Study On The Mechanism Of The Attenuate Effect Of Tauroursodeoxycholic Acid On Cisplatin-induced Ototoxicity By Inhibiting The Accumulation And Aggregation Of Unfolded Or Misfolded Proteins In The Endoplasmic Reticulum

Part I Tauroursodeoxycholic acid attenuate cisplatin-induced hair cells damageObjectives: To prove the antagonistic effects of tauroursodeoxycholic acid(TUDCA)on cisplatin-induced hearing loss in rats,and reveal the protective effects and the dose-effect relationships of TUDCA on cisplatin-induced hair cells damage.Methods:(1)In vivo: Twelve healthy adult male SD rats were injected with 12mg/kg of cisplatin intraperitoneally via a pump(duration>30 min).One hour before cisplatin administration,0.5 mg/m L of sterile TUDCA solution was injected into the right tympanic cavity via tympanic membrane puncture until the liquid fully filled the tympanum.The left ear was given an equal amount of sterile Phosphate Buffer Saline(PBS)in the same way.Auditory brainstem response(ABR)was used to test the threshold shifts in the left and right ears before and after the administration.(2)In vitro: The cochlear explants obtained from SD rats at postnatal day 3 were dissected and cultured,randomly divided into three groups: the control group,the cisplatin group and the cisplatin + TUDCA group.Hair cells were labeled by rabbit anti-rat myosin 7a.Observed the morphology of outer hair cells in each group by immunofluorescence.The number of OHCs in the apical,middle,and basal turns of the basilar membrane in each group were counted separately.TUNEL test was applied to compare the differences in the apoptosis ratio of outer hair cells between groups.The OC1 hair cell line was selected and treated with different concentrations of cisplatin for 24 hours.The cell proliferation rate was detected by CCK8 method.10?M cisplatin was selected to construct a cisplatin-induced damage model of OC1 cell line,and added different concentrations of TUDCA pretreating 0h,6h,12 h,24h respectively.The cell proliferation rate was detected by CCK8 method.The optimal time and concentration of TUDCA were screened out.Flow cytometry was used to detect the apoptosis of each group of OC1 cell lines at this concentration and time.Results:(1)In vivo experiments: the ABR threshold shifts of the right ear(TUDCA,2.50±2.79 d B SPL at click,23.33±5.52 d B SPL at 8 k Hz,20.00±6.09 d B SPL at 16 k Hz,28.33±5.16 d B SPL at 24 k Hz,42.08±4.50 d B SPL at 32 k Hz,and 36.25±3.84 d B SPL at40 k Hz)were lower than those of the left ear(PBS,9.17±3.93 d B SPL at click,35.83±5.32 d B SPL at 8k Hz,37.50±4.75 d B SPL at 16 k Hz,47.50±3.17 d B SPL at 24 k Hz,61.67±3.22 d B SPL at 32 k Hz,and 55.83 ±1.83 d B SPL at 40 k Hz).When the stimulus sound is click,the differences between groups is not statistically significant,when the stimulus sound is tone-bust,the differences in all frequencies were statistically significant(p<0.05).(2)In vitro experiments: under the condition of cochlear basement membrane culture in vitro,the outer hair cells of the control group had good morphology,the outer hair cells of the cisplatin group were disordered and missed.Compared with the cisplatin group(Apical turn: 198.00±20.17,middle turn: 141.75±12.91,and basal turn: 138.50±22.50),the outer hair cells of the cisplatin+TUDCA group were arranged neatly and had a higher survival rate(Apical: 232.50±24.36,middle: 201.25±7.51,and basal: 186.75±20.19).The morphology and survival rate of outer hair cells,treated with 1m M TUDCA alone cultured in vitro,were not significantly different from those of the control group.The TUNEL test results showed that the apoptosis rate of outer hair cells in the cisplatin group(apical:2.33±1.86,middle: 14.33±1.45,and basal: 11.33±0.33)was higher than that of the control group(no TUNEL-positive cells)and the cisplatin+TUDCA group(apical: 0.00±0.00,middle: 3.33±1.86,and basal: 2.33±1.45).In the OC1 cell line,as the concentration of cisplatin increased,the activity of OC1 cells gradually decreased.No attenuation effect was observed if TUDCA treatment began from 0h,6h,or 12 h before cisplatin administration.The survival rate of cells pretreated with 1.6m M TUDCA for 24 h before cisplatin administration(88.03%±6.53%)was significantly higher than that of the cisplatin group(57.37%±2.05%).The results of flow cytometry showed that the percentage of apoptosis in the cisplatin+TUDCA group was 1.68%±0.33%,which was significantly lower than that in the cisplatin group(8.21%±0.61%)(p<0.05).Conclusions:(1)TUDCA attenuated the increasement of cisplatin-induced ABR threshold shifts in rats.(2)TUDCA inhibited cisplatin-induced auditory hair cell apoptosis.Part II The mechanism of endoplasmic reticulum protein quality control system in the attenuate effect of tauroursodeoxycholic acid on cisplatin-induced hair cells damageObjectives: To reveal the expression changes of key proteins in the endoplasmic reticulum protein quality control(ERQC)system of cochlear hair cells under treated with TUDCA and/or cisplatin,and clarify the mechanisms of the ERQC system in tauroursodeoxycholic acid(TUDCA)alleviating cisplatin-induced hair cell damage.Methods:(1)The cochlear explants model of cisplatin-induced hair cell damage were prepared from SD rats at postnatal day 3,randomly divided into three groups: the control group,the cisplatin group and the cisplatin + TUDCA group.The cisplatin group was treated with 5 ?M cisplatin for 48 h,and the cisplatin+TUDCA group was treated with 1 m M TUDCA from 24 h before the cisplatin treatment.The expression differences of caspase12,CRT and UGGT1 between the groups,which were the key members of ERQC system,were detected by RT-q PCR and immunofluorescence.(2)Establishment of OC1 cell line model of cisplatin-induced hair cell damage,randomly divided into three groups: the control group,the cisplatin group and the cisplatin + TUDCA group.The cisplatin group: the OC1 cells were treated with 10?M cisplatin for 24 h.The cisplatin+TUDCA group: the OC1 cells were pretreated with 1.6 m M TUDCA for 24 hours before the treatment of TUDCA and cisplatin for 24 h.Detected the expression changes of key proteins between groups in the ERQC system by western blotting technology.Flow cytometry was used to detect the effect of TUDCA on antagonizing cisplatin-induced apoptosis after CRT,UGGT1,or OS9 silencing.(3)The concentration gradient of cisplatin was added to a 0.2% BSA solution with or without 5 m M dithiothreitol(DTT)and/or 10 m M TUDCA in a 96-well plate.The solutions were then incubated at 75 °C for 10 min,20 min,30 min,40 min,50 min,and 60 min.The increase in turbidity caused by protein aggregation was measured as the optical density at 492 nm.Results:(1)The results of RT-q PCR test showed that,compared with the cisplatin group,the expression of caspase-12,UGGT1 and OS9 was lower in the cisplatin+TUDCA group,while the expression of CRT was higher.The effect of TUDCA on the expression of UGGT1 was consistent with the results of RT-q PCR detection showed by immunofluorescence.The results of immunofluorescence showed that TUDCA had no statistical significance on the increase of CRT expression under the cisplatin treatment.(2)The results of Western Blot showed that the expression level of caspase-12 and UGGT1 were consistent with the results by RT-q PCR.Compared with the cisplatin group,the expression level of total ubiquitinated protein,especially large molecular weight ubiquitinated protein,was higher in the cisplatin+TUDCA group,while the expression level of CRT was not changed.CRT,UGGT1,or OS9 silencing can weaken the attenuate effect of TUDCA on cisplatin-induced ototoxicity.(3)Protein aggregation experiments showed that the 0.2% BSA solution without DTT did not show turbid precipitation after being heated at 75 ° C for 60 min,while the turbidity of the BSA solution containing 5 m M DTT gradually increased with increasing heating time.In the BSA solution containing 5 m M DTT and cisplatin,and under the same heating conditions,the turbidity of the solution decreased with the increase in the cisplatin concentration.The BSA solution containing 10 m M TUDCA did not exhibit turbid precipitation after being heated for 60 min;this phenomenon occurred independently of the existence and concentration of cisplatin.Conclusions: TUDCA inhibited the accumulation and aggregation of unfolded or misfolded proteins in the endoplasmic reticulum,improved the degradation ability of ubiquitin-dependent ERAD,attenuated cisplatin-induced hair cells damage.