Study On The Mechanism Of TAK1-mediated Necroptosis In Age-related Hearing Loss

Objective: Age related hearing loss(ARHL),also known as presbycusis,is the most common hearing disease in the elderly.The main clinical features are progressive,bilateral symmetrical sensorineural hearing loss.The main pathological features are the damage of cochlea hair cells(HCs),spiral ganglion neurons(SGNs)and Stria Vascularis(SV).Previous studies have shown that TAK1,as an important factor in the upstream of a variety of inflammatory pathways,plays an important role in regulating cell survival and death.The mechanism of TAK1-mediated inflammation has been intensively studied in a variety of age-related diseases,but the regulatory role of TAK1 in ARHL remains unclear.Therefore,in this study,DBA/2J mice were used as the animal model,D-galactose(D-gal)was used to construct a cell aging model,and plasmid transfection was used to interfere with TAK1 expression level,so as to explore the role of TAK1 in the regulation of ARHL cochlea hair cell death.Methods:(1)The audiological characteristics of DBA/2J mice at 2,4,8 and 16 weeks were detected by Auditory Brainstem Response(ABR).(2)Hematoxylin and Eosin(H&E)staining was used to observe the morphological characteristics of HCs,SGNs and SV in the scala middle of the cochlea of DBA/2J mice at 2,4,8 and 16 weeks.(3)The expression of TAK1 in brain,heart,liver,lung,kidney,muscle and cochlea tissues of DBA/2J mice at 2 weeks and the change trend of TAK1 in cochlea tissues of DBA/2J mice at 2,4,8 and 16 weeks were detected by q RT-PCR.(4)Immunofluorescence was used to determine the localization of TAK1 in the cochlear scala media of DBA/2J mice and HEI-OC1 cells at 2 weeks.(5)HEI-OC1 cells were stimulated with 0,10,20,30,40,and 50mg/ml D-gal for 24 h,48h,and 72 h,respectively.The concentration and time of D-gal cell aging model were determined by cell proliferation assay(CCK-8).The relative m RNA expression levels of P16 and P21 and the level of reactive oxygen species(ROS)were detected to verify the D-gal cell aging model.(6)q RT-PCR and Western blotting were used to detect the gene and protein levels of TAK1 in the D-gal cell aging model.The levels of apoptosis and necrosis were detected by flow cytometry.(7)Plasmid transfection was used to construct TAK1 knockdown group,and the relative m RNA expression levels of TAK1,NF-κB,CASP8,RIPK3 and MLKL in different groups of cells were detected by q RT-PCR.The protein levels of TAK1,RIPK3 and MLKL were detected by Western blotting.Cell apoptosis and necrosis were detected by flow cytometry.(8)Plasmid transfection was used to construct TAK1 overexpression group,and the related gene levels,protein levels,apoptosis and necrosis levels were detected again.Results:1.The results of ABR showed that the hearing of DBA/2J mice was basically normal at 2weeks of age,and the hearing decreased gradually with the increase of age.DBA/2J mice can be used as an animal model of ARHL.2.The results of H&E staining showed that HCs,SGNs and SV of DBA/2J mice were almost normal at 2 weeks.With the increase of age,the number of outer hair cells(OHCs),the density of SGNs and the width of SV gradually decreased.3.The results of q PCR showed that TAK1 was highly expressed in the cochlea of DBA/2J mice,and the expression of TAK1 decreased with age.4.Immunofluorescence results showed that TAK1 was expressed in HCs,SGNs and SVS in the scala middle of the cochlea of DBA/2J mice,and also in HEI-OC1 cells.5.CCK-8 results showed that the proliferation activity of cells treated with 40mg/ml D-gal for 48 hours was significantly decreased.The m RNA relative levels of P16 and P21 in cells were increased,and the level of ROS was increased.These results indicated that the D-gal cell aging model could be successfully established by treatment with 40mg/ml D-gal for 48 hours.6.In the D-gal cell aging model,the m RNA relative expression of TAK1 and protein level were significantly decreased,and the cells could undergo necroptosis.7.Compared with the normal group and the negative control group,the m RNA relative levels of NF-κB and CASP8 in the TAK1 knockdown group were decreased,the m RNA relative levels and protein levels of RIPK3 and MLKL were increased,and the cells underwent necroptosis.8.Compared with the normal group and the plasmid empty group,the m RNA relative levels of NF-κB and CASP8,the m RNA relative levels and protein levels of RIPK3 and MLKL were decreased in the TAK1 overexpression group,and the level of necroptosis was decreased.Conclusion:The expression of TAK1 gradually decreased with tissue aging and cell aging.TAK1 knockdown can mediate necroptosis leading to cochlear hair cell death,while TAK1 overexpression can reduce necroptosis and alleviate cochlear hair cell death.This study preliminarily confirmed that TAK1-mediated necroptosis plays an important role in the pathogenesis of ARHL.