Oxygen And Glucose Deprivation / Complex Sugars Reoxygenation Injury Of Nerve Cells And The Intervention Effect Of Salvianolic Acid B Mechanism

Ischemic cerebrovascular disease has the characteristics of high incidence,high mortality and high deformity.It has caused serious social problems and economic burden.Therefore,to deeply understand its pathogenesis and find a new target is the key issue to be solved.The immediate cause of ischemic cerebrovascular disease is a decrease in the cerebral blood flow,inducing the interruption of oxygen and energy as well as cell death.Cerebral blood flow restores after treatment,however,nervous system damages more seriously,which is called ischemia-reperfusion injury.In Chinese medicine theories,the etiological factors of stroke(here mainly refers to ischemic cerebrovascular disease) is related to the toxin hurts brain collaterals,the toxin is including wind,fire,sputum and stasis.The Chinese traditional herb- Salvia miltiorrhiza has the effects of activating blood circulation and dissipate blood stasis.It has been widely used in clinical treatment of ischemic cerebrovascular disease. Salvianolic acid B(Sal B) is a water-soluble active component extracted from Salvia miltiorrhiza.It is a phenolic acid compound,which is condensated by three molecules tanshinol and one molecule caffeic acid.Sal B exhibits higher antioxidant and radical scavenging activities.Nevertheless,the mechanisms Of its intervening effect during the cerebral ischemia and reperfusion have not yet been explained very clearly.The effects of Sal B on the cerebral ischemic mice have been proved in our laboratory. Under the instruction of Chinese medicine theories and our findings,in the present thesis,we established the oxygen-glucose deprivation/reintroduction model of primary neuron cultures. Then,the mechanisms of neuron injuries and the interventional effects of Sal B were systematically investigated in terms of excitotocitity,calcium overload,oxidant stress and neuron apoptosis.Therefore,the research work is divided into four parts:1,The establishment of the oxygen-glucose deprivation(OGD) model and the determination of effective dose of Sal B.Objective:To establish a stable and reliable OGD model;to determine the effective dose of Sal B.Methods:Primary cortical neuron cultures were preformed by using neonate rats.The neurons which grew for 8～10 d in vitro were treated by OGD for 2 h,3 h,4 h,5 h and the dissolved oxygen concentrations in the cultures media were measured by an oxygen measuring tester.The neurons vitality and survival rate were determined by MTT assay.Results:The primary cultures were in good conditions.The percentage of NSE-positive neurons reached 70～80%,Nissl's staining showed the Nissl's bodies were abundant in the neurons.The dissolved oxygen concentrations decreased as the time of OGD prolonged,the neurons vitality and survival rate also reduced.There was significant difference between the control group and the model group at various time points(P＜0.01).Since the cell damages were moreserious at OGD 4 h,we selected it as the duration of OGD in further experiments. After OGD 4 h,the neurons vitality declined(P＜0.01);the Sal B treatment group included eight different doses:10μg/L,100μg/L,1mg/L,5 mg/L,7.5 mg/L,10 mg/L,25 mg/L and 50 mg/L.The neurons vitality in the Sal B treatment group of 10 mg/L,25 mg/L and 50 mg/ L increased obviously compared with that in the OGD 4 h group(P＜0.01).There was a relationship between the dose of Sal B and the neurons vitality.In addition,normal neurons were treated by adding 10 mg/L,25 mg/L and 50 mg/L Sal B,respectively.Their vitalities had no significant difference,as compared with the control group.The final concentration of 10 mg/L was determined as the dosage of Sal B in the following experiments.2,The study of OGD induced neurons injury and the interventional mechanisms of Sal B.Objective:To study OGD induced neurons injury and the interventional mechanisms of Sal B.Methods:Primary cultured rat cortical neurons were randomly divided into the control group,the OGD 4 h group and the Sal B treatment group.The cell model was established by depriving of oxygen and glucose for 4 h.The cortical neurons vitality and survival rate were determined by MTT assay.The leakage rate of lactate dehydrogenase(LDH) and the content of glutamate in neuron medium were measured by chromatometry.The mitochondria membrane potential(MMP),the cytosolic free calcium concentration were quantitatively analyzed by flow cytometry.The neuronal morphous and ultrastructure were observed with an inverted phase contrast microscope,HE staining and a transmission electron microscope (TEM),respectively.Results:After OGD 4 h,the cortical neurons vitality,the survival rate and the fluorescence intensity of MMP reduced,the leakage rate of LDH,the content of glutamate in neuron medium and the cytosolic free calcium increased.There was significant difference between the OGD 4 h group and the control group(P＜0.01).In the Sal B treatment group the cortical neurons vitality,the survival rate and the fluorescence value of MMP were obviously higher than those in the OGD 4 h group,the cytosolic free calcium and the content of glutamate in the medium were significantly lower than those in the OGD 4 h group.Obvious difference was observed between the two groups(P＜0.05～0.01).In addition,the shapes of normal neuronal bodies were pyramidal,cell organs were abundant.In OGD 4 h group,most of the neuronal bodies and mitochondria were markedly swollen,the amount of cell organs decreased.The neuronal bodies of Sal B treatment group were swollen slightly,ultrastructure was still complete.3,The study of OGD-reintroduction(OGD-R) induced neurons injury and the interventional mechanisms of Sal B. Objective:To investigate OGD-R induced neurons oxidative injury and to confirm the interventional mechanisms of Sal B related to its antioxidation in vitro.Methods:Primary neuron cultures were randomly divided into the control group,the OGD-R group and the Sal B treatment group.The cell model was established by depriving of oxygen and glucose for 3 h and reintroducing for 1 h,3 h,6 h,12 h,18 h and 24 h, respectively.The neurons vitality and survival rate were determined by MTT assay.Then,the OGD-R 3 h and OGD-R 24 h were selected as the two timepoints for the following experiments.The leakage rate of LDH was measured by chromatometry.The level of cellular ROS was detected by fluorescent labeling method and spin trapping technique.The activities of neuronal Mn-SOD,CAT and GSH-PX were assaied by chromatometry.The morphologies were observed with an inverted phase contrast microscope.Results:The neurons vitality and the survival rate in the OGD-R group were lower than those in the control group at six different timepoints(P＜0.05～0.01).Except OGD-R 1 h and 12 h,the neurons vitality and the survival rate of Sal B treatment group were obviously higher than those in the OGD-R group(P＜0.05).After OGD-R 3 h,24 h,the leakage rate of LDH, the level of cellular ROS increased obviously compared with the control group(P＜0.05～0.01).In the Sal B treatment group the leakage rate of LDH,the level of cellular ROS were lower than those in the OGD-R group(P＜0.05～0.01).The activities of neuronal Mn-SOD,CAT and GSH-PX reduced in the OGD-R group at the two timepoints,there was obvious difference between the control group and the OGD-R group(P＜0.05～70.01).The activities of these antioxidases in the Sal B treatment group were higher than those in the OGD-R group (P＜0.05～0.01).Moreover,neuronal appearance changed after OGD-R 3 h,neurons shrinked and part of neurons died at OGD-R 24 h.The morphology of neurons in the Sal B treatment group changed slightly.4,The study of OGD-R induced neurons apoptosis and the anti-apoptosis mechanisms of Sal B.Objective:To study OGD-R induced neurons apoptosis and the anti-apoptosis mechanisms of Sal B.Methods:Primary neuron cultures were randomly divided-into the control group,the OGD-R 24 h group and the Sal B treatment group.The cell model was established by depriving of oxygen and glucose for 3 h and reintroducing for 24 h.The cytosolic free calcium was assessed using laser scanning confocal microscopy(LSCM).The MMP and the apoptosis rate were quantitatively analyzed by flow cytometry.The expression of Bcl-2,Bax were observed by immunohistochemical method.The release rate of Cytochrome C was detected by western blot.The morphology of neuronal nuclei was observed by Hoechst 33342 fluorescence staining.The neuronal ultrastructure was observed by TEM.Results:The fluorescence intensity of cytosolic free calcium and the apoptosis rate in the OGD-R 24 h group were higher than those in the control group,the fluorescence value of MMP in the OGD-R 24 h group was lower than that in the control group.There was signiflcant difference between the two groups(P＜0.01).Compared to the OGD-R 24 h group, the fluorescence intensity of cytosolic free calcium and the apoptosis rate in the Sal B treatment group were significantly decreased(P＜0.01),the fluorescence value of MMP in the Sal B treatment group was obviously increased(P＜0.01).The expression level of Bcl-2 decreased and the expression level of Bax increased in the OGD-R 24 h group,there was significant difference compared with the control group(P＜0.01).In the Sal B treatment group,the level of Bcl-2 expression was higher and the level of Bax expression lower than those in the OGD-R 24 h group(P＜0.05).The content of cytosolic Cytochrome C in the control group was very low,Cytochrome C existed in mitochondria mainly.In the OGD-R 24 h group the content of cytosolic Cytochrome C raised markedly compared with the control group(P＜0.01),this index decreased in the Sal B treatment group,there was obvious difference between the two groups(P＜0.05).In the OGD-R 24 h group the release rate of Cytochrome C was higher than that in the control group(P＜0.01),in the Sal B treatment group the release rate was lower than that in the OGD-R 24 h group,there was obvious difference between the two groups(P＜0.05).The nuclei showed adqulis blue fluorescence in the control group.Chromatin condensation,intensive blue fluorescence and nuclear fragmentation were observed in the OGD-R 24 h group.In the Sal B treatment group,most of the nuclei had light blue or blue fluorescence similar to the control group,only individual nuclei were stained densely and had bright blue fluorescence.In the OGD-R 24 h group, shrunken nuclei with condensed and marginated chromatin and the intact plasma membrane were observed by TEM,these showed the morphological changes of apoptosis.In the Sal B treatment group,the neuronal ultrastructures changed slighter,in comparison with these in OGD-R 24 h group.Conclusion:We successfully established a stable and reliable primary neuron cultures system. OGD-induced neuronal injury can be achieved by in vitro model.After OGD 4 h,the content of glutamate in neuron medium increased,cytosolic calcium overload and the fluorescence intensity of MMP reduced.The interventional mechanisms of Sal B in the OGD neurons would be due to the fact that Sal B can diminish the content of glutamate in neuron medium and inhibit calcium overload and maintain the MMP.OGD-R-induced neurons injury is concerned with oxidative stress,Sal B can protect neurons from OGD-R-induced injury through scavenging the cellular ROS and enhancing the activities of Mn-SOD,CAT,GSH-PX. Neurons apoptosis can be induced by OGD-R 24 h,Sal B can reduce neurons apoptosis through conserving the function of mitochondria,elevating the ratio of Bcl-2/Bax, diminishing the release of pro-apoptotic substance.To sum up,the pathogenesis of OGD is related to the excitotocitity,calcium overload, the pathogenesis of OGD-R is involved in oxidant stress and neuron apoptosis by the mitochondrial pathway.Sal B can protect the OGD neurons through relieve excitotocitity, inhibit cytosolic calcium overload,it also has neuroprotective effects on the OGD-R neurons by improving the cellular redox state,preserving mitochondria and resisting apoptosis.