Skin secretions of N. ventripunctata (n = 30; weight range 20–25 g) were collected as previously reported (Wu et al., 2018). Frogs were stimulated with volatilized anhydrous ether immersed in absorbent cotton, and their skin surface was seen to exude secretions. Skin secretions were washed with 0.1 M phosphate buffer (PBS), (pH 6.0, containing 1% protease inhibitor cocktail, Sigma, United States). The collected solutions containing skin secretions were quickly centrifuged (10, 000 × g for 10 min) and the supernatants were lyophilized.

The peptide purification procedures were performed according to the method described in our previous work (Wu et al., 2018). An aliquot (1 g) of lyophilized skin secretion was dissolved in 10 ml PBS and centrifuged at 5, 000 × g for 10 min. The supernatant was applied to a Sephadex G-50 (Superfine, Amersham Biosciences) gel filtration column (2.6 cm diameter, 100 cm length) equilibrated with 0.1 M PBS for preliminary separation. Elution was performed with the same buffer, collecting fractions of 3.0 ml. The absorbance of the eluted fractions were monitored at 280 nm. The anti-photoaging activity in mice was tested as described below. The fraction containing anti-photoaging activity was further purified by a C₁₈ reversed-phase high performance liquid chromatography (RP-HPLC, Gemini C₁₈ column, 5 μm particle size, 110 Å pore size, 250 mm length, 4.6 mm diameter) column. The elution is performed using a linear gradient of 0–80% acetonitrile containing 0.1% (v/v) trifluoroacetic acid in 0.1% (v/v) trifluoroacetic acid/water over 60 min as illustrated in Supplementary Figure S1B. UV-absorbing peaks were collected, lyophilized, and assayed for anti-photoaging activity. Peaks with anti-photoaging activity were collected and lyophilized for a second HPLC purification procedure using the same condition as illustrated in Supplementary Figure S1C.

N-terminal sequence of the purified peptide was determined by Edman degradation on an Applied Biosystems pulsed liquid-phase sequencer (model ABI 491). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify the purity of the isolated peptide. AXIMA CFR mass spectrometer (Kratos Analytical) was analyzed in linear and positive ion mode using an acceleration voltage of 20 kV and an accumulating time of single scanning of 50 s.

The experiment was performed according to the method described in our previous work (Wu et al., 2018). Total RNA was extracted from the skin of N. ventripunctata using RNeasy Protect Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions. An in-fusion SMARTer™ directional cDNA library construction kit was used for cDNA synthesis. The synthesized cDNA was used as template for PCR to screen the cDNAs encoding the purified peptide (antioxidin-NV). According to the sequence determined by Edman degradation, an antisense degenerate primer (antioxidin-NV-R1) was designed and coupled with a 5′ PCR primer (the adaptor sequence of 3′ PCR primer provided in the kit) to screen the 5′ fragment of cDNA encoding antioxidin-NV. Then, a sense primer (antioxidin-NV-F1) was designed according to the 5′ fragment and coupled with 3′ PCR primer from the kit to screen the full-length cDNAs. The PCR conditions were, 2 min at 95°C, and 30 cycles of 10 s at 92°C, 30 s at 50°C, 40 s at 72°C followed by 10 min extension at 72°C. The PCR products were cloned into pGEM^®-T easy vector (Promega, Madison, WI, United States). DNA sequencing was performed on an Applied Biosystems DNA sequencer, model ABI PRISM 377. Primers used in this research are listed in the supplementary material Supplementary Table S1.

Antioxidant-NV (GWANTLKNVAGGLCKMTGAA) and the scrambled version of antioxidin-NV called sNV (LTAGMAWNAKGKACTVGLGN), were synthesized by the peptide synthesizer Synpeptide Co. Ltd (Shanghai, China). The synthetic peptides were purified and then analyzed by HPLC and MALDI-TOF MS to confirm that the purity was higher than 98%.

Free radical scavenging activity was determined by measuring reduction of radical 2, 2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺) according to manufacture instruction of the kit GMS10114.4 (Genmed Scientifics INC, Shanghai, China). The total formation of products (i.e. the reduced form of ABTS and the purple antioxidin-NV modification) and the total consumption of ABTS radical were determined by linear regression analysis. The concentrations of ABTS and ABTS free radical were calculated by using ε₃₄₀ = 4.8 × 10⁴ M⁻¹cm⁻¹ and ε₄₁₅ = 3.6 × 10⁴ M⁻¹cm⁻¹, respectively (Yu et al., 2015). The purple antioxidin-NV modification was monitored at A₅₅₀.

Cytotoxicity against human skin fibroblasts (HSFs) (KCB 200537, Kunming Cell Bank, Chinese Academy of Sciences) and human HaCaT keratinocytes (KCB200442YJ, Kunming Cell Bank, Chinese Academy of Sciences) was determined by the MTT assay. Antioxidin-NV dissolved in serum-free DMEM medium was added to cells in 96-well plates (2 × 10⁴ cells/well), and the serum-free DMEM medium without antioxidin-NV was used as control. After incubation for 24 h, 20 μl of MTT solution (5 mg/ml) was added to each well, and the cells were further incubated for 4 h. Finally, cells were dissolved in 200 μl of Me₂SO₄, and the absorbance at 570 nm was measured. Rabbit erythrocyte suspensions were incubated with antioxidin-NV and then the absorbance of supernatant was measured at 540 nm. 1% (v/v) Triton X-100 and PBS were used as positive and negative controls, respectively (Mu et al., 2017).

The level of intracellular ROS generation was detected using 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH-DA) with an Ex/Em of 504/529 nm. After 24 h later with UVB irradiation and sample treatment, cells were stained with 30 μM 2′, 7′- DCFH-DA (Sigma, United States) for 30 min at 37°C in a CO₂ incubator. The cells were then analyzed by flow cytometry (FACSCaliburTM, Becton-Dickinson, CA, United States) and an inverted fluorescence microscope (Zeiss, Germany). Mitochondrial ROS production with an Ex/Em of 585/590 nm was detected using mitochondrial reactive oxygen ROS kit (CA1310, Solarbio, China) as described by the manufacturer’s instructions.

UVB irradiation and sample treatment were performed according to a method previously reported (Hwang et al., 2013a; Hwang et al., 2013b). When HaCaT or HSF cells were cultured in six-well culture plates (2 × 10⁶ cells/well) and reached over 80% coverage, cells were pretreated with serum-free DMEM for 12-h incubation, then cells were washed twice with phosphate buffered saline (PBS). The cells with thin layers of PBS were exposed to UVB lamps (JT8-Y20W, Philips, Netherlands) in the wavelength range of 280–320 nm and their irradiation intensity was measured with a UVB irradiometer (Shanghai Sigma High Technology Co. Ltd, Shanghai, China), controlling the total irradiation dose at 80 mJ/cm². After UVB irradiation, the cells were washed with warm PBS three times. The cells were immediately treated with antioxidin-NV (10, 20, and 40 μg/ml) or vitamin C (40 μg/ml, SCR, China) in serum-free medium conditions for 24 h. Control cells were maintained in the same culture conditions without UVB exposure.

DNA fragmentation was assayed by agarose gel electrophoresis. HaCaT cells were seeded in six-well plates and cultured as described above. After 24 h later with UVB irradiation and sample treatment, HaCaT cells DNA were extracted for DNA fragmentation analysis. Cellular DNA was extracted using cell genomic DNA extraction kit (Solarbio, China) as described by the manufacturer’s instructions. The DNA samples were mixed with the 6× loading buffer (TaKaRa, Japan) and stained with nucleic acid dye (ZEESAN, China), and then used 10 μl for 1% agarose gel electrophoresis and observed under UV light imaging system (Bio-Rad ChemiDoc™ XRS, United States).

After 24 h later with UVB irradiation and sample treatment, the cells were washed twice with ice-cold PBS and lysed with RIPA lysis buffer (Beyotime, China). The proteins were extracted for western blot analysis according to our previously described method (Wu et al., 2018). The concentration of protein was determined by the Bradford protein assay. Then the cellular proteins were separated on a 12% SDS-PAGE gel and electro blotted onto a polyvinylidene difluoride membrane. Primary antibodies against γH2AX, JNK, p38 MAPK, IkBα, NF-κB p65, caspase-3, cleaved caspase-3, Smad2 (1:2000; CST, United States), and β-actin (1:5000, Santa Cruz Biotechnology, United States) were used in western blot analysis.

HaCaT cells were seeded in 24-well plates (5 × 10⁵ cells/well) with cell crawling (Solarbio, China) and cultured as described above. After 24 h later with UVB irradiation and sample treatment, the cells were washed twice with ice-cold PBS, then treated with 0.5% Triton X-100 (Solarbio, China) for 15 min, and then blocked with 5% BSA (Solarbio, China) for 2 h, followed by an overnight incubation with a primary antibody against cleaved caspase 3 antibody (1:400, CST, United States), Phospho-Histone H2A.X (1:400, CST, United States) at 4°C, respectively. The experiments were conducted with anti-rabbit IgG-FITC (1:100, Solarbio, China) for 1 h at room temperature using DAPI-containing mounting tablets (Solarbio, China). The pictures were collected using an inverted fluorescence microscope (Zeiss, Germany).

Skin tissues in UVB-irradiated hairless mice were taken for tissue immunofluorescence staining. Primary antibodies against cleaved caspase 3, collage I (1:400, CST, United States) were used in tissue immunofluorescence analysis.

Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining was used to measure percentile of apoptosis in HaCaT cells. After 24 h later with UVB irradiation and sample treatment, the cells were re-suspended in 500 μl of 1× binding buffer and mixed with Annexin V-FITC/PI (Cat number APOAF, Sigma, United States). After incubation for 30 min, the cells were measured by Accuri C6 flow cytometry (Accuri, Ann Arbor, United States).

After 24 h later with UVB irradiation and sample treatment, culture supernatants were collected and assessed for transforming growth factor-β1 (TGF-β1) and IL-6 using ELISA kits (DAKAWE, Beijing, China).

Photo-aged skin tissue weighing 100 mg plus ice-cold PBS was fully ground into a 10% (m/v) tissue suspension. The suspension was processed by an ultrasonic disruptor (Saifei, China), centrifuged at 4°C for 10 min (3500 g/min), and the supernatant was collected. The supernatant was used to assay the level of TGF-β1 and IL-6 using ELISA kits (DAKAWE, Beijing, China).

Adult N. ventripunctata (n = 30; weight range 20–25 g) was collected from Shangri-La, Yunnan province of China. Adult male SKH-1 hairless mice were purchased from Labreal Laboratories and housed in the pathogen-free facility. At the termination of the study, mice were sacrificed by cervical dislocation under CO₂ anesthesia in accordance with the guidelines from the Care and Use of Medical Laboratory Animals (Ministry of Health, People’s Republic of China). All the animal study was reviewed and approved by the Institutional Animal Care and Use Ethics Committee of Kunming Medical University (IACUC approval number: KMMU2020063). All the animal experiments described in this study were conducted at Kunming Medical University.

Adult male SKH-1 hairless mice (n = 30, 6–8 weeks old, 20–30 g, Labreal Laboratories) were used. The mice were housed for at least 7 days prior to the experiments in a ventilated and temperature-controlled room and had access to water ad libitum. ASS-03AB UV phototherapy light source (Shanghai Sigma High Technology Co. Ltd, Shanghai, China) was used for UVB irradiation (wavelength 280–320 nm). The mice were randomized into five treatment groups (six mice per group): (Slominski et al., 2012) Sham (mice were covered with PBS); (Slominski et al., 2018) PBS (mice were covered with PBS after UVB exposure); (Portugal et al., 2007) NV (mice were covered with antioxidin-NV dissolved in PBS after UVB exposure); (Rinnerthaler et al., 2015) VC (mice were covered with vitamin C dissolved in PBS after UVB exposure, vitamin C, recognized as an antioxidant, is often used to prevent light-induced skin aging, therefore, vitamin C was selected as the positive control) and (Baek and Lee, 2016) sNV(the scrambled version of antioxidin-NV, mice were covered with sNV dissolved in PBS after UVB exposure). In the PBS, NV, VC and sNV group, mice were directly exposed to UVB radiation, then were treated with PBS, antioxidin-NV, vitamin C, or sNV (100 μl, 200 μg/ml) to the back, respectively. Mice were exposed to UVB radiation at 100 mJ/cm² (one minimal erythematal dose = 100 mJ/cm²) five times during the first week and then to 200 mJ/cm² three times a week for 12 weeks thereafter. After sacrifice, some of the skin tissues were snap frozen in liquid nitrogen and stored at −80°C, and others were fixed in formalin and embedded in paraffin for immunohistochemistry.

The tissues were fixed in 10% formalin. Then, the tissues were sectioned using a microtome and stained with hematoxylin and eosin (H&E) for histological analysis. The pathology slides were read in blindness, and the images were recorded.

The paraffin-embedded skin specimens were measured using Masson’s trichrome stain kit (Solarbio, China). The slides were stained with Bouin’s Fluid and Weigert’s iron hematoxylin working solution. Furthermore, the slides were differentiated in phosphomolybdic-phosphotungstic acid solution and stained with aniline blue solution. Finally, the slides were read in blindness, and the images were recorded.

The paraffin-embedded tissue sections were dried, deparaffinized, and rehydrated. Following a microwave pretreatment in citrate buffer (pH 6.0), the slides were immersed in 3% hydrogen peroxide for 20 min to block the activity of endogenous peroxidase. After extensive washing with PBS, the slides were incubated with γH2AX (1:480; CST, United States), Cleaved Caspase 3 antibody (1:200; CST, United States), Collagen I antibody (1:200; abcam, United Kingdom) or α-SMA antibody (1:100; abcam, United Kingdom) overnight at 4°C. The sections were then incubated with the secondary antibody for 1 h at room temperature, and the slides were developed using the UltraVision Quanto HRP detection kit (Thermo Scientific, United States). Finally, the slides were counterstained using hematoxylin. The slides were read in blindness, and the images were recorded.

Statistical differences were determined using Student’s t-tests or one-way ANOVA provided by GraphPad Prism software. Results are shown as mean ± SD from three independent experiments. A p value less than 0.05 was considered as statistically significant difference.

As shown in Supplementary Figure S1A, the skin secretions of N. ventripunctata were divided into five fractions after Sephadex G-50 gel filtration. The fraction containing anti-photoaging activity was pooled and subjected to a C₁₈ RP-HPLC column for further purification (Supplementary Figures S1B,C). The purified peptide was designated as antioxidin-NV (Supplementary Figure S1C). After Edman degradation, the amino acid sequence of antioxidin-NV was identified as GWANTLKNVAGGLCKMTGAA. MALDI-TOF MS analysis indicated that antioxidin-NV had a measured molecular mass of 1963.70 Da (Supplementary Figure S2), matching well with the calculated molecular mass of 1963.30 Da.

The cDNA clone encoding the precursor of antioxidin-NV was sequenced from the skin cDNA library of N. ventripunctata (GenBank accession number: MW114946). As shown in Figure 1, the deduced amino acid sequence of antioxidin-NV is completely consistent with that sequenced by Edman degradation. It is composed of 72 amino acid residues, including a predicted signal peptide (24 amino acid residue), an acidic peptide region (28 amino acid residue) that ends in a typical trypsin-like proteases processing site (-Lys⁵¹Arg⁵²-), followed by a mature peptide (20 amino acid).

ABTS⁺ free radical scavenging kinetics, owing to its relative stability, easy measurement, good reproducibility, ABTS⁺ radicals are commonly used to evaluate antioxidant capacity (Yang et al., 2009). We confirmed the antioxidant activity of antioxidin-NV by assessing its ability to scavenge ABTS⁺ free radical. The assay is based on decolorization by monitoring absorbance decreases at the characteristic wavelength of 734 nm. As illustrated in Figure 2A, antioxidin-NV could rapidly scavenge ABTS⁺ in a dose-dependent manner. It could get rid of ABTS⁺ immediately when it contacted with ABTS⁺. At the concentration of 80 μg/ml, antioxidin-NV scavenged 96% ABTS⁺ within 1 min, and scavenged nearly 99% ABTS⁺ within 8 min. Even the concentration down to 5 μg/ml, 40% ABTS⁺ was scavenged within 4 min by antioxidin-NV.

Then, we were interested to assay whether antioxidin-NV directly clear the ROS induced by UVB irradiation in HaCaT cells. As an indicator of ROS production, DCFH-DA fluorescence intensity was measured by flow cytometry. A progressive increment of intracellular ROS level was observed in the UVB-irradiated HaCaT cells, and the addition of antioxidin-NV significantly decreased the intracellular ROS level in HaCaT cells upon UVB irradiation (Figures 2B,C). The scavenging efficacy of UVB-induced intracellular ROS is comparable to the ROS inhibitor, N-acetyl-L-cysteine (NAC) (Figures 2B,C). Furthermore, antioxidin-NV effectively cleared the ROS in mitochondria induced by UVB irradiation (Figure 2D). The data indicate that antioxidin-NV had a strong ability to scavenge ROS induced by UVB irradiation, suggesting a strong antioxidant activity of antioxidin-NV.

UV-induced skin photoaging leads to the accumulation of intracellular ROS (Zhang et al., 2017), especially the stronger biological effect of UVB (Diffey, 2002). To evaluate the anti-phtoaging activity of antioxidin-NV, we established a UVB-induced skin photoaging mouse model to assay whether topical application of antioxidin-NV can inhibit skin photoaging in mice. As illustrated in Figure 3A, UVB irradiation obviously induced skin photoaging in hairless mice, but topical application of antioxidin-NV significantly suppressed UVB-induced skin photoaging in hairless mice with reduced skin erythema, hyperplasia, wrinkling, and roughness compared to PBS-treated mice. H&E staining of the dorsal skin showed that UVB-irradiation resulted in a reduction of the thickness of epidermal layers, but topical application of antioxidin-NV significantly reversed this reduction (Figures 3B,C). To our surprise, antioxidin-NV showed a better therapeutic efficacy against UVB-induced skin photoaging than vitamin C (VC, positive control) (Figures 3A–C). The scrambled antioxidin-NV (sNV, isotype control) had no significant therapeutic effects on UVB-induced skin photoaging, indicating that the therapeutic efficacy of antioxidin-NV against UVB-induced skin photoaging is due to its unique amino acid sequence (Figures 3A–C). Besides, antioxidin-NV did not exhibit cytotoxicity and hemolytic activity at an absolutely high concentration (Supplementary Figure S3), and no adverse effect on the body weight, general health or behavior of the mice were observed for the topical antioxidin-NV treatment, implying antioxidin-NV had low side effects.

Skin photoaging is closely associated with DNA damage (Zhang et al., 2017), and keratinocytes are the cells distributed in the outer layer of skin which can be directly irradiated by UVB. So we analyzed whether antioxidin-NV can suppress UVB-induced DNA damage. Agarose gel electrophoresis showed that UVB irradiation produced a typical ladder with clearly increased intensity of DNA fragmentation in HaCaT cells, and antioxidin-NV significantly reduced its formation in a dose-dependent manner (Figure 4A). Western blot analysis and immunofluorescence staining further showed that UVB irradiation resulted in a significant increment of p-Histone H2A.X (γH2AX) expression, a marker protein for DNA damage. However, antioxidin-NV significantly reduced γH2AX expression in HaCaT cells induced by UVB irradiation in a dose-dependent manner (Figures 4B,C). Furthermore, IHC analysis also showed that UVB irradiation significantly increased γH2AX expression in hairless mice, but topical application of antioxidin-NV reduced UVB-induced γH2AX expression (Figure 4D).

Cell apoptosis is a critical pathological process of skin photoaging (Liu et al., 2021). The therapeutic effect of antioxidin-NV against UVB-induced apoptosis was assayed by flow cytometry. As illustrated in Figure 5A, UVB exposure resulted in the apoptosis of HaCaT cells (43.26%), while antioxidin-NV (40 μg/ml) treatment reduced UVB-induced HaCaT cell apoptosis at both the early and late stages (14.44%). Immunofluorescence and western blot analysis showed that antioxidin-NV significantly reduced the expression of apoptotic protein cleaved caspase 3 (a marker protein for apoptosis) in the UVB-irradiated HaCaT cells in a dose-dependent manner (Figures 5B,C).

We further analyzed whether antioxidin-NV can suppress UVB-induced cell apoptosis in hairless mice. Immunofluorescence, IHC and western blot analysis showed that UVB irradiation markedly increased the expression of cleaved caspase-3 in the skin of hairless mice, indicating that UVB irradiation significantly resulted in cell apoptosis in the skin of mice (Figures 6A–C). But antioxidin-NV significantly inhibited the expression of cleaved caspase-3 in the skin of hairless mice induced by UVB irradiation, suggesting that it could inhibit UVB-induced cell apoptosis in the skin of hairless mice (Figures 6A–C).

Inflammation was found to enhance the epidermal hyperproliferative response to UVB and play a crucial role in promoting skin photoaging (Pillai et al., 2005). As illustrated in Figure 7A, UVB irradiation obviously increased the secretion of IL-6 in UVB-exposed HaCaT cells, but antioxidin-NV effiectively suppressed the secretion of IL-6 in a dose-dependent manner. Furthermore, UVB irradiation increased IL-6 production in the skins of hairless mice, but the topical application of antioxidin-NV significantly decreased the secretion of IL-6 compared to PBS treatment (Figure 7B).

In addition, inflammation-related receptor inhibitors were used to determine which receptor was involved in antioxidin-NV-mediated IL-6 down-regulation in UVB-irradiated HaCaT cells (Figures 7C–E). After the addition of TLR4 inhibitor MTS510 (10 μg/ml), antioxidin-NV-mediated IL-6 down-regulation in UVB-irradiated HaCaT cells was completely inhibited (Figure 7C). The purine receptor inhibitor KN-62 (2 μM) and G protein coupling receptor inhibitor PTX (10 μg/ml) had no significant effect on antioxidin-NV-mediated IL-6 down-regulation (Figures 7D,E). These results suggest that TLR4 is involved in the antioxidin-NV-mediated IL-6 down-regulation in UVB-irradiated HaCaT cells. In addition, after the treatment by TLR4 inhibitor MTS510, the inhibitory effect of antioxidin-NV on NF-κB p65 phosphorylation in UVB-irradiated HaCaT cells was completely inhibited (Figure 7F).

MAPKs and NF-κB signaling are known to be important signal transduction pathways activated by UVB irradiation (Subedi et al., 2017). Therefore, western blot analysis was performed to further explore the effect of antioxidin-NV on MAPK and NF-κB signaling pathway in HaCaT cells and skin tissues. As illustrated in Figures 8A,B, UVB irradiation markedly increased JNK, p38, IkBα and p65 phosphorylation in HaCaT cells, but antioxidin-NV significantly decreased UVB-induced JNK, p38, IkBα and p65 phosphorylation in a concentration-dependent manner. The same results were observed in UVB-irradiated hairless mice, antioxidin-NV also significantly decreased JNK, p38, IkBα and p65 phosphorylation (Figures 8C,D).

HSFs, which can synthesize and maintain the extracellular matrix of skin and reduce skin photoaging, are a very critical cell type in skin photoaging (Tobin, 2017). Given the above observation that antioxidin-NV significantly suppressed UVB-induced skin photoaging in hairless mice, we further explored the potential effect of antioxidin-NV on HSF cells survival rate and the accumulation of alpha smooth muscle actin (a-SMA) following UVB irradiation in vitro and in vivo. As illustrated in Figure 9A, UVB irradiation directly inhibited HSF survival rate, but antioxidin-NV obviously restored HSF cells survival rate post UVB irradiation in a concentration-dependent manner. The expression of α-SMA, a marker protein of HSF cells was examined in the skin tissue of UVB-irradiated mice using IHC staining. As illustrated in Figure 9B, UVB irradiation obviously reduced the expression of α-SMA in the skin of UVB-exposed mice, but a higher accumulation of a-SMA positive staining was observed in NV treatment when compared to PBS.

Collagen is derived from HSF cells and plays important role in maintaining the elasticity of skin (Lee et al., 2012). Considering that antioxidin-NV has strong ability to restore the accumulation of a-SMA in hairless mice following UVB irradiation, and a-SMA is also a marker of myofibroblast which has a higher capacity to synthesize collagen (Nakyai et al., 2018). We further investigated whether antioxidin-NV can promote collagen production in UVB-irradiated hairless mice. Masson’s trichrome staining was used to evaluate the presence and distribution of collagen. As shown in Figure 9C, the collagen fibers of mice without UVB irradiation (sham) were dense and regular, while the collagen fibers of mice became less dense and more erratically arranged after UVB irradiation, but antioxidin-NV treatment markedly increased the abundance and density of collagen fibers in UVB-irradiated skins compared to PBS treatment. Type I collagen, which is the major component of collagen fibrils, is the most abundant structural protein in the skin (Makrantonaki and Zouboulis, 2007). Therefore, we further explored the potential effect of antioxidin-NV on type I collagen expression in the skin. Collagen I expression level in the skin tissue was examined using IHC staining (Figure 9D), immunofluorescence (Figure 9E) and western blot (Figure 9F). The results showed that UVB irradiation markedly resulted in a reduction of collagen I deposition in the skin of hairless mice, while antioxidin-NV treatment significantly rescued collagen I production in hairless mice post UVB irradiation (Figures 9D–F).

Transforming growth factor-β (TGF-β) is an important cytokine that promotes collagen production (Mouw et al., 2014). In addition, the TGF-β/Smad pathway also promotes the differentiation of myofibroblasts (Guo et al., 2009). To determine whether antioxidin-NV affect TGF-β secretion in HSF cells upon UVB irradiation, we measured the effect of antioxidin-NV on TGF-β1 production in HSF cells using ELISA and western blot, respectively. UVB irradiation obviously suppressed TGF-β1 production in HSF cells, but antioxidin-NV treatment significantly increased TGF-β1 production in UVB-irradiated HSF compared with PBS treatment in a dose-dependent manner (Figures 10A,B). Furthermore, UVB exposure also suppressed TGF-β1 production in the skin of hairless mice, while topical application of antioxidin-NV significantly increased TGF-β1 production in the skin of UVB-irradiated hairless mice compared with PBS treatment (Figures 10C,D). Furthermore, Smad proteins, including Smad2, are essential components of downstream TGF-β signaling. As illustrated in Figure 10E, UVB irradiation markedly reduced Smad2 phosphorylation in HSF cells, but antioxidin-NV obviously increased Smad2 phosphorylation in UVB-irradiated HSF cells compared to PBS-treated cells in a dose-dependent manner, suggesting that antioxidin-NV rescued collagen production in hairless mice upon UVB exposure through restoring TGF-β1/Smad2 signaling.