The Mechanism Of Formyl Peptide Receptor 2 Mediating Cisplatin-induced Apoptosis Of Mouse Cochlear Cells

ObjectiveTo study the role and mechanism of formyl peptide receptor 2(FPR2)in cisplatin-induced apoptosis of mouse cochlear cells,so as to provide a new idea for clinical protection against cisplatin-induced deafness.Methods1.In vivo experimentSeventy-two 6-week-old BALB/c mice were randomly divided into control group,cisplatin group and cisplatin+Boc-2 group(Boc-2 is an inhibitor of FPR2).Mice in cisplatin group were intraperitoneally injected cisplatin solution(4.5mg/kg/d)for 5 consecutive days.Mice in cisplatin+Boc-2 group were intraperitoneally injected with Boc-2 solution(50μg/kg/d)30min before intraperitoneal injection of cisplatin solution(4.5mg/kg/d)for 5 consecutive days.Mice in control group were intraperitoneally injected with the same amount of normal saline for 5 consecutive days.Auditory brainstem response(ABR)is used to detect auditory function of mice.The localized expression of FPR2,cleaved caspase-3,p-ERK1/2 and p-p65 in mouse cochlea was detected by immunofluorescence(IF)staining and image analysis.quantitative reverse transcription polymerase chain reaction(q RT-PCR)was used to detect FPR2 m RNA expression in mouse cochlea.The expression level of FPR2 protein in mouse cochlea was detected by western blot(WB).Enzyme-linked immunosorbent assay(ELISA)was used to detect the secretion of TNF-α,IL-1β and IL-6 in serum of mice.2.In vitro experimentHEI-OC1 cells were divided into control group,cisplatin group,siRNA-NC+ cisplatin group and siRNA-FPR2+cisplatin group.siRNA-FPR2+cisplatin group was transfected with FPR2 sequence,cultured for 36 h,and then replaced with fresh medium containing 20μM cisplatin for 12 h.siRNA-NC+cisplatin group was transfected with negative control sequence,cultured for 36 h,and then replaced with fresh medium containing 20μM cisplatin for 12 h.Cisplatin group was cultured normally for 36 h,and then replaced with fresh medium containing 20μM cisplatin for 12 h.Control group was given normal medium for 48 h.After transfection for 48 h,the activity of HEI-OC1 cell was detected by CCK-8 method,and the protein levels of FPR2,ERK1/2,p-ERK1/2,p65,p-p65,IκBα and p-IκBα in HEI-OC1 cells were detected by WB technique.p65 kernel shift was detected by IF;The secretion of TNF-α,IL-1β and IL-6 in HEI-OC1 cells was detected by ELISA.The apoptosis of HEI-OC1 cell was detected by TUNEL assay.Results1.FPR2 mediated cisplatin-induced ototoxic injury in miceAfter cisplatin administration,ABR threshold was significantly increased at8 kHz,16kHz,24 kHz and 32 kHz in cisplatin group,and the expression level of cleaved caspase-3 in cochlear hair cells was significantly increased in cisplatin croup as compared with control group(P < 0.001).Combined administration of the FPR2 antagonist Boc-2 significantly reduced cisplatin-induced ABR threshold and cleaved caspase-3 high expression(P < 0.001,P < 0.05).FPR2 was highly expressed in hair cells,spiral ganglion cells and vascular striae cells of mice in cisplatin group,and the mRNA and protein levels of FPR2 in mice were significantly decreased after Boc-2 treatment as compared with cisplatin group(P < 0.05).Small RNA interference was performed on HEI-OC1 cells to silence FPR2 gene.The results showed that the expression level of FPR2 protein and cell viability of siRNA-FPR2+cisplatin group were significantly lower than those of cisplatin group and siRNA-NC+ cisplatin group after 12 h administration of cisplatin(P < 0.01).2.FPR2 mediated cisplatin-induced apoptosis of mouse cochlear cells through ERK/NF-κB signaling pathwayp-ERK1/2 and p-p65 were highly expressed in the hair cells,spiral ganglion and vascular striae cells of mouse cochlea in cisplatin group,and the expression levels of p-ERK1/2 and p-p65 in cochlea cells of mice were significantly decreased after Boc-2 treatment(P < 0.001).p-ERK1/2 and p-p65 were also highly expressed in cisplatin-treated HEI-OC1 cells.After targeted silencing of FPR2 gene,the protein expression levels of p-ERK1/2,p-IκBα and p-p65 in siRNA-FPR2+cisplatin group were significantly lower than those in cisplatin group and siRNA-NC+ cisplatin group(P < 0.01).The nuclei shift of p65 was also inhibited(P < 0.001).The contents of pro-inflammatory factors(TNF-α,IL-1β,IL-6)in serum of mice in cisplatin group and in cisplatin treated HEI-OC1 cells were significantly higher than those in control group(P < 0.05).After Boc-2 treatment and targeted silencing of FPR2 gene,the serum proinflammatory factor secretion level of mice was significantly decreased(P < 0.05),and the serum proinflammatory factor secretion level of siRNA-FPR2+ cisplatin group was significantly lower than that of cisplatin group and siRNA-NC+ cisplatin group(P < 0.01).After cisplatin treatment,the apoptosis rate of HEI-OC1 was significantly higher than that of control group(P < 0.001).After targeted silencing of FPR2 gene,the apoptosis rate of HEI-OC1 was significantly decreased(P < 0.001).ConclusionsFPR2 mediated ototoxic injury of mice induced by cisplatin.Gene silencing of FPR2 can effectively inhibit the high expression of ERK and NF-κB induced by cisplatin,then reduce the synthesis and release of pro-inflammatory factors of TNF-α,IL-1 and IL-6,so as to inhibit the apoptosis of cochlear cells and improve hearing function.