何首乌苷对对乙酰氨基酚致豚鼠听力损伤的保护作用

Protective Effect of 2,3,5,4’-Tetrahydroxy Stilbene-2-Ο-β-D-glucoside Against Acetaminophen-induced Hearing Impairment in Guinea Pigs

关键词:  何首乌苷  听力损伤  耳鸣耳聋  对乙酰氨基酚

Key words:  2,3,5,4’-tetrahydroxy stilbene-2-Ο-β-D-glucoside  hearing impairment  tinnitus and deafness  acetaminophen

摘要:

目的 探究何首乌苷(2,3,5,4’-tetrahydroxy stilbene-2-Ο-β-D-glucoside，TSG)对对乙酰氨基酚(4-acetaminophen，APAP)致豚鼠听力损伤的保护作用。方法 采用APAP诱导豚鼠耳损伤，TSG处理后，测试豚鼠的听性脑干反应(auditory brainstem response，ABR)、畸变产物耳声发射(distortion product otoacoustie emissions，DPOAE)等，判断听力损失情况，免疫荧光观察耳蜗毛细胞损伤情况，对豚鼠肝肾组织进行病理学观察，判断TSG对肝肾的毒性；以APAP致HEI-OC1听神经细胞损伤为耳损伤细胞研究，通过MTT法、细胞形态学观察、流式细胞仪检测细胞存活及凋亡情况，荧光定量PCR检测Birc5基因的表达。结果 与模型组相比，第14天时，豚鼠ABR听力阈值显著降低，DPOAE测试频率为2 kHz时差异更加显著(P<0.01)，听觉惊跳反射显示在100 dB时惊跳反射幅值显著降低(P<0.05)，间隙前脉冲抑制试验表明在10，40，50 ms时惊跳反射幅值降低；剥离豚鼠耳蜗，免疫荧光染色后观察毛细胞完整性，与模型组比较，TSG组外毛细胞排列整齐，细胞无缺失，内毛细胞排列基本整齐；病理学观察发现TSG具有一定的肝毒性而肾毒性不明显。细胞实验发现，APAP对HEI-OC1听神经细胞的半数抑制浓度为800 µmol·L^-1，10 µmol·L^-1TSG干预APAP诱导的细胞损伤时，凋亡率下降至16.0%，显著低于模型组(APAP)组的25.5%。APAP组HEI-OC1细胞Birc5表达水平明显下降，TSG处理可明显升高Birc5表达水平。结论 TSG 15 mg·kg^-1处理14 d可以对100 mg·kg^-1APAP诱导的豚鼠听力损失产生保护作用，该保护作用与TSG抑制APAP导致的听神经细胞凋亡及促进凋亡抑制因子Birc5的表达有关。

Abstract:

OBJECTIVE To explore the protective effect of 2,3,5,4’-tetrahydroxy stilbene-2-Ο-β-D-glucosid(TSG) on hearing injury in acetaminophen(APAP) induced guinea pigs. METHODS The ear injury of guinea pigs was induced by APAP, after treatment with TSG, the auditory brainstem response(ABR) and distortion product otoacoustic emission(DPOAE) of guinea pigs were tested to judge the hearing loss, the injury of cochlear hair cells was observed by immunofluorescence, and the liver and kidney tissues of guinea pigs were observed by pathology to judge the toxicity of TSG to liver and kidney. The damage of HEI-OC1 auditory nerve cells induced by APAP was used as the study of ear injury cells. The cell survival and apoptosis were detected by MTT method, cell morphology observation, and flow cytometry, the expression of Birc5 gene was detected by fluorescence quantitative PCR. RESULTS Compared with the model group, on the 14th day, the ABR hearing threshold of guinea pigs decreased significantly, the difference was more significant when the DPOAE test frequency was 2 kHz(P<0.01). The acoustic startle reflex test showed that the amplitude of startle reflex decreased significantly at 100 dB(P<0.05). The gap-prepulse inhibition experiment showed that the amplitude of startle reflex decreased at 10, 40, 50 ms. The guinea pig cochlea was peeled off and the integrity of hair cells was observed after immunofluorescence staining. Compared with the model group, the outer hair cells in TSG group were arranged orderly without loss, and the inner hair cells were basically arranged orderly. Pathological observation showed that TSG had certain hepatotoxicity, but the nephrotoxicity was not obvious. The cell experiment found that the half inhibitory concentration of APAP on HEI-OC1 auditory nerve cells was 800 µmol·L^-1. When 10 µmol·L^-1 TSG intervened in APAP induced cell injury, the apoptosis rate decreased to 16.0%, which was significantly lower than 25.5% in model(APAP) group. In APAP group, the expression level of Birc5 in HEI-OC1 cells decreased significantly, and TSG treatment could significantly increase the expression level of Birc5. CONCLUSION TSG 15 mg·kg^-1 can protect guinea pigs from hearing loss induce by 100 mg·kg^-1APAP for 14 d. The protective effect is related to TSG inhibiting APAP induces auditory nerve cell apoptosis and promoting the expression of apoptosis inhibitor Birc5.