3.1 Physical properties of OB and OBF
The relative molecular weights of OB and OBF were determined by gel permeation chromatography (GPC), and the elution curves are shown in Fig. 2A. Molecular weight measurements (Table 5) showed that the average molecular weights of OB and OBF were 5.626633×106 and 10.605001×106 Da respectively. The molecular weight of OBF was higher than that of OB, which indicates that the enzymes produced during microbial fermentation help to destroy the plant cell wall, thus promoting the release of active substances.
The structure and morphology of oat bran before and after fermentation were observed using scanning electron microscopy (SEM). Figure 2B shows the morphological characteristics of oat bran under different observation accuracies. From the figure, it can be seen that OBF has better structural homogeneity, and compared with OB, the surface structure of OBF is rougher and has better adhesion.
Table 5
Determination of OB and OBF molecular weights
|
OB
|
OBF
|
Mn (Da)
|
165
|
145
|
Mw (Da)
|
5.626633×106
|
1.0605001×107
|
Mz (Da)
|
4.3279738×107
|
4.93026158×108
|
Mw/Mn
|
3.409657620×104
|
7.300501671×104
|
Mz/Mw
|
7.69194
|
46.48997
|
3.2 Determination of OBF content
As shown in Fig. 3, total sugars were abundant in OB and OBF, and the content in OBF was more than 1.6 mg/mL. OB and OBF were also rich in total phenols and total proteins, with OBF containing more than 0.39 mg/mL of total phenols, 1.53 times more than OB. The content of proteins compounds in OBF was significantly higher than that of OB, 1.46 times higher than that of OB. Compared with unfermented oat bran, a significant increase in the contents of all three bioactives occurred after S. commune fermentation.
3.3 Determination of OBF in vitro antioxidant activity
When more free radicals are generated in the body than its own scavenging ability can handle, it will cause damage to human health, while antioxidants can protect the body from damage caused by free radicals.
The DPPH radical scavenging effect of OBF is shown in Fig. 4A. When the volume fraction of both OB and OBF samples increased, both samples had stronger DPPH radical scavenging ability. When the volume fraction of OBF was 100%, the best scavenging effect on DPPH radicals was 68.01%.
The hydroxyl radical scavenging effect of OBF is shown in Fig. 4B. It can be seen that both OBF and OB had a good scavenging effect on hydroxyl radicals, with similar scavenging effects. The scavenging effect of OBF at 50% volume fraction could reach more than 90% for hydroxyl radicals and was superior to that of OB at 100% volume fraction.
The smaller the IC50 value, the better the free radical scavenging ability of the sample. From Fig. 4C, it can be seen that OB and OBF were more effective in scavenging hydroxyl radicals and OBF was superior with a volume fraction of 19.17% scavenging 50% of hydroxyl radicals.
As can be seen in Fig. 4D, The scavenging capacity of OBF for ABTS + and the reducing capacity of Fe2+ were significantly higher than those of OB. The results of the ABTS method showed that OBF was 2.66 times more effective than OB in removing ABTS+, reaching 0.41 mM Trolox equivalent. The results of the FRAP method showed that the reduction capacity of OBF for Fe2+ was 1.23 times higher than that of OB, reaching 0.46 mM Trolox equivalent.
3.4 Effects of OBF on cell viability
The results of the experiments without UVB irradiation (Fig. 5A) showed that the cell viability of the OBF group was significantly improved compared with that of the OB, and the cell viability was always greater than 80% when the volume fraction of OBF was in the range of 0.625%-5%, which indicated that the OBF was not toxic to the cells in this volume fraction range. OBF with volume fractions of 1.25% and 2.5% had a proliferative effect on cells with cell survivals rates of 103.48% and 103.89% respectively.
It could be seen from Fig. 5B, a significant decrease in cell survival occurred in the model group after undergoing UVB damage. When the volume fraction of the sample was 2.5%, it had a significant reparative effect on the cells. The reparative effects of OB and OBF on UVB-damaged HaCaT cells decreased when the volume fraction of samples was greater than 2.5%, and the reparative effect of OBF on UVB-damaged model group was significantly better than that of OB.
By analyzing cell viability and status, we selected 2.5% as the volume fraction for both samples for subsequent cellular level and molecular level experiments.
3.5 Effects of OBF on cell migration capacity
Figure 6, shows the results of our measurements of cell migration capacity. After UVB irradiation, a significant decrease in cell migration ability occurred in group M, and the empty area region was significantly larger than that in group C, which was not irradiated by UVB. After UVB irradiation, the OB and OBF samples were added, and the sizes of the empty areas were calculated 24 h later. The sizes of the empty areas in the two sample groups underwent a significant decrease in comparison with that in group M, which suggests that OB and OBF at a volume fraction of 2.5% had a certain proliferative effect on cells and a certain reparative effect on damage caused to the cell migration ability by UVB irradiation.
3.6 Effects of OBF on cellular oxidative stress
Excess ROS cross cell membranes and undergo oxidative reactions with a variety of biomacromolecular substances, which in turn cause a state of oxidative stress in the body (Fernando et al. 2016). When excessive ROS are produced in the body, the ROS production-consumption balance is disrupted, and the excessive ROS promotes the release of inflammation-related factors in the body, leading to inflammation of the skin and further damaging the barrier function of the skin. Removing excess ROS is beneficial to the health of the skin. Heme oxygenase-1 (HO-1) is an enzyme with antioxidant and anti-inflammatory activities, which acts mainly by catalyzing the degradation of heme (Kim et al. 2021). NAD(P)H: quinone oxidoreductase-1 (NQO-1), a homodimeric flavoproteinase, is a representative detoxifying enzyme for phase II carcinogens (Natarajan et al. 2010).
The scavenging effect of 2.5% OBF on ROS contained in the cells is shown in Fig. 7A & D. As can be seen from the figures, the control group without UVB irradiation had less green fluorescence and lower brightness, indicating that the content of ROS in group C was lower. After UVB irradiation, the content of green fluorescence in the model group was significantly higher and brighter, indicating that the cells were damaged and the content of ROS increased after UVB irradiation. 2.5% OB and OBF were added after UVB irradiation to determine the reparative effects of the samples on HaCaT cells after UVB irradiation. The green fluorescence intensity of both the OB and OBF groups were lower than that of the model group, and the content of ROS was also reduced, which indicated that both OB and OBF had a certain reparative effect on cellular damage caused by UVB irradiation, whereas the lower green fluorescence intensity of the OBF group indicated that the effect of OBF in removing ROS was better than that of OB.
As can be seen in Fig. 7B & C, the levels of both antioxidant enzymes decreased after UVB irradiation compared to the non-UVB irradiated control group. An increase in the content of both HO-1 and NQO-1 occurred after the cells were treated with OB and OBF, and the content of both enzymes in OBF was higher than that in OB, 1.54 and 1.23 times higher than that in OB respectively.
3.7 Effects of OBF on UVB-induced inflammatory cytokines
Exposure to excessive UVB irradiation leads to a disruption of the dynamic balance of intracellular ROS production and clearance, further contributing to a state of oxidative stress in the cells and leading to skin inflammation. TNF-α and ILs are important indicators used to evaluate whether or not an inflammatory response is occurring. The effects of OB and OBF on the content of inflammatory chemokines and their expression levels are shown in Figs. 8 and 9.
From Fig. 8, compared with the control group that did not undergo UVB irradiation, a significant increase in the content of all four inflammatory factors can be seen in the model group.
From Fig. 9, after UVB irradiation, the relative mRNA expression of all four inflammatory factors underwent a significant increase, OB and OBF with a volume fraction of 2.5% had an inhibitory effect on the mRNA expression of all four inflammatory factors, and the inhibitory ability of OBF on the release of inflammatory factors was superior to that of OB.
3.8 Effects of OBF on UVB-induced skin barrier-related factor content
With the assistance of FLG monomer in the connection, keratin fibers regularly gather in the outermost layer of the epidermis, forming a strong physical barrier, thus preventing water loss and the entering of external irritants (Hoyer et al. 2022). AQP3 is expressed predominantly in the plasma membrane of epidermal keratinocytes and transports water and glycerol (Schrader et al. 2012). In immature skin, which lacks an effective skin barrier, AQP3 plays a role in epidermal hydration and transdermal water transport (Agren et al. 2010). Initially kallikrein-7 (KLK-7) was thought to be an enzyme involved in the degradation of intercellular cohesive structures in the stratified squamous epithelial stratum corneum that catalyzes the degradation of the skin's outermost bridging granules, resulting in the detachment of cells from the surface of the skin (Kumar et al. 2020).
In Fig. 10, the amount of both FLG and AQP3 proteins in the model group without samples after UVB irradiation was significantly reduced, and the enzyme activity of KLK-7 was significantly increased. After irradiation with UVB and treatment with OB and OBF at a volume fraction of 2.5%, the relative expression of FLG was significantly increased compared with that of the model group, which were 2.55 and 3.32 times higher than that of the model group respectively. After treating the cells with OB and OBF, the relative expression of AQP3 was higher than that of the model group and even higher than that of the control group which was not irradiated by UVB, and the effect of OBF was better than that of OB. After treating the cells with OB and OBF, the relative expression of KLK-7 was significantly reduced compared with that of the model group, which were 0.70 and 0.38 times that of the model group respectively, and the effect of OBF was better than that of OB.
3.9 OBF can act on the JAK/STAT pathway
In Fig. 11, after UVB irradiation, the relative expression of JAK1, STAT1 and STAT3 mRNA were significantly increased, and the relative expression of SOCS1 mRNA was significantly decreased. After treating the cells with OB and OBF at a volume fraction of 2.5%, a significant decrease in the transcript levels of JAK1, STAT1 and STAT3, and a significant increase in the transcript level of SOCS1 occurred, and the effect of OBF was superior to that of OB. This suggests that OBF may be able to exert a reparative effect on UVB-induced skin inflammation by inhibiting the activation of the JAK/STAT pathway in HaCaT cells.
3.10 Safety of OBF
Figure 12A & B show the effects of different concentrations of OB and OBF on the hemolytic capacity of rabbit erythrocytes. The experimental results showed that the effect of different mass concentrations of the samples on the hemolysis of rabbit erythrocytes were relatively small, which indicated that OB and OBF had high safety profiles.
The Fig. 12C shows the stimulating effect on blood vessels. The stimulation score of the positive control group (NaOH) was calculated to be 18.45, with obvious hemolysis and strong stimulation. The negative control group (NaCl) had a stimulation score of 0.03, and there was no hemolysis in the blood vessels. The stimulation scores of OB and OBF were 0.08 and 0.07 respectively, and there was no hemolysis in the blood vessels, indicating that OB and OBF did not stimulate the eyes.