艾塞那肽减轻卡那霉素加呋塞米诱导的小鼠螺旋神经元损伤

Exendin-4 Alleviates Spiral Neuron Injury Induced by Kanamycin and Furosemide in Mice

目的探讨高血糖素样肽-1受体(glucagon like peptide-1 receptor,GLP-1R)激动剂艾塞那肽(exendin-4)对卡那霉素加呋塞米诱导的小鼠螺旋神经元(spiral ganglion neurons,SGNs)损伤的作用。方法原代培养的小鼠SGNs进行免疫荧光染色、小鼠耳蜗切片免疫组化染色,验证GLP-1R在SGNs上的表达。将30只C57BL/6J小鼠分成3组,每组10只,卡那霉素加呋塞米组一次性腹腔注射硫酸卡那霉素1 g/kg及呋塞米0.4 g/kg;艾塞那肽组卡那霉素和呋塞米注射前3 d开始腹腔注射艾塞那肽400μg/kg,每天1次,直到卡那霉素和呋塞米给药后第7 d;对照组腹腔注射等量生理盐水。分组处理后,使用HE染色观察SGN损伤情况并计数骨螺旋板里SGNs数量;采用Western blot技术检测各组耳蜗组织GLP-1R及凋亡相关蛋白Bax、Bcl-2、Cleaved-caspase-3的表达。结果免疫荧光及免疫组化染色结果显示,GLP-1R表达于SGNs。HE染色显示,与对照组比较,卡那霉素加呋塞米组SGNs数量下降,艾塞那肽组SGNs数量有所恢复(P<0.05);Western blot结果显示,卡那霉素加呋塞米组GLP-1R蛋白表达下降,促凋亡蛋白Cleaved-caspase-3、Bax蛋白水平升高,抗凋亡蛋白Bcl-2表达水平下降;艾塞那肽组GLP-1R蛋白表达较卡那霉素加呋塞米组升高,Cleaved-caspase-3、Bax表达降低,Bcl-2表达升高(P<0.05)。结论GLP-1R激动剂艾塞那肽可能通过减轻细胞凋亡,拮抗卡那霉素加呋塞米诱导的小鼠SGNs损伤。

Objective To investigate the effect of exendin-4,a glucagon-like peptide-1 receptor agonist,on spiral ganglion neurons(SGNs)injury induced by kanamycin and furosemide in mice.Methods The expression of GLP-1R on SGNs was verified by immunofluorescence staining of primary culture mouse SGNs and immunohistochemical staining of mouse cochlea sections.Thirty C57BL/6J mice were divided into three groups with 10 mice in each group.Kanamycin and furosemide group was given kanamycin sulfate 1 g/kg and furosemide 0.4 g/kg intraperitoneally.Exendin-4 group was injected with exendin-4400μg/kg intraperitoneally 3 days before kanamycin and furosemide injection,and continued injection until 7 days of kanamycin and furosemide were given.The control group was given intraperitoneal injection of the same amount of normal saline.After group treatment,HE staining was used to count the number of SGNs in the spiral plates.The expressions of GLP-1R and apoptosis-related proteins Bax,Bcl-2,Cleaved caspase-3 were detected by Western blot.Results Immunofluorescence and immunohistochemistry showed that GLP-1R was expressed in SGNs.HE staining showed that the SGNs density decreased in kanamycin and furosemide treatment group,but recovered in exendin-4 treatment group(P<0.05).Western blot results showed that the expression of GLP-1R,cleaved-caspase-3 and Bax were increased in the kanamycin and furosemide group,Bcl-2 expression was decreased,and these results were partially reversed in the exendin-4 treatment group,compared with the kanamycin group.(P<0.05).Conclusion GLP-1R agonist exendin-4 may antagonize the SGNs injury induced by kanamycin and furosemide in mice by alleviating apoptosis.