| Object:To explore the expression of SIRT1 in rat cochlea.Methods:Hibateral cochleas were dissected from new-born rats, normal adult rats and mimetic aging rats with hypodermic 5% D-galactose injection for 8 weeks. Five objects were chosen randomly from each group. The exploration of the expression of SIRT1 was carried out by cochlear frozen section followed by immunofluorescent staining.Results:The abundance of SIRT1-specific-immunofluoresce was detected in a variety of tissues of rat cochlea, especially stria vascularis, spiral ganglion and Corti organ. The intensity of the immunofluoresce among the groups didn't make great difference.Conclusion:SIRT1 was expressed extensively in cochlea, especially the stria vascularis, spiral ganglion and the organ of Corti. There was no noticeable difference of the expression of SIRT1 among the groups by the immunofluorescent staining detection. Object:To explore the mimetic aging effect of D-galactose and establish the premature-aging cell model with notable mtDNA4834bp deletion.Methods:Cultured marginal cells (MCs) and spiral ganglion cells (SGCs) were treated with 0,2,4,6,8,10,12,14,16 g/L D-galactose respectively for 48h followed by MTT cell viability detection in order to find out the appropriate concentration of D-galactose for cell survival. The culture were then incubated with that very concentration of D-galactose for periods of 24h, 72h,120h,168h and 216h followed by mitochondrial common deletion assay using the Taqman RTqPCRs. To estimate the premature aging effect of D-galactose, we performed SA-B-gal staining for morphological assay, AnnexinⅤ-PI double stained or TUNEL for detection of apoptosis, JC-1 staining for measurement of mitochondrial membrane potential (MMP).Results:The concentration dependent devaluation of cell viability emerged in 14g/L and lOg/L D-galactose groups in MCs and SGCs respectively, so 12g/L and 8g/L D-galactose are recognized as the suitable concentration for MCs and SGCs survival and induction. Statistical significancant increase in mtDNA4834bp deletion frequency, SA-β-gal activity, apoptosis, and depolarization were found in MCs treated with 12g/L D-galactose. The same situation was found in SGCs treated with 8 g/L D-galactose for 120h.Conclusion:D-galactose could induce mtDNA4834bp deletion mutation and premature senescence effectively in vitro. The experimental premature senescence cell model with notable mtDNA4834bp deletion could be established successfully using cultured cells exposed to D-galactose. Object:To investigate the effect of SIRT1 in the formation of mtDNA 4834bp deletion, premature senescence and mitochondrial function, to explore the relationship of SIRT1 with NRF-1, mtTFA, PGC-1α, COXⅢ, UCP-2, and the effect of SIRT1 on free radical scavenger SOD activity, to research the mechanism of SIRT1 in the formation of mtDNA 4834bp deletion and senescence.Methods:When the explants grew to a confluent monolayer, cells were re-suspended and seeded in 6-well plates and then divided into four groups as follows:(1)SIR:in which 60μM sirtinol was added to the MEM/D-galactose (12g/L) complete culture medium (DMSO final concentration was 0.125‰); (2) RSV:in which 20μM resveratrol was added to the MEM/D-galactose complete culture medium (DMSO final concentration was 0.125‰); (3)DMSO:DMSO is a specific dissolvent of SIR and RES used as the vehicle, equivalent dosage of the dissolvent of SIR and RSV was added to the MEM/ D-galactose complete culture medium (DMSO final concentration was 0.125‰); (4) control (CONT):in which MCs were incubated in the MEM/ D-galactose complete culture medium. All the groups were incubated at 37℃, 5% C02 for further 168h. Thereafter SA-13-gal staining, AnnexinⅤ-PI double stained apoptosis assay, determination of MMP by JC-1 were carried out repectively. Taqman real-time quantitative PCR and real-time quantitative PCR were used to detect the relative mtDNA 4834bp deletion frequency, the relative mtDNA copy number and the mRNA level of SIRT1, NRF-1, mtTFA, PGC-la, COXIII and UCP-2. Western blot was performed to detect the expression of SIRT1, Ac-H3, UCP-2, PGC-1a and Ac-PGC-1a. WST-1 method was used for MnSOD and Cu/ZnSOD activity assay.Results:(1) Resveratrol could decrease the frequency of mtDNA 4834bp deletion mutation, the SA-β-gal positivity, apoptosis, depolarization of MMP induced by D-galactose, while sirtinol went to the contrary. (2) Resveratrol increased mitochondrial DNA content,while sirtinol repressed mitichondrial biogenesis. (3) The mRNA level of SIRT1, NRF-1, mtTFA, PGC-la and COXIII increased with resveratrol treatment. Decreases in NRF-1, mtTFA, PGC-la and COXIII mRNA were found in sirtinol group. The mRNA level of SIRT1 didn't change with sirtinol treatment. And none statistical significance was found in UCP-2 level among the four groups. DMSO group didn't show great disparity with control group. (4) Resveratrol contributed to the expression of SIRT1, PGC-1a, repressed the expression of Ac-H3, Ac-PGC-la and UCP-2, while the PGC-1a protein level was downregulated and Ac-H3, Ac-PGC-1a and UCP-2 protein level were upregulated with sirtinol treatment. There was no significant change in DMSO group compared with control group. (5) The activity of MnSOD increased in RSV, whereas decreased in SIR compared to DMSO and CONT. The activity of Cu/ZnSOD didn't make difference among groups.Conclusion:SIRT1 modulates the mitochondrial defect and premature senescence through the regulation of mitochondrial biogenesis, the primary line of defense against oxidant stress and the inner mitochondrial membrane anion carrier system.
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