Study On Function And Mechanism Of SIRT3 Regulates Neuronal Autophagy In Oxygen-glucose Deprivation-reperfusion Injury

Background:Ischemic stroke(IS)is a kind of disease of high incidence in our country.Epidemiological survey shows that the standardized prevalence,incidence and mortality of stroke in China in 2013 were 1114.8 / 100000,246.8 / 100000 and 114.8 / 100000.In recent years,the prevalence and mortality rate of stroke in Europe and the United States decreased year by year,while the incidence rate in our country increased by 8.7% annually,which was much higher than the overall annual incidence rate of stroke in the world.Stroke has become the largest cause of death in our nation nowadays.Among the newly diagnosed patients,ischemic stroke accounts for approximately 70% of the total diseases.The timely and efficient treatment of acute ischemic stroke is essential for Chinese residents,and its high incidence and fatality rate can cause a large number of disabled groups and bring a heavy burden to society and families.In recent years,although abundent research has been devoted to understanding the mechanisms of ischemic stroke,there is still no definite and efficient treatment.Studies have shown that,IS not only induce apoptosis and necrosis,but also leads to autophagy.However,the role of autophagy in neurological dysfunction after IS is still controversial.Additionally,the level of a class of mitochondrial sirtuins increased after neuronal injury,which may be involved in energy metabolism,apoptosis and survival signaling pathways.Previous studies have shown that SIRT3,as an important mitochondrial sirtuin,plays a protective role in senile cardiac hypertrophy,cancer and aging-related diseases.However,how SIRT3 functions in ischemic stroke and the associated mechanisms have not yet been clearly confirmed.Purpose:The aim of this study was to investigate:(1)To study the effects of different duration of oxygen deprivation and reperfusion on neuronal structure and function in OGD/R model;(2)To observe the spatial and temporal changes of autophagy related molecules(LC3、P62、PINK1、Parkin、Cleaved caspas-3、SIRT3);(3)Observe the relationship between neuronal autophagy and SIRT3 protein;(4)To investigate the effects of inhibite autophagy and increase autophagy on the cell activity and apoptosis of neurons after OGD/R injury;(5)To investigate the relationship between SIRT3,autophagy and injury after oxygen-glucose deprivation injury,and to find potential targets for the treatment of cerebral ischemic injury.Methods:1.Through culture of primary mouse cortical neurons in vitro,hypoxia environment was simulated by anaerobic incubator to establish OGD/R,an in vitro injury model of cerebral ischemia.2.Morphological changes of cortical neurons in mice were observed under bright field with microscope.The Cell Counting Kit-8(CCK-8)and lactate dehydrogenase(LDH)release assay were used to evaluate neuronal cell viability and injury;3.The protein expression of autophagy,mitophagy,apoptosis-related molecules and SIRT3 in OGD / R were detected by Western blot and immunofluorescence.4.Autophagy agonist RAPA and autophagy inhibitor 3-MA were used to regulate autophagy after OGD/R treatment and to observe the effect of autophagy on neurons;5.The expression of SIRT3 was upregulated by lentiviral transfection,and the model of OGD / R injury was established.Morphological changes were observed after injury.Cell viability and LDH release were measured to evaluate the neuronal damage.Terminal,terminal dUTP nick end labeling(TUNEL)was used to detect neuronal apoptosis.6.The levels of autophagy,mitophagy,apoptosis-related molecules and other related proteins were detected after SIRT3 overexpression during OGD/R by Western blot and immunofluorescence.7.Through overexpression of SIRT3 and application of autophagy agonist-RAPA and autophagy inhibitor 3-MA,after OGD/R treatment to observe the effect of autophagy on neurons.Results:The first part of the study found that:(1)The mouse cortical neurons are sensitive to ischemia and hypoxia,OGD / R can significantly change neuronal morphology,and as the OGD injury time prolonged,LDH release increased,the cell activity decreased,the differences between groups were significant(P<0.01).(2)Western blotting and immunofluorescence results showed that LC3-II expression was highest at OGD4 h and P62 increased at OGD2 h,4h started to decline,compared with normal control group,indicating autophagy were significantly higher(P <0.05).Parkin protein expression decreased overall after OGD/R injury(P <0.05).The expression of Cleaved caspase-3 increased significantly at the beginning of injury,and the platform stage appeared after OGD4 h,but the expression was still higher(P<0.01).the expression of LC3-Ⅱ,PINK1 and SIRT3 molecules were upregulated after OGD / R treatment and were upregulated in simply OGD treatment(3)The period of OGD 4h was chose for the time point for following research.Through autophagy inhibitor(3-MA)and autophagy agonist(RAPA),CCK-8 and Western blot results showed that,compared with the OGD4 h group,the inhibition of autophagy can decrease cell viability and incease apoptosis.Moreover,upregulation of autophagy can improve cell viability decreased expression of apoptosis to a certain extent(P <0.05)..This part of the study shows that: OGD / R treatment can activate neuronal autophagy;the time point of OGD 4h is the peak of autophagy;SIRT3 may modulate autophagy and reduce apoptosis.Within 4h,modest upregulation of autophagy can reduce apoptosis to a certain extent.The second part of the study found that:(1)Overexpression of SIRT3 can significantly improve the morphology and neuronal activity of primary cortical neurons after OGD/R and reduce the rate of apoptosis;(2)Western blot results showed that overexpression of SIRT3 after OGD/R treatment,compared with OGD4 h group,the expression of PINK1 increased and the expression of Cleaved caspase-3 decreased(P<0.05).Furthermore,the result of further immunofluorescence assay confirmed that SIRT3 expression was upregulated and the autophagy was increased and apoptosis was reduced.(3)CCK-8 detection and Western blot detection results,inhibiting autophagy decreased cell viability,increased injury and reduced apoptosis in neurons transfected lentivirus sirt3;enhancing autophagy increased cell viability,decreased apoptosis in neurons transfected lentivirus sirt3.This part of the study shows that: Overexpression of SIRT3 can significantly improve neuronal survival after OGD/R treatment,reduce apoptosis,exerting a protective role;inhibition of autophagy,SIRT3 can counteract the role of neurons and increase autophagy,which can increase antagonism of SIRT3 on OGD/R Injury.Conclusion:This study confirmed that OGD/R treatment can activate neuronal autophagy and the time point of OGD 4h is the peak of autophagy.In the early stage,moderately upregulating autophagy can reduce apoptosis and play a neuroprotective role.Overexpression of SIRT3 mouse cortical neurons resists OGD/R injury and enhances cell viability.SIRT3 may inhibit neuronal apoptosis by regulating autophagy and participate in antagonizing neuronal ischemic injury and exerting neuroprotective effects.