耳聋左慈丸上调SIRT1减轻老年性聋小鼠耳蜗螺旋神经节细胞损伤的作用机制研究  

Mechanisms of Erlong Zuoci Wan alleviating damage to cochlear spiral ganglion neurons by upregulation of SIRT1 in mice with presbycusis

作　　者：王艺蓉 阎希芮 欧阳朝明 李景全 闫黎 张新蕾 刘晴[2] 施建蓉[1] 楚敏[1] 董杨[1] WANG Yirong;YAN Xirui;OUYANG Zhaoming;LI Jingquan;YAN Li;ZHANG Xinlei;LIU Qing;SHI Jianrong;CHU Min;DONG Yang(School of Integrative Medicine Sciences,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;School of Traditional Chinese Medicine,Shanghai University of Traditional Chinese Medicine,Shanghai201203,China)

机构地区：[1]上海中医药大学中西医结合学院,上海201203 [2]上海中医药大学中医学院,上海201203

出　　处：《上海中医药杂志》2023年第8期81-88,共8页Shanghai Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(81774020);上海市卫健委上海市进一步加快中医药事业发展三年行动计划项目〔ZY(2021-2023)-0208〕;上海中医药大学预算内项目(2021LK030)。

摘　　要：目的探讨耳聋左慈丸对老年性聋小鼠耳蜗螺旋神经节细胞损伤的保护作用及其机制。方法①将27只C57BL/6J小鼠随机分为3组,即对照组、老年性聋组和耳聋左慈丸组,每组9只。耳聋左慈丸组小鼠从3月龄开始给予含有中药的饲料喂养至9月龄,老年性聋组同时喂养普通饲料;对照组采用2月龄小鼠。听性脑干反应(ABR)法检测小鼠听阈值的变化;苏木精-伊红(HE)染色法观察小鼠耳蜗螺旋神经节细胞形态学变化;免疫荧光染色法观察小鼠耳蜗螺旋神经节细胞微管相关轻链蛋白3(LC3)、选择性自噬接头蛋白(p62)和沉默信息调节因子1(SIRT1)蛋白的表达;转移酶介导的三磷酸脱氧鸟苷-生物素刻痕末端标记(TUNEL)法检测小鼠耳蜗螺旋神经节细胞的凋亡情况。②体外实验采用C57BL/6J小鼠耳蜗基底膜离体培养模型,分为空白对照组、过氧化氢(H2O2)模型组(0.3 mmol·L^(-1))和耳聋左慈丸低、中、高剂量组(5μg·mL^(-1)、10μg·mL^(-1)和50μg·mL^(-1))。神经丝蛋白200(NF200)免疫荧光染色法观察耳蜗基底膜螺旋神经节细胞形态变化;Western blot法检测SIRT1、LC3-Ⅱ、p62、切割的半胱天蛋白酶-3(cleaved Caspase-3)、半胱天蛋白酶-3(Caspase-3)的蛋白表达。结果①与对照组比较,老年性聋组小鼠(9月龄)听阈值显著升高(P<0.05),耳蜗底回螺旋神经节细胞数量显著下降(P<0.05),SIRT1蛋白表达明显下降(P<0.05),自噬相关蛋白LC3-Ⅱ和p62表达升高(P<0.05),TUNEL阳性细胞数表达增多(P<0.05)。耳聋左慈丸可明显降低老年性聋小鼠的听阈值(P<0.05),显著增加耳蜗底回螺旋神经节细胞的数量(P<0.05)。与老年性聋组比较,耳聋左慈丸组SIRT1蛋白表达显著增加(P<0.05),自噬相关蛋白LC3-Ⅱ和p62表达降低(P<0.05),TUNEL阳性细胞数显著减少(P<0.05)。②体外实验发现,与空白对照组干预措施比较,H2O2可致螺旋神经节细胞出现核碎裂或胞体皱缩,听神经纤维紊乱,SIRT1�Objective To study the protective effects of Erlong Zuoci Wan(ELZC)on cochlear spiral ganglion neurons(SGNs)in mice with presbycusis and explore its mechanisms.Methods①Twenty-seven C57BL/6J mice were randomly divided into three groups:control group,ARHL group and ELZC group,with nine mice in each group.The 2-month-old C57BL/6J mice were taken as control,and the 3-month-old C57BL/6J mice were randomly divided into two groups that were fed diets containing Erlong Zuoci Wan(ELZC group)or standard diets(presbycusis group)for 6 months.Auditory brainstem response(ABR)was employed to test changes in hearing threshold.HE staining was used to evaluate the morphologic changes in mouse cochlear spiral ganglion neurons.The expression levels of LC3,p62 and SIRT1 in SGNs were detected by immunofluorescence.TUNEL assay was used to detect the apoptosis of SGNs.②In the ex vivo study,cochlear basement membrane in C57BL/6J mice was used as an ex vivo culture model,which were divided into 5 groups:blank control group,H2O2 model group(0.3 mmol L^(-1))and ELZC groups with low,middle and high concentrations of ELZC(5μg·mL^(-1),10μg·mL^(-1),50μg·mL^(-1)).The morphological changes in SGNs were assessed by NF200 immunohistochemistry.Western blot was employed to analyze the protein expressions of SIRT1,LC3-Ⅱ,p62,cleaved Caspase-3,and Caspase-3.Results①The mice(9 months old)in presbycusis group had significantly higher hearing threshold(P<0.05),significantly fewer SGNs in cochlea(P<0.05),significantly lower expression of SIRT1 protein(P<0.05),higher expressions of autophagy related proteins LC3-Ⅱand p62(P<0.05),and more TUNEL positive cells(P<0.05)than mice in control group.ELZC could significantly lower hearing threshold(P<0.05)and significantly improve SGNs survival(P<0.05)in mice with age-related hearing loss.The mice in ELZC group showed significantly higher SIRT1 protein expression(P<0.05),lower LC3-Ⅱand p62 expressions(P<0.05),and significantly fewer TUNEL-positive cells(P<0.05)than those in presbycusis group.②In t

关 键 词：耳聋 经典名方 作用机制 自噬 凋亡 模型小鼠 中药研究 

分 类 号：R285.5[医药卫生—中药学]