Role Of Mitochondrial Fusion And Fission Regulated By AMPK In The Protective Effects Of P2X7 Receptor Inhibition Against Cerebral I/R Injury In Mice

Ischemic stroke is caused by insufficient blood supply to the brain.Currently,the main treatment method is to restore blood reperfusion in time.However,during reperfusion,the damage in ischemic area of brain tissue will be further aggravated,and this pathophysiological change is called cerebral ischemia/reperfusion(I/R)injury.Due to its complicated mechanisms,research on the pathophysiology of cerebral I/R injury and how to reduce this injury have become the focuses.Studies showed that inhibition of P2X7 receptor can reduce cerebral I/R injury,but its mechanisms still needs to be further explored.Mitochondria are dynamic organelles that provide energy to cells.In normal condition,mitochondria are in a dynamic equilibrium of fusion and fission.However,the dynamic balance of mitochondria would be destroyed and tended to fission when I/R injury occurred.Studies have shown that AMP-activated protein kinase(AMPK)can regulate mitochondrial fusion and fission.And in alcoholic liver disease,inhibition of P2X7 receptor can enhance AMPK activity.However,whether it has the similar effects in mice with cerebral I/R injury still unclear.Therefore,this study used in vivo and in vitro experiments to investigate the role of mitochondrial fusion and fission regulated by AMPK in the protective effects of inhibiting P2X7 receptor against cerebral I/R injury in mice.Objective: To investigate the role of mitochondrial fusion and fission regulated by AMPK in the protective effects of inhibiting P2X7 receptor against cerebral I/R injury in mice.Methods: 1.In vivo: adult male ICR mice were randomly assigned to sham group,cerebral ischemia/reperfusion(I/R)group,cerebral ischemia/reperfusion + BBG(Bright Blue G,the inhibitor of P2X7 receptor,I/R + BBG)group,cerebral ischemia/reperfusion + BBG + Dorsomorphin(the inhibitor of AMPK,I/R + BBG + Dorsomorphin)group.The model of cerebral I/R injury was prepared by middle cerebral artery occlusion(MCAO),ischemia for 1 h and reperfusion for 24 h.Longa score was used to evaluate the neurobehavioral deficits of mice;2,3,5-triphenyltetrazolium chloride(TTC)staining was used to detect the cerebral infarction volume of mice;transmission electron microscopy(TEM)was used to assess the mitochondrial morphology of brain tissue;western blot was used to determine the expression of p-AMPK,p-Drp1 and Mfn2.2.In vitro: primary neurons were obtained from cerebral cortex of ICR fetal mice.The model of hypoxia/reoxygenation(H/R)injury was introduced and neurons were divided into control group,H/R group,H/R + BBG group,H/R + BBG Dorsomorphin group.The survival rate of neurons were determined by Calcein-Am;The reactive oxide species(ROS)kit was used to detect the ROS content;The fusion and fission of mitochondria in each group were observed by the laser confocal microscope;western blot was used to determine the expression of p-AMPK,p-Drp1 and Mfn2.Results: 1.The Longa scores results showed that compared with sham group,the neurobehavioral scores of mice in I/R group increased significantly.Compared with I/R group,the neurobehavioral scores of mice decreased significantly after the mice pretreated with BBG.However,compared with I/R + BBG group,the neurobehavioral scores of mice increased significantly after the inhibitor of AMPK was used.2.TTC results showed that the percentage of cerebral infarction volume of mice in I/R group increased significantly.Compared with I/R group,BBG decreased the percentage of cerebral infarction volume in mice.But compared with I/R + BBG group,the inhibitor of AMPK attenuated the protective effects of BBG on reducing the percentage of cerebral infarction volume in mice.3.The results of TEM showed that compared with sham group,the mitochondrial perimeter,roundness and aspect ratio decreased significantly in I/R group.Compared with I/R group,the perimeter,roundness and aspect ratio of mitochondrial increased markedly after the mice pretreated with BBG.However,compared with I/R + BBG group,the mitochondrial perimeter,roundness and aspect ratio decreased significantly after the inhibitor of AMPK was used.4.The results of western blot showed that compared with sham group,the expression of p-AMPK,Mfn2 decreased while the expression of p-Drp1 increased in I/R group.Compared with I/R group,the expression of p-AMPK?Mfn2 increased while the expression of p-Drp1 decreased after the mice pretreated with BBG.Compared with I/R + BBG group,the expression of p-AMPK?Mfn2 decreased while the expression of p-Drp1 increased after the inhibitor of AMPK was used.5.The results of Calcein-AM showed that compared with control group,the mortality rate of neurons increased significantly in H/R group;compared with H/R group,BBG decreased the mortality rate of neurons;but compared with H/R + BBG group,the mortality rate of neurons increased markedly after the inhibitor of AMPK was used.6.The results of ROS showed that compared with control group,the level of ROS in H/R group increased markedly;compared with H/R group,the level of ROS decreased significantly after pretreated with BBG;but compared with H/R + BBG group,the inhibitor of AMPK alleviated the protective of BBG on reducing the level of ROS in neurons.7.The observation results of laser confocal microscope showed that compared with control group,the mitochondrial fission of neurons increased while mitochondrial fusion decreased;compared with H/R group,the mitochondrial fission of neurons decreased while mitochondrial fusion increased;but compared with H/R + BBG group,the mitochondrial fission of neurons increased while the mitochondrial fusion decreased after the inhibitor of AMPK was used.8.The western blot results showed that compared with control group,the expression of p-AMPK,Mfn2 decreased while the expression of p-Drp1 increased in H/R group.Compared with H/R group,the expression of p-AMPK,Mfn2 increased while the expression of p-Drp1 decreased after the neurons treated with BBG.Compared with H/R + BBG group,the expression of p-AMPK,Mfn2 decreased while the expression of p-Drp1 increased after the inhibitor of AMPK signaling pathway was used.Conclusions: 1.Inhibition of P2X7 receptor can alleviate cerebral I/R injury of mice by inhibiting mitochondrial fission and promoting mitochondrial fusion.2.Inhibition of P2X7 receptor can alleviate cerebral I/R injury of mice by increasing the expression of p-AMPK.3.Inhibition of P2X7 receptor can alleviate cerebral I/R injury of mice by regulating AMPK to inhibit mitochondrial fission and promote mitochondrial fusion.