The Regulatory Effect And Mechanism Of NF-κB Signaling Pathway On Radiation-induced Inner Ear Damage

Background and purposeNasopharyngeal carcinoma(NPC)is one of the most common head and neck cancers,and China has a high incidence of nasopharyngeal carcinoma in the world.Radical radiotherapy is the preferred treatment of nasopharyngeal carcinoma.Due to the anatomical location of the inner ear adjacent to the nasopharynx,the inner ear is inevitably irradiated during the radiotherapy of nasopharyngeal carcinoma,which leads to sensorineural hearing loss(SNHL)and vestibular dysfunction,resulting in a series of symptoms such as hearing loss,deafness,vertigo,abnormal position perception and so on.For most nasopharyngeal cancer patients,long-term survival can be achieved through multidisciplinary treatment,mainly radiotherapy.Therefore,radiation induced inner damage will seriously affect the quality of life of these long-term cancer survivors.Usually,the radiation exposure of the inner ear is considered to be the main factor causing radiation-induced damage to the inner ear.However,our previous series of related studies showed that the inflammation of the inner ear induced by radiotherapy of nasopharyngeal cancer also participated in the injury of the inner ear after radiotherapy of nasopharyngeal cancer.Nuclear Factor kappa-B(NF-κB),as a master switch of inflammatory response,regulates the expression of a variety of inflammation-related genes,such as interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),cyclooxygenase-2(COX-2),vascular cell adhesion molecule-1(VCAM-1),macrophage inflammatory protein-1β(MIP-1β),etc.And then regulate the inflammatory response.A large number of studies have confirmed that radiotherapy can activate the NF-κB pathway in tissues,but there are no reports about the activation of NF-κB pathway in inner ear tissues after radiotherapy and its involvement in the injury after radiotherapy.Therefore,combined with the role of radiotherapy induced inner ear inflammation in post-radiation injury and the central role of the NF-κB pathway in the inflammatory response,in this study,we explored the role and potential mechanism of NF-κB pathway in radiationinduced inner ear damage through animal experiments,hoping to provide a new theoretical basis for the prevention and treatment of radiation-induced inner ear damage.Methods1.Activation of NF-κB pathway after radiation of the inner ear:The radiotherapy model of the inner ear of mice was established by 6 MV-X radiation at a dose of 20 Gy on both sides of the inner ear.Eighteen healthy mice were selected,and the inner ear of mice was irradiated by radiotherapy model.Before radiation and at 2,4,6,8 and 10 h after radiation,three mice were randomly chosen to be killed and remove the inner ear tissue.The expression of p65(one of the important members of the NF-κB family,whose translocation in the nucleus and cytoplasm is an important sign of the activation of the NF-κB pathway)in the inner ear tissues of mice at different time points was detected by Western blotting technique.The activation of the NF-κB pathway and its activation time were analyzed.2.The role of NF-κB pathway in radiation-induced inner ear damage:JSH-23,a compound that can inhibit the NF-κB pathway by inhibiting nuclear shift of p65,was pretreated by intraperitoneal injection of JSH-23.Forty-eight mice were randomly divided into four groups:wild group,JSH-23 pretreated wild group,X-ray radiation group and JSH23 pretreated X-ray radiation group.On the 7th and 14th day after exposure,6 mice were killed in each group.Hematoxylin and Eosin(H&E)staining and Scanning Electron Microscope(SEM)were used to observe and compare the morphological changes of hair cells in the basement membrane of cochlea in the inner ear of mice.3.Potential mechanism of NF-κB pathway involved in radiation-induced inner ear damage:Using quantitative real-time polymerase chain reaction(qRT-PCR)and Western blotting techniques,the expression levels of inflammatory factors TNF-α,IL-6,COX-2 and inflammation-related proteins VCAM-1 and MIP-1β in the downstream of the NF-κB pathway were detected in the inner ear tissues of the above four groups on the 7th and 14th day after radiation,and the differences among the four groups were analyzed and compared.Results1.The NF-κB pathway was activated immediately after radiation,and reached its peak 2～6 h after exposure.2.Morphological analysis by H&E staining and SEM showed that the hair cells in the basement membrane of mice cochlea were seriously damaged at day 7 and 14 after radiation,but the damage was significantly reduced after pretreatment with NF-κB inhibitor JSH-23.3.Along with the above morphological changes of cochlear hair cells,the expression levels of inflammatory factors TNF-α,IL-6,COX-2 and inflammation-related proteins VCAM-1 and MIP-1β in the inner ear tissue of mice were significantly increased at day 7 and 14 days after radiation,but the expression levels were significantly decreased after JSH-23 pretreatment compared to radiation alone.ConclusionsThe activation of NF-κB pathway in the inner ear tissue induced by radiotherapy is involved in the radiation-induced inner ear damage.The mechanism may be through regulating the expression of some inflammatory molecules in the inner ear tissue,and inhibiting the activation of radiation-induced NF-κB helps to reduce the radiation-induced inner ear damage.