The inner ear is an important organ at risk (OAR) in patients with head and neck cancers receiving radiotherapy, and SNHL and vestibular dysfunction are often inevitable. Some studies suggest that the incidence of post-irradiation SNHL may be as high as 50% [20]. The main cause of radiation-induced SNHL is thought to be the direct action of irradiation on cochlea hair cells and the spiral ganglion, leading to damage to cell and mitochondrial membranes, lysosomal membrane lipid peroxidation and DNA damage and subsequently resulting in cellular metabolic disorder, hair cell degeneration, necrosis, and dysfunction [21, 22]. Damage to the vasculature can cause endolymphatic edema, which can also lead to hearing loss, tinnitus, ear distension, vertigo or balance disorders [22, 23]. In this study, we also observed hearing loss in mice, evaluated by ABR, along with HE staining, whole-mount staining and SEM showing various degrees of damage to the cochlea basal membrane hair cells and spiral ganglion cells after irradiation. Post-irradiation vestibular dysfunction has rarely been reported, and our previous study found an incidence of post-irradiation vestibular dysfunction of up to 60–70% in clinical practice [9], but the mechanism has not previously been reported. In this study, post-irradiation vestibular dysfunction in mice was also indicated by the results of relative balance ability tests, and HE staining, whole-mount staining and SEM all showed that damage to utriculus and sacculus hair cells after irradiation was the main cause of post-irradiation vestibular dysfunction.
The direct damaging effect of irradiation on the target organ is the main cause of post-irradiation inner ear damage, but another cause of post-irradiation inner ear damage is the irradiation-induced inflammatory response. Some studies have reported that various inflammatory factors significantly increased after radiotherapy, mainly proinflammatory cytokines such as IL-1, IL-6, IL-8, and TNF-α [24]. Our previous study found that IL-6 was significantly increased in nasopharyngeal carcinoma patients after radiotherapy and was associated with post-irradiation vestibular dysfunction [9], implying that the irradiation-induced inflammatory response participated in vestibular dysfunction. In this study, we directly observed a significant decrease in the mRNA level of IL-2 and significant increases in the mRNA levels of IL-6 and TNF-α in mouse inner ears at different time points after irradiation. These results confirmed the potential involvement of the inflammatory response in post-irradiation inner ear damage.
RvE1 is a trihydroxy derivative of the polyunsaturated fatty acid EPA, and is found in fish oil and sea food. RvE1 possesses a remarkable treatment effect for many inflammation-related diseases and conditions when administered via subcutaneous injection, intraperitoneal injection, intraplantar injection and intrathecal injection [11, 13–17, 25]. In this study, we adopted intraperitoneal injection with 1 µg RvE1 [11, 16] and achieved the expected results.
There have been no previous reports regarding whether RvE1 has a radioprotective effect on the inner ear. However, based on the fact that the local inflammatory response in irradiated tissues is a universal occurrence, and further, that RvE1 has remarkable effects in counteracting inflammation and promoting resolution of inflammation, we explored the radioprotective effect of RvE1 on the inner ear via experiments in a mouse model. To our surprise, we found that RvE1 considerably ameliorated irradiation-related damage to the inner ear. In this study, employing RvE1 pretreatment before irradiation, we not only observed the alleviation of hearing loss and vestibular dysfunction, but also observed the amelioration of microstructure damage associated with hearing loss and vestibular dysfunction. On the one hand, this result proved that the local inflammatory response in irradiated tissues is involved in tissue damage, and at the same time, also proved that RvE1 has a radioprotective effect on the inner ear.
The molecular mechanisms underlying the RvE1 anti-inflammatory activity and promotion of inflammation resolution are complicated [15, 17, 18, 26]. Among these complicated mechanisms, RvE1 modulation of inflammation-related cytokine expression plays an important role. Therefore, in this study, we investigated changes in the expression of the inflammatory factors IL-2, IL-6, IL-10, TNF-α, and IFN-γ after RvE1 pretreatment to explore the potential mechanisms via which RvE1 protects against irradiation-induced damage to the inner ear. Among these inflammatory factors, IL-2 and IL-10 are anti-inflammatory factors, which can inhibit the inflammatory cascade reaction by downregulating the expression of proinflammatory factors [19, 27]. IL-6, TNF-α, and IFN-γ are important proinflammatory factors that activate the inflammatory cascade reaction and cause damage to target organs [19, 27]. The influence of RvE1 on these inflammatory factors has been previously reported. Xu et al. reported that RvE1 ameliorates pulpitis by inhibiting the expression of the proinflammatory cytokines IL-6 and TNF-α in a chemerin receptor23 (ChemR23)-dependent manner [28]. Rey et al. reported that RvE1 decreased LPS-induced gene expression of proinflammatory cytokines (TNF-α and IL-6), suggesting that they exert proresolutive activity in microglia [29]. Wang et al. reported that RvE1 treatment improves allogeneic corneal graft survival by significantly reducing the mRNA expression of proinflammatory cytokines, including TNF-α, IL-2, IL-6 and IFN-γ, in corneal grafts, as well as the protein level of the proinflammatory cytokines TNF-a, IL-2, IL-6 and IFN-γ [30]. However no similar findings have been reported in the inner ear.
In this study, the results showed that the mRNA expression level of IL-2 significantly increased and those of IL-6 and TNF-α significantly decreased in the inner ear of mice at different time-points after RvE1 pre-treatment compared to irradiation alone. In contrast, the mRNA expression levels of IL-10 and IFN-γ did not show any obvious changes after irradiation following RvE1 pretreatment. Further analysis found that the changes in expression of IL-2, IL-6, and TNF-α were also associated with damage to cochlea hair cells, spiral ganglion cells, and vestibular sacculus and utriculus hair cells. All of these results imply that the inflammatory response induced by irradiation is involved in inner ear damage after irradiation, and that the radioprotective mechanism of RvE1 in the inner ear is also mediated through its anti-inflammatory effects via regulation of IL-2, IL-6, and TNF-α.
Regarding the way in which RvE1 regulates the expressions of IL-2, IL-6, and TNF-α, current consensus is that RvE1 mediates its effects via binding to the ChemR23 and BLT1 receptors. In particular, ChemR23 is widely expressed on monocytes, macrophages, dendritic cells and epithelial cells [31, 32] and is upregulated in response to tissue damage or inflammatory stimuli [33, 34]. Within the inflammatory microenvironment induced by irradiation, these cells are universally present [19], but in this study, it was unclear whether RvE1 regulates the expressions of IL-2, IL-6, and TNF-α in the inner ear via this mechanism. This question is worthy of study in the future.