Effect of High, Medium, and Low Molecular Weight Hyaluronan on Inflammation and Oxidative Stress in an In Vitro Model of Human Nasal Epithelial Cells

Abstract

IL-17A is involved in the activation of oxidative stress and inflammation in nasal epithelial cells. Hyaluronan (HA) in its high molecular weight form (HMW-HA) shows anti-inflammatory responses in contrast to low and medium molecular weight HA (LMW-HA and MMW-HA). The aim of this study was to investigate the pro- or anti-inflammatory biologic function of HA at different molecular weight in an in vitro model of nasal inflammation IL-17A mediated. We evaluated the ERK1/2 and IκBα phosphorylation, NF-κB signal pathway activation, ROS production, IL-8 and NOX-4 protein, and mRNA levels, in nasal epithelial cells RPMI 2650 stimulated with recombinant human (rh) IL-17A. Furthermore, the cells were treated with HMW-HA, MMW-HA, LMW-HA, and U0126. Our results showed that rhIL-17A increased the ERK1/2, IκBα phosphorylation and NF-κB signal pathway activation, ROS production, IL-8 and NOX-4 proteins, and mRNA levels. The addiction of HMW-HA or U0126 showed a significant downregulatory effect on inflammation due to the rhIL-17A stimulation in nasal epithelial cells. IL-17A is able to generate oxidative stress and inflammation via the activation of ERK1/2/NF-κB pathway in nasal epithelial cells. The HMW-HA might represent a coadjuvant of the classic anti-inflammatory/antioxidative treatment of nasal epithelial cells during IL-17A nasal inflammation.

Figure 1

Effect of U0126 inhibitor on ERK and IκBα phosphorylation in RPMI 2650 cells stimulated with rhIL-17A. The cells were stimulated with rhIL-17A (20 ng/mL) or PMA (50 ng/mL) for 30 min in absence or presence of U0126 (25 μM). (a) pERK and pIκBα protein expression were evaluated in the cell lysates by western blot. The results were expressed as ratio of band intensity and β-actin of 3 separate experiments. Representative gel images of pERK, pIκBα, and β-actin are shown. (b) The activation of ERK1/2 and NF-κB for each experimental condition was tested for the pERK1/2/total ERK1/2 ratio and for the pNF-κB/total NF-κB, respectively, by ELISA and normalized for protein content. ANOVA with Fisher's test correction was used for the analysis of the data. p < 0.05 was statistically significant.

Figure 2

Effect of U0126 inhibitor in RPMI 2650 cells stimulated with rhIL-17A. (a) The cells were stimulated with rhIL-17A (20 ng/mL) for 6 hrs in absence or presence of U0126 (25 μM). ROS production was evaluated in the cells by flow cytometry. The bars represent the mean ± SD of 3 separate experiments. Representative flow cytometry are shown; (b) the cells were stimulated with rhIL-17A (20 ng/mL) for 18 hrs in absence or presence of U0126 (25 μM). NOX-4 and IL-8 protein expression were evaluated in the cell lysates by western blot. The results were expressed as ratio of band intensity and β-actin of 3 separate experiments. Representative western blot is shown. ANOVA with Fisher's test correction was used for the analysis of the data. p < 0.05 was statistically significant.

Figure 3

Effect of HMW-HA, MMW-HA, and LMW-HA on ERK1/2 and NF-κB signal pathway in RPMI 2650 cells stimulated with rhIL-17A. The cells were preincubated with HMW-HA (100 μg/mL), MMW-HA (100 μg/mL), and LMW-HA (100 μg/mL) for 1 h and then stimulated with rhIL-17A (20 ng/mL) for 30 min; (a) pERK and pIκBα protein expression were evaluated in the cell lysates by western blot. The bars represent the ratio of band intensity and β-actin of 3 separate experiments. Representative gel images of pERK, pIκBα, and β-actin are shown; (b) the activation of ERK1/2 and NF-κB for each experimental condition was tested for the pERK1/2/total ERK1/2 ratio and pNF-κB/total NF-κB ratio by ELISA and normalized for protein content. ANOVA with Fisher's test correction was used for the analysis of the data. p < 0.05 was statistically significant.

Figure 4

Effect of HMW-HA, MMW-HA, and LMW-HA in RPMI 2650 cells stimulated with rhIL-17A. (a) The cells were preincubated with HMW-HA (100 μg/mL), MMW-HA (100 μg/mL), and LMW-HA (100 μg/mL) for 1 h and then stimulated with rhIL-17A (20 ng/mL) for 6 hrs. ROS production was evaluated in the cells by flow cytometry. The bars expressed mean ± SD of 3 separate experiments. Representative flow cytometry is shown; (b) the cells were preincubated with HMW-HA (100 μg/mL), MMW-HA (100 μg/mL), and LMW-HA (100 μg/mL) for 1 h and then stimulated with rhIL-17A (20 ng/mL) for 18 hrs. NOX-4 and IL-8 protein synthesis were evaluated in the cell lysates by western blot. The bars represent the ratio of band intensity and β-actin of 3 separate experiments. Representative western blot is shown. ANOVA with Fisher's test correction was used for the analysis of the data. p < 0.05 was statistically significant.

Figure 5

Effect of U0126, HMW-HA, MMW-HA, and LMW-HA on nuclear translocation of NF-κB in RPMI 2650 cells stimulated with rhIL-17A. The cells were preincubated (a) with U0126 (25 μM) or (b) with HMW-HA (100 μg/mL), MMW-HA (100 μg/mL), and LMW-HA (100 μg/mL) for 1 h and then stimulated with rhIL-17A (20 ng/mL) for 30 min; NF-κB was evaluated in nuclear cell lysate by western blot. The bars represent ratio of band intensity and β-actin of 3 separate experiments. Representative gel images of NF-κB and β-actin are shown. ANOVA with Fisher's test correction was used for the analysis of the data. p < 0.05 was statistically significant.

Figure 6

Effect of HMW-HA, MMW-HA, LMW-HA, and U0126 on IL-8 mRNA transcript. The cells were preincubated with HMW-HA (100 μg/mL), MMW-HA (100 μg/mL), LMW-HA (100 μg/mL), and U0126 (25 μM) for 1 h and then stimulated with rhIL-17A (20 ng/mL) for 18 hrs to study IL-8 mRNA. mRNA levels were quantified by quantitative Real-Time PCR (see Materials and Methods for details). The bars represent the mean ± SD of 3 separate experiments. ANOVA with Fisher's test correction was used for the analysis of the data. p < 0.05 was statistically significant.

Figure 7

Specific effect of HMW-HA in RPMI 2650 cells stimulated with rhIL-17A. HMW-HA was incubated with HAdase 2.5 units/100 μg (60°C for 72 hrs plus 65°C for 10 min). The cells were preincubated with HMW-HA (100 μg/mL) or HAdase treated HMW-HA for 1 h and stimulated with rhIL-17A (20 ng/mL) for 6 hrs for ROS or 18 hrs for NOX-4 and IL-8. (a) ROS production was evaluated in the cells by flow cytometry. Representative flow cytometry is shown. The results were expressed as the mean ± SD of 3 separate experiments; (b-c) NOX-4 and IL-8 protein synthesis were evaluated in the cell lysates by western blot. The bars represent ratio of band intensity and β-actin of 3 separate experiments. Representative western blot is shown. ANOVA with Fisher's test correction was used for the analysis of the data. p < 0.05 was statistically significant.

Figure 8

Graphical abstract of the study.