Expression Of Survivin And Caspase-3 During The Development Process And In The Protection Of Fosfomycin Sodium Against Cisplatin-induced Ototoxicity In Rat Cochlea

Since it was discovered that mechanosensory hair cells in birds can regenerate, men become focus on cytothesis in mammal inner ear again. It was reported that the regenerative process of hair cells actually repeated its embryonic development, and some regulators during the development play an important role in regenerative process. In many respects, rats are closer to human than mice, but few study in rats before. In 2003, cloning of rats was achieved, which would make rats as first selected rodents animal again. To explore mammal hair cell regeneration, the development characters in the rat cochlea and the effects of its regulators were studied in this project.As a member of the inhibitor of apoptosis protein (IAP) family, survivin can protect cells from apoptosis by binding specifical with the terminal effector cell death proteases, caspase-3 and -7 and inhibiting the processing of these zymogens into active caspases. Present during fetal development, survivin is undetectable in terminally differentiated adult tissues. Survivin is also involved in the regulation of cell division, specially expressed in G2/M stage.Caspas-3 is a key protein in the activating pathways of caspase cascade led to apoptosis, and it may be one of ultimate executors in apoptosis event. Caspase-3 is activated by the proteolytic processing of procaspase-3 into the mature form of the enzyme. Once activated, caspase-3 in turn directly activates specific DNases and cleaves other cellular proteins, resulting in some morphological hallmarks of apoptosis.The morphological development process of cochlea was observed in Wistar rat age between embryo day 13 to postnatal day 14 in this experiment. Survivin and caspase-3 were respectively detested at protein and mRNA levels.The results: Three phases could be partitioned in cochlear developing period. Embryo day 13 and14 are the epithelial proliferous period. Embryo day 15 and16 are the period of epithelial differentiation. From embryo day 17 to postnatal day 14 are the mature period. The organ of Corti was completely differentiated during the mature period that almost occupied 2/3 of total cochlear developing period. A stereotypic pattern of cochlear sensory cell development begins in the base of the cochlea and progresses towards the apex. In addition, the hair cell developmentprecedes the degradation of epithelial ridge, and the small epithelial ridge was missing earlier than the obsolescence of big epithelial ridge. These results indicate that the differentiation of organ of Corti is orderly developed from base to apex. The figure of organ of Corti is almost developed well in antenatal period. The organ of Corti continues growing until full-grown at 14 days after born. During the development, cell proliferation is the precondition, sensory cell differentiation is the key to decision, and the mature plays an important role to shape the complicated cochlear structure.The results: The expression of survivin and caspase-3 had the similar location and trend. In the first stage, survivin is poorly and generally expressed, and caspase-3 is hardly present. In the second stage, both of them is increased and limited in bottom cells of cochlear duct, but the former is stronger than the latter. In the third stage, caspase-3 is stronger than survivin in the sensory cell, but weaklier than the latter in supporting cells during differentiate. However, in adult cochlea, caspase-3 is not present and survivin is only expressed in organ of corti. This shows that both of them participate the cochlear morphological development. With their temporal and spatial expression, both of them have a crest-time, and survivin is prior to caspase-3. It is suggested that cell differentiation leads to appearance of cell phenotype, then expression of survivin is decreased. This will activate caspase-3 and finally results to apoptosis. Hence, it is inferred that the interaction between survivin and caspas-3 regulates apoptosis to take part in the cochlear morphological development. On the other hand, this experiment shows that the temporal and spatial character of survivin seems to be related to cell differentiation.Strit control of cellular proliferation is required to shape the complex structures of the developing embryo. This experiment result indicates that apoptosis during the development reduces the superfluous cells through cell proliferation to fill the need for fine structure formation. Thus, apoptosis is a supplement regulation of cell proliferation.Because survivin is expressed in adult cells of organ of corti, and cisplatin (CDDP) -induced ototoxicity results in hair cell apoptosis, we go on following experiment to explore the role of survivin in ototoxicity.CDDP widely used as a highly effective chemotherapeutic agent to treat many malignancies, including head-neck and urogenital system tumors, but it is limited by its significant and does dependent ototoxic and renal toxicity side effects. It is generally accepted that CDDP causes cochleotoxicity, and apoptosis is an important mechanism of cochlear hair cell loss following exposure to an ototoxic level of CDDP. It was reported that fosfomycin ameliorates the dose-limiting ototoxicity and nephrotoxicity of CDDP chemotherapy. Nonetheless, themechanisms that CDDP causes ototoxicity and fosfomycin can ameliorate remain poorly understood.To explore the role of survivin and caspase-3 in the cochlea of CDDP ototoxicity and fosfomycin amelioration, this study observed the characters of Wistar rat after peritoneal injections of CDDP, fosfomycin or the combination of both. Auditory brain-stem response (ABR) was tested and losing hair cells was counted by cochlear stretched preparation. At the same time, survivin and caspase-3 in the cochlea were examined by means of immunohistochemistry at protein level and by means of in-situ hybridization and RT-PCR at mRNA level to primarily approach their role.The results: It was observed that it has not effective on the physiological index, hearing and cochlear morphology in fosfomycin-treared group. The toxicity reaction was found in the CDDP-treated group. ABR threshold was markedly elevated. Same results also can be detected by morphology. The degree of damaged cell including the outer hair cells and spiral ganglion was gradually seriously from the apex towards the base of the cochlea and from the third to the first hair cell in the same turn. The fosfomycin in protective group markedly inhibits the elevation of ABR thresholds and simultaneous outer hair cell loss that results from CDDP administration. This result show that fosfomycin can protect against the ototoxicity of CDDP to some extend.In this study, the expression of caspase-3 and survivin have the similar change trend. Both hardly changed between the control and fosfomycin-treated groups, which shows that alone fosfomycin haven't effect on them. The expressions of them were obviously reinforced in two CDDP-treated groups, especially in the outer hair cells. But both decreased in the protective group. These results indicate that CDDP initiates apoptosis signal to activate caspase-3, at the same time, the expression of survivin is enhanced. So survivin can inhibit apoptosis by suppressing caspase-3 , While caspase-3 exceeds the limit of survivin, it will lead to apoptosis. The interaction between survivin and caspase-3 will decide cell fate.Organisma multicellularis with the complex structures requires more multiple and finer control to coordinate the function. The cell as the minimal unit in regulation network accepts various of informations and make the reaction. Individual cell death would also be decided by factors such as the availability of energy and the metabolic condition of the cell. It was reported that CDDP may cause mitochondrial release of cytochrome c and caspase-3 activation, then this activation leads to an irreversible commitment to apoptotic cell death to eliminate (apoptosis) of damaged sensory cells from adult inner ears after CDDP-treated that generates oxidative stress. Although apoptosis and necrosis are conceptuallydistinct forms of cell death with very different morphological and biochemical characteristics, these two types of demise may occur simultaneously in tissues or cell cultures exposed to the same CDDP insult. Thus, some CDDP-induced cells might die as a result of an unfinished apoptotic program that lacks some morphological and biochemical characteristics attributed to apoptosis. Otherwise, because CDDP is a nonspecific drug and reacts not only with DNA but also with proteins, it is possible that CDDP damage to proteins could induce apoptosis at the execution phase level. These may be the results of the interaction between survivin and caspase-3.On the other hand, this study displays that fosfomycin protects against the ototoxicity of CDDP by inhibiting the activation of caspase-3 and decreasing the diference between surviving and caspase-3. To study the role of caspase-3 inhibition and/or elevating survivin expression in cochleas against the CDDP-ototoxicity will contribute to understanding the molecular basis of CDDP-mediated apoptosis and could lead to interventional therapies to treat and rescue oxidative stress-damaged inner ear sensory cells from apoptosis. This will provide new strategies for improved therapeutic benefits.