The Signaling Pathways Study Of Poly-L-arginine To Promote IL-6 And IL-8 Release Of NCI-H292 Cells Induced By LPS

ObjectiveWe studied the signaling pathways by which PLA might increase IL-6, IL-8 production induced by LPS in cultured NCI-H292 cells and tested the levels of IL-6, IL-8.Methods1. Cell cultureNCI-H292 cells were cultured in the conventional medium containing 10% fetal bovine serum. Passage every 3 to 4 days according to the growth of the cells. Selected the cells that grew well seeded in 24-well culture plates in 3×105/500 μl.2. The measurement of the mRNA and protein of cytokine IL-6 and IL-8According to the experimental design, we set up control experimental group, LPS group (5μg/ml), PLA group (5μg/ml), LPS+PLA group, LPS+PLA+PD98059 group, LPS+PLA+SB203580 group, PD98059 group, SB203580 group, PLA+PD98059 group, PLA+SB203580 group and PD98059+SB203580 group. Based on the experimental grouping requirement, firstly, to add ERK1/2 inhibitor PD98059 and p38 MAPK inhibitor SB203580, incubate in 37℃ 5% incubator for 1 h, and then interfuse PLA and LPS. Incubation for 2 h on the experiment requirement, RNAiso Plus was used to extract total RNA of cells, the production of IL-6 and IL-8 mRNA were measured by real-time quantitative PCR, and the differences between different groups were compared. Co-cultured 24 hours later, collecting supernatant of cells, the production of IL-6 and IL-8 were detected by ELISA, and then the differences between the different groups were analysed.3. the detection of ERKl/2, p-ERKl/2, p38 MAPK and p-p38 MAPKAccording to experimental design, the well matched inhibitor of ERK1/2 and p38 MAPK, PD98059 and SB203580 are added and then incubated in 37℃ 5% incubator for 1 h ahead of PLA or LPS. After adding PLA and LPS, still co-cultured for 1 h, the protein of cells are extracted, the level of ERK1/2, p-ERKl/2, p38 MAPK and p-p38 MAPK are detected by Western Blot. P-actin was used as reference.4. Statistical processingAll parameters and comparisons were performed using SPSS 17.0. Data were expressed as means±SD. For multiple comparisons one-way Analysis of Variance (ANOVA) was used followed by the least significance difference when equal variances were assumed or Dunnett’s T3 when no equal variances were assumed. Differences were considered significant at P<0.05.Results1. Comparing with LPS, PLA and control group, the expression level of IL-6 and IL-8 mRNA are increased obviously in NCI-H292 cells by the combined effects of PLA and LPS.. The differences have statistical significance (P<0.05).2. Comparing with LPS, PLA and control group, the expression level of IL-6 and IL-8 are increased obviously in NCI-H292 cells by the combined effects of PLA and LPS. The differences have statistical significance (P<0.05).3. The mRNA and protein expression levels of IL-6 and IL-8 in LPS+PLA group were significant decreased after adding ERK1/2 inhibitor PD98059 in NCI-H292 cells (P<0.05).4. After adding p38 MAPK inhibitor SB203580, the mRNA and protein expression levels of IL-6 and IL-8 in NCI-H292 cells were lower than LPS+PLA group without inhibitor (P<0.05).5. The phosphorylation of ERK1/2 and p38 MAPK are elevated when NCI-H292 cells are induced by LPS and/or PLA, and the highest expression levels was in the LPS+PLA group. However, the phosphorylation of ERK1/2 and p38 MAPK could be significantly blocked by ERK1/2 inhibitor PD98059 or p38 MAPK inhibitor SB203580.Conclusions:1. PLA increased the production of IL-6 and IL-8 whose expression were induced by LPS in NCI-H292 cells.2. PLA increased the production of IL-6 and IL-8 whose expression were induced by LPS via ERK1/2 and p38 MAPK signaling pathways in NCI-H292 cells.