【作者】 董杨； 郭春荣； 丁越； 张永； 宋海燕； 彭垠婷； 张彤； 施建蓉；

【Author】 Yang Dong;Chun-rong Guo;Yue Ding;Yong Zhang;Hai-yan Song;Yin-ting Peng;Tong Zhang;Jian-rong Shi;Experimental Teaching Center,Shanghai University of Traditional Chinese Medicine;

【机构】 上海中医药大学教学实验中心；

【摘要】 目的:观察耳聋左慈汤对C57BL/6J小鼠老年性聋的作用及其机制。方法:采用听性脑干诱发电位检测小鼠听功能,HE染色观察耳蜗形态学改变,TUNEL法检测细胞凋亡。采用透射电镜观察耳蜗线粒体损伤情况。实时定量PCR检测p53和Bak基因表达,荧光免疫组化观察p53和Bak蛋白表达。结果:老年性聋组小鼠的听阈值明显高于青年对照组,耳聋左慈汤可降低老年性聋小鼠阈值的升高(P<0.001),提示耳聋左慈汤可以抑制C57BL/6J小鼠听功能的损失。HE染色显示,耳聋左慈汤可以抑制老年性聋小鼠耳蜗螺旋神经节的缺失和形态改变。TUNEL染色显示,老年性聋组小鼠耳蜗螺旋神经节凋亡细胞显著增加,耳聋左慈汤组有所降低。透射电镜观察显示,老年性聋组线粒体数量减少,且有明显的微丝断裂、空泡形成等形态学改变,耳聋左慈汤组有明显改善。实时定量PCR显示,老年性聋小鼠p53和Bak基因表达增高(P<0.05),耳聋左慈汤可降低老年性聋小鼠p53和Bak基因表达的增高(P<0.01,P<0.05)。荧光免疫组化显示,耳聋左慈汤可降低老年性聋小鼠p53和Bak蛋白表达的增高。结论:耳聋左慈汤可能通过抑制耳聋螺旋神经节细胞p53/Bak介导的线粒体凋亡途径防治老年性聋的发生。更多还原

【Abstract】 Objective To observe the effects of ELZCD on the ARHL in mice and explore the mechanisms.Methods Auditory brainstem response(ABR) was used to assess the hearing function of mice.The morphologic changes were observed by hematoxylin eosin(HE) staining.Apoptosis was tested by terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL) method.Mitochondrial damage was detected by transmission electron microscopy(TEM).Quantitative RT-PCR(qRT-PCR) was used to observe the mRNA expression of p53 and Bak.Fluorescence immunohistochemical technique was used to test the protein expression of p53 and Bak.Results The hearing threshold of ARHL group was higher than that of control group(PO.001) and ELZCD decreased the rise of hearing threshold levels of ARHL mice(P<0.001),which suggested ELZCD inhibited the hearing loss of ARHL mice.HE staining showed that ELZCD decreased the spiral ganglion(SG) cell damage and loss in ARHL.TUNEL test showed that the apoptotic SG cells increased in ARHL group compared to control group and decreased in ELZCD group compared to ARHL group.TEM observation showed that mitochondrial damage was obvious in SG cells of ARHL group and ELZCD inhibited the mitochondrial damage.qRT-PCR results showed that the mRNA expression of p53 and Bak in ARHL group increased compared to that of control group(P<0.05),and ELZCD reduced the elevated mRNA expression levels of p53 and Bak(P<0.01,P<0.05).In addition,ELZCD inhibited the increased protein expression(green fluorescence) of p53 and Bak.Conclusion ELZCD prevented ARHL in C57BL/6J mice by inhibiting p53/Bak mediated mitochondrial apoptosis of SG cells.更多还原