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. 2017 Nov;93(11):1257-1266.
doi: 10.1080/09553002.2017.1377360.

Role of NADPH oxidase in radiation-induced pro-oxidative and pro-inflammatory pathways in mouse brain

Hyung Joon Cho  1 , Won Hee Lee  2 , Olivia Min Ha Hwang  3 , William E Sonntag  4 , Yong Woo Lee  3
Affiliations

Role of NADPH oxidase in radiation-induced pro-oxidative and pro-inflammatory pathways in mouse brain

Hyung Joon Cho et al. Int J Radiat Biol. 2017 Nov.

Abstract

Purpose: The present study was designed to investigate our hypothesis that NADPH oxidase plays a role in radiation-induced pro-oxidative and pro-inflammatory environments in the brain.

Materials and methods: C57BL/6 mice received either fractionated whole brain irradiation or sham-irradiation. The mRNA expression levels of pro-inflammatory mediators, such as TNF-α and MCP-1, were determined by quantitative real-time RT-PCR. The protein expression levels of TNF-α, MCP-1, NOX-2 and Iba1 were detected by immunofluorescence staining. The levels of ROS were visualized by in situ DHE fluorescence staining.

Results: A significant up-regulation of mRNA and protein expression levels of TNF-α and MCP-1 was observed in irradiated mouse brains. Additionally, immunofluorescence staining of Iba1 showed a marked increase of microglial activation in mouse brain after irradiation. Moreover, in situ DHE fluorescence staining revealed that fractionated whole brain irradiation significantly increased production of ROS. Furthermore, a significant increase in immunoreactivity of NOX-2 was detected in mouse brain after irradiation. On the contrary, an enhanced ROS generation in mouse brain after irradiation was markedly attenuated in the presence of NOX inhibitors or NOX-2 neutralizing antibody.

Conclusions: These results suggest that NOX-2 may play a role in fractionated whole brain irradiation-induced pro-oxidative and pro-inflammatory pathways in mouse brain.

Keywords: Fractionated whole brain irradiation; NOX-2; ROS; inflammation.

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Conflict of interest statement

Declaration of Interest

The authors report no conflicts of interest.

Figures

Figure 1.
Figure 1.
Effect of fractionated whole brain irradiation on mRNA expression of TNF-α and MCP-1 in mouse brain. Compared with sham-irradiated controls, fractionated whole brain irradiation significantly up-regulated mRNA expression levels of TNF-α (A) and MCP-1 (B) in mouse brain. Data represent means ± SEM for each group (n=4). **p<0.001 compared to control.
Figure 2.
Figure 2.
Effect of fractionated whole brain irradiation on protein expression of TNF-α and MCP-1 in mouse brain. Immunoreactivities of TNF-α (A-D) and MCP-1 (F-I) were visualized in mouse brain. Fractionated whole brain irradiation significantly up-regulated protein expression levels of TNF-α and MCP-1 in mouse brain (E and J). (A and F) Sham-irradiation (Control); (B and G) 4 h post-irradiation; (C and H) 8 h post-irradiation; (D and I) 24 h post-irradiation; (E and J) Quantitative analysis of fluorescence intensity. Data represent means ± SEM for each group (n=4). *p<0.05; **p<0.001 compared to control. Scale bar: 200 μm.
Figure 3.
Figure 3.
Effect of fractionated whole brain irradiation on microglia in mouse brain. Immunoreactivity of Iba1 was visualized in mouse brain. Fractionated whole brain irradiation significantly increased microglial activation in mouse brain (E). (A) Sham-irradiation (Control); (B) 4 h post-irradiation; (C) 8 h post-irradiation; (D) 24 h post-irradiation; (E) Quantitative analysis of fluorescence intensity. Data represent means ± SEM for each group (n=4). *p<0.05 compared to control. Scale bar: 50 μm.
Figure 4.
Figure 4.
Effect of fractionated whole brain irradiation on reactive oxygen species (ROS) generation in mouse brain. The localization of red fluorescence demonstrates ROS generation in mouse brain (A-D). Fractionated whole brain irradiation significantly increased superoxide anion formation in mouse brain (E). (A) Sham-irradiation (Control); (B) 4 h post-irradiation; (C) 8 h post-irradiation; (D) 24 h post-irradiation; (E) Quantitative analysis of fluorescence intensity. Data represent means ± SEM for each group (n=4). *p<0.05 compared to control. Scale bar: 100 μm.
Figure 5.
Figure 5.
Effect of fractionated whole brain irradiation on protein expression of NADPH oxidase-2 (NOX-2) in mouse brain. Immunoreactivity of NOX-2 (A-D) was visualized in mouse brain. Fractionated whole brain irradiation significantly up-regulated protein expression level of NOX-2 in mouse brain (E). (A) Sham-irradiation (Control); (B) 4 h post-irradiation; (C) 8 h postirradiation; (D) 24 h post-irradiation; (E) Quantitative analysis of fluorescence intensity. Data represent means ± SEM for each group (n=4). *p<0.05 compared to control. Scale bar: 100 μm.
Figure 6.
Figure 6.
Effect of NADPH oxidase (NOX) inhibitors and NOX-2 neutralizing antibody on radiation-induced ROS generation in mouse brain. An increase in ROS generation in mouse brain 4 h after fractionated whole brain irradiation (FIR) was markedly and significantly attenuated in the presence of NOX inhibitors (FIR + APO or FIR + DPI) or NOX-2 neutralizing antibody (FIR + anti-NOX-2). (A) Sham-irradiation (Control); (B) 4 h after fractioned whole brain irradiation (FIR); (C) FIR + 1 mM apocynin (APO); (D) FIR + 20 μM diphenylene iodonium (DPI); (E) FIR + anti-NOX-2 antibody; (F) Quantitative analysis of fluorescence intensity. Data represents mean ± SEM for each group (n=4). **p<0.001 compared to control, #p<0.05; ##p<0.001 compared to irradiated brain (FIR). Scale bar: 100 μm.
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