Nitric Oxide Synthase Activity Correlates with OGG1 in Ozone-Induced Lung Injury Animal Models

Abstract

Background: NO is an important cellular signaling molecule which is derived from L-arginine by nitric oxide synthase (NOS) and the effects of NOS signaling in lung injury is conflicting. The present study was designed to observe the effect of NOS and Arginase signaling in the occurrence and development of lung injury and its mechanism. Methods: An ozone-stressed lung injury animal model was established by exposure to 2.0 ppm O₃ for 30 min every day for consecutive 12 day with or without the administration of NO precursor L-arginine or non-selective NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME). Then, the lung histopathology, the releases of inflammatory mediators and the production of ROS were assayed by immunohistochemistry, ELISA and flow cytometry respectively. The activities and expression of NOS and Arginase were assayed by biochemical methods and western blot. Correspondingly, the release of 8-oxoguanine glycosylase 1(8-OxoG) and 8-oxoguanine glycosylase 1 (OGG1) were assayed by ELISA and western blot. The correlation between NOS/Arginase signaling with 8-OxoG/ OGG1 was also analyzed by Pearson correlation coefficients and immunofluorescence in NOS deficient bronchial epithelial cells. Results: In ozone-induced rat lung injury models, lung inflammation as well as lung architecture was disrupted in a time dependent manner. Ozone treatment with L-arginine showed a substantial attenuation of adverse lung histopathological changes and treatment with L-NAME promoted the inflammation and remodeling. Importantly, the expression of NOS was promoted by L-arginine and inhibited by L-NAME and the expression of Arginase was promoted by L-NAME treatment. Further, we observed significantly higher levels of 8-OxoG and lower levels of OGG1 in ozone group which was reversed by L-arginine and promoted by L-NAME. The expression of NOS is closely related with 8-OxoG /OCG1. Conclusion: These findings give further evidence that the NOS signaling is related with base excise repair.

Keywords: 8-OxoG; NOS; OGG1; arginase; lung injury.

Figure 1

The lung injury induced by ozone and treated with L-arginine and L-NAME was assayed by immunohistochemistry (A, scale bar = 100 μM) and ELISA (n = 5, B). All groups were observed at Day 0, Day 4, Day 8, and Day 12 and 4 rats were observed at different time points. ^**P < 0.01 vs. Day 0 of ozone group; ^##P < 0.01 vs. the same time point of ozone group. Arrows represented α-SMA positive staining.

Figure 2

The production of ROS was assayed by flow cytometry (n = 4). (A), representative images. (B), the average MFI. All groups were observed at Day 0, Day 4, Day 8, and Day 12 and 4 rats were observed at different time points. ^**P < 0.01 vs. Day 0 of ozone group; ^##P < 0.01 vs. the same time point of ozone group.

Figure 3

The activities of Arginase (A) and NOS (B) were assayed by detecting the concentrations of reaction products (n = 5) and the expression and distribution of NOS were assayed by Western blot (C, n = 4) and immunohistochemistry (D, scale bar = 50 μM). All groups were observed at Day 0, Day 4, Day 8, and Day 12 and 4 rats were observed at different time points. ^**P < 0.01 vs. Day 0 of ozone group; ^##P < 0.01 vs. ozone group. The distribution of NOS was observed at Day 12. Arrows represented NOS positive staining.

Figure 4

The production of 8-ohdG (A) and OGG1 (B) was assayed by ELISA (n = 5) and the protein expression and distribution of OGG1 were assayed by Western blot (C, n = 4) and immunofluorences (D, scale bar = 50 μM). All groups were observed at Day 0, Day 4, Day 8, and Day 12 and 4 rats were observed at different time points. ^**P < 0.01 vs. Day 0 of ozone group; ^##P < 0.01 vs. ozone group. The distribution of OGG1 was observed at Day 12. Arrows represented OGG1 positive staining.

Figure 5

Correlation between NOS and 8-ohdG/OGG1 was analyzed by Pearson correlation coefficients and immunofluorences. (A,B): correlation between NOS and 8-ohdG/OGG1; (C,D): correlation between Arginase and 8-ohdG/OGG1; (E): correlation between NOS and OGG1 in NOS-deficient cells (scale bar = 50 μM). The results showed that NOS was closely associated with OGG1. Arrows represented NOS (green) and OGG1 (red) positive staining respectively.