The Role Of NADPH Oxidase-mediated Oxidative Stress In Adiponectin's Protection Against Cerebral Ischemia-reperfusion Injury And Its Underlying Mechanism

Stroke severely jeopardizes the health of the residents in China.It has high morbidity and high mortality.Due to its narrow treatment time window,it has brought great difficulties to treatment.Acute ischemic stroke is the highest incidence among various subtypes of stroke,and reperfusion of blood flow as soon as possible is the most important method for the treatment of acute ischemic stroke.With the advancement of thrombolysis and interventional therapy,the therapeutic effect of acute ischemic stroke has been greatly improved,but due to other factors such as narrow time window and large side effects,the treatment effect is still not satisfactory.One of the causes is ischemia-reperfusion injury.Restoration of brain tissue perfusion is a target for the treatment of acute ischemic stroke,but ischemia-reperfusion injury increases neuronal damage.A variety of mechanisms are involved in the pathophysiology of ischemia-reperfusion injury,including oxidative stress,inflammation,blood-brain barrier disruption,mitochondria-mediated mechanisms,and leukocyte infiltration.Oxidative stress is due to the supply of oxygen after reperfusion,which produces excessive free radicals.The amount of these free radicals exceeds the ability of the body's antioxidant enzyme system to scavenge,leading to the damage of the cells.NOX,or NADPH oxidase,is the major family of enzymes that produce ROS.Adiponectin is expressed and secreted by adipocytes.Initially,scientists found that adiponectin can enhance insulin sensitivity and play an important role in regulating systemic metabolism.Subsequent studies have suggested that adiponectin has a protective effect on ischemic stroke,but its specific mechanism remains to be elucidated.In the current study,the middle cerebral artery occlusion model was used to induce cerebral ischemia and the suture was removed for reperfusion.The subject was male C57/BL6 mice,and the protective effect of APN-P on brain I/R injury in mice was studied.To explore the role of NOX-mediated oxidative stress in APN-P's neuroprotection.This study will provide new evidence for the neuroprotective mechanism of APN-P and provide a theoretical basis for its clinical application.The study is divided into the following three parts:1.Expression level changes of oxidative stress-related molecules in cerebral ischemia-reperfusion injury in mice2.Protective effect of APN-P on cerebral ischemia-reperfusion injury in mice3.Mechanism of NOX-mediated oxidative stress in APN-P's neuroprotectionPart 1 Expression level changes of oxidative stress-related molecules in cerebral ischemia-reperfusion injury in miceMethodsThe MCAO model was established in Male C57BL/6 mice and were reperfused after ischemia.Mice were randomly divided into six groups:Sham group,I/R-6h group,I/R-12h group,I/R-24h group,I/R-48h group,I/R-72h group.The neurological scores of each group were evaluated first,and the ischemic penumbra tissue was extracted after reperfusion.The expression levels of gp91^phox,p47^phox,4-HNE and 8-OHdG were detected by Western blot.Results1.Compared with the Sham group,the neurological scores of each I/R group decreased significantly,but there was no significant difference between the I/R groups.2.After I/R,the expression levels of oxidative stress-related proteins(gp91^phox,p47^phox,4-HNE and 8-OHdG)increased,peaked at 24 h after reperfusion,and then decreased.ConclusionsAfter I/R,neurological damage occurs,and the expression of oxidative stress-related protein increases,peaks at 24 h after reperfusion,and then decreases.Part 2 APN-P attenuates I/R injuryMethodsThe MCAO model was established in Male C57BL/6 mice and were reperfused for24h after ischemia.Mice were randomly divided into six groups:Sham group,I/R group,Vehicle+I/R group,APN-P 2.5 mg/kg+I/R group,APN-P 5 mg/kg+I/R group.APN-P10 mg/kg+I/R group.The neurological scores of each group of mice were evaluated first,then the infarct volume was measured.Nissl staining,TUNEL staining and ROS staining were performed,and the ultrastructural changes of neurons were observed using electron microscopy.Results1.Compared with the Vehicle+I/R group,the neurological score and infarct volume of the APN-P 5 mg/kg+I/R group and the APN-P 10 mg/kg+I/R group were significantly decreased.We chose APN-P 5 mg/kg as the dose in the further study.2.The results of Nissl staining suggested that APN-P attenuated neuronal damage after cerebral I/R injury.3.The results of TUNEL staining suggested that APN-P attenuated neuronal apoptosis after cerebral I/R injury.4.Electron microscopy results suggested that APN-P attenuated neuronal ultrastructural changes after cerebral I/R injury.5.The results of ROS staining suggested that APN-P attenuated ROS production after cerebral I/R injury.ConclusionsAPN-P attenuates neurological damage and infarct volume after I/R injury,and reduces neuronal apoptosis,ultrastructural damage,and ROS production.Part 3 APN-P confers neuroprotection via attenuation of NOX-mediated oxidative stressMethodsThe MCAO model was established in Male C57BL/6 mice and were reperfused for24h after ischemia.The mice were randomly divided into six experimental groups:Sham group,I/R group,Vehicle+I/R group,APN-P+I/R group,Apo+I/R group,APN-P+Apo+I/R group.The expression levels of gp91^phox,p47^phox,4-HNE and 8-OHdG and the expression levels of apoptosis-related proteins were detected by Western blot.The apoptosis of neurons was detected by TUNEL staining.Results1.The expression levels of gp91^phox,p47^phox,4-HNE,8-OHdG were significantly decreased after treatment with APN-P or Apo?P<0.05 compared with Vehicle+I/R group?.At the same time,the protein levels of gp91^phox,p47^phox,and 4-HNE in the APN-P+I/R group were significantly lower than those in the Apo+I/R group?P<0.05?.There was no significant difference in protein levels of 8-OHdG between the APN-P+I/R and Apo+I/R groups.2.The expression levels of Bax and active caspase-3 were significantly down-regulated in the APN-P+I/R,Apo+I/R and APN-P+Apo+I/R groups?P<0.05compared with the Vehicle+I/R group?.In addition,the Bax and active caspase-3 protein levels were lower in the APN-P+I/R group?P<0.05 compared with the Apo+I/R group?.In contrast,the level of Bcl-2 was higher in the APN-P+I/R group?P<0.05compared with the Apo+I/R group?.The results of TUNEL staining showed that the proportion of neuronal apoptosis was significantly down-regulated in the APN-P+I/R,Apo+I/R and APN-P+Apo+I/R groups?P<0.05 compared with the Vehicle+I/R group?.Additionally,the level of apoptosis was lower in the APN-P+I/R group?P<0.05compared with the Apo+I/R group?.ConclusionsAPN-P attenuates NOX-mediated oxidative stress after ischemia-reperfusion,which is an important mechanism of its neuroprotective effect.