摘要
目的探讨耳聋左慈丸对H_(2)O_(2)致小鼠耳蜗基底膜损伤模型miR-34a的作用及其机制。方法采用C57BL/6J小鼠耳蜗基底膜的离体培养,实验分为对照组、H_(2)O_(2)模型组(0.3 mmol·L^(-1))、耳聋左慈丸组(5 mg·L^(-1)、10 mg·L^(-1)和50 mg·L^(-1))。q-RTPCR检测miR-34a表达,免疫荧光法检测沉默信息调节因子1(silent information regulator 1,SIRT1)、微管相关轻链蛋白3(microtubule-associated protein 1 light chain 3,LC3)和选择性自噬接头蛋白(p62/sequestosome 1,SQSTM1)在耳蜗基底膜毛细胞的表达;TUNEL法检测基底膜毛细胞的凋亡情况。结果与对照组相比,模型组小鼠耳蜗基底膜miR-34a表达明显升高(P<0.01),耳聋左慈丸组miR-34a表达下降(P<0.01);模型组小鼠耳蜗基底膜毛细胞SIRT1蛋白表达与对照组相比明显降低(P<0.01),LC3-Ⅱ和p62蛋白表达明显增加(P<0.05,P<0.01),TUNEL阳性细胞数增多;与模型组比较,耳聋左慈丸组SIRT1表达升高(P<0.05),LC3-Ⅱ和p62蛋白表达降低(P<0.01,P<0.05),TUNEL阳性细胞数减少。结论耳聋左慈丸可能通过作用于miR-34a,调节SIRT1蛋白,进而影响耳蜗毛细胞的自噬和凋亡,达到抑制H_(2)O_(2)致毛细胞氧化损伤的作用。
Aim To explore the effects of Erlong Zuoci Wan on miR-34a in H_(2)O_(2) damaged cochlear organotypic cultures and its mechanisms.Methods Cochlear organotypic cultures were treated with 0.3 mmol·L^(-1) hydrogen peroxide(H_(2)O_(2))either alone or with different concentrations of ELZC(5,10,50 mg·L^(-1)).Real-time qPCR technique was used to test the mRNA expression of miR-34a.Fluorescence immunohistochemical technique was used to test the protein expression of SIRT1,LC3-Ⅱand p62 in hair cells.Terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL)method was used to assess the apoptosis.Results The expression of miR-34a in model group increased compared to that of control group(P<0.01),and ELZC reduced the elevated miR-34a expression levels(P<0.01).Compared with model group,ELZC treatment increased SIRT1 protein expression(P<0.05)and decreased LC3-Ⅱand p62 protein expression(P<0.05,P<0.01).TUNEL test showed that the apoptotic hair cells increased in model group compared to control group,and ELZC decreased the numbers of apoptotic hair cells compared to model group.Conclusions ELZC has protective effects against H_(2)O_(2) damaged cochlear hair cells through regulating autophagy and inhibiting apoptosis,which might be related to modulating the expression of miR-34a and SIRT1 protein.
作者
楚敏
阎希芮
龚永昌
刘晴
施建蓉
董杨
CHU Min;YAN Xi-rui;GONG Yong-chang;LIU Qing;SHI Jian-rong;DONG Yang(Experimental Teaching Center,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Basic Medical College,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)
出处
《中国药理学通报》
CAS
CSCD
北大核心
2021年第12期1757-1762,共6页
Chinese Pharmacological Bulletin
基金
国家自然科学基金面上项目(No 81774020)。
