The Neuroprotective Effect Of MitoQ On Traumatic Brain Injury In Mice And Its Related Mechanism

Part 1 The role and mechanism of Mito Q in attenuating oxidative stress following traumatic brain injuryOxidative stress is one of the physiological reactions of secondary brain injury.The primary objective of this study was to investigate the role and mechanism of Mito Q in oxidative stress after TBI.Objective: To investigate the neuroprotection afforded by Mito Q in a mouse model of TBI,and the involvement of the Nrf2–ARE signaling pathway in the putative neuroprotective mechanism.METHODS: Male ICR mice were randomly allocated into four groups: Sham group,TBI group,TBI + vehicle group,and TBI + Mito Q group(n=24/group).A mouse model of TBI was made according to the modified Feeney method.After 24 h,brain samples were harvested for analysis.The water content of the brain was determined by dry-wet weight method.Contents of superoxide dismutase(SOD),glutathione peroxidase(GPX)and malondialdehyde(MDA)were detected using ELISA.The expression of Nrf2 in neurons was detected by immunofluorescence analysis.The expression of Nrf2 in plasma/nuclear protein and the expression of its downstream proteins HO-1 and Nqo1 were measured by Western blot;The expression of HO-1 and Nqo1 m RNA were determined by Real-time PCR analysis.Results: Compared with the Sham group,the NSS,the brain water content,and oxidative stress in the TBI group were remarkably increased in the TBI group,respectively.No obvious difference was observed between the TBI group and the vehicle-treated group;these effects of the brain injury could be reversed after Mito Q administration.Mito Q remarkably accelerated translocation of Nrf2 from cytoplasm to nucleus after TBI,upregulated the expression of Nrf2 downstream proteins,including HO-1 and Nqo1,and prevented the decline of antioxidant enzyme activities,including SOD and GPx.Conclusion: Mito Q attenuated the oxidative stress by enhancing the expression and activation of antioxidant enzymes and alleviate brain injury in mice with TBI,possibly by activating the Nrf2–ARE pathway.Part II The mechanism of Mito Q in alleviating neuronal apoptosis following traumatic brain injuryApoptosis plays a vital role in the process of secondary brain injury.Previous studies have shown that Mito Q exerts neuroprotective effects in the mouse model of TBI.However,no data are available about the effect of Mito Q on apoptosis following TBI and the underlying molecular mechanism of this effect of Mito Q remains unknown.Previous Studies have shown that the mitochondrial apoptotic pathway is activated after TBI and exerts neuroprotective effects by inhibiting mitochondrial apoptosis.The primary objective of this study was conducted to investigate the effect and mechanism of Mito Q on neuronal apoptosis through the mitochondrial apoptosis pathway.Objective: The prognosis of traumatic brain injury is closely associated with Mitochondrial Damage.Our study was to investigate the anti-apoptotic effect of Mito Q and its potential mechanism in the mouse model of TBI.Methods: Male ICR mice were randomly allocated into the following groups: sham,TBI,TBI + vehicle and TBI + Mito Q.The mice models of TBI were established by Feeney's method.Mice were sacrificed at 24 h after TBI,the ipsilateral cortex pericontusion was harvested for determining brain edema,measuring the expressions of cleaved caspase-3 in total protein,cytochrome c and Bax in the mitochondria and cytosol of the brain tissue by western blot,detecting the expressions of caspase-3 in brain tissue by immunohistochemistry,and evaluating the survival of the neurons and neuronal apoptosis in the cortical area by Nissl and TUNEL staining,respectively.Results: The brain water content,the content of MDA,cleaved caspase-3 expression and neuronal apoptosis index were markedly higher while the neuron survival remarkably lower in the TBI + vehicle group than in the sham group;The levels of mitochondrial and cytosolic Bax protein were increased and decreased,respectively,after TBI,compared with the sham group,whereas mitochondrial and cytosolic cytochrome c levels were decreased and increased,respectively,after TBI,relative to the sham group.These effects were reversed by Mito Q treatment.Conclusion: Mito Q exerts the neuroprotective effects in the mouse model of TBI,possibly by inhibiting the neuronal apoptosis through the mitochondrial pathway.Part III The role of Mito Q on autophagy and its regulation mechanism after traumatic brain injuryExperiment 1 Mito Q induced autophagy after traumatic brain injury Objective: This study was aimed to detect the modulation manner of MitoQ on autophagy following TBI.Methods: Male ICR mice were randomly allocated into the following groups: sham,TBI,TBI + vehicle and TBI + Mito Q.The mice models of TBI were established by Feeney's method.Mice were sacrificed at 24 h after TBI,the ipsilateral cortex pericontusion was harvested.The mice neurobehavior was assessed at 1 day,3 days and 7 days after TBI using a 10-point neurological severity score(NSS).We used western blot analysis to detect the expression of autophagy-related proteins LC3 and Beclin-1.Immunofluorescence analysis for LC3 and Neu N was performed to determine the expression of LC3 in neurons.Results: The expression of LC3-II and Beclin-1 upregulated in the TBI group when compared with the Sham group.The NSS scores of the TBI and TBI +vehicle groups were higher than the scores of the Sham group.Autophagosomes in neurons increase following TBI.However,the Mito Q treated group exhibited reduced neurological deficits,and further enhanced the LC3-II and Beclin-1 expression when compared with the TBI group.Conclusion: These results demonstrated that administration of Mito Q stimulated the expression of autophagy and improved neurobehavioral performance after traumatic brain injury in miceExperiment 2 Mito Q activated autophagy through modulation of PI3K/Akt/m TOR pathway in mice after traumatic brain injuryObjective: The aim of this study is to investigate the mechanism of Mito Q induced autophagy after TBI in mice?Methods: ICR mice were randomly allocated into the following groups: Sham,TBI,TBI + Mito Q and TBI + Mito Q+LY294002.The mice models of TBI were established by Feeney's method.Mice were sacrificed at 24 h after TBI,the ipsilateral cortex pericontusion was harvested.The expression of PI3K/Akt/m TOR pathway related proteins Akt,m TOR,p-Akt,and p-m TOR and the autophagy-related proteins LC3 and Beclin-1,were detected by the Western blot analysis.Results: The expression of p-Akt and p-m TOR were increased after TBI,compared with the Sham group.Administration with Mito Q significantly reduced the expression of p-Akt and p-m TOR,when compared to the TBI group.The expression of p-Akt and p-m TOR in the TBI + Mito Q + LY294002 group(PI3K kinase inhibitor)were further decreased,while the expression of LC3-II and Beclin-1 were further upregulated compare with the Mito Q treated group.Conclusion: MitoQ administration could inhibit the activation of the PI3K/Akt/m TOR pathway and Mito Q might induce autophagy through modulation of PI3K/Akt/m TOR pathway.