The Mechanism Of HMGB1/RAGE Signaling Pathway In Cisplatin-induced Ototoxic Injury Research

【Objectives】Sensorineural hearing loss(SNHL)is the major type of deafness,which can be caused by multiple factors such as noise over-exposure,ototoxic drugs,aging,and genetic factors.Cisplatin,widely used in chemotherapy,can treat various malignancies with high side effects,namely definite ototoxicity.The accumulation of ROS is considered to be the leading cause of cisplatin-induced hearing loss.However,using antioxidants as a single clinical treatment for cisplatin-induced hearing loss is not enough;it also requires strategies such as anti-inflammatory.Cisplatin-induced hair cell injury does not involve the entry of pathogens,which may be related to aseptic inflammation.Damage-associated molecular patterns(DAMPs)are intracellular molecules released in response to sterile injury and can activate innate immunity just like pathogen-associated molecular patterns(PAMPs).High mobility group box 1(HMGB1),a highly preserved nuclear DNA-binding protein,is one of the defined DAMPs.RAGE binds to DAMPs mainly through its V structural domain,resulting in RAGE-dependent NF-κB activation and mediating the inflammatory process activation.Therefore,our purpose of the study was to examine whether inhibitions of HMGB1/RAGE axis,Dipotassium glycyrrhizinate(DPG),and FPS-ZM1(FPS)have a significant ameliorative effect against ototoxicity in vivo and in vitro.This may provide a new effective protective or therapeutic strategy for hair cell protection.【Methods】In this study,we investigated the effects of DPG,an HMGB1 inhibitor,and FPS-ZM1,a RAGE inhibitor,in neonatal mouse cochlear explants and HEI-OC1 cell lines,and the protective effects of FPS-ZM1 in C57BL/6 mice.1、We identified apoptosis by Annexin-V FITC/PI staining,Cleaved Caspase-3,and TUNEL staining;assessed intracellular ROS levels by Mito SOX Red and Cell ROX Green staining;detected HMGB1,RAGE,MAPKs(P38,JNK,Erk),NF-κB and apoptosis-related proteins by western blotting;determined Hmgb1、Rage、Nf-κb and pro-inflammatory cytokines(Il-1β、Tnf-α)by q PCR;and inhibited HMGB1 and RAGE by knocking down HMGB1 and RAGE using si RNA.We randomly divided P 42 C57BL/6 mice into 4 groups,namely,control(Control),cisplatin-exposed(Cis),DPG-pretreated(DPG-Cis)and FPS-ZM1-pretreated(FPS-Cis)groups.Before cisplatin(5 mg/kg,sterile saline [0.9% Na Cl] reserve)was injected intraperitoneally for 2 hours,50 mg/kg DPG(sterile saline [0.9% Na Cl] reserve),or 20mg/kg FPS-ZM1(10% DMSO + 40% PEG300 + 5% Tween-80 + 45% [0.9%Na Cl])was injected intraperitoneally.Furosemide(200 mg/kg)was injected intraperitoneally 1hour after cisplatin injection.Auditory Brain Response(ABR)was measured in mice on day 14 after cisplatin injection.After the assay,mice were executed,and the survival of hair cells,synapses,and several nerve fibers in adult mice was observed by immunofluorescent staining of the cochleae.【Results】1,The expression of HMGB1 and RAGE was significantly upregulated in the cisplatin group compared with the control group in the cochlear explants and HEI-OC1 cell line,and the expression of RAGE-dependent nuclear transcription factor NF-κB in the nucleus was increased.The expression of pro-inflammatory cytokines(IL-1β,TNF-α)was also significantly upregulated.2,HMGB1 inhibitor DPG and RAGE inhibitor FPS-ZM1 were able to protect hair cells from cisplatin-induced ototoxic effects and inhibit cisplatin-induced apoptosis and excessive accumulation of intracellular ROS in hair cells.Pretreatment with DPG and FPS-ZM1 significantly reduced the cisplatin-induced increase in HMGB1 and RAGE expression,significantly reduced NF-κB phosphorylation into the nucleus,and in turn,the release of NF-κB-related inflammatory factors.3,The number of cochlear hair cells in neonatal cochlear explants after the knockdown of HMGB1 and RAGE by si-HMGB1 and si-RAGE,respectively,was significantly increased compared with the cisplatin group.4,The RAGE inhibitor FPS-ZM1 significantly reduced the cisplatin-induced ABR threshold shift and decreased the number of cisplatin-induced outer hair cell loss.Also,FPS-ZM1 inhibited cisplatin-induced neuronal fibers and synapse disruption and loss.【Conclusions】1,Cisplatin can cause cochlear hair cell death and release HMGB1,which activates the RAGE-NF-κB-related inflammatory pathway.2,HMGB1 inhibitor DPG and RAGE inhibitor FPS-ZM1 can protect hair cells from cisplatin-induced ototoxic effects and inhibit the activation of NF-κB-related inflammatory responses.3,The RAGE inhibitor FPS-ZM1 inhibited cisplatin-induced hearing loss in vivo.