| PartⅠCurcumin Attenuates Peroxynitrite-induced Neurotoxicity in Spiral Ganglion NeuronsObjectives:The present study was designed to investigate the neurotoxicity of peroxynitrite (ONOO-) on rat spiral ganglion neurons (SGN), as well as to explore the effect of curcumin on ONOO--induced damage in SGNs and the mechanisms underlying such effects.Methods:The primary cultured rat SGN were identified by immunostaining with anti-NSE antibody, then SGN were exposed to ONOO-with or without the pretreatment of curcumin, and cells in normal control group were not subjected to any treatment. Cell viability was measured by MTT assay. Apoptosis was determined by Ho.33342and propidium iodide (PI) double staining and flow cytometry (FCM). The cellular glutathione (GSH) content, superoxide dismutase (SOD) activity, and malonaldehyde (MDA) level were evaluated by spectrophotometer. The mRNA expressions of Apaf-1, Caspase-9, Caspase-3, Bcl-2, and Bax were examined by RT-PCR, while, the protein expressions of mitochondrial and cytosolic Cytochrome c, Caspase-9, Caspase-3, Bcl-2and Bax proteins were determined by western blot, respectively.Results:The cell viability was markedly reduced to59.57±3.02%of normal control group, whereas, the apoptotic rate increased significantly to50.27±3.26%(P<0.001) of normal control group after exposure of ONOO-(100μM) to SGN for24h. Examination of Ho.33342and PI labeled nuclei in damaged SGN revealed the occurrence of apoptotic cells as well as limited numbers of necrotic cells in response to ONOO-. Pretreatment of SGN with curcumin could protect the excitotoxicity elicited by ONOO" in a dose-and time-dependent manner and neuroprotection reached at the peak with the concentration of15μM curcumin pretreated for12h, as the cell viability was raised to97.64±2.04%and cell apoptotic rate was declined to7.31±0.99%(P<0.001) of control group respectively. Furthermore, Ho.33342and PI staining proved that pretreated with curcumin caused obvious reduction of apoptotic neurons and that the necrosis almost disappeared. Spectrophotometer showed that, after being treated with ONOO-, the activity of SOD and level of GSH in SGN were notably reduced to36.78±3.62%and45.15±7.03%of normal control group respectively, whereas, the MDA level was significantly increased to207.36±24.49%of control level. Pretreatment with curcumin improved the levels of SOD and GSH to86.19±3.13%and116.47±1.75%of control group respectively, and decreased the elevation of MDA to85.04±3.68%of control level (P<0.01). ONOO-induced Cytochrome c release from the mitochondria of SGN and subsequently activated Caspase-9, Caspase-3and cell apoptosis. On the other hand, the protein expression of Bcl-2was lowered while Bax expression was elevated in the process. Moreover, the mRNA expressions of Apaf-1, Caspase-9, Caspase-3and Bax were increased and that of Bcl-2was decreased significantly after exposure to100μM ONOO-for24h as evidenced by RT-PCR. On the other hand, pretreatment with curcumin abrogated Cytochrome c release, blocked activation of Caspase-3, and altered the expression of Bcl-2family triggered by ONOO-, as well as effectively eliminated the ONOO--induced changes in mRNA expressions of Apaf-1, Caspase-9, Caspase-3, Bcl-2and Bax respectively.Conclusions:To the best of our knowledge, this is the first study on the synergistic effects of ONOO-and curcumin on SGN. These findings from the present work prove that ONOO-could markedly reduce the cell viability of SGN, induce apoptosis and oxidative damage, and that the induction of apoptosis in SGN by ONOO" is related to the release of cytochrome c from mitochondria and subsequent activation of Caspase-3elevation. Conversely, curcumin abrogates Cytochrome c release or activation of Caspase3and alteres the expression of Bcl-2family protein, which, in turn, exerts protective action on ONOO’-induced in SGN. Therefore, curcumin can be potentially used as antioxidant for prevention or treatment of hearing loss due to the oxidative injury of SGN. Part IIIntranuclear Localization of Apoptosis-inducing Factor and Endonuclease G Involves in Peroxynitrite-triggered Apoptosis of Spiral Ganglion NeuronsObjectives:The present study was designed to determine whether or not the Caspase-independent apoptotic pathway participated in the cellular death of spiral ganglion neurons (SGN) after exposure to peroxynitrite (ONOO-), with particular attention given to the intranuclear translocation of mitochondrial apoptosis-inducing factor (AIF) and endonuclease G (Endo G) in this process.Methods:The rat SGN were isolated and primary cultured in vitro and were exposed to ONOO-with pretreatment of pan-Caspase inhibitor Z-VAD-FMK. Morphological changes of SGN were observed by acridine orange cytochemistry staining, and apoptosis was examined by flow cytometry. Cleaved-Caspase-3was measured directly in lysates by western blot to confirm whether the induced terminal Caspase activation was blocked by Z-VAD-FMK under the experimental conditions. The translocation of mitochondrial AIF and Endo G was detected by immunocytochemistry and western blot, the mRNA expressions of AIF and Endo G were examined by real-time PCR. The protein expressions of Bcl-2family in SGN exposed to ONOO-were determined by western blot.Results:Morphological features of apoptosis were observed in SGN exposed to ONOO"(100μM) with or without preincubation of Z-VAD-FMK by acridine orange staining. The results of flow cytometry analysis showed the apoptotic rate of ONOO"-Z-VAD-FMK group was24.25±6.35%of control level compared with the apoptotic rate of50.27±3.26%(P<0.01) in ONOO-group. Western blot showed that addition of Z-VAD-FMK was sufficient to block the generation of Cleaved-Caspase-3as the active fragment of Caspase-3was almost disappeared. The immunocytochemical analysis showed that AIF and Endo G labeling were marked in neuronal nuclei, while, the western blot demonstrated the intranuclear localization of AIF and Endo G in SGN treated with ONOO". Western blot analysis demonstrated that ONOO" increased the Bax expression while reduced Bcl-2expression, which was not prevented by pretreatment with Caspase-inhibitor. Real-time PCR showed that there was no detectable change of AIF and Endo G mRNA expressions among different groups.Conclusions:These data indicate that Caspase-blockage can inhibit, to a certain extent, but not fully prevent the occurrence of ONOO"-induced apoptosis in SGN, thereby revealing that the Caspase-independent pathway is, indeed, operated in SGN exposed to ONOO’. Then it is also suggested that the translocation of both AIF and Endo G to the nucleus is involved in this Caspase-independent apoptotic process. ONOO-regulates the expression of Bcl-2family proteins in SGN is not prevented by pretreatment with Z-VAD-FMK, indicating that mitochondrial Bcl-2and Bax are activated by ONOO-without any involvement of Caspase activation. |
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