Melatonin Attenuates Upregulation of Duox1 and Duox2 and Protects against Lung Injury following Chest Irradiation in Rats

Abstract

Objective: The Lung is one of the most radiosensitive organs of the body. The infiltration of macrophages and lymphocytes into the lung is mediated via the stimulation of T-helper 2 cytokines such as IL-4 and IL-13, which play a key role in the development of fibrosis. It is likely that these cytokines induce chronic oxidative damage and inflammation through the upregulation of Duox1 and Duox2, which can increase the risk of late effects of ionizing radiation (IR) such as fibrosis and carcinogenesis. In the present study, we aimed to evaluate the possible increase of IL-4 and IL-13 levels, as well as their downstream genes such as IL4ra1, IL13ra2, Duox1, and Duox2.

Materials and methods: In this experimental animal study, male rats were divided into 4 groups: i. Control, ii. Melatonintreated, iii. Radiation, and iv. Melatonin (100 mg/kg) plus radiation. Rats were irradiated with 15 Gy ⁶⁰Co gamma rays and then sacrificed after 67 days. The expressions of IL4ra1, IL13ra2, Duox1, and Duox2, as well as the levels of IL-4 and IL-13, were evaluated. The histopathological changes such as the infiltration of inflammatory cells, edema, and fibrosis were also examined. Moreover, the protective effect of melatonin on these parameters was also determined.

Results: Results showed a 1.5-fold increase in the level of IL-4, a 5-fold increase in the expression of IL4ra1, and a 3-fold increase in the expressions of Duox1 and Duox2. However, results showed no change for IL-13 and no detectable expression of IL13ra2. This was associated with increased infiltration of macrophages, lymphocytes, and mast cells. Melatonin treatment before irradiation completely reversed these changes.

Conclusion: This study has shown the upregulation of IL-4-IL4ra1-Duox2 signaling pathway following lung irradiation. It is possible that melatonin protects against IR-induced lung injury via the downregulation of this pathway and attenuation of inflammatory cells infiltration.

Keywords: Duox1; Duox2; Lung; Melatonin; Radiation.

Fig.1

Results of changes in the levels of IL-4 and IL-13 following irradiation with gamma rays and treatment with melatonin (MLT). A. IL-4 and B. IL- 13. a; Significant compared to control and b; Significant compared to radiation (Rad), ANOVA Tukey’s HSD post hoc, P<0.05.

Fig.2

The expression of IL4ra1, Duox1, and Duox2 following irradiation or melatonin treatment before irradiation in lung tissues of rats. A. IL4ra1, B. Duox1, and C. Duox2. a; Significant compared to control and b; Significant compared to radiation (Rad), ANOVA followed by Tukey’s HSD post hoc, P<0.05.

Fig.3

Histopathological investigation of the protective effect of melatonin on radiation-induced lung injury. Control and melatonin groups: no infiltration of macrophages and lymphocytes, as well as normal vascular and alveolar thickening, radiation: severe infiltration of macrophages and lymphocytes, as well as vascular thickening, while alveolar thickening mildly changed. A. Control; B. Melatonin, C. Radiation, D. Radiation+Melatonin (H&E staining ×100).

Fig.4

Results of trichrome staining showed a mild collagen deposition, while treatment with melatonin completely reversed collagen deposition. A. Control, B. Melatonin, C. Radiation, and D. Radiation+Melatonin (Masson’s Trichrome staining ×100).

Fig.5

Infiltration of mast cells following irradiation of lung tissues in rats. The administration of melatonin before irradiation could not significantly attenuate mast cell infiltration. A. Control, B. Melatonin, C. Radiation, and D. Radiation+Melatonin (Giemsa staining ×100).