10-Hydroxy-2-Decenoic Acid Prevents Ultraviolet A-Induced Expression of Lamin AÄ150 in Human Dermal Fibroblasts

Abstract

10-Hydroxy-2-decenoic acid (10-HDA) as the main component of royal jelly has pharmacological characteristics. But the influence of 10-HDA on skin photoaging and photo damage is poorly understood. In the present study, we used 10-HAD immediately after UVA exposure and tested the effects on the attenuation of LMNAÄ150 expression in cultured human dermal fibroblasts

Human dermal fibroblasts (cultured cells) were exposed to UVA irradiation. The mRNA level of LMNAÄ150 was determined by Taqman Real-Time PCR Assay.

Real-time PCR analysis of LMNAÄ150 transcripts indicated that the level of LMNAÄ150 transcripts was higher in the UVA exposed group than the group treated with 10-HAD after UVA exposure (>8.22-fold). The LMNAÄ150 expression is down-regulated in human dermal fibroblasts after treatment with 10-HDA.

It can be concluded that treatment with 10-HDA suppresses the UVA-induced gene expression of LMNAÄ150 and protects skin from UVA-induced photoaging and photo damage.

Keywords:10-HAD, UVA, LMNAΔ150, photoaging.

INTRODUCTION

Photo-damage and photoageing is under the control of several genetic and environmental risk factors such as UV index (1). The sun is the main source of environmental UV. The UVR consists of three bands of different wavelengths: one of 320-400 nm (UVA), an average wave length of 290-320 nm (UVB) and a wave length of 200-290 nm (UVC) (2). The UVC does not usually reach the surface of the Earth and is absorbed almost entirely in the upper stratosphere and only slightly passes through that ozone layer. About 95% of the solar radiation is UVA and ~ 5% is UVB (3). Intrinsic aging changes have been observed in areas permanently protected from sunlight, while additional exposure to sunlight is chronic as a result of aging of the skin. The main factor in the evolution of the extracellular matrix of the dermal layer in the skin is mainly due to UVA light. However, UVB rays reach the top of the dermal layer and can induce dermal changes through epidermal signaling to the dermis (4). In fact, exposure to sunlight induces cell damage and therefore accelerates the process of inherent aging (5).

Royal jelly is a yellow matter and a honeybee product. The specific components of royal jelly are fatty acids, 10-hydroxy-2-decenoic acid (10-HDA) and 10-hydroxy decanoic acid (HDAA). Royal jelly is well identified on behalf of its protective properties on reproductive health, neurodegenerative disorders, wound healing, and aging (6). Royal jelly decreases melanin synthesis and inhibits the expression of melanogensis-associated proteins and genes. 10-HAD (10-hydroxy-2E-decenoic acid) is the major lipid component of royal jelly, which is widely taken by human as a health food could be used to delay aging and onset of age-related diseases (7). 10-HDA is an unsaturated fatty acid produced from honeybees (8). 10-HDA has longevity-promoting properties in C. elegans (9). It down-regulates matrix metalloproteinases and inhibits VEGF-induced angiogenesis (10). 10-HDA promotes collagen construction in skin fibroblasts (11). Therefore, it may be a useful tool for the management and treatment of skin photoageing (12). Zheng et al (2013) indicated that 10-HDA could stop UVA-induced damage and reduce MMP-1 and MMP-3 genes transcriptional activity (13). Several literature data indicate the role of progerin in skin aging (14-16). Point mutation of cytosine to thymine at position 1824 in exon 11 of LMNA gene causes a truncated form of lamin A, which is defined as progerin. In humans, the A-type, lamin A (74 kDa) is encoded by a gene located on the chromosome 1q21.2-q21.3. Lamins A and C as intermediate filenames are encoded by LMNA gene. These nucleophilic proteins are isoforms and created by altered splicing of exon 10 of LMNA gene. To date, more than 40 mutations, mainly missense, have been reported in the LMNA gene, which results in variable phenotypes (17). Approximately 90% of cases with Hutchinson-Gilford progeria syndrome (HGPS) are caused by a de novo mutation within exon 11 of the LMNA gene (1824C->T) (18). The alternate splicing in exon 11 of the LMNA gene leads to low production of wild-type pre-lamin A transcripts but high production of mutant pre-lamin A transcript missing the latest 150 nucleotides (a truncated prelamin A). The mutated transcript encodes a mutant pre-lamin A protein with 50 amino acid deletion that is called proge- rin (19). The normal lamin A protein plays a major role in determining the form of the nucleus in cells. Alterations that cause the HGPS generate an unusual lamin A protein that causes instability of the nuclear coating and permanently damages the nucleus and DNA. Progerin accumulation not only causes the abnormal shape of the nucleus, but also disrupts the function of the nucleus, including altering histone modification patterns, abnormal chromatin regeneration, impaired nuclear transfer, delay in DNA repair response, shortening the length of the nucleus telomeres and increased activation of p53, which will ultimately lead to a reduction in cell lifetime due to early cellular aging (20). The disruption of laminar gene processing, which results in progerin production, is considered an aging cell biomarker (21). Proge- rin-associated nuclear envelope is involved in cellular aging associated with DNA damage repair (21).

The most of Iranian cities have a desert climate. It is essential to evaluate the risk of UV-associated health problems. In this study, we used 10-HAD immediately after UVA exposure and tested the effects on the attenuation of lamin AÄ150 expression in cultured human dermal fibroblasts.

MATERIALS AND METHODS

Cell culture

Human dermal fibroblasts were used in this investigation. The cells were cultured in Dulbecco’s modified Eagle’s media (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin antibiotics (Gibco, Scotland) at 37 °C in 5% CO2 atmosphere under 90-95% humidity. The cells were trypsinized after reaching 80% confluence using 0.25% trypsin-EDTA solution (Gibco, USA). The study was approved by the Clinical Research Ethics Committee of Urmia University of Medical Sciences, Urmia, Iran.

Ultraviolet irradiation and 10-HDA treatment

The cells were seeded into wells and were washed with PBS before radiation; a culture plate of cells was irradiated with Philips UVA lamp with a spectrum between 320 and 400 nm at room temperature. After irradiation, the cells were treated with DMEM with or without 10-HDA medium containing 10% FBS to escape from stress and recovery. 10-HDA was dissolved in PBS. The control cells followed the same medium change procedure.

Acridine orange/propidium iodide (AO/PI) assay

AO/PI assay were used for determination of cell viability.

RNA extraction and cDNA synthesis

Total RNA was isolated from cultured cells using the RNX Plus Solution Kit (SinaClon) (Catalog Number: RN7713C) with minor modifications. RNA pellets were dissolved in 50 microliters of nuclease-free water, quantified via a BioPhotometer (Eppendorf AG, Hamburg, Germany). Two microliters RNA was reverse transcribed with random hexamers using the revertaid first standard synthesis kit (Fermentas, Lithuania) according to the manufacturer’s instruction at 65 °C for 5 min, 25 °C for five minutes, 42 °C for 60 minutes and 70 °C for five minutes.

Taqman quantitative real-time PCR

Taqman quantitative real-time PCR method was performed for determining the progerin mRNA level in tested samples. Real-time PCR was carried out via Premix Ex Taq Probe qPCR (TaqMan) (TaKaRa), primer pairs (5’-actgcagcagctcgggg-3’ and 5’-tctgggggctctgggc-3’ for LMNAÄ150 in exon 11) (22), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. The real-time PCR settings were as follows: initial denaturation at 95 °C for 15 minutes, followed by 40 cycles of 95°C for 15 seconds and 58°C for 60 seconds. Reactions were performed in duplicate.

Statistical analysis

Analysis of relative gene expression data was performed using the 2-ÄÄCt Method (23). All parameters were reported as means±standard error (SE). Relative amounts of mRNA expression were compared between two groups using the independent sample t test. Statistical analyses were done by means of SPSS software for Windows (version 16, SPSS Inc. Chicago, IL, USA). Differences were considered to be significant at P-value < 0.05.

RESULTS

Cell viability/non-viability results are depicted in Figure 1.

RNA was extracted from cultured cells in tested groups. Taqman assay used for quantification of lamin AÄ150 transcripts in the tested samples. Real-time PCR analysis of lamin AÄ150 transcripts indicated that lamin AÄ150 was the most highly expressed in the UVA exposed group. The levels of AÄ150 transcripts in the UVA exposed group were higher, >8.22-fold, than the levels of AÄ150 transcripts in the group treated with 10-HAD after UVA exposure. It can be found that 10-HAD has protective role against the UVA irradiation, regarding to high expression of lamin AÄ150 transcripts in the exposed group. This may indicate that the lamin AÄ150 expression is up regulated after UVA irradiation in cultured human dermal fibroblasts. And also, the lamin AÄ150 expression is down-regulated after treatment with 10-HAD

DISCUSSION

UV energy divided in three components including UV-A, -B and -C. UV-A having the highest energy that influence a variety of cells and tissues. UV-A is proficient at production of reactive oxygen species to facilitate DNA damage. So, this leads to generation of mutations and any other modifications that have been associated with a variety of skin diseases including inflammation, aging and cancer (23). Progerin (prelamin AĢ150) is caused due to a point mutation within exon 11 of the LMNA gene (1824C->T) that alternate splice donor site. The splicing reduces wild-type prelamin A expression but increases the expression of a truncated prelamin A (progerin) (24). It has been demonstrated that expression of progerin is a biomarker for normal aging in human skin (25). Fong et al. reported that prelamin A was toxic to cells and its accumulation was responsible for the disease phenotypes in vivo. Also, reducing of prelamin A levels by as little as 50% runs a remarkable protection from disease (26). Progerin expression results in accumulation of ROS-mediated oxidative damage and consequently, protein oxidation and accumulation, that could not be resolved by cellular repair systems. Theses process results in accumulation of damaged cells and donate considerably to aging (27, 28). Large bodies of studies have been conducted to evaluate the influence of 10-HDA on skin photoageing and its potential molecular mechanisms. Some studies indicated the protective influence of 10-HDA on UVA-induced aging in human dermal fibroblasts (29-31).

In this study, our findings implied that 10-HDA could down-regulate UVA-induced aging in cultured human dermal fibroblasts and are in agreement with those from other reports (29-32). The molecular mechanisms of aging have not been completely identified. In this regard, the expression of the other transcripts at LMNA locus might be evaluated. In this study, there is no data on expression of lamins A and C. Future investigations with larger control groups considering more details from older cells might confirm our findings in aging models.

CONCLUSION

It can be concluded that 10-HDA may potentially protect the skin from UVA induced photoaging.

Conflict of interests: none declared

Financial support: This work was supported by Urmia University of Medical Sciences [grant number: 2134].

FIGURE 1.

a) Control group, healthy cells without change in nucleus of the cells, indicating that cells are viable (healthy cells show a green nucleus with intact structure); b-d) cells receiving UVA exposure show DNA fragmentation in cells undergoing apoptosis and condensation of the nucleus as dense orange areas. AO/PI assay were used for determination of cell viability.