Wen Yingying, Zong Shimin, Liu Tianyi, Du Peiyu, Li Hao, Xiao Hongjun
Department of Otorhinolaryngology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
Department of Otorhinolaryngology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
Toxicology. 2021 Apr 15;453:152736. doi: 10.1016/j.tox.2021.152736. Epub 2021 Feb 22.
Cisplatin-induced ototoxicity is one of the important reasons that limit the drug's clinical application, and its mechanism has not been fully elucidated so far. The aim of this study was to explore the attenuate effect of tauroursodeoxycholic acid (TUDCA), a proteostasis promoter, on cisplatin-induced ototoxicity in vivo and in vitro, and to explore its possible mechanism. Auditory brainstem response (ABR) was measured to identify the attenuate effects of TUDCA administered subcutaneously [500 mg/kg/d × 3d, cisplatin: 4.6 mg/kg/d × 3d, intraperitoneal injection (i.p.)] or trans-tympanically (0.5 mg/mL, cisplatin: 12 mg/kg, i.p. with a pump) in Sprague-Dawley (SD) rats subjected to cisplatin-induced hearing loss. The cochlear explants of neonatal rats and OC1 auditory hair cell-like cell lines cultured in vitro were used to observe the number of apoptotic cells and the endoplasmic reticulum (ER) stress in the control, cisplatin (5 μM for 48 h for cochlear explants, 10 μM for 24 h for OC1 cells), and cisplatin + TUDCA (1 mM for 24 h for cochlear explants, 1.6 mM for 24 h for OC1 cells) groups. Differences in the expression of key proteins in the ER protein quality control (ERQC) system were detected. The changes in the attenuate effect of TUDCA on cisplatin-induced ototoxicity after down-regulating calreticulin (CRT), UDP-glucose ceramide glucosyltransferase-like 1 (UGGT1), and OS9 ER lectin (OS9) were also measured. The effect of TUDCA (10 mM) on stabilizing unfolded or misfolded proteins (UFP/MFP) was analyzed in a cell-free 0.2 % bovine serum albumin (BSA) aggregation system in vitro. Both the subcutaneous and trans-tympanic TUDCA administration alleviated cisplatin-induced increase in ABR thresholds in rats. TUDCA was able to reduce cisplatin-induced apoptosis and alleviate ER stress in cochlear explants and OC1 cells. Under the cisplatin treatment, the expression levels of CRT, UGGT1, and OS9 in the auditory hair cell increased, and the expression of total ubiquitinated proteins decreased. TUDCA attenuated the effect of cisplatin on UGGT1 and OS9, and recovered the protein ubiquitination levels. After down-regulating CRT, UGGT1, or OS9, the protective effect of TUDCA decreased. In the cell-free experimental system, TUDCA inhibited the aggregation of denatured BSA molecules. In summary, TUDCA can attenuate cisplatin-induced ototoxicity, possibly by inhibiting the accumulation and aggregation of UFP/MFP and the associated ER stress.
顺铂诱导的耳毒性是限制该药物临床应用的重要原因之一，其机制迄今尚未完全阐明。本研究旨在探讨蛋白稳态促进剂牛磺熊去氧胆酸（TUDCA）对顺铂诱导的体内外耳毒性的减轻作用，并探讨其可能的机制。通过测量听性脑干反应（ABR）来确定皮下注射[500 mg/kg/d×3天，顺铂：4.6 mg/kg/d×3天，腹腔注射（i.p.）]或经鼓膜给药（0.5 mg/mL，顺铂：12 mg/kg，用泵腹腔注射）TUDCA对顺铂诱导听力损失的Sprague-Dawley（SD）大鼠的减轻作用。使用新生大鼠的耳蜗外植体和体外培养的OC1听觉毛细胞样细胞系，观察对照组、顺铂组（耳蜗外植体用5 μM处理48小时，OC1细胞用10 μM处理24小时）和顺铂+TUDCA组（耳蜗外植体用1 mM处理24小时，OC1细胞用1.6 mM处理24小时）的凋亡细胞数量和内质网（ER）应激情况。检测ER蛋白质量控制（ERQC）系统中关键蛋白的表达差异。还测量了下调钙网蛋白（CRT）、UDP-葡萄糖神经酰胺糖基转移酶样1（UGGT1）和OS9 ER凝集素（OS9）后TUDCA对顺铂诱导耳毒性减轻作用的变化。在无细胞的0.2%牛血清白蛋白（BSA）聚集系统中体外分析TUDCA（10 mM）对稳定未折叠或错误折叠蛋白（UFP/MFP）的作用。皮下和经鼓膜给予TUDCA均减轻了顺铂诱导的大鼠ABR阈值升高。TUDCA能够减少顺铂诱导的耳蜗外植体和OC1细胞凋亡，并减轻ER应激。在顺铂处理下，听觉毛细胞中CRT、UGGT1和OS9的表达水平升高，总泛素化蛋白的表达降低。TUDCA减弱了顺铂对UGGT1和OS9的作用，并恢复了蛋白泛素化水平。下调CRT、UGGT1或OS9后，TUDCA的保护作用降低。在无细胞实验系统中，TUDCA抑制了变性BSA分子的聚集。总之，TUDCA可能通过抑制UFP/MFP的积累和聚集以及相关的ER应激来减轻顺铂诱导的耳毒性。
