Protective Effect Of Mitochondrial Biogenesis Regulated By Butylphthalide Activating AMPK In Cerebral Ischemia/Reperfusion Injury

Objective: Ischemic stroke is one of the common chronic diseases.Besides age,the occurrence of ischemic stroke is also related to genetic factors,unhealthy lifestyle factors,other chronic diseases(hypertension,diabetes,atrial fibrillation,malignant tumor,etc.)and environmental factors.Research shows that there is a correlation between temperature,air pressure,humidity,air pollution in meteorological factors and the incidence of ischemic stroke.Short-term temperature change or extreme weather can increase the risk of stroke and death.Poor nutritional diet will also increase the risk of ischemic stroke,such as high salt and high-fat eating habits,heavy drinking and vitamin B12 deficiency.Due to the increasing aging of China’s population,poor control of disease-related risk factors and environmental pollution,the harm and economic burden of ischemic stroke in China will further increase.Therefore,the research on improving the therapeutic effect of ischemic stroke is particularly important.Dl-3-n-butylphthalide(NBP),abbreviated as butylphthalide,originated from celery seed extract.It is an ischemic stroke therapeutic drug independently developed in China and has multi-target neuroprotective effects.Although the protective effect of butylphthalide on mitochondria in the central nervous system has been confirmed,its specific mechanism in ischemia/reperfusion induced mitochondrial injury of nerve cells has not been fully clarified.The study of endogenous protective mechanism is helpful to explore the prevention and treatment strategies of ischemic stroke.Mitochondria are the power source of cells.They not only provide energy in the form of ATP,but also have many other important cellular functions.Mitochondrial dysfunction is considered to be one of the signs of ischemia/reperfusion injury leading to neuronal death.Maintaining mitochondrial function is very important to promote neuronal survival.Studies have found that the activation of adenosine 5’-monophosphate-activated protein kinase(AMPK)in cells can stimulate peroxisome proliferator activated receptor γ Peroxisome promoter activated receptor γ coactivator-1(PGC-1 α),regulate mitochondrial biogenesis,supplement damaged mitochondria,increase the number of mitochondria and respiratory function,and up regulate antioxidant defense system.We speculate that butylphthalide may regulate mitochondrial production by activating AMPK signal to protect against cerebral I/R injury.To verify the above conjecture: first,We used C57BL/6J male mice to establish a model of transient middle cerebral artery occlusion(MCAO)to study the protective effect of butylphthalide on neuronal mitochondrial injury induced by I/R injury in mice.Then,we used glucose oxygen deprivation/reoxygenation(OGD/R)model of primary cortical neurons to study the protective effect of butylphthalide on mitochondrial production of OGD/R damaged neurons;finally,compound C(CC),a targeted inhibitor of AMPK,was used to further verify the role of AMPK signal in the protection of mitochondrial production disorder of OGD/R neurons by butylphthalide.Methods:1.To study the protective effect of butylphthalide on neuronal mitochondrial injury induced by I/R injury in mice: screened adult male C57BL/6J mice were randomly divided into 4 groups,as follows: sham operation group,sham operation +NBP group,MCAO group and MCAO + NBP group,with 18 mice in each group respectively.MCAO model mice received 45 min cerebral ischemia and 14 d reperfusion.The behavioral indexes such as neurological function score,brain water content,cerebral infarction volume,apoptosis rate,cellular ROS,mitochondrial ROS,mitochondrial membrane potential,ATP production and mitochondrial formation were monitored.2.To study the protective effect of butylphthalide on mitochondrial formation of neurons injured by OGD/R: primary cortical neurons were randomly divided into five groups: control group(culture medium),100 μM NBP group,OGD-R group,OGD-R+ 50 μM NBP group and OGD-R + 100 μM NBP group,the treatment time of NBP was 24 h.Firstly,the activity of neurons treated with OGD/R for 0 h,1 h,2 h,3 h and 4h was detected by CCK8 reagent,so as to determine that the experimental condition was glucose oxygen deprivation for 3 h.Then,the morphology of neurons was observed and the indexes such as apoptosis rate,oxidative stress,mitochondrial dysfunction and mitochondrial formation were evaluated.3.To study the role of AMPK signal in the protection of OGD/R neurons against mitochondrial production disorder by butylphthalide: primary cortical neurons were randomly divided into five groups: control group(culture medium),10 μM CC group,OGD-R group,OGD-R + 100 μM NBP group,OGD-R + 100 μM NBP + 10 μM CC group.The glucose oxygen deprivation time was 3h,the reoxygenation time was 24 h,the 100 μM NBP treatment time was 24 h,and the CC pretreatment time was 1h.Firstly,the cell viability of neurons was detected,the morphology of neurons was observed,and then the apoptosis rate,oxidative stress and mitochondrial dysfunction of neurons were evaluated.Finally,mitochondrial production,phosphorylation level of AMPK and PGC-1α and other indicators were detected.Results: 1.NBP reduced the neurological damage caused by cerebral ischemia/reperfusion injury in mice,activated AMPK and promoted mitochondrial production.The neurological function score of MCAO group was lower than that of sham operation group.In the MCAO group,the motor ability decreased in the rotarod performance test,grip strength and weight-bearing test.In the open field experiment,the total moving distance,average speed,the moving distance and residence time in the central area decreased.In the gait analysis experiment,maximum contact area,swing speed,BOS,print length,and stride length decreased,meanwhile duty cycle,initial dual stance and terminal dual stance increased.The apoptosis rate increased,the levels of cellular ROS and mitochondrial ROS increased,the mitochondrial membrane potential and ATP production decreased,and the gene and protein expression levels of PGC-1α,NRF1 and TFAM decreased,and the copy number of mitochondrial DNA decreased in MCAO group.After NBP intervention,compared with MCAO group,MCAO+NBP group had higher neurological function score,higher motor ability,higher total moving distance and average speed in open field experiment.Maximum contact area,swing speed,print length,BOS and stride length increased,meanwhile duty cycle,initial dual stance and terminal dual stance decreased in the gait analysis experiment.The brain edema alleviated,cerebral infarction volume lightened and degree of degeneration decreased,the apoptosis rate decreased,the levels of cellular ROS and mitochondrial ROS decreased,the levels of mitochondrial membrane potential and ATP increased,the gene and protein expression levels of PGC-1α,NRF1 and TFAM increased,and the copy number of mitochondrial DNA also increased in the MCAO+NBP group.Butylphthalide alone had no effect on the above indexes of mice brain cells.2.NBP alleviated the cell damage caused by OGD/R in primary cortical neurons,activated AMPK and promoted mitochondrial production,and the protective effect was dose-dependent.Compared with the control group,in the OGD-R group,the cell viability decreased,the cell morphology and reticular structure were damaged,the apoptosis rate increased,the ratio of Bax/Bcl-2 and the expression of Cyt-C in cytoplasm increased,the levels of cellular ROS and mitochondrial ROS increased,the level of ATP production and mitochondrial membrane potential decreased,and gene and protein expression levels of PGC-1α,NRF1 and TFAM decreased,the copy number of mitochondrial DNA decreased,and the phosphorylation level of PGC-1α decreased in the OGD-R group;Compared with OGD-R group,butylphthalide treatment reduced the decline of cell viability caused by OGD/R,improved cell morphology and reticular structure,reduced the rate of apoptosis,decreased the ratio of Bax/Bcl-2 and the expression of Cyt-C in cytoplasm,reduced the levels of cellular ROS and mitochondrial ROS,increased the level of ATP production and mitochondrial membrane potential,and increased gene and protein expression levels of PGC-1α,NRF1 and TFAM,increased the copy number of mitochondrial DNA,significantly increased phosphorylation level of AMPK and PGC-1α,and this protective effect was dosedependent,100μM NBP has stronger protective effect.Butylphthalide alone had no effect on the above indexes of primary cortical neurons.3.NBP activated AMPK,promoted the mitochondrial production of primary cortical neurons and reduced the cell damage caused by OGD / R,after adding AMPK targeted inhibitor,this protective effect was reversed.Compared with OGD-R group,in the OGD-R+100μM NBP group,the cell viability of primary cortical neurons increased,the damage of cell morphology and reticular structure decreased,the apoptosis rate decreased,the ratio of Bax/Bcl-2 and the expression of Cyt-C in cytoplasm decreased,the levels of cellular ROS and mitochondrial ROS decreased,the level of mitochondrial ATP production and mitochondrial membrane potential increased,and gene and protein expression levels of PGC-1α,NRF1 and TFAM increased,the copy number of mitochondrial DNA increased,phosphorylation level of AMPK and PGC-1α also increased;After adding 10 μM CC,compared with OGDR+100 μM NBP group,the cell viability of primary cortical neurons decreased,the number of cells decreased,the destruction of reticular structure aggravated,the apoptosis rate increased,the ratio of Bax/Bcl-2 protein expression increased and the content of Cyt-C in cytoplasm raised,the levels of cellular ROS and mitochondrial ROS increased,ATP production decreased and membrane potential in mitochondria declined,meanwhile the protein and gene expression of PGC-1α,NRF1 and TFAM decreased,the copy number of mitochondrial DNA decreased,phosphorylation level of AMPK and PGC-1α also decreased significantly.Pretreatment with 10 μM CC alone had no effect on primary cortical neurons.Conclusion: Butylphthalide can reduce nerve injury and apoptosis induced by ischemia/reperfusion injury and improve neuronal reticular structure.By activating AMPK and up regulating the mitochondrial production of neurons after ischemia/reperfusion injury,butylphthalide can reduce cellular oxidative stress response and mitochondrial dysfunction,and has a protective effect on ischemia/reperfusion injury.