摘要
本研究通过大鼠卡那霉素耳毒性动物模型的建立,于在体和离体培养条件下,采用ABR检测、耳蜗铺片、离体器官培养以及免疫组织化学染色方法,从功能和形态学两方面分别观察了卡那霉素对耳蜗毛细胞及螺旋神经节神经元细胞的毒性作用;应用免疫荧光及Western blot等实验技术检测了卡那霉素干预的耳蜗组织中caspase 12及calpain的表达情况并定量测定其表达规律;同时,观察了应用calpain抑制剂对受损耳蜗组织毒性及caspase 12活性的影响;从分子和蛋白水平阐述了它们在卡那霉素诱导耳毒性中的作用机制;从而得出结博,caspase 12介导的内质网应激是卡那霉素导致药物性耳聋的机制之一,calpain抑制剂对卡那霉素耳毒性具有保护作用。应用calpain抑制剂有望成为治疗卡那霉素耳毒性的有效手段。
kanamycin is a highly effective antibiotics but with significant ototoxic side effects. Now the exact mechanism of kanamycin induced ototoxicity is not known.
Studies have shown that drug induced ototoxicity of cochlear hair cells and spiral neuron injury is closely related to apoptosis, but the the ways and molecular mechanism has yet to be clearly spelled out. Endoplasmic reticulum stress (ERS) activated apoptosis pathway was discovered only in recent years, a new apoptosis pathway, with a variety of diseases are closely linked. Caspase 12 is the only present in the cytoplasm of the surface of the endoplasmic reticulum Caspase members, is considered the key elements of to ERS.
experimental techniques such as organ culture in vitro, immunohistochemistry and Western blot will be used this study. ion of kanamycin intervention caspase 12 and calpain expression will be detected in cochlear treating with kanamycin; we will observe calpain inhibitor on the toxicity and caspase 12 activity; we attempt to show the mechanism of kanamycin induced hearing loss and provide new experimental data to the treatment of drug induced hearing loss.
Materials and Methods:
Healthy adult Wistar rats were given kanamycin 250 mg / kg, 500 mg / kg every day intramuscularly at different time line ABR testing. Animals were killed after testing. the cochleas were derived using cochlear technology and HE staining of paraffin section in order to inspect hair cell loss and neuronal death. Immunofluorescence technique was used on caspase 12 monoclonal antibody and calpain immunostaining to detect protein expression. We measured caspase 12 and the relative expression levels of calpain by Western Blot. The postnatal day 3 Wistar rats were used for in vitro treated with different concentrations of kanamycin, morphological change of hair cells and neurons were observed. Detection of intracellular calcium concentration ,calpain expression and the effect of calpain inhibitor were detected.
Results:
ABR test: following that kanamycin accumulation in the inner ear, ABR thresholds increased. ABR thresholds are statistically significant before and after kanamycin treatment. Cochlear basement membrane stretched: OHC damage from the bottom back to top back to gradually reduce. In the large dose group, OHC loss was significantly higher than other groups, in the bottom back up to 40%; shows a serious deformation of Corti devices, SGN reduction in the number of cells. monoclonal antibody showed that in kanamycin treatment group, caspase 12 and calpain were expressed. Calpain was detected in normal group and low dose group weakly, and high expression in high dose group. In calpain inhibitor group the number of hair cells and SGNs survival increased significantly. using calpain inhibitors can reduce the activity of caspase 12.
Conclusion:
(1) Kanamycin can induce hearing impairment, hearing threshold shifts before and after administration of a dose dependent effect of the change.
(2) Kanamycin induce hair cell loss in rats. OHC damage from the bottom to top gradually.
(3) In low dose group, SGN cells reduced slightly, but decreased significantly in high dose group.
(4) There is no expression of caspase 12 in Normal tissue.In kanamycin treated group, caspase 12 expressed mainly in the inner and outer hair cells and spiral neurons. although hair cells did not lost, We also detected the expression of caspase 12 protein In low dose group.
(5) the expression pattern of calpain and caspase 12 was different and only in high dose group calpain expressed.
(6) In kanamycin inducd cochlea in vitro, hair cells and neurons injury with time dose dependent effect
(7) Kanamycin increased intracellular calcium and calpain II expression in cochlear neurons, caspase 12, caspase 9, caspase 3 activity was enhanced.
(8) Calpain inhibitor reduced caspase 12, caspase 9, caspase 3 activity, and protected hair cells survival. the results suggest that caspase 12 activated ERS apoptosis pathway is involved in the mechanism of kanamycin ototoxicity
引文
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