Rhamnazin (see the chemical structure in Fig. 1), 2,7-dichlorofluorescein diacetate (DCFH-DA) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F-12) was obtained from GIBCO™ (Grand Island, NY, USA), and fetal bovine serum (FBS) was obtained from Life Technologies Inc. (Lenexa, KS, USA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and dihydrorhodamine 123 (DHR 123) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), and all other reagents were of the highest commercial grade available.

RAW264.7 cells, a macrophage like cell line, were purchased from the American Type Culture Collection (Rockville, MD, USA). The cells were cultivated in DMEM supplemented with 10% FBS, at 37℃ in a humidified atmosphere of 5% CO₂ in air. Treatment with rhamnazin and / or LPS (100 ng/mL) was carried out under serum-free conditions. All subsequent procedures were carried out under these conditions, and all the experiments were carried out in triplicate and repeated three times to ensure reproducibility.

Cells were treated with or without LPS (100 ng/mL) and various concentrations of rhamnazin (0~20 µM) for 18 h.15 After treatment, cell viability was determined using an MTT assay according to the method described by Mosmann.16

Total RS generation was determined in culture supernatant according to the method described by Paraidathathu et al.18 In short, following rhamnazin treatment for 24 h, 25 µM DCFH-DA was added to the cells that were incubated at 37℃ for 15 min, and changes in fluorescence were determined for 30 min at an excitation wavelength of 486 nm and emission wavelength of 530 nm. N-acetyl-L-cystein (NAC, 10 µM) was used as a positive control.

ONOO^–-dependent oxidation of DHR 123 to rhodamine 123 was assayed, according to the method described by Kooy et al.19 Samples were added to the rhodamine buffer (pH 7.4) consisting of 6.25 µM DHR 123 and 125 µM diethylenetriaminepentaacetic acid and incubated 5 min at 37℃. The absorbance was assessed at 500 nm, which is the absorbance of rhodamine 123. Trolox (10 µM), curcumin (10 µM) and penicillamine (10 µM) were used as positive controls.

NO^• levels were estimated by assessing the accumulation of nitrite in the conditioned medium by the Griess assay. Shortly after, 100 mL culture supernatant was allowed to react with 100 mL of the Griess reagent,20 and after that, it was incubated at room temperature for 5 min. The optical density of the samples at 540 nm was determined using a microplate reader. Trolox (10 µM), curcumin (10 µM) and penicillamine (10 µM) were used as positive controls.

The ^•O₂-scavenging activity was estimated by assessing the reduction in the ratio of the decrease of nitro blue tetrazolium (NBT), as described by Ewing and Janero.21 The culture supernatant was added to the reaction buffer [50 µM PBS with 125 µM EDTA, 62 µM NBT and 98 mM NADH] containing 33 µm 5-methylphenazium methyl sulfate. The absorbance at 540 nm, as an index of NBT reduction, was determined after 5 min. Trolox (10 µM), curcumin (10 µM) and penicillamine (10 µM) were used as positive controls.

To estimate the lipid peroxidation, the levels of TBARS was determined using slightly modified assay according to Buege and Aust.22 This method entailed the creation of a colored complex between the products of lipid peroxidation and thiobarbituric acid at a temperature of 100℃ in an acidic environment. The maximum absorption of this complex was estimated at a wavelength of 532 nm. NAC (10 µM) was used as a positive control.

Cells were cultivated in 100-mm culture dishes containing DMEM supplemented with 10% FBS in a humidified atmosphere of 5% CO₂ in air at 37℃. The cells were detached from the dish by 0.05% v/v trypsin-EDTA in PBS and subcultured in culture plates. After incubation for 24 h, cells were treated with rhmanazin, and then the cells were collected and sonicated for 3 min at 30-s intervals. Cell debris was pelleted, and the resulting supernatant was used for the enzyme activity assays. In short, catalase activity was assessed spectrophotometrically by direct estimation of the reduction of light absorption at 240 nm due to the decomposition of H₂O₂ by catalase.23 Catalase activity was calculated as nmole/min/g protein, and the enzyme activities were calculated by normalizing the results to the total protein concentration of the whole extract, as estimated by the bicinchoninic acid protein assay23 with NAC, (10 µM) as a positive control.

PGE₂ and LTB₄ levels were evaluated according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). Cells were transferred to a 48-well plate and allowed to adhere overnight. Cells were incubated for each time in an FBS-free medium in the presence or absence of rhamnazin, and the PGE₂ and LTB₄ levels present in the media were assayed utilizing an enzyme immunoassay kit.

All the experiments were performed in triplicate and repeated three times to ensure reproducibility. Data are presented as mean ± SEM. The effect of each parameter was assessed by the one-way analysis of variance. Individual differences between groups were determined using the Dunnett's test, and those at p < 0.05 were considered statistically significant.

To determine the protective effect of rhamnazin against LPS-induced cellular damage, cell viability was examined. As Fig. 1 shows, the LPS-treatment decreased cell viability. However, the rhamnazin treatment boosted cell proliferation, indicating that rhamnazin has a protective effect against LPS-induced cell damage.

As shown in MTT assay, rhamnazin showed cytoprotective effect. In order to investigate the underlying action of cytoprotective action by rhamnazin, total RS production was examined. As shown in Table 1, total RS generation was up-regulated after treatment with LPS, while the rhamnazin treatment decreased total RS generation.

To investigate the contributing factors of the cytoprotective effect against LPS exposure in greater detail, we estimated various RS, including ONOO^– , NO^• and ^•O₂, and found that rhamnazin reduced · ONOO^–, NO^• and ^•O₂ levels (Table 2) in LPS-treated macrophages. These results indicate that various scavenging activities by rhamnazin are one of the potent, contributing underlying actions that protect against LPS-induced cellular damage.

As Fig. 3A shows, LPS enhanced TBARS levels, while rhamnazin reduced TBARS levels. These data confirm that rhamnazin achieves its anti-oxidative properties by not only scavenging various RS such as ^•O₂, NO^•, and ONOO^–, but also by suppressing lipid peroxidation.

To determine the effect of rhamnazin on the regulation of an antioxidant enzyme during LPS-induced oxidative insult, catalase activity was assessed. As shown in Fig. 3B, catalase activity decreased after treatment with LPS. However, rhamnazin treatment enhanced catalase activity. These data indicate that rhamnazin possesses a protective effect against LPS-induced oxidative insult by up-regulating antioxidant enzyme activity.

To examine whether rhamnazin's cytoprotective property is associated with the modulation of inflammation, key pro-inflammatory enzymes, COX and 5-LO activities were estimated. As Fig. 3C and Fig. 3D display, rhamnazin reversed both COX and 5-LO activities with LPS treatment. These results indicate that rhamnazin suppresses inflammation through inhibition of both COX and 5-LO, implying that the dual modulation of COX and 5-LO might be a crucial underlying action in rhamnazin's protection against LPS-induced cytotoxicity.