| BackgroundCerebral ischemia is the second most common cause of death over the age of 60,and the second most common cause of dementia.In high-income countries,payment for stroke accounts for 3-7%of the health budget.As China’s aging population continues to increase,the number of patients with cerebral ischemia in recent years is also increasing.Cerebral ischemia has become one of the most important public health problems in China.Therefore,it is of extremely important to actively carry out the theoretical and clinical research on cerebral ischemia,to further understand the pathogenesis and search effective treatment,to improve patient survival and reduce morbidity,for patients and their families and society.For a long time,people have always thought that as long as the timely recovery of blood supply,which can reduce damage caused by ischemic to a minimum.However,this theory was questioned in the 1970s,and studies suggest that the sudden recovery of oxygen in the hypoxia tissue can lead to a unique injury response and is different from the hypoxic condition of the injury response.Therefore,cerebral ischemia and reperfusion injury was found.Cerebral ischemia reperfusion injury(CIR)means the brain tissue ischemia,hypoxia after a certain period of time caused by tissue damage,than be treated actively to restore brain blood supply,but the degree of brain damage not only did not reduce but increased.There have been more serious brain dysfunction,and even cause multiple organ damage phenomenon.However the current treatment of cerebral ischemia is still as soon as possible to restore blood supply according to the clinical consensus,to reduce the degree of cerebral ischemia.Therefore,reperfusion injury can not be avoided,and lacking of effective treatment.Therefore,the study of reducing the treatment of reperfusion injury has important clinical significance.Astragaloside iv is a monomer extracted from astragalus.Modern pharmacological studies have found that Astragalus has a variety of medicinal value,such as anti-inflammatory,immune regulation,anti-oxidation,anti-cancer,anti-diabetic,myocardial protection,liver protection,anti-virus function.Studies have shown that astragaloside has a good protective effect of the brain that by inhibiting the expression of peripheral benzodiazepine receptors in the penumbra region after ischemia,reducing the penumbra area,protecting the brain tissue,and prevent the memory impairment and neurological inflammation in bilateral common carotid artery occlusion.The protective mechanism is to reduce the expression of TLR4 and its downstream receptor protein,including MyD88,TRIF and TRAF6,and inhibit the phosphorylation of NF-κB.Some studies have shown that the protection of astragaloside on cerebral ischemia-reperfusion is due to the ability to improve the function of the blood-brain barrier and to reduce the permeability of the blood-brain barrier in pathological conditions.In this study,we found that astragaloside can inhibit the endoplasmic reticulum stress and reduce mitochondrial damage,reduce the apoptosis of cerebral vascular endothelial cells,thus protecting the structure and function of the blood-brain barrier,to protect the brain tissue and reduce the infarct size.Experiment I:Pre-protection Effect of Astragaloside Ⅳ on Blood Brain Barrier of MCAO RatsObjectives:Study the preconditioning effect of astragaloside on blood-brain barrier after cerebral ischemia-reperfusion,and determine the stress-related protein of endoplasmic reticulum to determine its mechanism.Method:There were 140 SPF grade SD male rats,aged 6 weeks.Rats were randomly divided into 5 groups.The administration was administered 3 weeks before intragastric administration.The rats were sacrificed by ischemia for 2 hours and reperfusion 24 hours after ischemia.At the end of reperfusion,TTC,Evans blue,HE staining,Nissl staining,SOD,MDA and protein electrophoresis were used to detect protection.Result:1.Neurological deficit scoreIn addition to sham group,compared the other four groups of animals score,there was no significant difference.2.TTC staining experimentThe infarct size of the vehicle group was significantly higher than that of the other groups(sham group:p = 0.002,other groups:p<0.05),and the infarct size of the three groups was significantly less than that of the vehicle group(ASL:p = 0.018;ASM:p = 0.006;ASH:p = 0.003).3.Evans blue staining experimentUnder the direct microscope,the right brain of the vehicle group was darker than the others.(ASL:p = 0.010;ASM:p = 0.001;ASH:p<0.001)in the infarcted brain of the vehicle group was significantly higher than that in the sham operation group(p<0.001).Compared with the left brain,there was no statistically significant difference(P>0.05)between the five groups.4.HE staining resultsSham group showed normal cortical structure of the brain,cell integrity,rich cytoplasm,nucleolus clear.The HE staining of the infarcted cortex in the vehicle group and the treatment group showed that the pathological changes of the infarcted cerebral cortex were observed in the infarcted cerebral cortex(the necrotic neurons were seen in the infarct area and the number of cells was less.While the drug group compared with the vehicle group,cell damage,eosinophil staining is also relatively reduced.5.Nissl dyeing resultsSham group showed that the cortical neonatal bodies arranged neatly,the number of more.While the vehicle group of rats with infarcted cortical Nissl body arrangement sparse,the number was significantly reduced.Compared with vehicle group,the infarcted cortical neonatal body injury was lighter,arranged neatly and more.6.Effects of AS on SOD and MDA in the infarcted brainThe levels of SOD in the right brain of the vehicle group were significantly decreased(p<0.01),and the level of MDA was significantly increased(p<0.001).(ASL:p = 0.014;ASH:p = 0.001),and decreased the level of MDA(ASL:p = 0.001;ASM:p)was significantly higher than that in the control group(ASL:p = 0.040;ASM:p = 0.014;ASH:p = ＝ 0.001;ASH:p&It;0.001).7.Effect of AS on SOD and MDA in BloodThere was no significant difference in blood MDA level between AS group and AS group(ASL p = 0.909,ASH p = 0.924,ASH p = 0.902).The activity of SOD in the blood of vehicle group was lower than that of sham group(p = 0.008).The activity of SOD in blood of AS 10uM group was higher than that of vehicle group(p = 0.041).There was no significant difference between the other two groups(ASL p = 0.253,ASM p = 0.065).8.Effects of AS on stress-related proteins in endoplasmic reticulum(P<0.05);P-PERK/PERK:p<0.001;P-eif2α/eif2α:p = 0.001;CHOP:p ＝0.025;P-PERK/PERK:p<0.001;P-eif2α/eif2α:p ＝ 0.003).AS could inhibit the expression of BIP,CHOP,P-PERK and P-eif2 a protein,and there was significant difference between the two groups(BIP:ASL p = 0.038,ASM p =0.015,ASH p = 0.014 P-eif2 a/eif2 a:ASL p = 0.029,ASH p = 0.007;P-PERK/PERK:ASL p = 0.039,ASM p = 0.028,ASH p = 0.004;ASM p = 0.026,ASH p ＝0.006).9.Anti-apoptotic effect of ASThe expression of Bax protein and the expression of Bcl-2 protein were inhibited in the cerebrovascular endothelial cells of vehicle group(p<0.001).(ASL p = 0.047,ASH p = 0.037,ASH p = 0.002),and the difference was statistically significant(ASL p = 0.047,ASM p = 0.037,ASH p = 0.002),and the difference was statistically significant(ASL p = 0.047,ASM p = 0.037,ASH p = 0.002).Vehicle group produces a higher level of caspase-3 protein expression in cerebrovascular endothelial cells(p<0.001).(ASL p = 0.045,ASM p = 0.031,ASH p = 0.011)was significantly higher than that of vehicle group(P<0.05).Conclusion:The expression of caspase-3 protein in endothelial cells of ischemic reperfusion cells is significantly lower than that in vehicle group(ASL p = 0.045,ASM p = 0.031,ASH p = 0.011).Experiment II:Pre-protection effects of Astragaloside Ⅳ on endothelial cells after oxygen-glucose deprivation/reoxygenation(OGD/R)Objectives:Study the protective effect of astragaloside on endothelial cells by inhibiting endoplasmic reticulum stress and reducing mitochondrial damage in endothelial cells after oxygen-glucose deprivation/reoxygenation.Method:The brain microvascular endothelial cells(BMECs)were divided into CON group,OGD/R group,OGD/R + AS 2.5uM group,OGD/R + AS 5uM group,OGD/R + AS lOuM group.MTT,SOD,MDA,protein electrophoresis,ATP,JC-1 were used to detect the protection.Result:1 The effect of astragaloside on normal endothelial cells and OGD/R-induced endothelial cell injuryMTT assay was used to detect the toxicity of astragaloside in normal endothelial cells.The results showed that AS 2.5 uM(p = 0.044)and 5 uM(p＝0.008)and 10 uM(p = 0.002)had the same effect on endothelial cells Good pre-protective effect,of which lOuM concentration of the most stable efficacy.2 Effects of Astragaloside Ⅳ on SOD Activity and MDA Level in OGD/R-induced Endothelial CellsOGD/R group compared with CON group,MDA level was higher,SOD activity decreased,the difference was statistically significant(p = 0.002).AS can reduce the level of MDA,increase the activity of SOD.(AS 5 p = 0.019,AS 10 p = 0.011)in the OGD/R + AS 5uM group and the OGD/R + AS lOuM group,and the increase in SOD activity was statistically significant only the OGD/R +AS 10uM group showed a statistically significant difference(AS 10 p = 0.01).3 Effects of Astragaloside Ⅳ on Expression of Apoptosis-related Proteins Compared with CON group,the expression of Bcl-2 protein was up-regulated and the expression of Bax protein was up-regulated in OGD/R group(p = 0.005).AS could significantly increase the expression of Bcl-2 protein and decrease the expression of Bax protein(AS 2.5 p = 0.047,AS 5 p = 0.019,AS 10 p ＝0.011).Compared with CON group,the expression of caspase-3 protein was up-regulated in OGD/R group,the difference was statistically significant(p=0.003).AS can significantly reduce caspase-3 protein expression(AS 2.5 p = 0.034,AS 5 p = 0.015,AS 10 p = 0.012).AS can play a significant anti-apoptotic effect.4 Effects of Astragaloside Ⅳ on Expression of Stress-related Proteins inEndoplasmic ReticulumThe expression of BIP,CHOP,P-PERK and P-eif2α protein was higher in OGD/R group than in CON group(BIP:p = 0.006;CHOP:p = 0.008;P-PERK/PERK:p＝0.028;P-eif2?/Eif2?:P = 0.003).AS Bp = 0.012,AS 10 p = 0.012;CHOP:AS 2.5 p=0.039,AS 5 p = 0.032,AS 10&It;RTI ID = 0.0>P = 0.027;P-PERK/PERK:AS 2.5 p = 0.048,AS 5 p = 0.015,AS 10 p = 0.008;P-eif2α/eif2 a:AS 2.5 p = 0.033,AS 5 p = 0.009,AS 10 p = 0.005)4 Effects of Astragaloside IV on ATPOGD/R group compared with CON group,ATP content was significantly reduced(p<0.001).AS 10uM group significantly increased ATP content(p = 0.005).There was a significant difference between the AS 2.5uM and the AS 5uM group and the OGD/R group(p = 0.610;p = 0.218).6 Effects of Astragaloside IV on Mitochondrial Membrane PotentialCompared with CON group,the green fluorescence of OGD/R group was significantly increased by fluorescence inverted microscope,which indicated that MMP decreased significantly.AS 2.5uM,AS 5uM group and AS lOuM group,compared with OGD/R group,red fluorescence more,indicating that AS can significantly improve mitochondrial MMP levels,improve mitochondrial function.Quantitative analysis was performed by flow cytometry.MMP levels were significantly lower in OGD/R group compared with CON group(p<0.001).AS10uM group can relieve the decrease of MMP and improve mitochondrial function(p=0.002).There was no significant difference between AS 2.5uM and AS 5uM groups compared with OGD/R(p = 0.148;p = 0.09)Conclusion:(1)Astragaloside can up-regulate the expression of Bcl-2 and down-regulate the expression of Bax and caspase-3,which can effectively reduce the apoptosis of endothelial cells caused by cerebral ischemia/reperfusion.(2)Astragaloside can down-regulate the expression of BIP,CHOP,P-PERK and P-eif2α protein and inhibit the endoplasmic reticulum stress.(3)Astragaloside can increase the content of ATP,increase the mitochondrial membrane potential,and effectively reduce the damage of mitochondria caused by cerebral ischemia/reperfusion.(4)Astragaloside can effectively protect the blood-brain barrier,reduce the area of cerebral infarction,to protect the brain.(5)Astragaloside can reduce the mitochondrial damage and inhibit the endoplasmic reticulum stress,to achieve brain protection.But the role of mitochondrial damage and endoplasmic reticulum stress relationship played in cerebral ischemia/reperfusion was still unclear,which needed to be further experimental study. |
