Protective Role of Alginic Acid and Fucoidan from Nitric Oxide-Induced Oxidative Stress

The protective activities of alginic acid and fucoidan from oxidative stress (OS) were investigated under cellular system using LLC-PK1 renal epithelial cells that are widely used for study cellular OS. The cellular OS in LLC-PK1 cells was induced by the free radical generators. The treatment of pyrogallol, sodium nitroprusside or 3-morpholinosydnonimine led to the production of superoxide anion (O2), nitric oxide (NO) and peroxynitrite (ONOO), respectively. The free radical generators decreased cell viability and elevated lipid peroxidation compared OSwith non-treated cells. However, the treatment of alginic acid and fucoidan attenuated OS significantly induced by NO, O2 and ONOO, resulting in the significant increase of cell viability and inhibition of lipid peroxidation in concentration-dependent manner. In particular, fucoidan showed the stronger protective effect from oxidative stress than alginic acid. These results indicated that alginic acid and fucoidan played the protective role from NO-induced OS. The present study suggests the promising antioxidative agents of alginc acid and fucoidan with protective activity from free radical-induced OS.


Introduction
The oxidative stress (OS) caused by nitric oxide (NO) has attracted much attention because the metabolic products, such as peroxynitrite (ONOO -) and the hydroxyl radical (•OH), as well as NO itself have been suggested to be important pathogenic causative agents of cellular damage, alterations of biomolecules and organ dysfunction.The toxicity and damage caused by NO in tissues and cells are multiplied enormously if NO reacts with the superoxide anion (O 2 -) to yield ONOO -, an extremely reactive radical (Akaike, Suga, & Maeda, 1998;Radi, Beckman, Bush, & Freeman, 1991).The reactions between ONOO -and biological molecules mediate toxic oxidative and nitrosative reactivity, leading to impaired function, toxicity and alterations in signaling ~ 13 ~ pathways that are implicated in diverse forms of free radical-induced tissue injury.The pathophysiological importance of NO suggests that regulation of NO formation may be an efficient intervention strategy to improve or alleviate these pathological conditions.Therefore, antioxidant therapy to reduce the toxicity of ONOO -as well as that of NO is considered to be a new avenue of therapeutic intervention and may have beneficial effects by ameliorating the damage and lesions involved in various pathological conditions.Unfortunately, only very few studies on the effects of antioxidants against NO-induced oxidative damage have been carried out.Although several synthetic antioxidants have been suggested for the prevention and treatment of diseases, their side effects and toxicities have become issues during or in post-treatment period.Therefore, dietary natural antioxidants have attracted much attention as preventive and therapeutic agents attenuating oxidative damage because of their safe role and effectiveness.Edible seaweeds have been studied for their various biological effects.Although the active components from brown seaweeds have not been clearly elucidated, they contain abundant alginate and fucoidan which are soluble dietary polysaccharide fibers (Ruperez, Ahrazem, & Leal, 2002).Alginic acid consists of alternating units of mannuronic and guluronic acids.Several reports have shown biological functions of alginic acid such as hypocholesterolemic, antidiabetic, antimutagenic, anticancer and antioxidative activities (Torsdottir, 1991) and antiviral (Feldman, Reynaldi, Stortz, Cerezo, & Damonte, 1999) activities.However, the protective activities of alginic acid and fucoidan from NO-induced OS have not been clearly elucidated.In particular, under the oxidative cellular system using LLC-PK1 renal epithelial cells that are widely used for the study on the protective effect from OS, the antioxidative activity of fucodian and alginic acid has not been investigated.In this study, we investigated the protective role of fucoidan and alginic acid under cellular OS model using LLC-PK1 renal epithelial cells.

Cell Culture
Commercially available LLC-PK1 cells were maintained at 37℃ in a humidified atmosphere of 5% CO 2 in culture plates with 5% FBS-supplemented DMEM/F-12 medium.The cells were subcultured weekly with 0.05% trypsin-EDTA in calcium-and magnesium-free phosphate buffer.

Treatment of Radical Generator
After confluence of LLC-PK1 cells had been reached, the cells were seeded into 96-well culture plates at 10 4 cells/mL.Two hours later, 1.2 mM of pyrogallol, 1.2 mM of SNP or 1.0 mM of SIN-1 were treated for 24 hr, and then alginic acid or fucoidan was added for 24 hr in the test wells at various concentrations.(

MTT Assay
Cell viability was determined using a MTT colorimetric assay (Mosmann, 1983).Fifty microliters of MTT (1 mg/mL) solution was added to each well.After incubation for 4 hr at 37℃, MTT solution was removed from the medium.The resultant formazan crystals in the renal cells were solubilized with 100 μL of dimethylsulfoxide.The absorbance of each well was then read at 540 nm using a microplate reader (model SPECTRAmax 340PC, Molecular Devices, Sunnyvale, CA, USA).

Thiobarbituric Acid Reactive Substances (TBARS)
The level of lipid peroxidant released from the cultured cells was estimated as TBARS according to the methods of Yagi (1976) and Yokode et al. (1988) with a slight modification.One aliquot of medium was mixed with 1.5 mL of 0.67% TBA aqueous solution and 1.5 mL of 20% trichloroacetic acid, and boiled at 95～100℃ for 45 min.The mixture was cooled and shaken vigorously with 3.0 mL of n-butanol.After centrifuging at 4,000 ×g for 10 min, the fluorescence of the n-BuOH layer was measured at an excitation wavelength of 515 nm and an emission wavelength of 553 nm using a fluorescence spectrophotometer.

Statistical Analysis
The results for each group were expressed as mean±SD values (n=3).Data were analyzed by one way ANOVA between control and sample treated groups using SAS software (SAS Institute Inc., Cary, NC, USA).Significant differences were determined among groups at p<0.05.viability was elevated by their treatments concentration-dependently, showing the cell viability of 63.90% and 76.70% at the concentrations of 100 μg/mL of alginic acid and fucoidan, respectively.In addition, the treatment of SNP elevated MDA production markedly, whereas alginic acid and fucoidan led to the reduction the levels as concentration-dependent manner (Figure 2).At the treatment of concentration of 25, 50 and 100 μg/mL of alginic acid, lipid peroxidation was decreased from 0.80 nmole/mg protein to 0.71, 0.56 and 0.42 nmole/mg protein, respectively.In addition, fucoidan at 25, 50 and 100 μg/mL lowered the MDA level to 0.63, 0.52 and 0.42 nmole/mg protein, respectively.

Protective Activity against Pyrogallol-Induced OS
Figure 3 represents the result on the cell viability under OS induced by pyrogallol.By the treatment of alginic acid and fucoidan at the concentration of 100 μg/mL, cell viability was increased from 30.56% to 65.70% and 73.10%, respectively.Moreover, MDA generation was significantly elevated by pyrogallol, but the treatment of alginic acid and fucoidan showed the significant and dose-dependent declines of lipid peroxidation (Figure 4).At 100 μg/mL of alginic acid and fucoidan treatment, MDA level was decreased from 0.79 nmole/mg protein to 0.28 nmole/mg protein and 0.27 nmole/mg protein.Figure 5 and 6 show the protective activities of alginic acid and fucoidan from ONOO -.The treatment of SIN-1 declined cell viability to 23.14%, whereas the treatment of alginic acid and fucoidan at 100 μg/mL led to the increase in cell viability to 68.50% and 70.40% (Figure 5).In addition, the elevated lipid peroxidation by SIN-1 was significantly decreased by the two antioxidants (Figure 6).The treatment of alginic acid at 25, 50 and 100 μg/mL inhibited lipid peroxidation from 0.81 nmole/mg protein to 0. 68, 0.50 and 0.38 nmole/mg protein, respectively.In addition, fucoidan also exerted the protective effect from lipid peroxidation by SIN-1, indicating a decline trend in MDA level of 0.65, 0.45 and 0.31 nmole/mg protein at the concentrations of 25, 50 and 100 μg/mL.Fucoidan had stronger protective effect than alginic acid from ONOO -.

Discussion
The over production of NO results in oxidative damage and contributes to disorder conditions (Halliwell, 1987;Halliwell, Zhao, & Whiteman, 1999).In addition, reaction of O 2 -with NO leads to the formation of ONOO -which is far more reactive and toxic than its precursors (Beckman, Beckman, Chen, Marshall, & Freeman, 1990; Patel et al., 1999).This reaction is extremely rapid, and it can generate the most toxic and reactive radical, •OH, that can react with unsaturated fatty acids of membrane phospholipids to generate free radicals, which in turn react quickly with oxygen to form peroxides (Blough, & Zafiriou, 1985).
Free radical-induced OS has been implicated in a variety of degenerative diseases as well as in the aging process (Bokov, Chaudhuri, & Richardson, 2004;Scott, & King, 2004;Gibson, & Huang, 2005).Therefore, antioxidants that scavenge free radicals and prevent free radical induced damage have attracted much attention, and there have been a great deal of effort to identify safe and effective therapeutic agents for OS-related diseases.Edible seaweeds are rich sources of dietary fibers, minerals, vitamins, proteins and antioxidants (Cahyana, Shuto, & Kinoshita, 1992; Yan, Chuda, Suzuki, & Nagata, 1999).In particular, fucoidan and alginic acid have been known as the main active compounds with various health beneficial effects including hypocholesterolemic, antidiabetic, anticancer, antiviral and antioxidative activities (Torsdottir et al., 1991 Lee et al., 2004).In the present study, the protective role of alginic acid and fucoidan from free radical-induced OS using LLC-PK 1 cells, which are vulnerable to OS induced by free radicals.
The cellular model of OS using LLC-PK 1 cells is well established and it is useful for searching for agents that can provide effective protection from free radicals (Yokozawa et al., 2000;Yokozawa et al., 2005;Piao et al., 2005).Free radical generators such as SNP, pyrogallol, and SIN-1, were treated to induce cellular OS in LLC-PK 1 cells.NO and O 2 -generated by SNP and pyrogallol, respectively, led to significant decline in cell viability and elevation of lipid peroxidation compared to non-treated cells.It indicated that the cellular OS was induced by the generators of NO and O 2 -.However, the treatment of alginic acid and fucoidan showed the significant protective effects from cellular loss and lipid peroxidation induced by NO and O 2 -.Peroxynitrite derived from NO and O 2 -is a strong oxidant and nitrating agent and can lead for the production of the most toxic •OH radical (Beckman et al., 1990).SIN-1 simultaneously generates both NO and O 2 -, which then combine rapidly to form ONOO − .The treatment of SIN-1 led to the significant decline of cell viability and elevation of lipid peroxidation compared with non-treated cells.However, alginic acid and fucoidan attenuated the OS by increase of cell viability and decrease of lipid peroxidation.These results supported that alginic acid and fucoidan played the protective role from NO-induced OS.

Figure 1 .
Figure 1.Protective effect of alginic acid and fucoidan on cell viability of LLC-PK 1 cells treated with SNP Figure 1 shows the effects of alginic acid and fucoidan under SNP-induced OS in LLC-PK1 cells.Treatment of SNP led to decrease in cell viability from 100% to 26.18%.However, the treatment of alginic acid and fucoidan exerted protective activity against SNP-induced cellular damage.The cell

Figure 2 .Figure 3 .
Figure 2. Protective effect of alginic acid and fucoidan on lipid peroxidation in LLC-PK 1 cells treated with SNP Data are presented mean±SD (n=3) a~h Mean values with different superscripts are statistically different from each other at p<0.05