Protective effect of oat bran extracts on human dermal fibroblast injury induced by hydrogen peroxide

Protective effect of oat bran extracts on human dermal fibroblast injury induced by hydrogen peroxide 1 2 first-author 2 3 1 3 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:weishaomin@jahwa.com.cn †E-mail:weishaomin@jahwa.com.cn weishaomin@jahwa.com.cn School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, China School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, China R&D Center, Shanghai Jahwa United Co., Ltd., Shanghai 201702, China R&D Center, Shanghai Jahwa United Co., Ltd., Shanghai 201702, China Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200433, China Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200433, China Oat contains different components that possess antioxidant properties; no study to date has addressed the antioxidant effect of the extract of oat bran on the cellular level. Therefore, the present study focuses on the investigation of the protective effect of oat bran extract by enzymatic hydrolysates on human dermal fibroblast injury induced by hydrogen peroxide (H 2 O 2 ). Kjeldahl determination, phenol-sulfuric acid method, and high-performance liquid chromatography (HPLC) analysis indicated that the enzymatic products of oat bran contain a protein amount of 71.93%, of which 97.43% are peptides with a molecular range from 438.56 to 1 301.01 Da. Assays for 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity indicate that oat peptide-rich extract has a direct and concentration-dependent antioxidant activity. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay and the TdT-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay for apoptosis showed that administration of H 2 O 2 in human dermal fibroblasts caused cell damage and apoptosis. Pre-incubation of human dermal fibroblasts with the Oatp for 24 h markedly inhibited human dermal fibroblast injury induced by H 2 O 2 , but application oat peptides with H 2 O 2 at same time did not. Pre-treatment of human dermal fibroblasts with Oatp significantly reversed the H 2 O 2 -induced decrease of superoxide dismutase (SOD) and the inhibition of malondialdehyde (MDA). The results demonstrate that oat peptides possess antioxidant activity and are effective against H 2 O 2 -induced human dermal fibroblast injury by the enhanced activity of SOD and decrease in MDA level. Our results suggest that oat bran will have the potential to be further explored as an antioxidant functional food in the prevention of aging-related skin injury. Oat bran Extraction Antioxidant Human dermal fibroblasts Cell injury Introduction 1 Avena sativa (oat) is a usually consumed as a whole-grain cereal and is distinct among the cereals due to its multifunctional feature and nutritional profile (Masood et al., 2008 2008 ). Recent advances in food and nutrition have revealed that oat contains many types of phytochemicals that have antioxidant properties, such as tocotrienols, flavonoids, phenolic acids, sterols, and phytic acid (Collins et al., 1991 1991 ; Peterson, 2001 2001 ) and that oat and its products have proven helpful in the treatment of diabetes and cardiovascular disorders. In particular, oat bran is a good source of B-complex vitamins, protein, fat, and minerals, as well as the heart-healthy soluble fiber β-glucan (Beck et al., 2009 2009 ). Recently, Ren et al. ( 2011 2011 ) reported the effect of avenanthramide-rich extract (ARE) from oat bran processing on the activity of antioxidant enzymes in D -galactose-induced oxidative-stressed mice. Administration of D -galactose markedly lowered not only the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) but also the gene expression of manganese SOD, copper-zinc SOD, GPx, and lipoprotein lipase (LPL) messenger RNA (mRNA) in mice. Although there are reports that oat bran contains at least 17.1% protein (Marlett, 1993 1993 ), and our previous study has presented a method of extracting polypeptides from oat bran using enzymatic hydrolysates (Feng et al., 2007 2007 ; Wang et al., 2008 2008 ), no study has addressed the effect of oat peptide-rich extract (Oatp) from oat bran on cell injury protection or antioxidative enzymes, particularly at the cellular level. So that, investigation of the antioxidant effect of the protein component in oat bran using enzymatic hydrolysates could increase the understanding of the efficacy of oat bran. Fibroblasts are essential cellular components of human skin and are the major cell type in the dermis. They play a pivotal role in the regulation of skin physiology and pathology because they generate and compose the extracellular matrix of the dermis. Additionally, fibroblasts carry out communications with each other and with other cell types in the skin (Gillitzer and Goebeler, 2001 2001 ). Because fibroblasts release multiple cytokines and growth factors through paracrine and autocrine interplay, they also play a significant role in inducing cell apoptosis or stimulating cell proliferation. In addition, they are involved in wound repair, immune processes, and inflammatory responses in the dermis (Niu et al., 2008 2008 ; Wiegand and Hipler, 2009 2009 ). Therefore, an investigation of the mechanisms by which fibroblasts modulate cell proliferation or apoptosis is critical to our cognition of dermal physiology and for skin’s pharmacological and clinical studies. The normal cellular environment with oxidation-reduction is important for cell physiological function. Oxidative stress has the capacity to cause cellular damage or death via diverse mechanisms, which depend on the nature of the oxidant and the environment by which it was produced (Klamt and Shacter, 2005 2005 ; Klamt et al., 2009 2009 ). Although oxidative stress products include radicals, such as hydroxyl radicals and superoxide anions, it has been proposed that cell damage induced by oxidative stress is mainly associated to the production of hydrogen peroxide (H 2 O 2 ), because H 2 O 2 is more stable and, thus, more able to diffuse throughout a larger tissue area. In the past few years, the cytotoxic effects of H 2 O 2 on human fibroblasts have been studied (Yuan et al., 2003 2003 ; Ohshima, 2004 2004 ). H 2 O 2 -induced oxidative injury provides a model for studying the effects of cellular protection of antioxidants. In this study, we first obtained the peptide-rich extract from oat bran by enzymatic hydrolysates. We then tested its effect on H 2 O 2 -induced dermal human fibroblast injury. The results demonstrated that Oatp possess antioxidant activity and is effective against H 2 O 2 -induced human dermal fibroblast injury through the enhanced activity of SOD and a decrease in the malondialdehyde (MDA) level. In addition to β-glucan which is known to have antioxidant effect, oat bran contains a variety of components whose role has not been fully understood. Our results revealed, for the first time, that the antioxidants effect of oat bran might be derived from Oatp, not only by β-glucan. Therefore, oat bran has the potential for further exploration as an antioxidant-functional food in the prevention of aging-related skin injuries. Moreover, Oatp may be used as additives in skin creams for anti-aging purposes and to help prevent skin damage induced by ultraviolet (UV). Materials and methods 1 Oatp preparation and analysis 2 The oat bran was prepared from naked oats provided by the Institute of Oats, Chinese Academy of Agricultural Science (Hebei, China). It was sieved through a 20–60 mesh screen. The oat bran (10 g) was diluted by 1:15 with 150 ml of 1 mol/L sodium hydroxide for 1 h at 55 °C and then reacted for 4 h with an alcalase enzyme at the temperature recommended by the enzyme manufacturer (55 °C). The enzyme to substrate ratio was 5%. After hydrolyzing, the enzyme in the hydrolysate was inactivated in a water bath at 100 °C for 20 min. Next, the solution was added to 1% β-glucan enzyme (0.01 g/ml; Novozymes, Denmark) and 1% saccharifying enzyme (0.01 g/ml; Novozymes, Denmark) and incubated for 1 h. The suspension was centrifuged at 4 000 r/min for 10 min in order to recover the soluble proteins and peptides. The protein and polysaccharide contents of Oatp from oat bran were quantified using the Kjeldahl method. The distribution of the molecular weight of Oatp was analyzed using high-performance liquid chromatography (HPLC) with electrochemical detection (ECD), as described by Lahogue et al. ( 2010 2010 ). Human dermal fibroblast culture 2 Primary cultures of skin fibroblasts from a healthy boy’s foreskin left over from surgery were grown in plastic flasks under standard conditions, as described previously (Feng et al., 2012 2012 ). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (0.1 g/ml) calf serum (CS), 100 U/ml penicillin, 100 U/ml streptomycin, and 0.25 mg/ml amphotericin at 37 °C in a humidified atmosphere with 5% CO 2 . Isolated cells were plated onto 35-mm-diameter petri dishes, with a seeding density of 5×10 4 cells/well and were grown to confluence over 24 h. The skin tissue was approved by Shanghai TCM-integrated Hospital with the written informed consent of the patient’ parents. MTT assay 2 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, a commonly used method to assess cell viability, measured mitochondrial activity in viable cells as described by Hansen et al. ( 1989 1989 ). The cells were plated at a density of 5×10 3 cells/well in 96-well plates. After 24 h of incubation, the cells were treated with different concentrations of oat extract (0.1–5.0 mg/ml) and exposed to H 2 O 2 (0.8 mmol/L). The MTT was at a concentration of 5 mg/ml in phosphate buffered saline (PBS). MTT was applied to the culture medium at a terminal concentration of 1 mg/ml and incubated for 4 h again at 37 °C. Finally, 150 μl of dimethyl sulfoxide (DMSO) was added and its absorbance at 570 nm wavelength ( A 570 ) was measured by an enzyme-labeling instrument. Propidium iodide (PI) staining 2 Cell viability was evaluated by counting live cells (phase-bright cells) under phase-contrast microscopy, and the number of dead cells (PI-labeled nuclei) was counted under a fluorescence microscope. The number of surviving or dead cells was assessed by averaging counts from 32 fields of view in four independent experiments. The viability was calculated as the ratio of the live cells to total cells—that is, phase-bright+PI-stained—and related to values obtained from the untreated control group. TUNEL assay evaluation of apoptosis 2 The TdT-mediated digoxigenin-dUTP nick-end labeling (TUNEL) was used to detect the apoptosis of human dermal fibroblasts with a commercially available kit (Diagnostics GmbH, Germany). The cells were fixed in 4% (40 g/L) paraformaldehyde for 10 min and washed twice with PBS for 5 min each at 23–25 °C. The staining technique of the TUNEL was performed in accordance with the manufacturer’s protocol. TUNEL-positive cells were marked using diaminobenzidine, a substrate for the kit’s horseradish peroxidase. The stained cells were viewed with a microscope. The images were transferred using a digital camera. The proportion of apoptotic cells was calculated by counting the number of TUNEL-positive cells based on five randomly selected areas across the horizon, and comparing it with the total number of cells. Assay for DPPH radical scavenging activity 2 A 1 ml sample at different concentrations was added to 1 ml of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical solution (12 mg/100 ml). Ultrapure water was added to 4 ml. The mixture was shaken energetically for 30 s and left to stand for 5 min in a dark room at 23–25 °C. Its absorbance was observed at 517 nm ( A 517 ). The effect of DPPH radical scavenging of the sample was based on the following formula: DPPH scavenging effect=[1−( T − T 0 )/( C − C 0 )]×100%, where T 0 is the absorbance of the contrast sample and T is the absorbance of the sample. C is the absorbance of the positive control and C 0 is the absorbance of the negative control. Determination of SOD activity and MDA levels 2 The concentration of MDA and the activities of SOD were determined using commercially available kits purchased from the Jiancheng Biological Engineering Academy (Nanjing, China). All procedures were based on the manufacturer’s instructions. The assay for SOD activity was according to its ability to reduce the oxidation of hydroxylamine by O 2 − · generated from the xanthine-xanthineoxiase system. One unit of SOD activity was defined as the amount necessary to reduce the absorbance at 550 nm by 50%. The MDA level was measured at a wavelength of 532 nm by response with thiobarbituric acid to form a stable chromophoric product. The MDA levels are reported in units per milligram of protein. Data analysis 2 SAS 6.0 was used for statistics analysis. Results are expressed as mean±standard error of the mean (SEM). The analysis of variance (ANOVA) test was used for comparison among the multiple groups. The mean values between two groups were compared using Student’s t -test. A P -value < 0.05 indicated a statistical significance. Results and discussion 1 Determination of the main components of the enzymatic products of oat bran and Oatp’s antioxidant capacity 2 Using DNA samples isolated from 40 barley accessions as templates, polymorphic DNA fragments were amplified from 29 among 35 simple sequence repeat (SSR) primer pairs selected in this study, including three out of five SSRs (60%) located on chromosome 2H, four out of five (80%) on chromosomes 1H, 4H, 5H, and 6H, and all (100%) on chromosomes 3H and 7H, respectively. Using the method described in the materials and methods section, the polypeptide and protein contents of the enzymatic products of oat bran can reach 71.93%, and the polysaccharide content can be reduced to 23% using the Kjeldahl method. The enzymatic products of oat bran were then quantified by HPLC. The results shown in Fig. 1 1 demonstrate that the distribution of their molecular weights ranged between 438.56 and 1 301.01 Da, which accounted for the total distribution of over 97.43% of the enzymatic products of oat bran. Moreover, two major peaks were estimated to correspond to molecular weights of 1 301.01 and 921.82 Da based on the calculated calibration curve, which accounted for 54.70% and 31.09% of the total enzymatic products of oat bran, respectively. Because we added β-glucanase to avoid the effect of β-glucan (Beck et al., 2009 2009 ) during the enzymatic hydrolysis process, the enzymatic products of oat bran are low-molecular-weight peptide-rich extracts (Oatp). Two major peaks with estimated molecular weights of 1 301.01 and 921.82 Da were observed Previous studies in vivo or in vitro reported that oat bran has antioxidant properties (Ren et al., 2011 2011 ). We first determined whether the oat bran extracts have antioxidant activity using a scavenging DPPH radical assay. (−)-Epigallocatechin-3-gallate (EGCG), which has been reported in previous studies to exhibit certain antioxidant activity (Jung et al., 2008 2008 ), was used as the positive control. The results shown in Fig. 2a 2a illustrate that Oatp significantly increased the scavenged DPPH radicals in a concentration-dependent manner. When the concentration of Oatp was more than 0.5 mg/ml, 29% to 37% of the DPPH radicals were scavenged during a 20 min treatment. Although the effect of Oatp on DPPH radicals scavenged is lower than that obtained from the EGCG treatment (68%, Fig. 2b 2b ), Oatp’s potential ability to scavenge DPPH radicals was observed. The results indicate that Oatp possesses antioxidant activity. (a) Oatp significantly increased the scavenged DPPH radicals in a concentration-dependent manner. (b) EGCG that exhibited certain antioxidant activity in previous research was used as the positive control. * P (0.05 when comparing different concentrations of Oatp or EGCG by one-way ANOVA. Data are presented as the mean±SEM obtained from three independent experiments Pretreatment with Oatp to protect human dermal fibroblasts against H 2 O 2 -induced cell injury 2 To detect the antioxidant activity of Oatp, we first set up an oxidation damage model for human dermal fibroblasts incubated with different concentrations of H 2 O 2 to determine whether Oatp can protect human dermal fibroblasts against injury caused by oxidant stress. Cell viability was assessed using PI staining and MTT colorimetric assay; damaged cell nuclei, which indicated apoptotic cells, were detected with the TUNEL assay. As shown in Fig. 3a 3a , little PI staining and few TUNEL-positive cells were present in untreated control fibroblasts. After exposure to H 2 O 2 for 6 h, the cells staining with PI and the numbers of TUNEL-positive nuclei were significantly enhanced in a dose-dependent manner. The statistical analysis indicated that 0.4, 0.6, and 0.8 mmol/L of H 2 O 2 reduced the cell survival rate to (75.2±9.3)%, (71.8±8.5)%, and (45.4±15.2)% of the control, respectively (Fig. 3b 3b ). In contrast, the number of TUNEL-positive nuclei was significantly enhanced to (10.7±0.8)%, (16.2±1.1)%, and (32.3±2.9)% of the control (Fig. 3c 3c ). This result indicates that approximately 30% of the injured cells were apoptotic when the cells were treated with 0.8 mmol/L H 2 O 2 for 6 h. In addition, a concentration-dependent reduction in cell viability was examined by the MTT assay. The result shown in Fig. 3d 3d indicate that H 2 O 2 in a 0.1 to 1.0 mmol/L concentration range significantly reduced cell viability; when the H 2 O 2 concentration was higher than 1.0 mmol/L, only a few viable cells were observed. Moreover, the MTT assay result showed that the concentration that induced 50% cell death was 0.8 mmol/L H 2 O 2 for 6 h. (a) Cell viability and damaged cell nuclei detected by PI staining and TUNEL assay after exposure of human dermal fibroblasts to different concentrations of H 2 O 2 . (b, c) The statistical analyses of the reduced survival rates and TUNEL-positive cells were resulting from treatments with different concentrations of H 2 O 2 . (d) Statistical analysis of cell death rate measured by an MTT colorimetric assay with different concentrations of H 2 O 2 and treatment time. * P < 0.05 according to one-way ANOVA. Data (mean±SEM) are obtained from five independent experiments We then determined the effect of oat bran extracts on H 2 O 2 -induced dermal fibroblast injury using MTT assay and TUNEL detection. The concentration of Oatp was between 0.1 and 5.0 mg/ml (Fig. 4a 4a ). Pre-incubation of human dermal fibroblasts in Oatp for 3–12 h before H 2 O 2 injury did not significantly increase cell viability in human dermal fibroblasts exposed to 0.8 mmol/L H 2 O 2 for 6 h, except for a slight increase (8%) in cell viability observed in human dermal fibroblasts pre-incubated in 1.0 mg/ml of Oatp for 12 h (Figs. 4b and 4c 4b and 4c ). When the pre-incubation time with Oatp was increased to 24 h before H 2 O 2 injury, oat bran extracts exhibited a significant effect against H 2 O 2 -induced cell damage (Fig. 4d 4d ). The cell viability rate increased from (34.9±0.19)% in the absence of H 2 O 2 to (51.8±2.12)%, (69.1±1.61)%, and (78.0±1.79)% in the presence of 0.1, 0.5, and 1.0 mg/ml oat bran extracts, respectively. Interestingly, when Oatp and H 2 O 2 treatments were given simultaneously without pre-incubation, oat bran extract’s protective effect against cell injury did not occur (Fig. 4a 4a ), suggesting that the protection mechanism of oat bran extract against H 2 O 2 -induced cellular injury did not directly affect the chemical reaction of H 2 O 2 or the free radical absorption, although Oatp showed the effect on DPPH radicals scavenged like EGCG. However, we noticed that when the concentration of Oatp was higher than 2.5 mg/ml, cell viability decreased instead. This result suggests that in a concentration range of 0.1 to 1.0 mg/ml, pre-incubation of human dermal fibroblasts with Oatp for 24 h reduced cell injury induced by H 2 O 2 treatment. An Oatp concentration higher than 2.5 mg/ml may lead to cell toxicity. (a–d) Statistical analyses of the effect of oat bran extracts on viability rates of human dermal fibroblasts undergoing pre-incubation time of 0–24 h before H 2 O 2 injury. The viability rates were measured with an MTT colorimetric assay. * P < 0.05; ** P (0.01 according to the Student’s t -test, compared with H 2 O 2 alone. Data (mean±SEM) are obtained from five independent experiments. (e) Effect of oat bran extracts on H 2 O 2 -induced apoptosis detected by the TUNEL assay. (f) Statistical analysis of the effect of oat bran extracts on the apoptotic rate induced by H 2 O 2 . * P < 0.05 according to Student’s t -test, compared with H 2 O 2 alone Because H 2 O 2 -induced dermal fibroblast injury includes cell apoptosis, we also examined the effect of Oatp on H 2 O 2 -induced apoptosis with the TUNEL assay. In the control group, few morphological signs of nuclear damage or chromatin condensation were observed (Fig. 4e 4e ). Once injured by H 2 O 2 , cells exhibited condensed nuclei. Pre-incubation with 1.0 mg/ml oat bran extracts for 24 h before H 2 O 2 injury significantly reduced the number of TUNEL-positive nuclei of fibroblasts exposed to 0.8 mmol/L H 2 O 2 for 6 h. Statistical analysis indicated that the number of TUNEL-positive nuclei was reduced from (8.9±0.7)% to (14.5±0.9)% of the control (Fig. 4f 4f ). Oatp intervention in H 2 O 2 -induced fibroblast damage with increased SOD activity and lower MDA formation 2 Lipid peroxidation is one of the main events in free radical-induced cell injury. MDA is a byproduct of lipid peroxidation and is widely used as a biomarker of oxidative stress (Cini et al., 1994 1994 ). However, antioxidant enzymes, such as SOD and glutathione (GSH)-Px, are considered to effectively augment cells’ antioxidant defenses (Luo and Xia, 2006 2006 ). In our study, Oatp’s ability to protect fibroblasts against H 2 O 2 -induced cell damage required a longer pre-incubation time before the H 2 O 2 injury. Therefore, we determined the SOD and GSH-Px activities and the level of MDA formation to address whether Oatp prevented cell apoptosis due to increased antioxidant enzymatic activity and decreased intracellular MDA. The results show that exposing human dermal fibroblasts to 0.8 mmol/L H 2 O 2 for 6 h significantly decreased SOD activity from (25.4±1.35) U/mg protein of the control to (14.8±1.17) U/mg proteins, resulting in a reduction of (41.7±1.31)% compared to the control (Fig. 5a 5a ). Consistent with the pretreatment of human dermal fibroblasts with Oatp for 24 h before H 2 O 2 injury, SOD activity exhibited a concentration-dependent recovery. When 1.0 mg/ml of Oatp was used in the pre-incubation medium, SOD activity reached (25.0±0.88) U/mg protein, close to the level of the control. In contrast, Oatp significantly reduced the level of intracellular MDA. Exposing the human dermal fibroblasts to 0.8 mmol/L H 2 O 2 for 6 h significantly increased the level of intracellular MDA from (10.3±0.77) nmol/mg protein of the control to (14.4±1.02) nmol/mg proteins, resulting a raise of (28.5±1.21)% compared to the control (Fig. 5a 5a ). Pretreatment of human dermal fibroblasts with Oatp for 24 h before H 2 O 2 injury significantly inhibited the level of intracellular MDA (Fig. 5b 5b ). Moreover, the experimental results of the SOD activity and the level of intracellular MDA are consistent with the results observed for cell survival: the effect of Oatp on SOD activity and the level of intracellular MDA occurred only after pretreatment with Oatp for 24 h before H 2 O 2 injury. (a) The statistical analyses of the effect of oat bran extracts on antioxidant enzymes SOD of human dermal fibroblasts undergoing pre-incubation time of 0–24 h or no pre-incubation before H 2 O 2 injury. (b) The statistical analyses of the effect of oat bran extracts on the production of lipid peroxidation MDA of human dermal fibroblasts undergoing pre-incubation time of 0–24 h or no pre-incubation before H 2 O 2 injury. (c) The statistical analyses of the effect of oat bran extracts on antioxidant enzymes GSH-Px of human dermal fibroblasts undergoing pre-incubation time of 0–24 h or no pre-incubation before H 2 O 2 injury. The data (mean±SEM) for this statistical analysis were obtained from four independent experiments. * P < 0.05 by Student’s t -test, compared with H 2 O 2 alone It is notable that the activity of another antioxidant enzyme, GSH-Px, was not significantly affected by Oatp (Fig. 5c 5c ). Exposing the human dermal fibroblasts to 0.8 mmol/L H 2 O 2 for 6 h significantly decreased the level of intracellular GSH-Px from (101.7±2.11) nmol/mg protein of the control to (51.4±1.91) nmol/mg protein, a decrease of (49.5±1.97)% compared to the control. However, pretreatment of human dermal fibroblasts with Oatp for 24 h before H 2 O 2 injury did not lead to a significant recovery of the level of intracellular GSH-Px, whether low or high concentrations of Oatp were used (Fig. 5c 5c ). It is well known that SOD serves as the first gatekeeper in the antioxidant defense system designed to scavenge superoxide anions (Reiter et al., 2000 2000 ), while GSH-Px is a catalyzer that activates the reaction of lipid hydroperoxides with reduced glutathione to form glutathione disulfide (Venukumar and Latha, 2002 2002 ). An avenanthramides (AVE)-enriched mixture extracted from oat was reported to increase human plasma GSH-Px activity by 30%–35% (Chen et al., 2007 2007 ). In this study, both SOD and GSH-Px activities were significantly downregulated in H 2 O 2 -treated groups. However, pretreatment with Oatp restored only the activity of SOD, not GSH-Px. Therefore, we presumed that Oatp was able to prevent H 2 O 2 -induced oxidative stress in human dermal fibroblasts by selectively enhancing SOD activity, rather than GSH-Px activity. Previous research has revealed that lipid peroxidation occurs when ROS attacks the membrane polyunsaturated fatty acids, leading to their disintegration and the formation of short-chain alkyl radicals and aldehydes. MDA is a major reactive aldehyde that is formed during the final stages of lipid peroxidation of biological membrane polyunsaturated fatty acid (Cini et al., 1994 1994 ). Our results demonstrated that the administration of H 2 O 2 caused an increase in the MDA concentration of the human dermal fibroblasts, while the application of Oatp decreased the MDA level, suggesting that Oatp was effective in scavenging free radicals and suppressing the production of MDA. However, because the effect of Oatp on SOD and MDA required a longer pre-incubation time, further studies are needed to confirm whether it involves the expression of antioxidant enzymes and the details of its mechanism. Conclusions 1 In summary, the present study has shown that the extract obtained from oat bran by enzymatic hydrolysates is rich in low-molecular peptides. Administration of H 2 O 2 to human dermal fibroblasts caused cell damage and apoptosis, decreased antioxidants’ enzyme activity, and increased the MDA level. Pretreatment with Oatp could restore the antioxidant defense system by increasing SOD activity. Therefore, oat bran has the potential to be explored further as an antioxidant-functional food in the prevention of aging-related skin injury. Moreover, Oatp may be used as additives in skin creams for anti-aging purpose and to help prevent skin damage induced by UV. References EJ Beck , SM Tosh , MJ Batterham , LC Tapsell , XF Huang . 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