INTRODUCTION

The ageing process is the accumulation of oxidative damage to cells and tissues, which is associated with a progressive increase in the chance of morbidity and mortality (Beckman & Ames 1998). Hu­man ageing can be studied in vitro, spe­cifically with normal human diploid fi­broblasts (HDFs) which undergo a li­mited number of cell divisions in culture and progressively reach a state of irre­versible growth arrest, a process termed as rep­licative senescence (Trougakos et al. 2006). Senescent cells exhibit a gra­dual loss of replicative potential that re­sults in reduced harvest cell densities and cell saturation densities (Cristofalo et al. 1998) and expressed senescence as­sociated ß-galactosidase enzyme (SA b-gal) which can be detected by SA ß-ga­lactosidase staining (Dimri et al. 1995). Since senescent cells have been shown to accumulate with age in human tissues, it has been proposed that they contribute to organismal ageing (Campisi 2000).

HDFs offer a typical model for stress induced premature senescence (SIPS) as it exhibits a finite potential of differen­tiation (Campisi et al. 2007). SIPS which shares the common features of replica­tive senescence can be defined as sus­tained effects of subcytotoxic stress on proliferative cell types, including irre­versible growth arrest of the cell popula­tion. Various oxidative agents such as hydrogen peroxide (H2O2) and ultraviolet light have been used to induce SIPS and H2O2 appeared to be the most commonly used agent.

Skin is the largest and outermost organ of the human body. Exposure to the envi­ronment resulted in the cellular compo­nents such as lipid, protein and DNA be­ing targets to oxidative agents and sus­ceptible to oxidative damage. Collagens are the main protein found in the skin of which collagen type I is the most abun­dant. It is incorporated into collagen type III, which is an important substance in reticular tissues in the dermis while col­lagen type IV is a crucial structure most commonly found in the basement mem­branes and is responsible for the intrinsic cohesiveness of functional basement membranes (Varani 2006). Three distinct collagenases are responsible for de­grading connective tissues namely MMPI, MMPII and MMPIII (Shingleton et al. 1996). MMPI is also called collage­nase 1 or interstitial collagenase which degrades type I collagen while MMPII is known as gelatinase A and readily di­gests the denatured collagens type I, II, III and gelatins. MMPIIIwhich is also known as stromelysin I, digests extra­cellular matrix and activates proMMPI (Nagase & Woessner 1999).

Skin tissue is frequently and directly exposed to a pro-oxidative environment, including ultraviolet radiation (UV), drugs and air pollutants. Besides external in­ducers of oxidative attack, the skin has to cope with endogenous generation of reactive oxygen species (ROS) and other free radicals, which are continuously be­ing produced and caused skin ageing (Suzanne et al. 2003).

Skin ageing is defined as structural changes of the skin as the age in­creases. It is indicated as the inability to balance the important functions of the skin leading to cell death (Chandrasoma & Taylor 2001). During the ageing process, there is an accumulation of oxi­dation products such as oxidized pro­teins, DNA adducts and lipid metabolites. In addition, there is a significant de­crease in the defense system of the living cell, including a significant decrease in the antioxidant defense system (Beckman & Ames 1998). Balance be­tween oxidants and antioxidants are needed to minimize molecular, cellular and tissue damage. However, if the bal­ance is upset in favour of the oxidants, oxidative stress could occur and results in oxidative damage. Reactive oxygen species (ROS) are known to cause oxid­ative modification of DNA, proteins, lipids and small cellular molecules (Kang et al. 2005). To counteract the harmful effects of ROS, the various compartments of the skin are equipped with layer specific an­tioxidant systems to maintain equilibrium between ROS and antioxidant systems and thus prevent oxidative stress (Thiele & Ekanayake-Mudiyanselage 2007).

The unicellular algae C. vulgaris con­tains many bioactive substances such as carotenoids, chlorophyll, toco­pherols, ubiquinone, and protein. C.vulgaris is capable of inhibiting lipid peroxidation and interacts with enzymatic and non-enzymatic antioxidants system attenuat­ing and resisting the oxidative stress and further lipid peroxidation. A recent study has shown that C. vulgaris has great potential as an anti­oxidant with the ability to exert anti tumor effects against liver cancer (Mukti et al. 2009).

The ethanolic extract of C. vulgaris ex­pressed significant antioxidant activity against naphthalene-induced oxidative stress in rat model (Vijayavel et al. 2007) while Chlorella dichloromethane extract ameli­orates NO production and iNOS expres­sion through the down-reg­ulation of NFκB activity mediated by sup­pressed oxidative stress in RAW 264-7 macro­phage (Park et al. 2005). Another study reported that administration of C. vulgaris inhibited MMPI activity (Cherng & Shih 2006).

Since skin tissue is frequently and di­rectly exposed to a pro-oxidative envi­ronment such as ultra violet radiation which results in oxidative damage and further causes cellular ageing, therefore in this study C. vulgaris was eva­luated for its antioxidant properties in preventing skin ageing. Primary culture of HDFs which was exposed to H2O2 was treated with C. vulgaris and the expression of COL and MMP genes which are involved in skin ageing was determined.

MATERIALS AND METHODS

Cell culture, induction of senescence and treatment with C. vulgaris

Primary culture of HDFs was derived from circumcised foreskin of 9-12 year-old boys, from a local clinic. Written in­formed consents were obtained from all subjects. The samples were aseptically collected and washed several times with 75% alcohol and phosphate buffered sa­line (PBS) containing 1% antibiotic–anti­mycotic (PAA, Austria). After removing the epidermis, the pure dermis was cut into small pieces and transferred into a falcon tube containing 0.03 % collage­nase type I solution (Worthington Bio­chemical Corporation, USA). Pure dermis was digested in an incubator shaker at 37ºC for 6-12 hours. Then, cells were rinsed with PBS before being cultured in Dulbecco Modified Eagle Medium (DMEM) containing 10% fetal bovine se­rum (FBS) (PAA, Austria) and 1% antibi­otic–antimycotic at 37ºC in a 5% CO2 humidified incubator. After 5–6 days, the cultured HDFs were harvested by trypsi­nization and culture-expand into new T25 culture flasks (Nunc, Denmark) with ex­pansion degree of 1:4. When the sub­cultures reached 80–90% confluence, serial passaging was done by trypsiniza­tion and the number of population doublings (PDs) was monitored.

HDFs were divided into three treatment groups viz; i) untreated control  ii) H2O2-induced oxidative stress, SIPS (10 μM of H2O2 exposure for two weeks) iii) cells pretreated with C. vulgaris extract before oxidative stress induction. Treatment with C. vul­garis extract was started at pas­sage 4 at dose 400 mg/ml based on the optimal cell viability that has been estab­lished by the Department of Biochemi­stry, Faculty of Medicine, Universiti Ke­bangsaan Malaysia. It was a prolonged treatment whereby at passage 6, HDFs were in­duced with H2O2 and treated with C. vulgarisextract.

Preparation of C. vulgaris hot water extract

In this study, a green unicellular microal­gae C. vulgaris Beijerinck grown in Bold’s Basal Media was used. Dried C. vulgaris cells were suspended in distilled water at a concentration of 10% w/v, boiled at 100°C for 20 minutes and then centri­fuged at 10000 rpm for 20 minutes. The supernatant was subse­quently lyophi­lized to obtain C. vulgaris extract which was then added to cell culture growth medium as supple­mentation.

Morphology analysis and senescence-associated β-galactosidase (SA-β-gal) staining.

HDFs positive for SA-β-gal activity was determined as described by Dimri et al. (1995). SA-β-gal staining was performed with a senescent cell staining kit (Sigma, USA) according to the manufacturer’s instructions. A total of 1X105 cells were seeded in six-well plates and incubated with fixation buffer (2% formalde­hyde/0.2% glutaraldehyde) for 6-7 mins at room temperature. Cells were then rinsed three times with PBS and incu­bated with 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside at 1 mg/ml in a buffer containing 40 mM citric acid/phosphate (pH 6.0), 5 mM K3FeCN6, 5 mM K4FeCN6, 150 mM NaCl, and 2 mM MgCl2 for 4 h at 37oC in the absence of CO2. Blue staining was visible after incubation and the percentage of blue cells observed in 100 cells under a light microscope was calculated.

Primer design

The expression level of each targeted gene was normalized to the housekeep­ing gene which is GAPDH. Primer 3 software was used to design the primers for each targeted gene and GADPH which were further blasted with GenBank database sequences from National Center of Bio­technology Information in order to obtain primers with high speci­ficity. The se­quence of the primers is shown in Table 1.

Total RNA extraction

Total RNA from HDFs of each group was isolated using TRI Reagent (Molecular Research Centre, Inc.) according to the manufacturer’s instruction. Polyacryl car­rier was added to each extraction to pre­cipitate the RNA and centrifuged for 8 mins at 12000g at 4°C. The pellet was washed with 75% ethanol. After another centrifugation, the extracted RNA pellet was left to dry at room temperature for 15 mins and then dissolved in RNase and DNase free distilled water. Total RNA was stored at -80°C immediately after extraction.

Quantitative real-time RT-PCR

Gene expression level of COLI, COLIII, COLIV, MMPI, MMPII, andMMPIII was quantitatively analysed with one-step real time RT-PCR technique. The expression level of each targeted gene was then normalized to GAPDH. Real-time PCR reaction was performed with 100 ng of total RNA, 400 nM of each primer and iScript One-Step RT-PCR kit with SYBR Green (Biorad, Canada) according to the manufacturer’s instruction. Reactions were run in Bio-Rad iQ5 with reaction profile as follows; cDNA synthesis for 20 mins at 50°C; pre-denaturation for 4 mins at 95°C; PCR amplification for 38 cycles with 10 secs at 95°C, 30 secs at 61°C. This was followed by a melt curve analy­sis to determine the reaction specificity. Agarose gel electrophoresis was per­formed for confirmation of the PCR prod­ucts.

Statistical analysis

Each experiment was carried out in dup­licates. Data were reported as mean ± SD. Comparison between groups was made by Student t-test (two-tailed). p<0.05 was considered statistically sig­nificant.

RESULTS

Cell morphology and senescence-associated β-galactosidase expression.

Morphological changes were observed with ageing of the HDFs. Young HDFs displayed the normal spindle shape cha­racteristic of fibroblast cells. However, in SIPS, the original fibroblastic shape was lost and HDFs became larger and flat­tened with accumulation of cytoplasmic granular inclusions. Similar cell morphol­ogy was observed for SIPS cells which were treated with C. vulgaris (Figure 1).

A positive blue stain of SA-β-galap­peared mainly in H2O2-treated HDFs (SIPS cells) suggesting the presence of senescent cells. Quantitative analysis showed the percentage of cells positive for SA-β-gal staining was increased (p<0.05) in SIPS compared to young un­treated HDFs (Figure 2). Similar results were observed for senescent cells at passage 30 and SIPS treated with C. vulgaris (p<0.05).

Specificity of primers and real-time RT-PCR

Six senescence-associated gene primers were designed. Agarose gel electropho­resis showed that each PCR product ap­peared as single bands (Figure 3). The melting curve analysis showed a single and narrow peak of each PCR product (Figure 4) indicating that the primers de­signed and the real-time RT-PCR proto­cols were specific.

Expression of COL and MMP genes

Treatment with H2O2 caused a downre­gulation of COLI and COLIII genes (Fig­ure 5) and upregulation of MMPI gene (Figure 6) compared to untreated control HDFs (p<0.05). H2O2-treated HDFs (SIPS cells) which were incubated with C. vulgaris extract showed downregula­tion of COLI gene and upre­gulation of MMPI gene compared to un­treated con­trol HDFs (p<0.05). COLIII gene was upregulated with C. vul­garis treatment compared to H2O2-treated HDFs (p<0.05).

DISCUSSION

The present study evaluated the effects of C. vulgaris extract in possibly mod­ulating H2O2-induced cellular senes­cence (SIPS) in skin HDFs. Our results showed that when HDFs reached senes­cence, there were clear changes in cell mor­phology, decreased cell proliferation and increased senescence associated β-ga­lactosidase activity. Morphological changes are typical features of senes­cent phenotype that can occur at both cellular and organism level (Cho et al. 2004). Senescent HDFs showed mor­phological changes of flattened and enlarged cell shapes. This shift is accom­panied by changes in nuclear structure, gene expression, protein processing and metabolism (Campisi 2000).

Bayreuther et al. (1988) stated that progressive morphological changes oc­cur while cells aged in vitro. Senescent cells are bigger and a senescent popula­tion has more diverse morphotypes than cells at earlier cumulative population doublings. In fact, a confluent senescent culture has a smaller cellular density than a confluent young culture, though this also occurs because senescent cells are more sensitive to cell-cell contact inhibi­tion. These changes are concomitant with increased activity of β-galactosidase measured at pH 6 (Dimri et al. 1995; Krishna et al. 1999). In the present study, C. vulgaris extract however was not able to show any morphological effects on H2O2-induced cellular senes­cence.

Extracts of C. vulgaris has pre­viously been reported to show antitumour effect in fibrobsarcomas (Konishi et al. 1985; Hasegawa et al. 2002) and liver can­cer (Mukti et al. 2009). The algal ex­tract has also been shown to exhibit possible anti­cancer properties in hepato­cellular carci­noma cell lines, HepG2 (Llovet et al. 2003; Md. Saad et al. 2006). There was also a report of using C. vulgaris as a potential thera­peutic agent against ad­vanced glycation end product (AGE), in which AGE has long been rec­ognized and implicated in the develop­ment of age related disorders such as atherosclerosis and diabetes (Yamagishi et al. 2005). C. vulgaris also had been shown to mod­ulate H2O2-induced DNA damage and telomere shortening of hu­man fibroblasts derived from different aged individuals whereby it exhibited bi­oprotective effects against free radical attacks (Makpol et al. 2009).

Our data showed that the mRNA ex­pression of COLI and COLIII decreased in H2O2-induced cellular senescence of skin HDFs while the expression of matrix metalloproteinase 1 mRNA (MMP1) was increased when cells aged. Previous study reported among the changes in gene expression in senescent cells, the over expression of matrix metalloprotei­nases (MMPs) was striking resulting in lost of proteins maintaining ultra struc­tural shape (Fisher et al. 2008). Relative overproduction of collagenase in ageing cells was proposed as the matrix-de­grading phenotype of senescent cells (Mawal-Dewan et al. 2002). In addition of that, it has been reported that senes­cent HDFs had decreased expression of sev­eral extracellular matrix components such as collagen I-1α , collagen III-1α and elastin besides increased expression of collagenase and stromelysin; two en­zymes that serve to breakdown the extracellular matrix (Fisher et al. 2008).

In this study, prolonged C. vulgaris treatment was not able to upre­gulate the mRNA expression of COLI or downre­gulate the mRNA expression of MMP1 in H2O2-induced senescent HDFs even­though the expression of COLIII was in­creased. These findings could be due to the irreversible effects of H2O2 in skin HDFs. According to studies by Chen et al. (2004) H2O2 induced senescent mor­phological changes and irreversible ar­rest in cell replicative capacity in HDFs F65 cells. With H2O2 treatment, the cells could not enter the cell cycle again even with mitogen stimulation. The effects of H2O2 were cumulative and irreversible. This might explain the reason why C. vulgaris could not reverse the effects of H2O2 in our study. In addition to that, H2O2 was reported to increase the steady-state mRNA levels of collagenase or MMPI in HDFs. It was an important intermediate in the downstream signaling pathway which leads to the induction of increased steady state MMPI mRNA le­vels (Olsen et al. 1989).

Compared to other antioxidants, rela­tively few researches had been done to explore the components of C. vulgaris. Therefore the chemical compo­sition of C. vulgaris and its benefi­cial potential espe­cially in preventing skin ageing have not been fully recognized. Various bioactive compounds in C. vulgaris may react to inhibit growth in cells that had been damaged by the oxidative stress induced by H2O2. C. vulgaris was reported to in­duce apoptosis in order to maintain nor­mal cell homeostasis (Md Saad et al. 2006).

In our study, the bioprotective effect of C. vulgaris extractin preventing skin ageing was determined. H2O2 was addedat passage 6 to induce cellular ageing of the skin while at the same time HDFs were treated with C. vulga­ris extract.Unfortunately there was no extensive study on the influence of H2O2 towards the effectiveness of prolonged C. vulgaris extract treatment. Therefore for future studies, we would like to suggest that nor­mal replicative senes­cence model shall be used in order to elucidate the protec­tive effects of C. vulgaris extract against skin ageing.

CONCLUSION

In skin ageing, COLI and COLIII genes were downregulated while MMPI was upregulated. C. vulgaris extract didmod­ulate the expression of COL and MMP genes by downregulating COLI and upregulating COLIII and MMPI but it did not exert anti-ageing effect.

ACKNOWLEDGMENTS

This study was funded by Universiti Ke­bangsaan Malaysia grant UKM-GUP-SK-07-21-043.