| Stroke is one of the leading causes of mortality and morbidity. Ischaemic stroke accounts for approximately80-85%of all cases and it is characterised by the disruption of cerebral blood flow and lack of oxygen to the affected area. Ischemia/reperfusion injury (IRI) is crucial in the pathology of ischaemic stroke. Oxidative stress has emerged as a key deleterious factor in brain ischemia and reperfusion. Reactive oxygen species are implicated in a number of disease processes. They mediate damage to cell structures, including lipids, membranes, proteins, and DNA. Blood-brain barrier (BBB) leakage and brain edema are critical parts of stroke pathophysiology. Oxidative stress closely relates with the active of matrix metallo proteinase (MMPs), which will damage brain microvascular endothelial cells and their tight junctions to cause BBB dysfunction such as edema, hemorrhage and inflammation.Therefore, these findings suggest that antioxidants or inhibition of the pathway of cytoplasmic oxidases, such as cyclooxygenase, may be a possible therapeutic strategy of cerebral ischemia. Anti-ischemia drugs, such as Edaravone, have already been marketed in Japan and China. If we can screen out a drug which not only have the effect of antioxidant but also the inhibition effect of the cytoplasmic oxidases, such as cyclooxygenase, NADPH, NOS, MMPs, those drug may be more effective. We aim at screening out the antioxidant from Traditional Chinese Medicine which may be effective on one or more target pathways of ischemia/reperfusion injury.This study aims at establishing the model of brain ischemia in mouse brain slices and PC12cells. These models will be used to investigate the following questions on oxygen-glucose deprivation/reperfusion induced injury and their possible mechanism.1. To investigate the protective effects of selective COX-2inhibitors on oxygen-glucose deprivation/reperfusion induced injury and possible mechanism in mouse brain slices and PC12cells.2. To investigate the protective effects of antioxidantsts such as Ginsenoside Rgl, Paeonol, Quercetin, Astragaloside, Andrographolide, resveratrol, chlorogenicacid, Baicalin and Huanglian-Jie-Du-Tang on oxygen-glucose deprivation/reperfusion induced injury in mouse brain slices and PC12cells.3. To investigate the neuroprotective effects of hydroxysafflor yellow A (HSYA) on oxygen-glucose deprivation/reperfusion induced injury, and and possible mechanism in PC12cells.Part1Establishment and improvement of mouse brain slices and injury model of PC12induced by oxygen-glucose deprivation and reperfusionObjective To establish the model of brain ischemia in mouse brain slices and PC12cells. To investigate the protective effects of selective COX inhibitors on oxygen-glucose deprivation/reperfusion induced injury and possible mechanism in mouse brain slices and PC12cells.Methods In mouse brain slices, the activity after OGD and reperfusion was assessed by the formation of formazn, a red product of2,3,5-trihenylterzolium chloride (TTC). In PC12cells, the cell viability and damage after OGD and reperfusion were assessed by MTT reduction and LDH. The expression of COX-2protein was assessed by immunocytochemisry.Results In mouse brain slices, selective COX-2inhibitors (Cel and Eto) significantly attenuated OGD injury while non-selective COX inhibitor Ind had no influence. In PC12cells, Cel and Eto protected the cells from OGD-induced injury at10-160μmol/L. Ind also protected PC12cells at1-10μmol/L. The latest selective COX-2inhibitor Eto increased cell viability and attenuated LDH release obviously in OGD-induced PC12cells. Expression of COX-2protein on OGD PC12cells was also inhibited by the treatment of Eto.Conclusion The model of oxygen-glucose deprivation/reperfusion induced injury in mouse brain slices and PC12can be used to screen out anti-ischemia drug effectively. Selective COX-2inhibitors, especially the latest selective COX-2inhibitor Eto, have concentration-dependent protective effect on OGD injury in vitro. The possible mechanism may be mediated by the inhibition of COX-2protein expression. Part2Protective effect of free radical scavenger from Traditional Chinese Medicine on oxygen-glucose deprivation induced injury in mouse brain slices and PC12cellsObjective To investigate the protective effects of antioxidants from traditional chinese medicine on oxygen-glucose deprivation induced injury in mouse brain slices and PC12cells.Methods In mouse brain slices, the activity after OGD was assessed by the formation of formazn, a red product of2,3,5-trihenylterzolium chloride (TTC). In PC12cells, the cell viability and damage after OGD were assessed by MTT reduction.Results In mouse brain slices, antioxidants (Astragaloside, resveratrol, chlorogenic acid, Baicalin and safflor yellow A) significantly attenuated OGD injury whereas antioxidants (Ginsenoside Rgl, Paeonol, Quercetin and Andrographolide) had no influence. In PC12cells, Ginsenoside Rgl and Andrographolide protected the cells from OGD-induced cell injury at1μmol/L. Baicalin and safflor yellow A protected PC12cells at0.1-10μmol/L. Resveratrol also protected PC12cells at10μmol/L. Antioxidants (Astragaloside, Paeonol, quercetin and chlorogenic acid) had no influence.Conclusion Antioxidants (Baicalin and hydroxy safflor yellow A) significantly attenuated OGD induced injury both in mouse brain slices and PC12cells.Part3Protective effect of hydroxy safflor yellow A on oxygen-glucose deprivation and reperfusion induced injury and its mechanismObjective To investigate the neuroprotective effects of hydroxyl safflor yellow A (HSYA) on oxygen-glucose deprivation and reperfusion induced injury, and its effect on the expression of cyclooxygenase and metrix metalloproteinase-9(MMP-9) in PC12cells.Methods The PC12cells were exposed to oxygen-glucose deprivation (OGD), and reperfusioned for different times. The PC12cell viability and lactate dehydrogenase (LDH) release were seperately detected by MTT assays and LDH detecting kits. The apoptotic cells were double stained by propidumiodid (PI) and Hoechst33342. The expression of COX-2was detected by immunocytochemistry and western blot. The expression of MMP-9was detected by immunocytochemistry.Results Compared to the control group, in OGD4h-R2h group, cell viability significantly decreased to (75.5±3.7)%(P<0.01). HSYA increased cell viability (90.8±7.6)%and reduced releasing of LDH to (109.5±7.0)%(P<0.01) at the concentration of10μmol/L.The result of PI and Hoechst33342showed that HSYA also markedly inhibited the apoptosis of PC12cell induced by OGD/RP, which decreased to (19.7±7.5)%in HSYA1μmol/L group (P<0.01). Meanwhile, the expression of active MMP-9induced by OGD/RP decreased after the treatment of HSYA, similar to positive control, while the expression of active COX-2induced by OGD/RP was not affected.Conclusion It is proved that HSYA protects the injury and inhibits the apoptosis induced by OGD/RP in PC12cells, and the mechanism may be related to the inhibition of active MMP-9expression. |
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