A unique radioprotective effect of resolvin E1 reduces irradiation-induced damage to the inner ear by inhibiting the inflammatory response

Abstract

Background: In addition to the direct effects of irradiation, the induced inflammatory response may play an important role in the damage to the inner ear caused by radiotherapy for the treatment of head and neck cancers. Resolvin E1 (RvE1) has anti-inflammatory activity, acting by reducing neutrophil infiltration and proinflammatory cytokine expression. Therefore, in this study we sought to confirm whether the inflammation induced by irradiation was involved in damage to the inner ear after radiotherapy and to investigate the protective effect and underlying mechanism of RvE1 using mouse models.

Methods: A dose of RvE1 was delivered by intraperitoneal injection to mice before irradiation. Changes in the auditory brainstem response (ABR), relative balance ability, inner ear morphology and the expression levels of inflammatory factors in the inner ear were analyzed on days 7 and 14 after irradiation and compared among different experimental groups.

Results: Changes of ABR and relative balance ability showed the inner functions of experimental mice presented severe damage after irradiation, but the damage was significantly alleviated after RvE1 pretreatment compared to irradiation alone. Morphological analysis of the inner ear showed severe damage to the cochlea and vestibule after irradiation. In contrast, damage to the cochlea and vestibule was significantly reduced in the RvE1-pretreated group compared to that in the irradiation alone group. Along with these functional and morphological changes, the mRNA expression level of anti-inflammatory factors interleukin-2 was significantly increased, while those of proinflammatory factors interleukin-6 and tumor necrosis factor-α were significantly decreased in the inner ear of mice after RvE1 pretreatment compared to irradiation alone.

Conclusions: We believe that inflammation induced by irradiation is involved in the damage to the inner ear caused by radiotherapy, and that RvE1 reduces the damage caused by irradiation to the inner ear by regulating the induced inflammatory response.

Keywords: Inflammatory response; Inner ear; Proinflammatory cytokine; Radioprotective effect; Resolvin E1.

Fig. 1

One-way analysis of variance for hearing threshold and relative balance ability showed the changes among the different groups after irradiation and resolvin E1-pretreatment. ^*, P ≤ 0.05; ^**, P ≤ 0.01; a. Hearing changes after irradiation and resolvin E1-pretreatment. Irradiation-induced hearing loss, which was obvious on days 7 and 14 after irradiation; resolvin E1 (RvE1) mitigated the irradiation-induced hearing loss. The red asterisk indicates a comparison between the Sham/WT and Rad groups. The black asterisk indicates a comparison between the Rad and Rad+RvE1 groups. b. Relative balance ability changes after irradiation and resolvin E1-pretreatment. The relative balance ability in Sham/WT, RvE1, Rad and Rad+RvE1 groups were respectively 1.154 ± 0.171, 1.149 ± 0.133, 0.869 ± 0.085, and 1.039 ± 0.115 on day 7 and 1.137 ± 0.151, 1.134 ± 0.161, 0.927 ± 0.066, and 1.048 ± 0.094 on day 14. Irradiation induced damage in relative balance ability (compared between the Sham/WT and Rad groups) on days 7 and 14 after irradiation (P = 0.0045 and P = 0.0107), and RvE1 mitigated this damage (comparison between the Rad and Rad+RvE1 groups, P = 0.0155 on day 7 and P = 0,0270 on day 14)

Fig. 2

Hematoxylin-eosin staining showed the changes to the hair cells of the basement membrane after irradiation and resolvin E1-pretreatment. The arrows indicate the points of the loss of basement membrane hair cells. The loss could be observed in the apical, medial and basal gyrus of the cochlea on days 7 and 14 after irradiation. Pretreatment with resolvin E1 (RvE1) clearly reduced this damage. An obvious reduction in the number of hair cells can be observed at the points indicated by the arrows in the Rad+RvE1 group compared to the Rad group

Fig. 3

Whole-mount staining showed changes to the hair cells of the basement membrane after irradiation and resolvin E1-pretreatment. The asterisks indicate the points of the loss of hair cells, and the number of asterisks represents the frequency of damage. The loss was observed on days 7 and 14 after irradiation, but this damage was markedly reduced by pretreatment with resolvin E1

Fig. 4

Scanning electron microscopy showed changes to the hair cells of the basement membrane after irradiation and resolvin E1-pretreatment. The asterisks indicate the points of the loss of hair cells, and the number of asterisks represents the frequency of damage, which could be observed on days 7 and 14 after irradiation. However, pretreatment with resolvin E1 obviously reduced this damage

Fig. 5

Hematoxylin-eosin staining showed changes to the spiral ganglion cells and hair cells of the utriculus and sacculus after irradiation and resolvin E1-pretreatment. The arrows indicate the points of the loss of spiral ganglion cells and utriculus and sacculus hair cells, and the number of arrows represents the frequency of damage, which could be observed on days 7 and 14 after irradiation. Meanwhile, pretreatment with resolvin E1 mitigated the damage. The loss of utriculus and sacculus hair cells represents that of vestibular hair cells

Fig. 6

Whole-mount staining (a) and scanning electron microscopy (b) showed changes to the vestibular hair cells after irradiation and resolvin E1-pretreatment. The arrows (a) indicate the points of the loss of vestibular hair cells. The red asterisks (b) indicate the points of cilia fusion and blue asterisks (b) indicate the points of cilia loss of vestibular hair cells, while the numbers of arrows and asterisks represent the frequencies of damage, which could be observed on days 7 and 14 after irradiation. Pretreatment with resolvin E1 obviously reduced this damage of cells

Fig. 7

One-way analysis of variance for inflammatory factor expression showed the changes among the different groups after irradiation and resolvin E1-pretreatment. ^*, P ≤ 0.05; ^**, P ≤ 0.01; a. The expression level changes of IL-2 among the different groups. The expression level of IL-2 in the modiolus was significantly decreased on days 7 and 14 after irradiation (compared between the Sham/WT and Rad groups). b. The expression level changes of IL-6 among the different groups. The expression levels of IL-6 in the basal membrane and vestibule were significantly increased on days 7 and 14 after irradiation (compared between the Sham/WT and Rad groups). c. The expression level changes of TNF-α among the different groups. The expression levels of TNF-α in the modiolus and vestibule were significantly increased on day 7 after irradiation (compared between the Sham/WT and Rad groups), and a similar significant change was observed only in the modiolus on day 14 after irradiation (compared between the Sham/WT and Rad groups). d and e. The expression level changes of IL-10 and IFN-γ among the different groups. The expression of IL-10 and IFN-γ did not significantly change among the different groups. All these significant changes after irradiation could be ameliorated by resolvin E1 (RvE1) pretreatment (compared between the Rad and Rad+RvE1 groups)