| Objective:To investigate the effects of resveratrol(Res)pretreatment on focal cerebral ischemia-reperfusion(I/R)injury-induced mitochondrial damage and the underlying mechanisms.Methods:1.40 Adult male C57BL/6J mice were randomly divided into 5groups: Sham,I/R and I/R+Res(7.5,15,30 mg/kg).Transient focal cerebral ischemia was introduced into mice by right middle cerebral artery occlusion(MCAO)technique.After 1 h of MCAO,the suture was removed.At 24 h of reperfusion,ischemic degrees in each group were detected according to cerebral infarct volume and neurological score assay.Western blot was used to detect the expression levels of SIRT1 and indicated proteins involved in mitophagy(p62,LC3B-Ⅱ/Ⅰ and PHB2)and mitochondrial biogenesis(NRF1 and TFAM).2.80 Adult male C57BL/6J mice were randomly divided into 4groups: Sham,I/R,I/R+Res and I/R+Res+Mdivi-1.After 1 h of MCAO,the suture was removed.At 24 h of reperfusion,ischemic degrees in eachgroup were detected according to cerebral infarct volume and neurological score assay.Western blot was used to analyse the expression levels of indicated proteins involved in mitochondrial fission(Drp1),mitophagy(p62,LC3B-Ⅱ/Ⅰ and PHB2),mitochondrial biogenesis(NRF1 and TFAM)and the intracellular localization of cytochrome C(CytC).A microplate reader was employed to detect the cellular adenosine triphosphate(ATP)level.RT-PCR was used to analyze mitochondrial DNA(mtDNA)content.3.Murine hippocampal neuronal HT22 cells were randomly divided into 6 groups: Control and Res(1.25,2.5,5,10,20 μM)cultured under the normal conditions for 26 h.HT22 cells were randomly divided into 7groups: Control,oxygen and glucose deprivation-reperfusion(OGD/R)and OGD/R+Res(1.25,2.5,5,10,20 μM).After 2 h of oxygen and glucose deprivation,the cells were replaced into normal medium and normoxic incubator for 24 h.HT22 cells were randomly divided into 4 groups: Res and Res+Mdivi-1 cultured under the normal conditions for 26 h,OGD/R+Res and OGD/R+Res+Mdivi-1 under the OGD/R conditions.A microplate reader was employed to detect the cell viability and LDH leakage.4.HT22 cells were randomly divided into 4 groups: Control,OGD/R,OGD/R+Res and OGD/R+Res+Mdivi-1.Western blot was used to detect the expression levels of indicated proteins involved in mitochondrial fission(Drp1),mitophagy(p62,LC3B-Ⅱ/Ⅰ and PHB2),mitochondrial biogenesis(NRF1 and TFAM)and the distribution of CytC.Flow cytometry was employed to measure mitochondrial membrane potential(MMP).A microplate reader was used to detect the cellular ATP level.RT-PCR was performed to analyze mitochondrial DNA(mtDNA)content.Transmission electron microscopy(TEM)was used to investigate the alternation of the mitochondria.5.HT22 cells were randomly divided into 4 groups: Control,OGD/R,OGD/R+Res and OGD/R+Res+Mdivi-1.The translocation of PINK1 and Parkin between cytosol and mitochondria were evaluated by Western blot.Results:1.Compared with the I/R group,Res(30 mg/kg)obviously reduced infarct volume,improved neurological function,increased the expression of SIRT1,promoted mitophagy with LC3B-Ⅱ/Ⅰ down-regulated,and p62,PHB2 up-regulated and simultaneously promoted mitochondrial biogenesis with NRF1 and TFAM up-regulated.2.Compared with I/R+Res group,Res+Mdivi-1 treatment increased infarct volume and neurological deficit score,reversed the Res-induced distribution of Drp1 in cytosol and mitochondria,attenuated mitophagy and mitochondrial biogenesis.It influenced the effects of Res on mitochondrial function,including promoting CytC release,degenerating ATP.But,there was no significant difference between I/R+Res and Res+Mdivi-1 in mtDNA content.3.Compared with the control group,Res and Res+Mdivi-1 treatment had no effects on cell viability and LDH leakage under normal conditions.For the OGD/R experiment,10 μM Res increased cell viability and decreased LDH leakage.However,extra Mdivi-1 application reversed these effects.4.Compared with the OGD/R group,Res treatment promoted the distribution of Drp1 from cytosol to mitochondria,and increased mitophagy and mitochondrial biogenesis.But,extra Mdivi-1 application reversed these expressions.Meanwhile,Mdivi-1 blocked the mitochondrial protection of Res with MMP depolarization,CytC release and ATP degeneration.But,there was no significant difference between I/R+Res and Res+Mdivi-1 in mtDNA content.5.Compared with the OGD/R group,Res treatment promoted the translocation of PINK1 and Parkin from cytosol to mitochondria.However,extra application of Mdivi-1 completely reversed this effects.Conclusion:Res pretreatment obviously reduced the cerebral I/R injury and ODG/R injury,and ensured the functional and structural integrity of mitochondria.The mechanisms may ascribe to the regulation of mitochondrial quality control system and the activation of PINK1/Parkin mitophagy pathway. |
