High-glucose Dulbecco's modified Eagle's medium (DMEM) and trypsin were obtained from Gino Biomedical Technology Co., Ltd. (Hangzhou, China). Fetal bovine serum was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Rat intestinal crypt cells (IEC-6) were from the American Type Culture Collection (Manassas, VA, USA). EPA, DHA and dimethylsulphoxide (DMSO) were from Sigma-Aldrich (St. Louis, MO, USA), and all other chemicals were of analytical grade.

IEC-6 cells were cultured in high-glucose DMEM supplemented with 10% heat-inactivated fetal bovine serum (at pH 7.2), and placed in a 5% CO₂-humidified incubator at 37°C. The culture medium was replaced every 48 h. CuSO₄, CrCl₃ and Pb(CH₃COO)₂·3H₂O were dissolved in high-purity water at a concentration of 100 mmol/l as a stock solution, and was filtered prior to use. Stock solutions (20 mmol/l) of EPA and DHA were prepared in anhydrous ethanol, respectively, and diluted with sterile water prior to use.

The IEC-6 cells in the logarithmic growth period were seeded in 96-well plates at a density of 104 cells/well. After 4-h culture, the cells were treated with 10 µl of different concentrations of DHA, EPA and metal ions (Cu2+, Cd3+ and Pb2+) and maintained for 48 h. Following this, 20 µl of MTT solution (5 mg/ml) was supplemented at the end of the incubation period. Cells were incubated for another 3 h at 37°C before being removed from the cultured medium. Subsequently, 150 µl of DMSO was added to each well, and the formazan crystals were dissolved on a horizontal oscillator. The optical density was detected at the 570 nm wavelength by an ELISA microplate reader and cell viability was calculated as follows.

By contrast, the effect of preincubation with 10 µl of different concentrations of metal ions for 1 h followed by further exposure to various doses of DHA and EPA was investigated.

IEC-6 cells were seeded in 96-well plates at a density of 2×105 cells/well. Subsequently, heavy metal-exposed IEC-6 cells were treated with ω-3 PUFAs. Subsequently, IEC-6 cells and culture medium were collected for the measurement of ROS, EGF and IL-6 using the LXA₄ ELISA kit, according to the manufacturer's protocol (Shanghai Lianshuo Biological Technology Co., Ltd., Shanghai, China). The results were corrected by the protein level based on the reference manual of the bicinchoninic acid protein assay kit (Shanghai Beyotime Biotechnology, Shanghai, China).

All the data are expressed as mean ± standard deviation, and was analyzed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). Significant differences of analyses between varying groups were analyzed by analysis of variance.

In the present study, to evaluate the damage effect of heavy metals (Cu2+, Cd3+ and Pb2+) on IEC-6 cells, cell viability was detected by the MTT assay. Supplementation of Cu2+ (100–500 µM), Cd3+ (5–30 µM) and Pb2+ (0.25–1.5 mM) to the IEC-6 cells resulted in a significant dose-dependent decrease in cell viability when compared with the control group (Fig. 1). According to the demonstrated results, concentrations of 500, 250 and 125 µM of Cu2+ with 30 and 15 µM of Cd3+, and 7.5 µM of Cd3+ with 1, 0.5 and 0.25 mM of Pb2+ were chosen for the subsequent experiments. Similarly, IEC-6 cells were incubated with DHA, ranging from 20 to 150 µM and EPA from 40 to 200 µM for 48 h, respectively. Simultaneously with the increase of concentration, cell viability decreased gradually (Fig. 2). According to these results, 60 µM DHA and 80 µM EPA were chosen for the subsequent experiments, as they exhibited no significant effect on IEC-6 cell viability.

In order to study the effect of ω-3 PUFAs on IEC-6 cells injured by heavy metals, the IEC-6 cell culture was supplemented with the two ω-3 PUFAs and a metal ion. Following pre-incubation of cells with 250 µM of Cu2+ for 1 h followed by further exposure to various doses of DHA and EPA, cell viability increased 14.78 and 14.29%, respectively (Figs. 3 and 4). By contrast, pre-incubation of cells with 10 µl DHA and EPA for 1 h prior to an addition of 250 µM Cu2+, the cell viability was enhanced 15.83 and 16.94%, respectively (Figs. 3 and 4). Furthermore, while DHA or EPA with 250 µM of Cu2+ were treated simultaneously, IEC-6 viable cells were increased by 15.05 and 14.74% (Figs. 3 and 4). These results enable us to conclude that DHA and EPA could significantly improve the cell viability of IEC-6 cells despite being inflicted by Cu2+.

The protective effect on the IEC-6 cells following DHA, EPA and Cd3+ exposure was also investigated. DHA was shown to have no significant effect on the 15 µM Cd3+-injured IEC-6 cells (Fig. 5). By contrast, EPA had a significant effect on the cell viability, with increases of 18.59, 16.88 and 19.71%, respectively (Fig. 6). Therefore EPA, but not DHA, has a protective effect on the IEC-6 cells injured by Cd3+.

Cell viabilities of 1 mM Pb2+-inflicted IEC-6 cells increased to 10.16 and 7.65% following treatment with DHA and EPA (Figs. 7 and 8). Additionally, the cell viabilities were enhanced by 20.73 and 9.76%, respectively, as cells were pre-incubated with 10 µl DHA and EPA for 1 h prior to supplementation with 1 mM of Pb2+ (Figs. 7 and 8). When the cells were simultaneously treated with DHA or EPA and 1 mM of Pb2+, the cell viabilities were observed to increase by 9.83 and 7.08%, respectively (Figs. 7 and 8).

The IEC-6 cell lines were exposed to various concentrations of DHA or EPA and Cu2+. After 48 h, the levels of ROS, EGF and IL-6 secreted by IEC-6 cells were detected. The amounts of ROS, EGF and IL-6 generated by the IEC-6 cells incubated with Cu2+ were significantly higher in comparison with the untreated control (**P<0.01). By contrast, incubation of the IEC-6 cells with DHA and EPA resulted in a substantial decrease of the IL-6 level when compared with the control group (P>0.05) (Figs. 9 and 10). Regardless of the additional sequence of DHA or EPA and Cu2+, DHA and EPA significantly reduced the expression levels of ROS, EGF and IL-6 in the IEC-6 cells (**P<0.01; *P<0.05) (Figs. 9 and 10). Therefore, DHA and EPA had a protective effect on the IEC-6 cells damaged by Cu2+ by reducing the expression levels of ROS, EGF and IL-6.

Similarly, incubation of IEC-6 cells with DHA and EPA for 48 h induced no significant variation in the secretion of ROS, EGF and IL-6; however, the level of ROS, EGF and IL-6 substantially increased after treatment of Cd3+ (Figs. 11 and 12). DHA treatment exhibited changes in the secretion level of ROS, EGF and IL-6 in the Cd3+-injured IEC-6 cells (Fig. 11). Notably, when IEC-6 cells were supplemented with EPA, as shown in Fig. 12, there was an evident reduction in the secretion of ROS, EGF and IL-6 levels.

Supplementation of DHA and EPA to IEC-6 cells did not significantly affect the expression levels of ROS, EGF and IL-6 (P>0.05). Conversely, a substantial increase was observed as Pb2+ was added (**P<0.01) (Figs. 13 and 14). This result indicates that treatment with DHA and EPA induced a similar decrease in the content of ROS, EGF and IL-6 in Pb2+-injured IEC-6 cells.