Protective Effects And Mechanisms Of DL0108 On Neurovascular Unit After Cerebral Ischemia In Rats

Stroke, a brain attack, is a problem that affects >15 million people world-wide, is the leading cause of disability and third leading cause of death in major industrialized countries. It has been estimated that -6 million people have died from stroke in 2005 and that >90% of these deaths will have occurred in less affluent countries.Recent epidemiologic data suggest that the decline in both stroke incidence and mortality reached a nadir in the early 1990s and is now rising because of the aging population. Stroke patients must not only survive the acute stages of infarction, but must then cope with significant mental, physical, and economic stresses associated with neurological impairment. Considering the cost in loss of life, physical and mental disability and subsequent self-esteem and productivity, the need for effective therapeutic interventions is obvious.Ischemic stroke is by far the most frequent type of stroke, accounting for 83% of all stroke cases. Ischemic stroke results from an obstruction, typically a blood clot. Cerebral ischemia may be either transient followed by reperfusion, or essentially permanent. In patients, both types of focal ischemia can occur. In transient occlusion, reperfusion injury also adds to brain damage. After cerebral ischemia, perturbations in neurovascular functional integrity initiate several cascades of injury. Upstream signals such as oxidative stress and glutamate excitotoxity together with upregulation of matrix metalloproteinases (MMPs), tissue plasminogen activator (tPA) and other proteases, which degrade matrix, lead to blood brain barrier (BBB) disruption. Inflammatory products which infiltrates through the damaged BBB amplify brain tissue injury. Additionally, disruption of cell matrix homeostasis and activation of microglia can also trigger cell death pathways in both vascular and parenchymal compartments.DL0108 (Pinocembrin, 5,7-dihydroxyflavanone, Fig.7) is the most abundant flavonoids in propolis. The compound was reported to have multiple actions including anti-inflammatory, antioxidant, antimicrobial, anti-allergic activities. Our previous study also found that DL0108 induced relaxation of rat aortic rings through an endothelium-dependent and -independent pathway, and that DL0108 could improve rat cognitive impairments induced by chronic cerebral hypoperfusion, which contributed to its improvement of regional cerebral blood flow (rCBF) and mitochondria structure and function. In the present study, we further examined the protective effects and mechanisms of DL0108 on neurovascular unit (NVU) injury after stroke.Partâ… Neurovascular unit protective action of DL0108 against permanent cerebral ischemia in ratsVascular- and neuroprotective effects of DL0108 were evaluated in a rat model of focal cerebral ischemia. Focal cerebral ischemia was induced by the occlusion of middle cerebral artery (MCA) for 24h. DL0108 (3, 10, or 30 mg/kg), intravenously injected at 0h, 8h, 16h after MCA occlusion, reduced the cerebral infarct volumes by 47%, 39%, and 37% respectively, as visualized by 2,3,5- triphenyltetrazolium chloride (TTC) staining (P<0.01). Treatment with DL0108 also reduced brain swelling and improve behavioral deficits significantly (P<0.01, P<0.05). To evaluate the effect of DL0108 on BBB disruption, Evans Blue (EB) and sodium fluorescein (NF) mixture were intravenously injected immediately after MCAO. Global NF/EB uptake and fluorescence imaging of local BBB disruption were measured. DL0108 treatment reduced the leakage of both dyes, manifesting a preventive action in BBB integrity. The electronic microscopy study of neurovascular unit showed that, in the identical areas of affected cortex of DL0108-treated groups, there is alleviated swelling of astrocyte end foot processes and endothelial cell cytoplasm, fewer numerous capillaries with compressed lumina, mild vacuolization of neuropil, no obvious migration of pericytes from the capillary wall basal lamina to the surrounding parenchyma and fewer apoptotic and pycnotic cells. These findings demonstrate that DL0108 has neurovascular protective action against permanent cerebral ischemia. Partâ…¡The study of protective mechanisms of DL0108 on NVU after cerebral ischemia--Protective mechanisms of DL0108 on BBB disruptionAdministration of DL0108 (3, 10, or 30 mg/kg) after the onset of ischemia in rats reduced infarction and improved cerebral blood flow. To understand the mechanism of protection, the involvement of inflammation in ischemic brain injury was examined. Treatment with DL0108 reduced the expression of tumor necrosis factor-a (TNF-a), interleukin-1β(IL-1β), and iNOS; inhibited the activation of microglia and astrocyte; and downregulated the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), aquaporin-4 (AQP-4) and matrix metalloproteinase (MMP-9) in the ischemic brain. mRNA expression of tight junction component proteins--occludin and ZO-1. The results indicated that DL0108 elevated their expression, which contributing to the preservation of BBB integrity. Further, the anti-inflammatory effect of DL0108 was also determined in cortical neuron-glial co-cultures stimulated by LPS (10ng/ml) for 12 h. NO and TNF-a in culture medium were reduced by DL0108 administration. From the above results, we conclude that DL0108 protects the neurovascular unit after ischemia partly through an anti-inflammation pathway.Partâ…¢The study of protective mechanisms of DL0108 on NVU after cerebralischemia--Effects and mechanisms of DL0108 prevents glutamate-inducedapoptosis in SH-SY5Y neuronal cellsTo assess the protective effects of DL0108 on neuronal cells, SH-SY5Y neuronal cells were treated for 12 h with glutamate (2mM). Cell viability was determined by (3, 4, 5-dimethylthiazol-2-yl)-2, 5-diphenylte-trazolium bromide (MTT) assay, and apoptosis was confirmed by cell morphology, capillary zone electrophoresis, and flow cytometry assay. Cell morphology was evaluated with Hoechst33258/PI dye. Treatment with DL0108 (10^-5, 10^-6, 10^-7mol/L) increased cell viability dose-dependently, inhibited LDH release and attenuated apoptosis. Intracellular free [Ca²⁺] was increased after glutamate exposure, and this increase was attenuated in cells treated with DL0108. Bax and bcl-2 mRNA expression were detected by RT-PCR analysis. Bax mRNA expression increased remarkably following glutamate exposure and DL0108 treatment manifested a reduction effect. Bcl-2 mRNA expression changes were not detected in groups with or without DL0108. Western blotting results indicated that DL0108 treatment reduced the expression of Bax and had no effect on Bcl-2, thus decreased the Bax-Bcl-2 ratio, which is consistent with the gene expression result. DL0108 could also down-regulate the expression of p53 protein, and inhibit the release of cytochrom c from mitochondria to cytosol. Thus we conclude that DL0108 exerts its neuroprotective effects in glutamate injury model partly by decreasing intracellular free [Ca²⁺], p53 expression, bax-bcl-2 ratio, and inhibiting the release of cytochrom c.Partâ…£The study of protective mechanisms of DL0108 on NVU after cerebralischemia--Protective effects of DL0108 on cultured rat cortical neurons subjectedto oxygen-glucose deprivation / reperfusionThis study was undertaken to find out the effects of DL0108 on cortical neurons in primary culture subjected to oxygen-glucose deprivation/reperfusion. According to three concentrations (10μmol·L^-1, 1μmol·L^-1, 0.1μmol·L^-1) of DL0108 exposed to OGD/24h-reperfusion, neuron cultures were randomly divided into 5 groups. Each group was observed by inverted phase contrast microscope, also stained with Hoechst 33258; neuronal viability was measured by the reduction of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT); the activity of lactate dehydrogenase was detected by LDH assay kit through a microplate reader; the production of ROS and NO was monitored by 2'7'-dichlorofluorescein diacetate (DCFH-DA) and Griess reagent respectively through a microplate spectrophotometer. As a result, the injured neurons were protected, and both degeneration and apoptosis were alleviated in treatment groups of DL0108. DL0108 increased neuronal viability and decreased LDH release in high, middle and low concentration treatments. The overproduction of NO and ROS was inhibited in the groups treated with DL0108 after 24h reperfusion. Our results demonstrated that DL0108 could protect the cortical neurons from oxygen-glucose deprivation/reperfusion in rats by alleviating the morphological damage, increasing neuron viability, decreasing LDH activity, inhibiting overproduction of ROS and NO in primary cortical cultures. Conclusion:1. The results of in vivo and in vitro experiments showed that, DL0108 has an obvious protective action in a focal cerebral ischemia model in rats.2. The action is due to its protection on various components of NVU, including BBB integrity, neurons and extracellular marix.3. The protective mechanisms of DL0108 on NVU can be illustrated as anti-inflammation, preventing the degradation of TJ proteins, downregulating MMP-9, inhibiting neurons apoptosis and anti-oxidantion.