| BackgroundStroke is a global health problem that has been paid close attention to all over the world.Stroke included ischemic stroke and hemorrhagic stroke.Ischemic stroke is the main clinical type of stroke,accounting for 60%-80%.Cerebral ischemia reperfusion injury is the main pathogenesis of ischemic stroke.Oxidative damage induced by oxidative stress is an important mechanism of ischemic brain injury.In the pathological process of cerebral ischemia reperfusion injury,oxidative stress produced large amounts of reactive oxygen species.It could lead to the damage of DNA,protein and lipid and could directly affect the unsaturated fatty acid in biomembrane.Excessive reactive oxygen species could destroy the normal function of cells,reduced the activity of several enzymes and the stability of DNA and RNA,induced oxidative damage to cellular molecules such as proteins,DNA and lipids,at last lead to cell apoptosis.In the process of cerebral ischemia reperfusion injury,there were many sources of reactive oxygen species.NADPH oxidase(NOX)was thought to be a key factor in the abnormal elevation of reactive oxygen species after cerebral ischemia reperfusion.A large number of studies showed that up-regulation of NADPH oxidase expression in ischemic brain tissue could aggravate cerebral ischemia reperfusion injury and increase neuronal death.The present study demonstrated that NOX2 played an important role in ischemic stroke in NADPH oxidase.It was closely related to ischemic stroke secondary disease.Inhibition of NOX2 activity could reduce cerebral ischemia reperfusion injury.Pulse is the house of blood,Qi is the commander of blood.Benifiting qi could promote blood producing.Removing blood stasis could promote new blood producing.Promoting blood circulation could promote muscle producing.Promote blood,new and muscle producing included the meaning of producing new pulse and new collaterals.Therefore,the newborn pulse and collaterals could not leave the qi and blood.So the medicine benefit qi and nourish could producing new blood vessels.Benefiting qi could produce pulse.For stroke,both ancient and modern physicians all took promoting brain collaterals unobstructed as the ultimate goal.Benefiting qi and promote pulse producing could make the brain work normally,brain collaterals unobstructed,and could restore the qi and blood running in brain.Therefore the qi mechanism in every internal organs and tissues is easy and smooth,physiological function returned to normal,so as to achieve the purpose of promoting stroke recovery.Modern medicine promotes angiogenesis by increasing angiogenesis,stimulating angiogenesis in ischemic region and peripheral zone and establish collateral circulation.This corresponds to the "pulse producing" theory of Chinese Medicine.Deng zhan sheng mai capsule is the representative medicine which could benefiting qi and promote pulse for the treatment of cerebral ischemic diseases such as cerebral infarction,had remarkable curative effect.Deng zhan xi xin was the main medicine of Deng zhan sheng mai capsule.Scutellarin is one of the main effective ingredient extracted from Deng zhan xi xin.Scutellarin has been widely used in the prevention and treatment of ischemic cardiovascular and cerebrovascular diseases.Recent studies had shown that scutellarin has protective effects on ischemic cerebrovascular disease.What is the mechanism of action of scutellarin on cerebral ischemia reperfusion injury?Our study used the model of oxygen glucose deprivation in rat model of middle cerebral artery occlusion,explore the possible mechanism of scutellarin on cerebral ischemia reperfusion injury.ObjectivesWe used the cell model(astrocyte hypoxia/reoxygenation model)and the whole animal(rat models of middle cerebral artery occlusion,tMCAO)to explore the effect of scutellarin on NOX2 and oxidative damage,to clear the protection mechanism of scutellarin on cerebral ischemia reperfusion injury after nerve signal.Methods1.The primary cultured astrocytes were used to prepare the model of oxygen glucose deprivation(OGD)injury.The astrocytes were divided into normal control group,oxygen glucose deprivation group,scutellarin low concentration(2μM)+ OGD group,medium concentration(10μM)+ OGD group,high concentration(50μM)+ OGD group,different concentrations of apocynin(NOX2 inhibitor)+ OGD group.Detect reactive oxygen species in astrocytes by reactive oxygen species kit.Detect NOX2 protein expression by Western Blot.We used docking study technology to study the scutellarin and NOX2 protein by molecular docking.2.An animal model of focal cerebral ischemia/reperfusion(I/R)injury was established by middle cerebral artery occlusion in rats.SD rats were randomly divided into sham operation group(sham)group,I/R group,scutellarin low concentration(50mg/kg)+ I/R group,scutellarin high concentration(100mg/kg)+ I/R group,different concentration of apocynin+ I/R group.Bederson 5 scoring method was used to evaluate the neurobehavioral score of rats.Changes of cerebral infarction area in rats was detected by TTC staining.Immunohistochemistry was used to detect the apoptosis of brain tissue in rats.Changes of oxidative stress indicators were detected by ELISA.Western Blot was used to detect the expression of NOX2 protein.Results1.The purity of primary cultured astrocytes was>98%.The survival rate of astrocytes decreased significantly after 12 h and 24 h,and the decreased survival rate of 24 h cells was maximum.After 24 h of oxygen and glucose deprivation and reoxygenation for 8 h,scutellarin(10,50 μM)significantly improved the survival rate of cells.Molecular docking revealed that scutellarin and NOX2 could be effectively docked as their active pockets were complementary.Western Blot analysis showed that the expression of NOX2 in astrocytes after OGD(2 h)was significantly higher than that in the normal control group.Scutellarin(2,10,50 M)pretreated with OGD 2 h and reoxygenation for 2 h showed a significant decrease in the level of NOX2 expression in the astrocytes than solvent control group at the same time point.OGD induced a significant increase in ROS levels in cells,whereas scutellarin(10,50 M)significantly reduced the level of intracellular ROS.2.The neurobehavioral scores,infarct area,brain cell apoptosis and oxidative stress indictors(8-OHdG,4-HNE,3-NT)in rats showed that compared with the sham group,the scores of neurobehavioral,infarct area,positive cell rate and oxidative stress indicators of I/R group were significantly higher.Compared with the I/R group,the neurobehavioral score,infarct area percentage,immunohistochemistry positive cell rate and oxidative stress related indicators of the scutellarin(low and high concentration)+ I/R group rats were significantly lower.3.The brain cell apoptosis and oxidative stress indictors(8-OHdG,4-HNE,3-NT)in rats showed that compared with the sham group,the positive cell rate and oxidative stress indicators of I/R group were significantly higher.Compared with the I/R group,the immunohistochemistry positive cell rate and oxidative stress related indicators of the scutellarin(low and high concentration)+ I/R group rats were significantly lower.The percentage of Caspase-3 positive cells in sham operation group and I/R 3 d group was 12.20±1.62%,55.45±4.99%,respectively.The 8-OHdG of sham operation group and I/R group were 100%and 177.2%±31.2%,respectively.4-HNE was 100%and 163.6%±28.70%,respectively,.3-NT was 100%and 159.3%±32.1%,respectively.Compared with I/R group,Scu(low,high concentration)in I/R group,the positive rate of immunohistochemistry and the related indexes of oxidative stress were significantly lower.The percentage of Caspase-3 positive cells in Scu low concentration group and high concentration group of Scu was 22.36±3.37%,11.70±1.79%.Scu in low concentration group and high concentration group of 8-0HdG were 113.1%±33.1%and 99.9%±11.30%,respectively,and the 4-HNE value was 112%±32.1%and 95.2%±18.9%,respectively,and the 3-NT was 85.6%±31.3%,90%±20.5%,respectively.4.Detection of NOX2 protein with Western Blot showed that the Expre-ssion of NOX2 protein in 3 day was significantly higher in the I/R group than in the sham group.The expression of NOX2 in scutellarin(low and high concentration)+ I/R group at 3 day were significantly decreased than the I/R group.The expression level of NOX2 in apocynin + I/R group at 3 day also decreased significantly.Conclusion1.Glucose and oxygen deprivation reduced the survival rate of astrocytes.Scutellarin could protect the injury induced by Glucose and oxygen deprivation.Molecular docking showed that the active pocket shapes of scutellarin and NOX2 protein were in good agreement with each other,and the molecular structure of 3D of the two groups was in good agreement.Scutellarin could inhibit the expression of NOX2 protein in brain tissue of rats and reduce the level of reactive oxygen species.Scutellarin played the role of NOX2 inhibitor.2.Scutellarin could protect cerebral ischemia reperfusion injury,reduce the neurological behavioral score,infarct size,oxidative damage and apoptosis after cerebral ischemia in rats.Scutellarin played the role of NOX2 inhibitor.3.Protective effects mechanism of scutellarin on cerebral ischemia reperfusion injury was related to the regulation of oxidative damage induced by NOX2. |
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