Biochemical Mechanism of Thai Fermented Soybean Extract on UVB-Induced Skin Keratinocyte Damage and Inflammation

Abstract

Ultraviolet B (UVB) radiation is a key factor contributing to photodamage in epidermal cells. This study investigated the protective effects of Thua Nao, a Thai fermented soybean product, against UVB-induced damage in human epidermal keratinocytes (HaCaT) and the underlying mechanisms. Thua Nao extract fractions were prepared using a solvent partition method. We found that the dichloromethane fraction (TN-DC), along with its isoflavones daidzein and glycitein, significantly protected against UVB-induced HaCaT cell death. This protection involved inhibiting caspase-9 and caspase-3 activation, thus preventing apoptosis. Additionally, treatment with TN-DC, daidzein, and glycitein suppressed the UVB-induced production of inflammatory mediators, including interleukin-6 (IL-6), IL-8, inducible nitric oxide synthase, and cyclooxygenase-2. These protective effects were associated with reduced intracellular reactive oxygen species and enhanced the levels of antioxidant enzymes, including superoxide dismutase and glutathione peroxidase 4. Signaling pathway analysis revealed that TN-DC activated the pro-survival ERK1/2 and Akt pathways while decreased the phosphorylation of JNK in UVB-exposed cells. On the other hand, daidzein and glycitein enhanced ERK1/2 activation and reduced the phosphorylation of JNK and p38 MAPKs. The involvement of ERK1/2 and Akt activation in cell survival was confirmed using specific inhibitors. Thus, TN-DC and its isoflavones protects keratinocytes from UVB-induced oxidative damage and inflammation by modulating MAPKs and Akt signaling.

Keywords: HaCaT keratinocyte; UVB; anti-apoptosis; fermented soybean; skin damage.

Figure 1

Effects of Thua Nao extract fractions on cell viability in UVB-irradiated HaCaT cells. HaCaT cells were treated with various concentrations of Thua Nao extract fractions for 48 h and the cell viability was measured by SRB assay (A). The protective effects of Thua Nao extract fractions on UVB-induced HaCaT cell death. The cells were pretreated with the fractions for 6 h and then irradiated with UVB at 15 mJ/cm². After incubation for 24 h, cell viability was measured using the SRB assay (B). The protective effects of different concentrations of TN-DC on UVB-induced damage in HaCaT cells (C). * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001, as compared to the UVB-irradiated alone group; ### p < 0.005 and #### p < 0.001 as compared to the non-UV group. The experiments were repeated with three biological replicates (n = 3) for each condition. The data represents three independent experiments and are presented as mean ± SD.

Figure 2

The chemical structures of daidzein, glycitein, and genistein. (A) The cytoprotective effects of major isoflavones in TN-DC against UVB-induced damage in HaCaT cells. The cells were pretreated with various concentrations of daidzein (B), glycitein (C), and genistein (D) for 6 h before UVB-irradiation; after 24 h, the cell viability was measured by SRB assay. * p < 0.05, ** p < 0.01, and **** p < 0.001 as compared to the UVB-irradiated alone group. #### p < 0.001 as compared to the non-UV group. The experiments were repeated with three biological replicates (n = 3) for each condition. The data represent three independent experiments and are presented as mean ± SD.

Figure 3

The antiapoptotic effects of TN-DC and its active compounds on UVB-irradiated HaCaT cells. Flow cytometric analysis of apoptosis, including representative histograms (A) and quantification (B), was performed to evaluate the effects of TN-DC and its active compounds on UVB-irradiated HaCaT cells. The cells were pretreated with TN-DC and its active isoflavones, followed by UVB irradiation at 18 mJ/cm². After 24 h of incubation, apoptosis was assessed using FITC Annexin V/PI flow cytometry. The upper left quadrant represents necrotic cells (PI-positive and Annexin-negative). The upper right quadrant shows late apoptotic cells (Annexin-positive and PI-positive). The lower right quadrant indicates early apoptotic cells (Annexin-positive and PI-negative). The lower left quadrant represents viable cells (Annexin-negative and PI-negative). The effect of TN-DC and its active compounds on MMP. HaCaT cells were pretreated with TN-DC and its isoflavones for 6 h, followed by UVB irradiation. After 18 h of post-irradiation incubation, the mitochondrial membrane potential was assessed using MitoView™ 633 staining. The results are presented as a histogram (C) and a bar graph (D). The flow cytometry histogram represents the MMP of the cells. The red histogram indicates the population of cells that have lost MMP, whereas the green histogram represents the population of cells with normal MMP. The effects of TN-DC and its bioactive compounds on the UVB-induced expression of apoptotic proteins were analyzed by Western blot at 24 h post-irradiation (E). Densitometric and statistical analysis of apoptotic protein expression levels, normalized to β-actin were performed (F). All experiments were repeated at least three times. * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001 compared to the UVB-irradiated group. ### p < 0.005 and #### p < 0.001 compared to the control group. The experiment of flow cytometry was repeated with two biological replicates (n = 2) for each condition. The data represents three independent experiments and is presented as mean ± SD.

Figure 4

The inflammation-modulating effects of TN-DC and its active compounds on UVB-irradiated HaCaT cells. HaCaT cells were treated with various concentrations of TN-DC and its active compounds for 6 h prior to UVB irradiation. Culture supernatants were collected 24 h post-irradiation, and pro-inflammatory cytokine levels were measured using ELISA. The production of IL-6 (A) and IL-8 (B) was quantified. Additionally, the effects of TN-DC and its bioactive compounds on the UVB-induced expression of pro-inflammatory enzymes, iNOS, and COX-2 were analyzed by Western blot analysis at 24 h post-irradiation (C). Densitometric and statistical analyses of relative protein expression levels were normalized to β-actin (D). All experiments were repeated at least three times. * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001 compared to the UVB-irradiated group. ### p < 0.005 and #### p < 0.001 compared to the control group. The ELISA experiment was repeated with two biological replicates (n = 2) for each condition. The data represents three independent experiments and is presented as mean ± SD.

Figure 5

The antioxidant potential of TN-DC and its active compounds on UVB-induced damage in HaCaT cell. The level of intracellular ROS in UVB-irradiated HaCaT cells was investigated by DCF-DA fluorescent dye (A). The cells were pretreated with TN-DC and active isoflavones for 6 h and incubated with DCF-DA for 30 min before being UVB irradiated (at 15 mJ/cm²). The fluorescence was immediately measured at an excitation/emission of 485/525 nm using a fluorescence microplate reader, after UVB exposure for 1 h. The effect of TN-DC and its bioactive compounds on the expression of antioxidant proteins SOD and GPx4 were evaluated by Western blot (B). Densitometric and statistical analyses of protein quantification data, normalized to β-actin, are presented as histograms (C). The ABTS and DPPH free radical scavenging activity results are shown in Table 2. All experiments were repeated at least three times. * p < 0.05 and ** p < 0.01 compared to the UVB-irradiated group. # p < 0.05 and ### p < 0.005 compared to the control group. The DCF-DA experiment was repeated with three biological replicates (n = 3) for each condition. The data represents three independent experiments and are presented as mean ± SD.

Figure 6

The effect of TN-DC and its active compounds on the modulation of the MAPK and Akt signaling pathways. HaCaT cells were pretreated with TN-DC and its active compounds overnight, followed by UVB irradiation for 1 h. The cells were then collected and subjected to Western blot analysis to assess the activation of MAPKs (A) and Akt (C). Densitometric and statistical analyses were performed to determine the relative phosphorylation levels of the MAPK (B) and Akt (D) signaling pathways. To evaluate the role of ERK1/2 and Akt activation in the photoprotective effects of TN-DC and its active compounds against UVB-induced cell damage, the cells were pretreated with TN-DC or its active compounds in the presence of an ERK1/2 inhibitor (PD98056) (E) or an Akt inhibitor (LY294002) (F), and cell viability was subsequently measured by SRB assay. * p < 0.05, ** p < 0.01, and *** p < 0.005 compared to the UVB-irradiated group treated with the tested compounds. ### p < 0.005 compared to the UVB-irradiated alone group. The experiment on cell viability was repeated with three biological replicates (n = 3) for each condition. The data represents three independent experiments and are presented as mean ± SD.