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Effect of Sirtuin3 on Hypoxic Preconditioning with Its Gene Knocked Down

LI Chao-ying1,2,HU Jun3,ZHAO Jia4,WANG Ying1,TAO Yu-qian1,LI Ling1(1.Department of Neurology,First Affiliated Hospital,Sun Yat-Sen University,Guangzhou 510080,China;2.The Central Hospital of Jiangmen,Jiangmen 529030,China;3.Peiking University Shenzhen Hospital,Shenzhen 518036,China;4.Department of Neurology,Second Affiliated Hospital,Sun Yat-Sen University,Guangzhou 510120,China)  
【 Objective】 To investigate the effect of Sirtuin3(SIRT3) on hypoxic preconditioning(Hyp) with its gene knocked down.【Methods】 Empty vectors(pGCSIL-GFP) were used to select the suitable MOI.pGCSIL-PUR-SIRT3-vectors were used in formal experiments.Cells transfected with empty vectors or SIRT3-RNAi-LV were divided into control,hypoxic preconditioning + Oxygen-glucose deprivation(Hyp+OGD) and OGD respectively.MTT assay was used for testing the cells viability.The ATP level was evaluated using ATP Fluorometric Assay Kit.The expression of SIRT3,Peroxisome proliferator-activated receptor-γ coactivator-1α(PGC-1α) and Manganese superoxide dismutase(MnSOD) were assessed at the protein level by Western blot.【Results】 After OGD,MTT values decreased more in SIRT3-/-compared with Vector(0.430 ± 0.009 vs 0.562 ± 0.140,P 0.01),but were increased in Hyp + OGD respectively(P 0.05),resulting in no significant difference between SIRT3-/-and Vector in Hyp+OGD(P = 0.311 0.05).After OGD,The ATP levels decreased more in SIRT3-/-compared with Vector(0.334 ± 0.006 vs 0.453 ± 0.015,P0.01),but were increased in Hyp+OGD respectively(P 0.05),resulting in no significant difference between SIRT3-/-and Vector in Hyp+OGD(P = 0.866 0.05).The expression of PGC-1α and MnSOD were declined significantly in the three groups of SIRT3-/-compared with that in Vector respectively(P 0.05),but were both increased in Hyp+OGD and OGD compared with control(P 0.05),and were increased more in Hyp+OGD compared with OGD(P 0.05).【Conclusion】 The expression of PGC-1α and MnSOD were decreased with SIRT3 gene knocked down and SIRT3 was one of the ways that hypoxic preconditioning or hypoxia up regulated the expression of MnSOD through PGC-1α pathway.
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