| Objective Herein, we aimed to study the signaling mechanism whereby lipopolysaccharide(LPS) had synergistical effect on poly-L-arginine(PLA) to promote the release of IL-6 and IL-8 in NCI-H292 cells.Methods 1. Cell culture NCI-H292 cells were cultured and propagated in RPMI-1640 medium supplemented with 10% fetal bovine serum in a humidified atmosphere of 5% CO2/95% air at 37°C. The cells were subcultured every 3 to 4 days according to their growth status. Optimal growth state of the cells were selected and seeded into 24-well culture plates at a density of 3×105 cells/500μl and then cultured. 2. The detection of the expression level of phosphorylated JNK through Western Blot According to experimental design, NCI-H292 cells were divided into groups of cells treated with PLA, LPS or PLA+LPS, specifically, each group was treated with 5μg/ml PLA or/and 5μg/ml LPS for 0, 15, 30, 45, 60, 90, or120 min. We detected the JNK and p-JNK at different points in each groups and compared the p-JNK/JNK. β-actin was used as reference. NCI-H292 cells were divided into control group, PLA(5μg/ml) group, LPS(5μg/ml) group, PLA(5μg/ml) + LPS(5μg/ml) group, we detected the JNK, p-JNK in each groups and analyzed the effect of SP600125(30μM), which is the specific inhibitor of JNK signaling pathway on the phosphorylation of JNK. 3. The measurement of IL-6 and IL-8 through ELISA According to experimental design, NCI-H292 cells were divided into control, PLA(5μg/ml), LPS(5μg/ml), PLA(5μg/ml) + LPS(5μg/ml), PLA(5μg/ml) + SP600125(30μM), LPS(5μg/ml) + SP600125(30μM), PLA(5μg/ml) + LPS(5μg/ml) +SP600125(30μM) and SP600125(30μM) groups. Prior to stimulation with LPS or/and PLA in a humidified atmosphere of 5% CO2/95% air at 37°C for 24 h, NCI-H292 cells were incubated with SP600125 for 2h. Then, cell culture supernatants were collected. ELISA commercial kits were used to detect the levels of IL-6 and IL-8 in samples according to the manufacturer’s instructions. 4. Statistical processing All the experiments were repeated for three times. Statistical analyses were perfored using SPSS version 17.0. All values were displayed as means ± standard error of the mean. One-way Analysis of Variance(ANOVA) was applied for comparisons of more than two groups, and LSD was used when equal variance was assumed between groups or Dunnett’s T3 was used when no equal variance was observed. P-values less than 0.05 were considered to denote statistically significant differences.Results 1. Compared with the control group, the expression level of phosphorylated JNK in NCI-H292 exposed to 5μg/ml-PLA for 15, 30, 45, 60, 90, and 120 min were increased, and the differences had statistical significance(P<0.01). And the expression level of phosphorylated JNK increased with the extension of time, it peaked at 45 min, then decreased(P<0.001).2. The expression level of phosphorylated JNK in NCI-H292 exposed to 5μg/ml-LPS for 15, 30, 45, 60, 90, and 120 min were increased compared with the control group, and the differences had statistical significance(P<0.05). There was a similar trend with PLA. And the peak time of LPS was 45min(P<0.01), the p-JNK begun to rise until 45 min and than fell. 3. The expression level of phosphorylated JNK in NCI-H292 exposed to 5μg/ml-PLA+5μg/ml-LPS for 15, 30, 45, 60, 90, and 120 min were markedly increased compared with the control group, and the differences had statistical significance(P<0.001). The peak time of PLA+LPS was advanced to 30min(P<0.01), the expression level of phosphorylated JNK decreased at 45 min than 30min(P<0.001), and increased at 60 min again then decreased(P<0.001). 4. The PLA+LPS group showed an increased level of p-JNK, which was greater than that of LPS or PLA alone, and the differences between PLA+LPS group and PLA group or LPS group had statistical significance(P<0.001). 5. Increased levels of p-JNK in NCI-H292 cells stimulated with PLA, LPS or PLA+LPS were blocked by SP600125, which is an inhibitor of the JNK signaling pathway(P<0.001). 6. Compared with the control group, the expression level of IL-6 and IL-8 were obviously increased in PLA group, LPS group or PLA+LPS group(P<0.01), and PLA + LPS stimulated significantly more IL-6 and IL-8 production than either PLA or LPS alone(P<0.001). Thus, the synergistic effects of PLA+LPS on the release of IL-6 and IL-6 could be blocked by SP600125(P<0.01).Conclusions 1. PLA or LPS activates the JNK signaling pathway in NCI-H292 cells, and there is a synergistic effect between PLA and LPS. 2. LPS has synergistic effect on PLA to promote the release of IL-6 and IL-8 in NCI-H292 cells. 3. The JNK signaling pathway contributes to the release of IL-6 and IL-8, which is stimulated by the synergistic actions of PLA and LPS in NCI-H292 cells. |
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