Experimental Study On The Mechanism Of PD-1/PD-L1 In Apoptosis Of Guinea Pig Spiral Ganglion Cells Induced By Sodium Salicylate

Objective To elucidate the possible mechanisms of sodium salicylate ototoxicity and find new drug targets for the treatment of tinnitus,a model of ototoxicity in guinea pigs was established,it will be clear whether sodium salicylate(SS)can induce apoptosis in guinea pig cochlear spiral ganglion neuron(SGN)and explore the expression and significance of programmed cell death protein 1(PD-1)and programmed death ligand 1(PD-L1)in it.Methods Screening 30(60 ears)healthy guinea pigs,whose auditory brainstem response(ABR)was normal(ABR threshold<40dBSPL)and distortion product otoacoustic emissions(DPOAEs)tests had passed.Using a random number table method,they were divided into five groups,six in each group.Group A:normal control group(administered saline);group B:SS administration 2 hours group;group C:SS administration 3 days group;group D:SS administration 7 days group;Group E:SS administration 14 days group.Among them,the dosage was 400mg/kg/d.Both saline and SS are administered intraperitoneally.Each group of guinea pigs were tested for ABR and DPOAE after administration,and then immediately anesthetized by excessive anesthesia,and the cochleas on both sides were taken out.The paraffin sections of left cochleas of each group were tested as follows:? Hematoxylin-eosin(HE)was used to observe the number and size of SGN cells;?Immunohistochemical techniques were used to detect the expression of PD-1 and PD-L1 in the cochlea;?TdT-mediated dUTP nick end labeling(TUNEL)was used to study the apoptosis of SGN.The arrangement and number of hair cells were observed by silver nitrate-stained basement membrane in cochlea on the right side of each group.Finally,the experimental datas were analyzed and processed using statistical product and service solutions(SPSS)20.0.Results 1.Auditory test results:the ABR threshold of each wave was analyzed.There was no significant difference between before and after administration in group A(p>0.05);The ABR thresholds in group B,C,D and E were higher than those before administration,and the differences were statistically significant(p<0.05);however,there was no significant difference among groups B,C,D and E after administration(p>0.05).DPOAE results,except for the waveforms that could be normally extracted in the left and right ears of group A,the amplitudes of B,C,D,and E groups were reduced or the waveform did not pass.2.HE staining:Under the microscope,the number of SGN cells in the A group were large,arranged in order,and the cell structure were clearly identifiable.The blue-stained nuclei was large and round,and the red-stained cytoplasm was full and stained uniformly.In group B,SGN cells were still very large,and the nucleus and cytoplasmic morphology were inferior to those in group A.In group C,the number of SGN cells were still large,but the nuclear staining was uneven,and nuclear fragmentation and nuclear dissolution occurred.In group D,the number and morphology of SGN cells were significantly changed compared with the previous groups(group A,B,and C).The cells were small and few,showing nuclear pyknosis and dissolution.In group E,SGN cells were small,irregular in shape,and reduced in number.The surrounding tissue is sparse,and there was a phenomenon of nuclear pyknosis,dissolution and even disappearion.3.TUNEL results:Under the microscope,there was no obvious apoptotic SGN cells in group A.A small number of positively stained SGN cells were seen in group B.More apoptotic SGN cells appeared in group C.The apoptotic cells of SGN in group D and E increased significantly.Comparison between groups,the apoptotic index increased with the prolongation of SS administration time,and the difference was statistically significant(p<0.05).4.Immunohistochemistry results:under the microscope,the positive expression of PD-1 and PD-L1 in SGN cells in group A was obvious.Compared with group A,the positive expressions of PD-1 and PD-L1 in SGN cells were decreased in group B,C,D and E,respectively.And the differences were statistically significant(p<0.05).5.Silver nitrate staining of the basement membrane of the cochlea:in group A,the hair cells were arranged neatly and the number was not missing;in group B,the outer hair cells were arranged in disorder,and the inner hair cells did not change significantly;small loss of outer hair cells in group C;increased number of missing outer hair cells in group D;large loss of outer hair cells in group E,the cell outline was unclear,and there was no significant change in inner hair cells.Conclusion 1.SS administration causes an increase in the ABR threshold of guinea pigs.2.SS induces apoptosis in guinea pig SGN cells,and PD-1/PD-L1 may be involved in this apoptotic process.3.SS administration causes loss of cochlear hair cells and structural damage,which is related to the time of administration.