Functional Study Of GSTM1/GSTT1 In Mouse Model With Cisplatin-induced Ototoxicity

Hearing loss induced by ototoxic drugs is one of the most common sensory deficits in human diseases and is an important factor in deafness,known as drug-induced deafness（DID）.Cisplatin,a clinical chemotherapy drug widely used to treat malignant tumors,can cause severe hearing loss in patients when used inappropriately as an anti-cancer drug.Studies have confirmed that oxidative stress,apoptosis,and inflammatory responses in animal experiments play key roles in the physiopathology of cisplatin-induced deafness.Therefore,it is particularly important to explore the dose-and time-limiting nature of cisplatin-induced deafness.The Gstt1 and Gstm1 genes encode two members of the soluble cytoplasmic glutathione S-transferase（GST）protein superfamily,GST-theta 1（GSTT1）and GST-mu 1（GSTM1）,which serve as important detoxification and antioxidant systems in the body for the metabolism and removal of GST functions as an important detoxification and antioxidant system in the body,metabolizing and scavenging endogenous and exogenous toxic compounds.Numerous studies have reported that Gstm1/Gstt1 gene polymorphisms are continuously identified in the population and are associated with an increased risk of many complex diseases,most notably cancer.Recently,it has been clinically reported that Gstt1/Gstm1 gene polymorphisms are closely associated with ototoxicity susceptibility in patients receiving cisplatin chemotherapy,leading to different degrees of hearing loss in different individuals;therefore,GSTM1/GSTT1 plays an important role in the detoxification process of cisplatin ototoxicity.Based on the successful construction of an animal model,this study aims to investigate the detoxification function of GSTM1/GSTT1 in the auditory system of mice,to investigate whether cisplatin affects the survival of inner ear hair cells through a certain pathway,and thus to elucidate the mechanism of action of Gstm1/Gstt1 knockout mice that are more susceptible to cisplatin ototoxicity.Gstt1 and Gstm1 systemic knockout mouse models(Gstm1^-/-and Gstt1^-/-mice)with CBA/CaJ strain as background were constructed by CRISPR-Cas9 gene editing technology in the early stage of this project,and then a double knockout mouse model（Gstm1/Gstt1-DKO mice）was generated by mating as the subsequent experimental The Gstm1/Gstt1 mRNA and protein expression were detected by Western Blot and real-time fluorescence quantitative PCR,and the hearing phenotypes of knockout mice before and after cisplatin intraperitoneal injection were further analyzed by ABR and DPOAE audiometry.In order to find the potential mechanism of hearing threshold difference between Gstm1/Gstt1-DKO mice and wild mice after cisplatin injection,the cochlea was firstly microdissected,followed by HE staining to observe the overall morphology of cochlear cross-section,including Cortical apparatus,vascular pattern and spiral ganglion,and secondly combined with immunofluorescence and scanning electron microscopy techniques to observe hair cells and synaptic loss,cilia morphology,etc.,from oxidative stress,apoptosis,immune inflammation,etc.To analyze the causes of cisplatin-induced hair cell loss leading to differential hearing,and finally to verify in knockout rats whether antioxidant drugs could rescue the redox imbalance mechanism caused by cisplatin.Statistical analysis of all the above data led to the following main results:（1）Gene sequencing showed that the base sequences of double knockout mice were missing and Gstm1/Gstt1 mRNA was significantly reduced and protein was no longer expressed in the cochlea,confirming the successful construction of the Gstm1/Gstt1-DKO mouse model.（2）CBA/CaJ wild mice induced by intraperitoneal injection of cisplatin showed a significant increase in the expression of GSTM1 and GSTT1 in the inner ear,a severe loss of outer hair cells at the location of the bottom gyrus,and a significant decrease in hearing and body weight of the mice,indicating that cisplatin is directly toxic to the inner ear or nerves,thus,also demonstrating the successful modeling of cisplatin ototoxic mice.（3）Under normal conditions,all histomorphological and audiological phenotypes of the cochlea of Gstm1/Gstt1-DKO mice were normal,indicating that GSTT1 and GSTM1 are nonessential for maintaining hearing function in mice.（4）After cisplatin injection,Gstm1/Gstt1-DKO mice showed significantly higher hearing thresholds and more death of extracochlear hair cells compared to wild-type mice,but no significant differences in vascular striae,spiral ganglia,cilia morphology,or synapse numbers,indicating that GSTM1/GSTT1 slowed the ototoxicity of cisplatin and played a facilitative role in maintaining hair cell survival and protecting hearing.（5）After cisplatin injection,the level of oxidative stress in the cochlea of Gstm1/Gstt1-DKO mice was higher than that of wild-type mice,and the degree of DNA destruction and protein oxidative damage was more severe,and the expression of related detoxification enzymes increased and imbalance of GSH/GSSG redox state,and the analysis indicated that GSTM1/GSTT1 had a certain detoxification effect on cisplatin,and such detoxification enzymes reduced the cisplatin-induced generation of reactive oxygen radicals.The analysis showed that GSTM1/GSTT1 had a detoxifying effect on cisplatin,and such detoxifying enzymes reduced the level of oxidative stress by reducing the cisplatin-induced generation of reactive oxygen species（ROS）mechanism,and partial hearing of mice could be rescued by intraperitoneal injection of the antioxidant drug NAC.（6）The number of apoptotic cells in Gstm1/Gstt1-DKO mice was significantly increased and PAkt content was significantly decreased after cisplatin injection,suggesting that the significant difference in hair cell mortality due to cisplatin is due to the involvement of GSTM1/GSTT1 in the apoptotic mechanism.In conclusion,GSTM1/GSTT1 is closely related to cisplatin ototoxicity,and it has certain detoxification function in cisplatin ototoxic mouse model,reduced levels of cisplatin-induced oxidative stress in the cochlea,slowing down the process of cisplatin-induced apoptosis of hair cells and appropriately protecting hearing,which may provide theoretical basis and new ideas for clinical research on GSTM1/GSTT1 gene polymorphism and the use of cisplatin ototoxic drugs.