Preliminary Study On The Impact Of Apocynin Pretreatment To Cerebral Infarction And Brain Tissue Damage

Objective In addition to the nerve injury caused by ischemia itself，acute cerebralinfarction also includes the further injury of the brain tissue caused by ROS comingfrom the excessive oxidative stress after the damage．Among them，oxidative stressbasically works by NADPH oxidase（NOX）．NOX composed of several subunits NOXenzyme complex．It plays an important part in maintaining normal functioning ofoxidase and the process of producing the oxygen ROS．Related researches find that theNOX activity increases under the effect of various factors．So excess reactive oxygenspecies involve in the death of neurons after cerebral ischemia through the signaltransduction pathways in or between the cells．The recent researches have found thatoxidative stress and ROS were proved to play an important role in inducing neuronaldysfunction after stroke．The researchers found that NOX had serious adverse effects onthe process of brain functional recovery after ischemic stroke．So the selective targetingnerve protectant therapeutic strategy is used as a targeted treatment plan．Currently，nonselective free radical scavenge（rsuch as apocynin）in clinical research provides somebeneficial results to alleviate nerve function damage after cerebral infarction．Althoughfurther studies are needed to better evaluate whether the treatment can be applied toclinic．But on the whole，it provides a direction for the future drug selection．Thisresearch applies permanent middle cerebral artery embolism（MCAO）model in mice to observe the changes of NOX after apocynin pretreatment and the impact of cerebralinfarction size．It observes the significance of the treatment of apocynin to ischemiccerebral apoplexy，and tries to explore its mechanism of action．Methods1．Select the health clean level of male mice（ICR weight，28-35g）fromthe Laboratory AnimaI Centre in Dalian Medical University．The mice were randomlydivided into4groups：the saline control group（Vehicle）10mice，the apocyninpretreatment groups were divided into10mg/kg，20mg/kg and50mg/kg，each10mice．All male mice were induced to middle cerebral artery occlusion by line switchmethod．After24hours，the experimental animals were beheaded and sectionedvertically from coronary position2mm．Specimens were2%triphenyl four azolenitrogen （TTC） staining and10%formalin fixed．Each slice was scanned with AdobePhotoshop program（version7.0）．Calculate the infarction area percentage．2．①The mice were randomly divided into4groups： the normal group（Sham）3mice，0.5hours，1.5hours and4hours after the model building，each3mice．Keep the cerebral infarction area specimens．The specimens were analyzed byp47phox，p67phox and gp91phox Western imprinting analysis to make a qualitativeevaluation if the inhibiting effect of apocynin is done through the inhibition of NOX．Dothe experiment indexes and time correlation detection after infarction respectively．②The mice were randomly divided into the following5groups：the normal group（Sham）3mice，the saline control group（Veh）3mice，the apocynin pretreatment groups weredivided into10mg/kg，20mg/kg and50mg/kg，each3mice．Select the time point，1.5hours and p67phox as the representative to compare with the protein expressions ot the10mg/kg，20mg/kg apocynin groups and50mg/kg apocynin pretreatment group．Results1．Apocynin pretreatment before middle cerebral artery infarction molding inmice．The infarction area of the control groups percentage was19.1±0.7％（10cases）．Inject10mg/kg apocynin30minutes earlier than middle cerebral arteryocclusion molding，the infarction area percentage was14.9±0.9％（10cases），decreased significantly compared with the infarction area in the control groups （P<0.05）．Increased to20mg/kg，the infarction area percentage was19.1±0.8％（10cases），there was not significant difference compared with the control groups （P>0.05）．Increased to50mg/kg，the infarction area percentage was9.2±1.0％（10cases），significantly lower compared with the infarction area in the control groups（P<0.01）．2．Western blot analyses the protein expression changes of the mice cortical in thenormal groups and the mice ischemic cortex extracts of gp91phox，p67phox andp47phox after the middle cerebral artery embolism of mice of each point in time（0.5hours，1.5hours and4hours）．The results showed that the protein expressions ofp91phox，p67phox and p47phox all increased．The protein expression of4hoursdecreased．Observe the protein expression of p67phox after giving the apocyninpretreatments of10mg/kg，20mg/kg and50mg/kg．The result is the protein expressiondecreased significantly when50mg/kg apocynin was added．Conclusion1．NADPH oxidase（NOX）subtype p47phox，p67phox and gp91phoxprotein in ischemic brain tissues show a significant rising．It leads to the raise of theNADPH oxidase activity which promotes ROS． It could be one of importantmechanisms of brain tissue damage after ischemia．2．Apocynin pretreatments of10mg/kg and50mg/kg reduce free radical damage aftercerebral infarction by inhibiting the NADPH oxidase activity in ischemic brain tissues．...