Protective Effects of C24 Ceramide From the Seeds of Brassica napus L. Against Ultraviolet B-Induced Photoaging in Normal Human Dermal Fibroblasts

Abstract

Ultraviolet (UV) radiation induces skin photoaging, which is associated with the elevation of matrix metalloproteinase-1 (MMP-1) and the decrease of collagen. Brassica napus plays a well-known role in the treatment of canola oil through their anti-oxidative and DNA protective properties. However, their skin protective activity toward UVB-induced damage remains unclear. In the present study, we investigated the protective effect of C24 ceramide from B. napus on UVB-irradiated normal human dermal fibroblasts. Our results show that C24 ceramide enhanced NHDFs cell migration. The UVB-induced increase in MMP-1 and decrease in type I procollagen were ameliorated by C24 ceramide treatment. Taken together, our data strongly suggest that C24 ceramide from B. napus could reduce UVB-induced photoaging.

Aging skin is a complex causes process that is affected by both intrinsic factors, such as hormonal changes and metabolic processes, and extrinsic factors, such as solar radiation, pollution, and chemical exposure. ¹ Among various complex factors, ultraviolet (UV) radiation is a major extrinsic factor that is divided into 3 wavelength range: 315 to 400 nm (UVA), 280 to 315 nm (UVB), and 100 to 280 nm (UVC). ² Ultraviolet B is mostly absorbed by the upper epidermis; severe and chronic UVB irradiation causes photoaging by damage to cell structures, ³ which induces matrix metalloproteinase-1 (MMP-1) expression and type 1 procollagen degradation. Specifically, MMP-1, an interstitial collagenase or fibroblast collagenase, degrades transforming growth factor-β1, collagen, and elastin in the extracellular matrix (ECM). ⁴ Brassica napus is one of the world’s most economically important products as oilseeds. This has been used in China to treat benign prostatic hyperplasia for over decades. In the previous studies, the ethyl acetate extract of the B.napus isolated sterols, terpenoids, flavones, long chain hydrocarbons, and brassinolide. ⁵ Sphingolipids, eg, ceramides and cerebrosides, are important constituents of cellular membranes and are emerging as important second messengers for various cellular processes such as cell cycle arrest, differentiation, senescence, and apoptosis. ⁶ Although B. napus is widely known for its supplements and cosmetic actions, its effects on skin aging have not been investigated. Therefore, we investigated the efficacy of an C24 ceramide from B. napus on UVB-irradiation-induced aging in NHDFs.

C24 ceramide (Figure 1) was obtained as an amorphous white powder. Its molecular formula was determined as C₄₂H₈₁NO₅ by high resolution electrospray ionization mass spectroscopy (HR-ESI-MS) [M+H]⁺, m/z 680.6173 (calcd for C₄₂H₈₂NO₅: 680.6188). Compared the 2D-Nuclear magnetic resonance (NMR) (Table 1.) and the HR-ESI-MS data. Similar chemical degradation of C24 ceramide further confirmed that the length of the chain and the location of the double bond were the same as in cerebroside (1-O-(β-d-glucopyranosyl)-(2S,3S,4R,8E)-2-[(2′R)-2′-hydroxytetracosenoilamino]-8-octadecene-1,3,4-triol). ⁷ The structure of C24 ceramide was determined to be (2S,3S,4R,8E)-2-[(2′R)-2′-hydroxytetracosenoilamino]-8-octadecene-1,3,4-triol). Normal human dermal fibroblasts (NHDFs) were exposed to UVB irradiation (144 mJ/cm²) and then treated with C24 ceramide (1, 5, and 10 µM). Following UVB irradiation, NHDFs density decreased noticeably, with a cell viability of only 74.3% (Figure 2) of the normal control (UVB nonirradiation). At the indicated concentrations, C24 ceramide had no significant cytotoxic effect on the cells. In fact, treatment with C24 ceramide improved the cell viability of UVB-irradiated NHDFs. The MMPs are a family of calcium-dependent, zinc-containing endopeptidases that can degrade all kinds of proteins in the ECM. After UVB irradiation, the secretion of MMP-1 increased to 301.5% of that found in normal NHDFs cells. Treatment with 10 µM of C24 ceramide after exposure to UVB suppressed the expression of MMP-1 by 43.2% (Figure 3). We also found that C24 ceramide recovered the collagen degradation induced in NHDFs by UVB-irradiation. In the UVB-irradiated cells, type I procollagen secretion decreased by 50.6% compared to untreated control cells (Figure 3). However, C24 ceramide (10 µM) significantly reversed the decrease by 85.4%. In this study, 140 mJ/cm² of UVB resulted in significantly decreased cell viability. Treatment with C24 ceramide did not cause obvious alterations to cell viability. NHDFs play a key role in healing skin wounds. They proliferate and migrate to the wound surface where they synthesize ECM. ⁸ We found that C24 ceramide markedly improved the migratory ability of NHDFs. Drugs derived from natural products can eliminate free radicals to prevent UVB-induced photoaging with few or no side effects. ⁹ The MMPs are a family of Ca²⁺-dependent, Zn²⁺-containing endopeptidases that can break down most proteins within the ECM. ¹⁰ Ultraviolet B irradiation can activate MMP-1, which then cleaves all types of collagen. ¹¹ Ultraviolet B irradiation increased the production of MMP-1, whereas decreased the production of type I procollagen. However, we found that C24 ceramide (10 µM) downregulated MMP-1 secretion and upregulated type I procollagen synthesis in UVB-exposed NHDFs. C24 ceramide shows higher UVB protective activity, which also demonstrated that the molecule plays an important role in the UVB protective activity of sphingolipids. In summary, the present study indicates that C24 ceramide protected in vitro NHDFs cells against UVB-induced photoaging. This preliminary study demonstrates the protective effects of C24 ceramide against UVB-induced photoaging. Further investigation of the C24 ceramide and its function in photoaging in in vivo animal models is warranted.

Figure 1

Chemical structures of C24 ceramide from the seed of Brassica napus L.

Figure 2

Effects of C24 ceramide on cell viability. Cell viability after 72 hours with or without ultraviolet B (140 mJ/cm²) and C24 ceramide (1, 5, and 10 µM). *P < 0.05 vs the nonirradiated control.

Figure 3

Effect of C24 ceramide on the secretion of the matrix metalloproteinases-1 and type I procollagen. The production of (a) matrix metalloproteinase-1 and (b) type I procollagen in non- and ultraviolet B-irradiated NHDFs. Values are means ± standard deviation. *P < 0.05 and ***P < 0.001 vs the nonirradiated control. #P < 0.05 vs the ultraviolet B-irradiated control.

Table 1

¹H and ¹³C NMR Spectral Data of C24 Ceramide in Dimethyl Sulfoxide-d ₆ (TMS, δ in ppm, J in Hz).

No.	1H(m, J, Hz)	13C	No.	1H(m, J, Hz)	13C
1a	3.60(m)	68.9	1′		173.3
1b	3.75(m)		2′′	3.85(m)	70.3
2	4.05(m)	49.9	3′a	1.46(m)	34.2
3	3.40(m)	74.3	3′b	1.56(m)
4	3.36(m)	70.4	14′,17′	1.93(m)	26.2
5a	1.46(m)	31.8	15′	5.29(m)	129.1
5b	1.57(m)		16′	5.29(m)	29.2
6	1.46(m)	25.7	CH2	1.22–1.30(m)	22.1–31.3
7	1.91(m)	32.3	24′	0.83(t)	13.4
8	5.35(m)	130.2	1″	4.12(d,7.4)	103.2
9	5.36(m)	129.6	2″	2.95(m)	73.4
10	1.935(m)	32.5	3″	3.14(m)	76.2
11–15	1.31(m)	28.6–29.1	4″	3.05(m)	70.1
16	1.23(m)	31.3	5″	3.11(m)	76.3
17	1.22(m)	22.4	6″a	3.424(d,11.6)	61.8
18	0.85(t)	13.9	6″b	3.63(d,11.5)	-

Experimental

General

The seeds of B. napus L. was purchased in November 2018 in G-market, Seoul, South Korea. A voucher specimen (Skedrm20181105) has been deposited at the raw material room, SKEDERM cosmetic R&D center, South Korea. NMR spectra were recorded on a Varian Inova-400 FT-NMR spectrometer (CA, United States) with Tetramethylsilane (TMS) as an internal standard, δ in ppm, and J in Hz. HR-ESI-MS were measured on Bruker APEXII mass spectrometer in m/z. EI-MS were measured on a VG ZABHS mass spectrometer at 70 eV. Silica gel (200-300 mesh) was obtained from Merck Co. Ltd. Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin, and fetal bovine serum (FBS) were all purchased from Gibco (Grand Island, NY, United States). 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, potassium persulfate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, United States). An enzyme-linked immunosorbent assay (ELISA) kit for type I procollagen and MMP-1 was purchased from ABcam (Cambridge, United Kingdom).

Extraction and Isolation

The air-dried seeds of B. napus L. (2 kg) was extracted with 70% EtOH (3 × 20 L, 7 days each) at room temperature and the EtOH was removed under reduced pressure to give a residue (612 g), which was suspended in distilled water and extracted with n-hexane and EtOAc. The EtOAc extract (36 g) was subjected to column chromatography over silica gel (200-300 mesh, 2500 g) and eluted with CHCl₃; CHCl₃-MeOH (95/5), (90/10), (85/15), (80/20), (70/30), (50/50), (30/70), and (10/90); and MeOH to yield 10 fractions (Fr.1-Fr.10) . Frs.2-4 were subjected to a silica gel column eluting with CHCl₃-MeOH (19/1) to give compound 2 (26 mg).

Cell Culture, UVB Irradiation, and Sample Treatment

Normal human dermal fibroblast cells (NHDFs) were obtained by skin biopsy from a healthy young male donor (MCTT Bio, Inc., Seoul, Korea). ¹² The cells were cultured in DMEM containing 1% penicillin-streptomycin and 10% FBS at 37°C in a 5% CO₂ humidified incubator. When the cells reached 80%-90% confluency, the NHDFs were subjected to UVB (140 mJ/cm²) irradiation using a Bio-Link BLX-312 (Vilber Lourmat GmbH, Vilber Lourmat, Marne-la-Vallée, France). ¹³ Afterward, the cells were rinsed with phosphate-buffered saline and immediately treated with C24 ceramide (1, 5, and 10 µM) in serum-free DMEM. Nonirradiated control cells were fed with serum-free DMEM medium.

Cell Viability

The viability of NHDFs treated with C24 ceramide after UVB irradiation was evaluated using the MTT assay, as described previously. ¹⁴ NHDFs cells (2 × 10⁵/35 mm² dish) were exposed to UVB (140 mJ/cm²) and then treated with NHDFs (or not for UVB-irradiated control cells). After 72 hours of treatment, 1 mL of MTT (100 µg/mL) was added and incubated at 37°C for 3 hours. Then, the MTT solution was discarded, and the purple formazan crystals were resolved in 800 µL of sterile DMSO. Finally, the dishes were shaken for 10 minutes at room temperature. The optical density values of 100 µL portions of formazan dissolved in DMSO were documented using a microplate reader (Molecular Devices FilterMax5; San Francisco, CA, United States).

Determination of MMP-1 and Type I Procollagen

After 72 hours of C24 ceramide treatments, commercial ELISA kits were used to measure the production of MMP-1 and type I procollagen in the culture supernatant. All of the experimental procedures were performed following manufacturer protocols.

Statistical Analysis

All of the data are presented as means ± standard deviation. Statistical analyses used the one-way analysis of variance followed by Dunnett’s test. *P < 0.05, **P < 0.01, and ***P < 0.001 were considered to be statistically significant.