Based On The JNK Pathway To Explore The Intervention And Mechanism Of Tetramethylpyrazine Restricting The Occurrence Of Ototoxicity Caused By Gentamicin

ObjectiveBased on the functionality of JNK pathway played in the occurrence of ototoxicity caused by gentamicin,explore the intervention mechanism of tetramethylpyrazine and provide a supplementary method to the treatment of ototoxic deafness.MethodsInitially,48 healthy and hearing responsive white red-eye guinea pigs were screened;then randomly divided them into four groups named normal saline(NS)group,gentamicin(GM)group,tetramethylpyrazine(TMP)group and GM+TMP group respectively.In the GM group all guinea pigs did GM intramuscular(IM)injection based on the amount of 120mg·kg-1·d-1;in the TMP group all guinea pigs did TMP intraperitoneal(IP)injection based on the amount of 30mg·kg-1·d-1;in the GM+TMP group,all guinea pigs did GM IM injection and TMP IP injection based on the amount of 120mg·kg-1·d-1 and 30mg·kg-1·d-1 respectively;in the NS group,all guinea pigs did NS IM injection based on the same amount as that the GM group did.All injections required being last for 13 days.Before injection and the first day after injection,80dB nHL click ABR test were performed.Once the test finished,all guinea pigs' cochleas were prepared to be the formalin-fixed paraffin-embedded format.After that the hearing threshold(HT)of all guinea pigs was estimated from the amplitude and latency of ABR Wave ?:cochlear slices and HE dye were done;the structure of Corti's organ and inner cell in the spiral ganglion were observed;Western blot method was used to examine JNK and p-JNK expression level;RealTime-PCR was applied to check out JNK mRNA expression level.ResultsFrom the results got from click ABR test,the HT of all guinea pigs from each group has no significant differences before injection(P>0.05).After injection,compared with the HT got form NS group,the HT got from the GM group and GM+TMP group increased significantly(P<0.01);there was no significant difference between the HTs got from NS group and TMP group(P>0.05);compared with the NS group,although the HT got from GM+TMP and GM group increased,there were also significant differences(P<0.01)between both groups with the HT got from GM+TMP group increased less.From the observation of cochlear slices,the histomorphology of cochlea got from NS group was normal;the Corti's organ got from GM group was severely deformative with OHC swelling and deformation and lots of cells missing and cavitation in the spiral ganglion;however,from the GM+TMP group,although there were some damage,the situation was much better;in the TMP group,the structure of Corti's organ and cells in the spiral ganglion were normal but with some slight change.From the western blot results,after one-way analysis of variance and comparison between groups,it can be seen the density of JNK in different groups were GM group>TMP group?NS group?GM+TMP group;form the aspect of 54KD in the expression level of JNK2(one subtype of JNK),the density of it in all groups were GM group>GM+TMP group?TMPgroup?NS group;the distribution of JNK 1(one subtype of JNK)in 46KD had no significant differences between all groups(P>0.05).After injection,compared to the NS group,the expreesion of p-JNK in the GM and GM+TMP group incresed significantly(P<0.05);there was also significant difference(P<0.05)between GM and GM+TMP group with the expression level of p-JNK(phosphorylated JNK)in the GM group was higher.For the expression of p-JNK 1(one subtype of p-JNK),all groups showed no significant difference(P>0.05).The distribution of p-JNK 2(one subtype of p-JNK)was as same as that of JNK2.From the results of RealTime-PCR,the JNK gene expression level from GM group was significantly different from that of the rest groups(P<0.05).There was no significant difference between the JNK gene expression level from NS,TMP and GM+TMP group(P>0.05).Based on the correlation analysis,it can be seen the expression level of JNK2,p-JNK2,p-JNK and JNK mRNA were linear correlation to the increase of HT(P<0.05).There were no linear correlations between the expression level of JNK1,p-JNK1,JNK and HT(P>0.05).Conclusion:GM is ototoxic and it can lead to the abnormal of the cochlear histomorphology,the increase of ABR latency and decresee of ABR amplitude,JNK2?p-JNK2 and JNK mRNA abnormally expressed;the expression level of JNK2,p-JNK2 and JNK mRNA can correlate to the increase of HT,which can also reflect from the cochlear histomorphology;TMP can improve HT,inhibit the phosphorylation of JNK2,decrease JNK expression level from molecular aspects and may effect JNK pathway and play a treatment role in ototoxic deafness.