N-Terminal 5-Mer Peptide Analog P165 of Amyloid Precursor Protein Repairs Skin Photodamage Induced by UVB through the Nrf2 Signaling Pathway.

BACKGROUND: Acute photodamage is an acute inflammatory reaction of the skin after ultraviolet (UV) irradiation. Many drugs have been successfully used for the treatment and prevention of photodamage. AIMS: To evaluate the molecular mechanism of N-terminal 5-mer peptide analog P165 of amyloid precursor protein in repairing photodamaged rat skin.
MATERIALS AND METHODS: We establish a rat model of acute UVB photodamage. The ratskin was treated with or without 250, 500, and, 1000 μM P165. Histological analysis was performed by hematoxylin and eosin staining. Apoptotic cells were analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The kits were used to measure the levels of protein carbonyl (PC), malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione (GSH). Western blotting was used to measure Nrf2.
RESULTS: P165 repaired UVB-induced cutaneous erythema and edema, and reduced apoptosis of skin cells. The levels of PC, MDA, and 8-OHdG in 250 and 500 μM P165 groups were all lower than those in the solvent group. Activities of SOD, CAT, and GPx, and the level of GSH in P165 groups were higher than those in the solvent group. Nrf2 expression in the solvent group was higher than that in the negative group, whereas in the 500 μM P165 group was higher than in the solvent group.
CONCLUSIONS: Our findings suggest that P165 repairs the rat skin with acute photodamage by reducing oxidative stress. These activities may be mediated by promoting the Nrf2 signaling pathway. Thus, P165 may be a promising agent for the treatment of acute photodamage, which may be used in cosmetics and postsun repair. Copyright:

Introduction

As the largest sensory organ, the skin is the first line of defense of the human body. Photodamage of the skin caused by ultraviolet (UV) radiation has been a hotspot in the skin research field in recent years. Acute photodamage is an acute inflammatory reaction of the skin after UV irradiation, which is characterized by erythema, edema, pigmentation, dryness, and desquamation.[12] Short-term UV irradiation causes oxidative damage, inflammation, and apoptosis of keratinocytes, leading to the destruction of epidermal tissue and loss of the barrier function, which is the main cause of acute photodamage.[3]

When the body is stimulated by UV, cell metabolism changes and reactive oxygen species (ROS) produced by skin tissues accumulate, which disrupts the balance between oxidation and antioxidation, and leads to oxidative stress damage of tissues and cells, one of the major mechanisms of acute photodamage.[4] ROS is the main inducer of acute photodamage, which attack almost all cell structures and molecules, causing body damage. For example, lipid peroxidation generates malondialdehyde (MDA), protein oxidation generates carbonyl carbon, and DNA base modification generates 8-hydroxy- deoxyguanosine (8-OhdG).[5] ROS activate multiple signaling pathways at the transcriptional level, such as MAPK, NF-kB, Nrf2, TGF-β/Smad, and JAK/STAT. Among them, the Nrf2-ARE pathway is involved in the clearance of intracellular ROS. After activation of this pathway, it promotes the synthesis of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx). These enzymes play an antioxidative stress role to reduce ROS-induced damage to tissues and cells.

In the recent years, plant extracts and compounds have been successfully used for the treatment and prevention of photodamage and photoaging. Amyloid precursor protein (APP) is a single-pass transmembrane protein that is widely distributed in tissues and cells of the whole body, which produces toxic β-amyloid protein after cleavage by proteases. The N-terminal 17-mer peptide of amyloid precursor protein (APP17 peptide) is a small molecule polypeptide synthesized in our hospital, which is the 319–335 peptide fragment at the N-terminal of β-amyloid protein. APP17 peptide scavenges free radicals in hippocampal nerve cells and suppresses oxidative stress.[6] The N-terminal 5-mer peptide of amyloid precursor protein (APP5 peptide) is the active sequence of the APP17 peptide, which has similar antioxidant and antiapoptotic properties as full-length APP.[7] Our previous studies have reported that the N-terminal 5-mer peptide analog P165 of amyloid precursor protein (P165) plays a photoprotective role in fibroblasts by inhibiting ROS production and downregulating MMP-1 expression through the MAPK pathway.[89] However, there is little information regarding the effects of P165 on animal skin with acute photodamage. We hypothesized that UVB induces acute photodamage-related signaling pathways, and P165 might repair the oxidative stress–induced damage. Thus, this study aimed to verify the effects of P165 on Wister rats with acute photodamage and investigate the possible mechanisms of P165 in regulating the expression of antioxidant enzymes.

Materials and Methods

Animals and P165

The study complied with the National Institutes of Health guidelines for Care and Use of Laboratory Animals and was approved by the Bioethics Committee of Capital Medical University. All animals were managed in accordance with the guidance of the Animal Care and Use Committee of Capital Medical University. Thirty-six male Wister rats aged 6–8-weeks and weighing 25–30 g were used in the study. The rats were purchased from the Experimental Animal Center of Xuanwu Hospital and housed in an environment at a constant temperature (22–25°C) and humidity, and a 12 h light/dark cycle. The animals were provided a standard laboratory diet and water. All animals were acclimatized to the laboratory conditions for at least 1 week before experiments. P165 was synthesized by the Department of Polypeptide Synthesis Laboratory, Xuanwu Hospital, Beijing, China. P165 was dissolved in saline and diluted to 250, 500, and 1000 μM. The study was approved on December 1, 2019.

UVB irradiation and Wister rat treatments

The rats were randomly divided into six groups (n = 6 per group): negative control group (NC), UVB irradiation group (UC), solvent treatment group following UVB irradiation (SC), and P165 treatment group following UVB irradiation at 250, 500, 1000 μM (P1, P2, and P3). Hair was removed from the dorsal skin area (3 × 3 cm2) using a shaver. The rats were exposed to UVB radiation using an SH4B ultraviolet phototherapy instrument (SIGMA, Shanghai, China) with peak emission at 312 nm except for the negative group. We used a VL × 312 radiometer equipped with a UVB sensor (SIGMA, Shanghai, China) to measure UVB irradiation. To establish an acute photodamage model, the rats were placed 15 cm below the lamp and exposed to 1600 mJ/cm2 UVB, which is five times the minimal erythemal dose (320 mJ/cm2). After UVB irradiation, the dorsal skin of each rat was covered with a cotton sheet soaked in saline or 250, 500, and 1000 μM P165. Irradiation was repeated at 12 and 24 h after the first irradiation. All rats were anesthetized for 36 h to collect skin tissues.

H and E staining

Skin tissues were fixed in 10% formalin, embedded in paraffin, sectioned at 4–5 μm thicknesses, and mounted on slides. The sections were deparaffinized, rehydrated in an ethanol gradient, stained with H and E, and observed under an optical microscope (Olympus, Japan). The pathological sections of skin tissues were independently diagnosed by different pathologists.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining

Apoptotic epidermal cells were assessed using a TUNEL staining kit (Keygen, Nanjing, China). The positive cells were observed and counted under the microscope. The apoptotic index was calculated by the percentage of apoptotic cells among the total number of cells in the visual field. Two independent observers randomly selected three fields for each specimen to calculate the apoptotic cell number and apoptotic index.

Measurement of PC, MDA, 8-OHdG, SOD, CAT, GPx, and GSH levels in the skin

Tissue samples were harvested, homogenized in normal saline, and centrifuged at 4°C for 10 min at 859 g. The supernatant was collected to measure the indicators with kits per the manufacturer's protocols. The protein concentration was measured by using a BCA Protein Assay Kit (Beyotime, China). The kits were PC (Cat No. A087-1-2), MDA (Cat No. A003-1-2), 8-OhdG (Cat No. H165-1-2), SOD (Cat No. A00-1-2), CAT (Cat No. A00-1-1), GPx (Cat No. A005-1-2), and GSH (Cat No. A006-1-1) purchased from Jiancheng (Nanjing, China).

Western blotting

The skin tissue was homogenized in precold Radioimmunoprecipitation assay buffer (RIPA) lysis buffer containing a protease and phosphatase inhibitor cocktail. The protein concentration was quantified by a BCA kit (Jiancheng). Equal amounts of proteins (25 μg) were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis) (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% dry non-fat milk in tris-buffered saline with Tween 20 (TBST) buffer at room temperature for 1 h, the membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies were as follows: rabbit anti-Nrf-2 antibody (1:1000, Abcam) and anti-β-actin antibody (1:1000, Abcam). After washing three times for 10 min with TBST buffer, PVDF membranes were incubated with peroxidase-conjugated anti-rabbit IgG (1:5000, Abcam) for 1 h at room temperature, followed by three washes for 10 min with TBST buffer. Target proteins were visualized by an enhanced chemiluminescence system. Gray value analysis was performed using ImageJ software.

Statistical analysis

Statistical analysis was performed using SPSS for Windows version 16.0 (SPSS, Chicago, IL, USA). The data are shown as the mean ± standard error of the mean. Between-group differences were assessed by analysis of one-way of variance (ANOVA) or the student's t-test. P < 0.05 was considered statistically significant.

Results

P165 repairs UVB-induced cutaneous erythema, edema, and formation of sunburnt cells

Short-term UVB irradiation can cause acute skin photodamages such as erythema and edema. As expected, the skin of rats in the negative group had no erythema or edema. Conversely, a macroscopic change in the skin of rats was clearly seen in the UVB and solvent groups, such as erythema formation, edema, and mild congestive inflammation. Compared with UVB and solvent groups, the skin of rats in the P165 groups showed varying degrees of erythema and edema reductions. Histology showed no sunburnt cells or edema in the negative group, and P165 groups showed fewer sunburnt cells and lighter interstitial edema compared with UVB and solvent groups [Figure 1].

Figure 1

P165 repairs UVB-induced epidermal damage in rats (n = 6). #Groups: NC (negative control group), UC (UVB control group), SC (solvent control group), P1 (low P165 dose group), P2 (intermediate P165 dose group), and P3 (high P165 dose group)

P165 reduces UVB-induced apoptosis of skin cells

Acute UVB radiation causes sunburn of epidermal cells, which leads to apoptotic keratinocytes.[10] TUNEL staining is used to detect nuclear DNA fragmentation during the early stage of apoptosis. In our study, apoptotic cells in the epidermal layer of rat skin were observed by TUNEL staining to further explore whether P165 had a protective effect on epidermal cells. The P165 group showed significantly fewer apoptotic cells compared with UVB and solvent groups. The reduction of apoptotic cells was more obvious in 500 and 1000 μM P165 groups compared with the 250 μM P165 group [Figures 1 and 2h].

Figure 2

Activities of antioxidant enzymes [SOD (a), CAT (b), and GPx (c)] and levels of PC (e), MDA (f), 8-OHdG (g), and GSH (d). (h) Apoptotic index. #Groups: NC (negative control group), UC (UVB control group), SC (solvent control group), P1 (low P165 dose group), P2 (intermediate P165 dose group), and P3 (high P165 dose group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Effects of P165 on biological macromolecules in UVB-induced skin photodamage

ROS produced by UVB irradiation of skin induces oxidative stress, that damages tissues and cells. To assess whether P165 reduced UVB-induced oxidative stress and damage to biological macromolecules, we measured the contents of protein carbonyl (PC), MDA, and 8-OHdG in rat skin tissues. The contents of PC, MDA, and 8-OHdG in UVB and solvent groups were all significantly higher than those in the negative group, which indicated successful modeling. There was no statistically significant difference between the above three indexes in UVB and solvent groups, which indicated that the solvent had no effect on the contents of PC, MDA, or 8-OHdG in rat photodamaged skin tissues.

Our results showed that the contents of PC, MDA, and 8-OHdG in 250 and 500 μM P165 groups were all significantly lower than those in the solvent group. Moreover, the contents of PC, MDA, and 8-OHdG in 250 and 500 μM P165 groups showed a gradual decline with the increase in P165 concentration, but the differences were not statistically significant. In the 1000 μM P165 group, the contents of MDA and 8-OHdG were higher than those in the 250 and 500 μM groups with no statistically significant differences [Figure 2e-g].

P165 may regulate the antioxidant response through the Nrf2 Pathway

The activities of SOD, CAT, and GPx and the level of GSH in UVB and solvent groups were all significantly lower than those in the negative group, which indicated successful modeling. There was no statistically significant difference between UVB and solvent groups, which indicated that the solvent had no effect on the levels of SOD, CAT, GPx, or glutathione (GSH). SOD activity in the 250, 500, and 1000 μM P165 groups was higher than that in the solvent group, but the differences between the three P165 groups were not statistically significant. CAT activity in the 250, 500, and 1000 μM P165 groups was higher than that in the solvent group, but only the differences between the 250 and 1000 μM P165 groups and the solvent group were statistically significant. GPx activity in the 250, 500, and 1000 μM P165 groups was higher than that in the solvent group, but only the differences between 500 and 1000 μM P165 groups, and the solvent group were statistically significant. The GSH level in the 1000 μM P165 group was significantly higher than that in the solvent group [Figure 2a-d].

The results suggested that the influence of P165 on various indicators was not closely related to its concentration. Western blotting showed that Nrf2 expression was significantly higher in the negative group compared with the solvent and 500 μM P165 groups, and significantly higher in the 500 μM P165 group compared with the solvent group [Figure 3].

Figure 3

(a) Western blot analysis by the gray level. (b) Statistical analysis of the gray level

Discussion

In this study, we applied P165 to a Wister rat model of acute photodamage. At the macro level, P165 reduced skin erythema and edema in rats with acute photodamage. At the microscopic level, P165 reduced sunburnt cells, interstitial edema, and epidermal cell apoptosis. At the molecular level, P165 may increase the expression of antioxidants and antioxidant enzymes by upregulating the Nrf2 signaling pathway. Additionally, P165 effectively reduced the contents of PC, MDA, and 8-OHdG. These findings suggest that APP5 peptide analog P165 reduces the oxidative stress response of skin with acute photodamage, decreases oxidative damage of tissue cells, and has a certain repair effect on the skin with acute photodamage.

P165 suppresses neurotoxicity induced by hyperglycemia, glutamate, and streptozotocin, and increases cell metabolism, promotes the growth of neurogliocyte, and enhances the expression of antiapoptotic signaling proteins.[11] Xu et al.[12] found that P165 may enhance hippocampus-dependent spatial learning and memory in a rodent model of cognitive decline by regulating the insulin signaling pathway. P165 has also improved the impairment of insulin signal transduction in a diabetes mellitus rat model.[13] Wang et al.[9] have shown that P165 reduces UVA-induced photodamage of fibroblasts, including cytotoxicity and MMP-1 expression. Additionally, P165 reduces the expression of MMP-1 by inhibiting the MAPK signaling pathway and reduces the degradation of collagen, thereby reducing UVA-induced photodamage of fibroblasts.[8] This is the only relevant report on P165 in the skin field. Wang et al.[9] demonstrated the light protection effect of P165 in fibroblasts and our study verified the therapeutic effect of P165 against light damage in animal skin.

Excessive ROS produced by skin cells after UV irradiation damage the structures of lipid membranes, enzymes, and DNA, and affect their functions.[14] ROS directly or indirectly attack proteins in various manners, which leads to protein modifications including the peroxidation of amino acids, oxidation of sulfur-containing groups, disulfide bond formation, and glutathione. Indirect modification refers to the combination of proteins and fatty acid peroxide lysates to form carbonylated proteins. Additionally, lipid peroxidation generates MDA, and the cytotoxic activity of MDA exacerbates oxidative damage. Our study measured PC and MDA contents in skin tissues of rats, and found that PC and MDA contents of the UVB group were significantly higher than those of the negative and solvent groups, which indicated successful modeling. After P165 treatment, the PC and MDA contents of the P165 groups were lower than those of the solvent group, which indicated that P165 reduces the oxidative attack of ROS on proteins and lipids, thereby reducing the production of PC and MDA. DNA damage caused by UVB radiation appears to be caused by the direct attack of nucleotides by UVB.[15] Additionally, after photons of UV irradiation are absorbed by cells, the generated energy is transferred to DNA or molecular oxygen, which increases the oxidative stress response and leads to indirect DNA damage.[16] Our study showed that 8-OHdG content in the negative group was significantly lower than that in the UVB and solvent groups, which indicated successful modeling. The 8-OHdG content in the 500 μM P165 group was lower than that in the solvent group, which suggests that P165 facilitates the reduction of oxidative damage of DNA caused by ROS.

Nrf2 is an important transcription factor involved in the inflammatory response, DNA repair, antioxidant reaction, and autophagy activation, all of which are related to maintaining the skin barrier function.[1718] It regulates production of mitochondrial ROS.[19] Under physiological conditions, Nrf2 binds to Keap1 and is anchored in the cytoplasm. When UV irradiation or oxidative stress occurs, Keap1 is modified, so that the affinity of Nrf2 and Keap1 is weakened, and they separate.[1620] The separated Nrf2 transfers from the cytoplasm to nucleus, forms a heterodimer with sMaf protein, recognizes and binds to antioxidant response elements (AREs), and initiates expression of ARE-regulated phase II detoxification enzymes and antioxidant enzymes such as SOD, GPx, and CAT.[2122] These enzymes eliminate ROS, improve the ability of cells to resist oxidative stress, and maintain the redox balance in the body.

Pharmacological activation of Nrf2 is a potential method to treat acute photodamage.[23] Vega et al.[24] reported that a bixin formulation reduces epidermal hyperproliferation and oxidative DNA damage, and inhibits UV-induced light damage in Nrf2+/+, but not Nrf2-/-, SKH1 mice. Lin et al.[25] reported that an elderberry extract activates the Nrf2 signaling pathway, weakens oxidative stress injury, inhibits the NF-kB signaling pathway, downregulates the release of inflammatory factors such as IL-6, and reduces the UVB-induced inflammatory response. Kim et al.[21] have shown that Nrf2 the signaling pathway may be negatively regulated by the expression of inflammatory mediators in the early stage of the inflammatory response. In our study, the skin pathology of rats treated with P165 showed less erythema, edema, and epidermal cell apoptosis, which may be related to upregulation of the Nrf2 signaling pathway by P165 activation and the reduction of inflammatory mediator release. Our results are essentially consistent with these previous studies. Lu et al.[26] reported that paeoniflorin inhibits UVA-induced photodamage via Nrf2/HO-1/NQ-O1 signaling and lipid-binding protein perilipin 2. Similar to HO-1 and NQ-O1, SOD, GPx, and CAT are all antioxidant enzymes. We found that expression of Nrf2 in the P165 group was higher than that in the UVB and solvent groups, and SOD, GPx, and CAT contents in the P165 group were also increased compared with those in UVB and solvent groups. Therefore, our results are consistent with previous reports because P165 acted as an agonist of Nrf2. P165 may accelerate the transmission of Nrf2 signals to the nucleus, thereby upregulating the expression of Nrf2 in acute photodamaged rat skin and promoting its downstream antioxidant enzymes. These antioxidant enzymes eliminate ROS and maintain the cell oxidation balance.

This study preliminarily demonstrated that P165 reduces oxidative stress damage of Wister rat skin induced by acute UVB irradiation. The ROS-clearing ability of P165 may repair acute photodamaged skin, which may be regulated by the Nrf2 signaling pathway. Therefore, P165 may be a promising agent for the treatment of acute photodamage, which may be used in cosmetics and postsun repair, but further animal studies are required.

Financial support and sponsorship

This paper was supported by Beijing Natural Science Foundation (Grant No7172092).

Conflicts of interest

There are no conflicts of interest.