF21013 was shown to attach to the intestine without producing toxins through the LDH assay and can help intestinal health (Fig. S3 and S4). Therefore, ABF21013 is expected to adhere to intestinal epithelial cells and contribute to maintaining the gut microbiota and homeostasis, including skin health. Also, the in vivo results suggested that ABF21013 effectively mitigates UVB-induced skin damage, with evidence of attenuation in wrinkle formation, extracellular matrix degradation, and skin barrier function. ABF21013's protective mechanism appears to involve the modulation of key molecular signals. Compared to reports by Tirka, P.S.W. et al. (2023), and Feng, C. et al. (2024), ABF21013 markedly downregulated the expression of matrix metalloproteinases, MMP-1 and MMP-13, renowned contributors to ECM degradation and wrinkle formation [15]. Simultaneously, ABF21013 restored collagen type 1 alpha 1 (COL1A1) levels, further supporting its role in mitigating UVB-induced damage while exhibiting parallel effects, according to the findings by Kim, H.M. et al. (2014) [16]. The observed dose-dependent effects of ABF21013 on MMP-1 and COL1A1 suggest that higher concentrations may offer enhanced protection. The early studies done by Ito, T., et al. (2003), had already investigated the effects of sequential skin barrier disruption (via tape-stripping) and UVB exposure on skin immunity in BALB/c mice, highlighting complex interactions between barrier disruption and UVB in skin immune responses[17]. Subsequently, Sharma, M.R. et al. (2020) elucidated that UVB irradiation activates an inflammatory response, accompanied by increased MMP-13 expression and decreased COL1A1 expression, thereby promoting collagen degradation. However, in the same study, under conditions where the inflammatory response was suppressed, although MMP-13 expression decreased, a relative predominance of MMP-1 expression and an additional decrease in COL1A1 expression were observed. Consequently, collagen fragmentation and a reduction in mature collagen were further exacerbated. Direct comparison of these results with existing studies on the regulation of MMP-1, MMP-13, and COL1A1 expression suggests that inhibiting the inflammatory MMP-13 pathway alone is insufficient to effectively limit collagen degradation. Instead, a balance between MMP-1-mediated matrix degradation activity and COL1A1 transcriptional regulation appears to play a key role in maintaining collagen homeostasis [18].

McAleer, M.A. and A.D. Irvine (2013), reported that the stratum corneum (SC), the outermost epidermal layer, acts as a critical barrier against water loss and exogenous stress through its organized lipid-protein matrix and filaggrin-dependent structure. Filaggrin, derived from PR filaggrin, aligns keratin filaments, controls SC hydration through its metabolites, known as NMF components, and regulates epidermal pH for enzyme and barrier function. Loss-of-function FLG mutations dysregulate filaggrin production, leading to disrupted barrier integrity and variations in filaggrin copy number among populations [19]. Meanwhile, in a recent study, Wang, Z., et al. (2024), proved recombinant filaggrin (rFLG) improved tight junction protein expression, including LOR, ZO-1, and caspase-14, cell migration, and epithelial resistance in vitro, while alleviating UVB-induced epidermal damage, inflammation, and oxidative stress in mice, ultimately improving the barrier function [20]. In the current study, ABF21013 also upregulated the expression of filaggrin by rejecting the hypothesis that ABF21013 doesn’t affect the barrier function and improvement of the skin's physical structure. While the effects on hyaluronan synthase 1 (HAS1) and HAS2 were less pronounced, the increased hyaluronic acid content following ABF21013 treatment suggests a potential role in improving skin hydration. This observation is consistent with the known function of HA in maintaining skin moisture, which many past studies have proved. Seo, E., et al., 2023, demonstrated that UVB-induced photoaging in mice was mitigated by oral administration of mixed probiotics, which improved skin hydration, reduced transepidermal water loss (TEWL), erythema, and epidermal thickness, and significantly suppressed wrinkle formation by downregulating hyaluronic acid amount (ng/ml), MMPs and inflammatory cytokines via the MAPK pathway. It also decreased ERK, JNK, and p38 protein phosphorylation [21].

The study also explored the impact of ABF21013 on upstream signaling pathways. ABF21013 restored the mRNA expression of EGFR and AMPKα, which play crucial roles in cellular repair and homeostasis. Even though the study is not related to UVB-induced skin deformities, Wu, Y. et al. (2023), proved that Lactobacillus plantarum (LP)-derived postbiotics induce AMPK-mediated autophagy to in order to suppress inflammation and NLRP3, which can be beneficial in skin barrier function [22]. Furthermore, Hasegawa, T., M. Nakashima, and Y. Suzuki (2016), reported the inhibition of DNA damage repair by knockdown of XPA, a main component of nucleotide excision repair system, by inducing cyclobutane pyrimidine dimer (CPD) accumulation had reversed the suppression of inflammasome in human keratinocytes [23]. These results may suggest that AMPK-mediated regulation of ABF21013 can suppress photoaging in mouse models by inhibiting the NLRP3 inflammasome, and this will be further investigated in future studies. According to the reports by Lee, J.-O., et al. (2021), the expressions of filaggrin, transglutaminase-1, claudin, occludin, and HAS-1, 2, and 3 were enhanced with the treatments in improving the skin barrier and hydration properties, suggesting that recovery process was regulated through Src/AKT/NF-κB and MAPK signaling pathway [24]. However, the upregulation of heparin-binding EGF-like growth factor by ABF21013, despite no significant change after UVB exposure, warrants further investigation to elucidate its potential involvement in the repair process. Nevertheless, Stoll, S.W. et al. (2012) have reported the importance of HB-EGF, showing that the overexpression of HB-EGF reduced human keratinocyte growth by > 90%, even though HB-EGF mRNA expression was not affected by UVB irradiation in our study [25].

Finally, the increased pro-collagen expression following high-dose ABF21013 treatment further reinforces its ability to improve ECM structure and counteract UVB-induced damage. This finding, coupled with the observed increase in HA content, suggests a comprehensive protective effect of ABF21013 on skin structure and function.

In conclusion, the current demonstrates the protective effects of LAB, ABF21013 alongside UVB-induced photoaging proven in the female mouse model, indicating the capability of attenuating the extracellular matrix (ECM) and skin barrier dysfunction to restore wrinkle formation against UVB-induced photoaging. In the mechanistic evaluation, we discovered modulation of key molecular signals, including downregulation of MMP-1 and MMP-13, upregulation and restoration of COL1A1, upregulation of filaggrin, epidermal growth factor receptor (EGFR), and AMP-activated protein kinase α (AMPKα). Moreover, ABF21013 improved the hyaluronic acid (HA) levels, promotion of skin hydration through improving transepidermal water loss (TEWL) and skin moisture content across the epidermis, improvement of epidermal structural barrier properties through maintaining the skin elasticity, pro-collagen recovery after UVB irradiation. These findings suggest that ABF21013 is a promising therapeutic candidate in addressing UVB-induced photoaging through its capability of improving skin barrier integrity and hydration properties. Here, we further suggest implementing trials with ABF21013 to evaluate synergistic effects with probiotics, postbiotics, or another topical agent to improve therapeutic effects to enhance HAS-1 and HAS-2-mediated regulation of epidermal recovery. We suggest future studies on the long-term effects of ABF21013 over 12 weeks to discover the impact on collagen homeostasis, chronic inflammation, and epigenetic and posttranslational modifications.

Supplemental Materials

Supplementary data for this paper are available on-line only at http://jmb.or.kr.