The oxidant-antioxidant imbalance was involved in the pathogenesis of chronic rhinosinusitis with nasal polyps

Abstract

Background: Although oxidative stress is involved in the pathophysiological process of chronic rhinosinusitis with nasal polyps (CRSwNP), the specific underlying mechanism is still unclear. Whether antioxidant therapy can treat CRSwNP needs further investigation.

Methods: Immunohistochemistry, immunofluorescence, western blotting and quantitative polymerase chain reaction (qPCR) analyses were performed to detect the distribution and expression of oxidants and antioxidants in nasal polyp tissues. qPCR revealed correlations between oxidase, antioxidant enzymes and inflammatory cytokine levels in CRSwNP patients. Human nasal epithelial cells (HNEpCs) and primary macrophages were cultured to track the cellular origin of oxidative stress in nasal polyps(NPs) and to determine whether crocin can reduce cellular inflammation by increasing the cellular antioxidant capacity.

Results: The expression of NOS2, NOX1, HO-1 and SOD2 was increased in nasal epithelial cells and macrophages derived from nasal polyp tissue. Oxidase levels were positively correlated with those of inflammatory cytokines (IL-5 and IL-6). Conversely, the levels of antioxidant enzymes were negatively correlated with those of IL-13 and IFN-γ. Crocin inhibited M1 and M2 macrophage polarization as well as the expression of NOS2 and NOX1 and improved the antioxidant capacity of M2 macrophages. Moreover, crocin enhanced the ability of antioxidants to reduce inflammation via the KEAP1/NRF2/HO-1 pathway in HNEpCs treated with SEB or LPS. Additionally, we observed the antioxidant and anti-inflammatory effects of crocin in nasal explants.

Conclusion: Oxidative stress plays an important role in the development of CRSwNP by promoting various types of inflammation. The oxidative stress of nasal polyps comes from epithelial cells and macrophages. Antioxidant therapy may be a promising strategy for treating CRSwNP.

Keywords: CRSwNP; Nrf2; macrophages; nasal epithelial cells; oxidative stress.

Figure 1

The expression of oxidase was increased in ECRSwNP compared with control. (A) The location of NOS2 and NOX1 was detected in control, ECRSwNP and nECRSwNP by IHC staining. (B) NOS2 and NOX1 protein expression was determined by Western blotting in control subjects, ECRSwNP patients and nECRSwNP patients. (C) Relative protein levels of NOS2 and NOX1 were normalized to GAPDH in control (n=4), ECRSwNP (n=4) and nECRSwNP (n=4). (D) Real-time quantitative PCR results for NOS2 in control subjects (n=14), ECRSwNP (n=27) and nECRSwNP (n=12) and for NOX1 in control subjects (n=13), ECRSwNP (n=22) and nECRSwNP (n=12). The Kruskal−Wallis test was used for comparisons among multiple groups. *p<0.05, **p<0.01, ****p<0.0001. NS, Not significant.

Figure 2

The expression of antioxidases was increased in ECRSwNP compared with control. (A) The location of HO-1 and SOD2 in control tissues and nasal polyps as detected by IHC. (B) Western blotting analyses of HO-1 and SOD2 protein levels in control subjects, ECRSwNP patients and nECRSwNP patients. (C) HO-1 and SOD2 relative protein levels were normalized to GAPDH in control subjects (n=4), ECRSwNP patients (n=4) and nECRSwNP patients (n=4). (D) qPCR results for HO-1 in control subjects (n=9), ECRSwNP (n=16) and nECRSwNP (n=8) and for SOD2 in control subjects (n=12), ECRSwNP (n=17) and nECRSwNP (n=12). The Kruskal−Wallis test was used for comparisons among multiple groups. *p<0.05, ***p <0.001, ****p <0.0001.. NS, Not significant.

Figure 3

Correlations between oxidase and antioxidase expression and inflammatory cytokines in CRSwNP. Spearman analyses on the correlation of IL-6, IL-8, IL-5, IL-13, IFN-γ mRNA levels and the mRNA levels of NOS2 (A), NOX1 (B), HO-1 (C), SOD2 (D) in nasal polyps tissue from CRSwNP.

Figure 4

Coexpression of oxidase, antioxidase and CD68 in human nasal tissues. Representative immunostaining photomicrographs show colocalization of CD68 with NOS2 (A), NOX1 (B), HO-1 (C), and SOD2 (D) in control, ECRSwNP and nCRSwNP tissue samples. Green staining indicates NOS2, NOX1, HO-1 and SOD2. Red and blue staining indicates CD68 and DAPI (nuclei), respectively.

Figure 5

The damage of antioxidant enzymes is related to M2 polarization. M0 were pretreated with IL-4 (20 ng/ml) with or without crocin (20 µM) for 24 h. (A-C) qPCR was used to detect the mRNA expression of CCL24, MRC1, NOS2, NOX1, HO-1 and SOD2. (D) Western blotting was used to evaluate the protein expression of NOS2, HO-1, SOD2 and KEAP1. Data were obtained in three independent experiments. One-way ANOVA was used to analyze the differences between multiple groups. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.. NS, Not significant.

Figure 6

Crocin treatment attenuates oxidative injury and inflammation in HNEpC. (A, B) HNEpC were pretreated with or without crocin (20 µM) for 24 h and incubated with or without SEB (1 μg/mL) for 2 h. Cells were visualized by immunostaining with anti-NF-κB (green) and anti-NRF2 (green) antibodies. Nuclei were stained with DAPI (blue). (C) HNEpC were incubated with SEB (1 μg/mL) with or without crocin (20 µM) for 24 h followed by nucleocytoplasmic separation. (D) Western blotting showed changes in NOS2, HO-1, SOD2 and KEAP1 protein levels. (E) qPCR showed changes in NOS2, HO-1 and IL-33 mRNA levels. Data were obtained in three independent experiments. One-way ANOVA was used to analyze the differences between multiple groups. ***p<0.001, ****p<0.0001.

Figure 7

Crocin reduces the inflammation of nasal polyp explants induced by SEB or LPS. (A–D) Nasal polyp explants were incubated with SEB (500 ng/ml) with or without crocin (20 µM) for 24 h. (A) Protein expression of NOS2, NOX1, HO-1, SOD2 was assessed by Western blotting. (B) mRNA expression of NOS2 and NOX1 was measured by qPCR. (C) mRNA expression of HO-1 and SOD2 were measured by qPCR. (D) Significant downregulation of IL-6, IL-8, IL-5, IL-13, IL-25, IL-33, IL-1β and IFN-γ mRNA levels after crocin treatment. (E, F) Nasal polyp explants were treated with LPS (100 ng/ml) with or without crocin (20 µM) for 24 h. (E) mRNA expression of NOX1, HO-1 and SOD2 was measured by qPCR. (F) mRNA expression of IL-6, IL-8, IL-25 and IL-33 was measured by qPCR. One-way ANOVA was used to analyze the differences between multiple groups. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.. NS, Not significant.