Postbiotic Effects of Pediococcus acidophilus LS for Anti-Melanogenesis, Photoprotection, and Wound Repair

Abstract

Skin health is significantly impacted by factors such as melanin production, UV-induced photodamage, and wound healing. Excessive melanin leads to hyperpigmentation, while UVA radiation accelerates skin aging and oxidative stress. This study investigated the multi-functional dermatological potential of S strain LS-derived cell-free supernatant (CFS-LS) to address these concerns. Our findings demonstrate that CFS-LS effectively inhibits melanogenesis in B16F10 cells. It significantly reduced α-MSH-induced melanin synthesis, comparable to arbutin, by downregulating key melanogenic enzymes (tyrosinase, TRP-1, and TRP-2) and regulatory proteins (p-CREB, MITF, SOX9, and SOX10). Mechanistically, CFS-LS suppressed the phosphorylation of MEK, ERK, p38, and JNK, indicating a dual inhibitory effect on both PKA/CREB and MAPK pathways. Furthermore, CFS-LS mitigated UVA-induced photodamage in HaCaT cells by significantly reducing intracellular reactive oxygen species and suppressing the downstream phosphorylation of p53 and α-MSH levels. It also restored UVA-suppressed Nrf-2 and HO-1 expression, enhancing cellular antioxidant defenses. Lastly, CFS-LS promoted skin wound healing by significantly enhancing HaCaT cell migration in a scratch assay, associated with increased p-MEK1/2 and p-ERK1/2 levels, and notably elevated collagen type I synthesis. Collectively, these results highlight CFS-LS as a potent multi-functional agent for skin protection and repair, with significant potential for cosmetic and therapeutic applications. The active components of CFS-LS warrant further investigation.

Keywords: Pediococcus acidophilus LS; cell-free supernatant; melanogenesis; photodamage; wound healing.

Figure 1

Growth curves of LS cultured in MRS and mMRS broth at 37 °C for 24 h. Data represent mean ± SEM (n = 3).

Figure 2

Effect of CFS-LS on melanin production in B16F10 cells, evaluated via melanin content (A) and Fontana–Masson staining (B). Cells were co-treated with α-MSH (1 μM), CFS-LS, or arbutin (300 μM) for 24 h. After treatment, melanin content was quantified, and stained melanin (black) and nuclei (pink) were visualized under an inverted light microscope (100×, scale bar = 50 μm). Data represent mean ± SEM (n = 3). * p < 0.05 vs. α-MSH group.

Figure 3

Effect of CFS-LS on melanogenesis-related protein expression in B16F10 cells after 24 h (A) and 48 h (B) of treatment. Cells were co-treated with 1 μM of α-MSH, indicated concentrations of CFS-LS, or 300 μM of arbutin. Protein expression levels were analyzed via immunoblotting. Immunoblots are representative of three independent experiments.

Figure 4

Effect of CFS-LS on MAPK signaling in B16F10 cells. Cells were co-treated with 1 μM of α-MSH, indicated concentrations of CFS-LS, or 300 μM of arbutin for 24 or 48 h. MEK expression was assessed at 24 h, and ERK, JNK, and p38 at 48 h via immunoblotting. Immunoblots are representative of three independent experiments.

Figure 5

Effect of CFS-LS pretreatment on UVA-induced ROS levels in HaCaT cells. Cells were pretreated with indicated concentrations of CFS-LS for 24 h, followed by UVA exposure (5 J/cm²). Intracellular ROS levels were measured after exposure. Data represent mean ± SEM (n = 3). * p < 0.05 vs. UVA group without CFS-LS.

Figure 6

Analysis of protein expression (A) and α-MSH levels (B) in UVA-irradiated HaCaT cells. The cells were seeded in six-well plates (3 × 10⁵ cells/well) and incubated for 24 h, then pretreated with various concentrations of CFS-LS for an additional 24 h, followed by UVA irradiation at 5 J/cm². After irradiation, they were returned to the incubator for 30 min. Total protein was extracted and analyzed via immunoblotting, while culture supernatants were collected for α-MSH level measurement. The representative blots in (A) show protein expression changes. The data in (B) are presented as mean ± SEM (n = 3). * p < 0.05 vs. UVA group without CFS-LS.

Figure 7

Effect of CFS-LS on wound healing in HaCaT cells. (A) Representative images, and (B) quantification of the cell-free area. Cells were pretreated with various concentrations of CFS-LS for 24 h, then scratched and incubated for an additional 24 h. Cell migration was assessed with light microscopy (50×, scale bar = 250 μm). Wound closure was quantified by measuring the remaining cell-free area, expressed as a percentage of the initial wound area. Data are presented as mean ± SEM (n = 3). * p < 0.05 vs. untreated (0%) group.

Figure 8

Effect of CFS-LS on MAPK signaling (A) and collagen type I synthesis (B) in HaCaT cells. For MAPK analysis, cells were pretreated with various concentrations of CFS-LS for 24 h, scratched, and then incubated for 10 min before lysis. Protein levels were assessed via immunoblotting, and representative blots showing changes in protein expression are presented in (A). For collagen analysis, cells received the same pretreatment; culture media were collected, and collagen type I levels were measured with ELISA. Data in (B) represent mean ± SEM (n = 3). * p < 0.05 vs. the untreated (0%) group.