α-硫辛酸对卡那霉素致毒小鼠耳蜗p-p38MAPK和p-JNK表达的影响

发布时间：2018-01-19 22:29

本文关键词： α-硫辛酸卡那霉素耳蜗 p38丝裂原活化蛋白激酶 c-Jun氨基末端激酶　出处：《辽宁医学院》2012年硕士论文　论文类型：学位论文

【摘要】：目的研究α-硫辛酸（alpha-lipoic acid，LA）对卡那霉素（kanamycin，KM）致毒小鼠耳蜗磷酸化p38丝裂原活化蛋白激酶（phosphorylated p38mitogen-activated protein kinase， p-p38MAPK）和磷酸化c-Jun氨基末端激酶（phosphorylated c-Jun N-terminal kinase，p-JNK）表达的影响，探讨LA对KM耳毒性损伤的防护作用及其机制。方法 56只健康BALB/c小鼠随机分成对照组、KM组、KM＋LA组和LA组，每组14只。各组动物均每天皮下注射2次，连续给药14d。应用免疫组织化学SABC法、显微图像分析技术以及蛋白质印迹检测观察小鼠耳蜗中p-p38MAPK和p-JNK的表达；同时结合听脑干反应（auditory brainstem response，ABR）测试，观察用药前后小鼠听力的变化。结果 1．连续用药14d后，，在各刺激频率下，KM组小鼠ABR阈移明显增大，并且与对照组比较差异显著（P＜0.01）；KM＋LA组小鼠ABR阈移虽然在用药后也有增大，但较KM组明显减小（P＜0.01）。 2．对照组耳蜗外毛细胞、螺旋神经节及血管纹中均可见p-p38MAPK和p-JNK表达，其阳性免疫反应产物呈棕黄色，分布于胞浆及胞核内。KM组、KM＋LA组和LA组小鼠耳蜗中p-p38MAPK和p-JNK阳性反应产物的分布与对照组大致相同，但KM组的阳性染色比对照组明显加深；而KM＋LA组的阳性染色则较KM组明显减弱。显微图像分析结果显示，KM组小鼠耳蜗p-p38MAPK和p-JNK表达较对照组明显增强（P＜0.01）；而KM＋LA组小鼠耳蜗p-p38MAPK和p-JNK的表达则明显弱于KM组（P＜0.01）。 3．蛋白质印迹检测结果显示，对照组小鼠耳蜗p-p38MAPK和p-JNK的电泳条带较弱，而KM组小鼠耳蜗p-p38MAPK和p-JNK的电泳条带均明显强于对照组，KM＋LA组小鼠耳蜗p-p38MAPK和p-JNK的电泳条带则较KM组明显减弱。半定量分析结果显示，KM组小鼠耳蜗p-p38MAPK和p-JNK的蛋白表达水平均较对照组明显增高（P＜0.01）；而KM＋LA组小鼠耳蜗p-p38MAPK/β-actin比值和p-JNK/β-actin比值则均较KM组明显减小，即p-p38MAPK和p-JNK的蛋白表达水平表达较KM组明显降低。结论 p38MAPK和JNK介导了KM对BALB/c小鼠的耳毒性损伤，LA可通过显著抑制KM所致p-p38MAPK和p-JNK的高表达，从而有效防护KM的耳毒性，这可能是LA发挥防护作用的机制之一。
[Abstract]:Purpose To study the effect of alpha-lipoic acid lac (伪 -lipoic acid) on kanamycin (kanamycin). KM- induced phosphorylation of p38 mitogen-activated protein kinase (p38) in the cochlea of mice. Phosphorylated p38mitogen-activated protein kinase. P-p38 MAPK) and phosphorylated c-Jun N-terminal kinase. To investigate the protective effect of LA on km ototoxicity and its mechanism. Method 56 healthy BALB/c mice were randomly divided into control group (km + LA) and LA group (14 rats in each group). The expression of p-p38 MAPK and p-JNK in mouse cochlea was observed by immunohistochemical SABC method, microimage analysis and Western blotting. At the same time, the auditory brainstem response (ABR) and auditory brainstem response (ABR) test were used to observe the hearing changes of mice before and after treatment. Results 1.After 14 days of continuous administration, the ABR threshold shift of mice in KM group increased significantly at different stimulation frequencies, and the difference was significant compared with that of control group (P < 0.01); The threshold shift of ABR in KM+LA group increased after administration, but decreased significantly compared with km group (P < 0.01). 2. The expression of p-p38 MAPK and p-JNK was found in the cochlear outer hair cells, spiral ganglion and stria vascularis in the control group. The positive immunoreactive products of p-p38 MAPK and p-JNK were brown and yellow. The distribution of p-p38 MAPK and p-JNK positive products in the cochlea of the cytoplasmic and cytoplasmic. Km + LA group and LA group was approximately the same as that in the control group. But the positive staining of km group was significantly deeper than that of control group. The positive staining in KM+LA group was significantly lower than that in km group. The expression of p-p38 MAPK and p-JNK in km group was significantly higher than that in control group (P < 0.01). The expression of p-p38 MAPK and p-JNK in KM+LA group was significantly weaker than that in km group (P < 0.01). 3. The results of Western blot analysis showed that the electrophoretic bands of p-p38 MAPK and p-JNK were weak in the cochlea of control mice. The electrophoretic bands of p-p38 MAPK and p-JNK in km group were significantly stronger than those in control group. The electrophoretic bands of p-p38 MAPK and p-JNK in cochlea of KM+LA group were significantly lower than those in km group. The expression of p-p38 MAPK and p-JNK protein in km group was significantly higher than that in control group (P < 0.01). However, the ratio of p-p38 MAPK / 尾 -actin and p-JNK/ 尾 -actin in cochlea of KM+LA group was significantly lower than that in km group. The protein expression of p-p38 MAPK and p-JNK was significantly lower than that of km group. Conclusion P38 MAPK and JNK mediated the ototoxic injury induced by km in BALB/c mice and the high expression of p-p38 MAPK and p-JNK was significantly inhibited by p38 MAPK and JNK. Thus, the ototoxicity of km can be effectively protected, which may be one of the protective mechanisms of LA.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R764.43;R965

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