Effects of EPS on UVA-irradiated HSF cell viability
In this study, a CCK8 assay was used to determine the cytotoxicity of EPS and the protective effects of EPS addition on HSF under UVA irradiation. As shown in Fig. 1, EPS has no toxic effects on cells at concentrations between 10 and 1000 µg/mL. At the same time, the cell activity rate increased significantly with the increase in EPS concentration, which suggests that EPS has a proliferation effect on cells. Meanwhile, cells were obviously damaged after UVA radiation and cell viability increased with the increase in EPS concentration. This phenomenon further confirmed that EPS has certain protective effects and helps to restore damage caused by UVA to HSF cells. In view of the protective effect of EPS on cells after UVA irradiation, EPS concentrations of 100, 250, and 500 µg/mL were used for subsequent tests.
Effects of different concentrations of EPS on cell apoptosis
FITC-Annexin V and PI Apoptosis staining was used to stain HSF. The experimental results were shown in Fig. 2, EPS showed obvious inhibition of HSF apoptosis under normal conditions. The apoptosis rate was about 10% when the EPS concentration was 100, 250, and 500 µg/mL. After irradiation with UVA, the apoptosis rate of the model group was significantly increased, reaching nearly 50%. After pretreatment with EPS, the apoptosis rate of the cells decreased. It can be seen that when EPS concentration was 500 µg/mL, the apoptosis rate of the cells decreased to 23.8%, which played an obvious inhibitory effect on the apoptosis of the cells.
Effects of EPS on ABTS, ROS, and MDA in UVA-irradiated HSF cells
EPS measured the antioxidant capacity, ROS content and lipid oxidation of the cells under normal conditions, and it was found that the total antioxidant capacity (3A) and lipid oxidation level of the sample group and the control group were not significantly different (3C). EPS significantly reduced the content of reactive oxygen species (3B) at concentrations of 250 µg/mL and 500 µg/mL. EPS can reduce the oxidative stress of cells under UVA irradiation and thus alleviate oxidative damage. First, we detected the antioxidant capacity of the HSF cells (Fig. 3D), which decreased significantly after UVA irradiation. With the increase in EPS concentration, the antioxidant capacity of the cells increased, presenting an apparent dose-dependent relationship. Afterwards, the ROS content in the HSF cells was detected (Fig. 3E), and it was found that ROS increased significantly after UVA irradiation. Treating HSF with EPS can reduce the content of ROS produced under UVA irradiation, and the ROS content can be basically restored to the same level as that of the control group. Cellular oxidative stress was accompanied by lipid oxidation, and the degree of lipid oxidation was judged by measuring the MDA content (Fig. 3F). The MDA content was significantly decreased in HSF treated with EPS.
Effects of EPS on CAT, SOD, and GSH-px activity in UVA-irradiated HSF cells
CAT, SOD, and GSH-px are common antioxidant enzymes in the antioxidant system of cells. Antioxidant enzymes protect the organelles from oxidative damage by removing free radicals and ROS produced in cells. The contents of CAT, SOD, and GSH-px were measured by EPS under normal conditions, and the relative mRNA expression levels of these three antioxidant enzymes were also detected. The experimental results showed no significant difference in the contents of antioxidant enzymes and relative mRNA expression levels between the sample group and the control group (Additional file 1). After UVA irradiation, it can be seen from Fig. 4A that the CAT content in cells decreased significantly, while EPS could weaken the trend of CAT decline in HSF cells. When combined with Fig. 4D, it can be seen that EPS could induce HSF cells to increase the expression of CAT mRNA, and the CAT in cells has a significant upward trend when the concentration of EPS increased. It can be seen from Figs. 4B and 4E that the SOD content in cells decreased significantly after UVA irradiation. When the EPS concentration was 500 µg/mL, the relative expression of SOD mRNA in the cells was significantly increased, and the SOD content in the cells was also increased. It can be seen from Fig. 4C that the GSH-px content in cells was significantly reduced after UVA irradiation, and when the EPS concentration was 500 µg/mL, the GSH-px content was significantly increased (P < 0.001). As can be seen from Fig. 4F, the GSH-px mRNA expression level was significantly increased under different concentrations of EPS. In conclusion, EPS can promote the increase of the mRNA relative expression level of antioxidant enzymes in HSF after UVA irradiation and repair oxidative stress damage by increasing the activity of CAT, SOD, and GSH-px.
Effects of EPS on UVA-irradiated COL-I, MMP-1, and caspase-3 contents
A major factor in skin aging is the loss of collagen and the degradation of the extracellular matrix, while the latter can affect the synthesis of collagen. In this study, COL-I and MMP-1 contents and mRNA relative expression levels were measured to evaluate whether EPS had a certain delaying effect on cell senescence. Caspase-3 is one of the key enzymes in the process of cell apoptosis. After aging, cells will go on the road of apoptosis. We determined whether EPS has an inhibitory effect on cell apoptosis by measuring caspase-3. We measured the contents of COL-I, MMP-1 and caspas-3 of EPS in HSF under normal conditions, and found that the contents of COL-I (5A) and MMP-1 (5B) did not change significantly, and the relative mRNA expression levels (5D and 5E) were not significantly different. However, the activity of caspase-3 (5C) gradually decreased with the increase of concentration, and its mRNA relative expression level (5F) was lower than that of control group. It can be seen from Fig. 5G, the content of COL-I in HSF were significantly reduced. Under UVA irradiation after EPS pretreatment, the content of COL-I in HSF were significantly increased. It can be seen from Fig. 5J, EPS obviously increased the mRNA expression level of COL-I in UVA-induced cells. This indicates that EPS can promote the generation of collagen after oxidative stress. As shown in Fig. 5H that the content of cell matrix metalloenzyme increased significantly after UVA irradiation, indicating that oxidative stress can promote the accelerated degradation of the extracellular matrix, while EPS can inhibit the generation of MMP-1. When combined with Fig. 5K, it can be seen that when the concentration of EPS was 500 µg/mL, MMP-1 mRNA expression was significantly decreased (P < 0.001). As shown in Fig. 5I, after UVA irradiation, the content of caspase-3 in cells significantly increased, while the content of caspase-3 in cells pretreated with EPS showed a downward trend. When combined with Fig. 5L, it can be found that the relative expression level of caspase-3 mRNA in cells decreased significantly after EPS pretreatment. These results indicated that EPS could inhibit the expression of caspase-3 to some extent. In conclusion, EPS can improve premature aging caused by UVA irradiation, delay skin aging, and inhibit cell apoptosis.
Effects of EPS on UVA-irradiated cell senescence and apoptosis pathways
Based on the above experimental data, we finally chose to detect the mRNA relative expression levels of the HSF senescence and apoptosis pathways under normal conditions and after UVA irradiation when the EPS concentration was 500 µg/mL. Under normal conditions, EPS has no obvious effect on the relative mRNA expression levels of HSF senescence and apoptosis pathways (Additional file 2). Under UVA irradiation, it can be seen from Fig. 6A that the relative expression level of Bax mRNA in HSF cells was up-regulated, while it was significantly down-regulated and obviously lower than that of the control group after EPS pretreatment. It can be seen from Fig. 6B, the relative expression level of Bcl-2 mRNA in HSF were down-regulated after UVA irradiation, while it was significantly up-regulated after EPS pretreatment to 2.7 times that of the control group. As can be seen from Fig. 6C that the relative expression level of SIRT1 mRNA in HSF were down-regulated after UVA irradiation, while it was significantly up-regulated and obviously higher than that of the control group after EPS pretreatment. It can be seen from Fig. 5D, the relative expression level of p16 mRNA in HSF cells was obviously up-regulated after UVA irradiation, while it was significantly down-regulated after EPS pretreatment and obviously lower than that of the control group. It can be seen from Fig. 6E, the relative expression level of p53 mRNA in HSF cells was up-regulated after UVA irradiation, while it was down-regulated after EPS pretreatment to 0.4 times that of the control group. Figure 6F shows that the relative expression level of AKT mRNA in HSF cells was down-regulated after UVA irradiation, while it was significantly up-regulated after EPS pretreatment. As can be seen from Fig. 6G, the relative expression level of p21 mRNA in HSF cells was down-regulated after UVA irradiation, while it was significantly up-regulated after EPS pretreatment to four times that of the control group. As can be seen from Fig. 6H, the relative expression level of FOXO mRNA in HSF cells was significantly up-regulated after UVA irradiation, while it was significantly down-regulated after EPS treatment. In conclusion, when the EPS concentration was 500 µg/mL, the expression of key genes in the UVA-induced HSF cell senescence and apoptosis pathways was regulated to repair the oxidative stress damage to cells.
Transcription factor forkhead box protein O (FOXO) is a downstream effector of AKT, and the expression of FOXO target genes regulates cell growth, apoptosis, and senescence (Hou et al. 2016). FOXO is phosphorylated and degraded by AKT (Wolfgang et al. 2009), and the inhibition of AKT phosphorylation in the presence of apoptotic factors facilitates the translocation of dephosphorylated FOXO into the nucleus and triggers the expression of apoptosis-related genes (Bax, p16, p21, and p53, etc.), which leads to cell growth cycle arrest and apoptosis (Orabi Sahar Hassan et al 2020; Zhixue et al. 2019; Jesel et al. 2019; Jose and Kurup 2017). Among them, the p21 gene can resist apoptosis, and at the same time it inhibits proliferation and promotes apoptosis. On the one hand, p21 can cause cell cycle arrest, giving cells extra time to repair damage; on the other hand, p21 upregulation promotes apoptosis, so it has an antagonistic duality (Jose and Kurup 2017). However, SIRT1 can inhibit the expression of apoptotic factors through the deacetylation of p53 and FOXO, and promote the expression of genes related to cell damage repair and growth (SOD, CAT, and Bcl-2, etc.) (XiaoHu et al. 2021; Lulu et al. 2019).
According to other studies, excessive UV radiation promotes phenomena, such as apoptosis and stress damage, which lead to skin aging. To investigate and analyze the value of Lactobacillus reuteri SJ-47 strain EPS in skin protection, a UVA-induced HSF model was established to investigate the oxidative stress protective and anti-aging effects of EPS on HSF at the biochemical, cellular, and molecular levels, and our findings are consistent with this conclusion. The downregulation of AKT and SIRT1 expression and upregulation of FOXO expression in HSF under UVA stimulation at 18 J/cm2 indicated the activation of the FOXO apoptotic pathway. This was evidenced by the marked upregulation of the expression levels of downstream pro-apoptotic factors Bax, p16 and p53. Fortunately, the level of the activated FOXO apoptotic pathway was extremely significantly decreased in EPS-treated HSF when they were stimulated by UVA again. In particular, the expression levels of AKT, SIRT1, and anti-apoptotic factor Bcl-2 were extremely significantly up-regulated, which largely inhibited the nuclear displacement and acetylation levels of FOXO, thereby reducing the expression of apoptotic factors, while the upregulation of p21 causes cell cycle arrest to repair cell damage, and ultimately promotes cell growth and oxidative stress repair, as evidenced by the increased activity and expression levels of cellular antioxidant enzymes and increased cell survival rate. For example, under UVA stimulation, the survival rate of HSF incubated with EPS increased to 64% from 32% without incubation, and its HSF apoptosis rate decreased from 47 to 24% without incubation, while the intracellular antioxidant enzymes (SOD, CAT, and GSH-px) showed a significant increase in enzyme activity and expression level. Under the incubation of 500 µg/mL EPS, the antioxidant capacity of cells was significantly increased, and the contents of ROS and MDA were obviously decreased. In addition, we found that the expression of MMP-1 in HSF after EPS incubation was inhibited and the corresponding enzyme activity was significantly reduced, while the expression level of Type I procollagen and extracellular COL-I content were significantly up-regulated, all of which resulted in the optimal effects of EPS at 500 µg/mL, effectively alleviating skin aging caused by the degradation of the extracellular matrix due to UVA irradiation.
In conclusion, Lactobacillus reuteri SJ-47 strain EPS have strong photoprotective effects on UVA-irradiated HSF, giving them great significance in the regulation of the cell senescence and apoptosis pathways, and great potential value in cosmetic applications. However, further experiments are required to analyze the single components of Lactobacillus reuteri SJ-47 strain EPS and explore their physical and chemical properties. At the same time, in addition to the antioxidant effects of Lactobacillus reuteri SJ-47 strain EPS, we can also study their other effects on cells and further determine whether EPS can become a functional raw material for cosmetics (Additional file 1 and 2).