Neuroprotective Effect And Mechanisms Of Vitexin On Focal Cerebral Ischemia/reperfusion Injury In Mice

Part I Study on the intestinal absorption properties of Vitexin in ratsObjective:Vitexin is a flavonoid compound characterized by protecting cardiac hypertrophy, inhibiting platelet aggregation, preventing hypertensive and inflammatory effects. To establish an in situ single-pass intestinal perfusion rat model, we examined the intestinal absorption characteristics of vitexin and provided the experimental basis for the dosage forms design, the rational and safety use in clinic.Methods:Experiments was divided into two components to observe the absorption of same concentration (80 μg/ml) vitexin and rhamnosylvitexin in four segments of rat intestine, duodenum, jejunum, ileum and colon and different concentrations of (20,40,80,160 μg/ml) vitexin in jejunum via the in situ rat single-pass intestinal perfusion model. Perfusate was collected at different time points after perfusion. High performance liquid chromatography (HPLC) system was used to detect the level of vitexin and rhamnosylvitexin. The effective permeability coefficients (Peff) at different conditions was calculated.Results:1. Under the condition of the same concentration, vitexin was absorpted in the four intestinal sections and there were no significant differences in the effective permeability coefficients (Peff) of vitexin between the four intestine segments. Rhamnosylvitexin could be absorpted in the four intestinal sections and there were no significant differences in the Peff of vitexin between the four intestines. Moreover, the Peff in vitexin and rhamnosylvitexin at the same intestine had no statistical significant difference.2. Under the condition of different concentration of vitexin at 20,40,80,160μg /ml, there were no significant differences in the Peff of vitexin in the jejunum section.Conclusion:There is not a preferential absorption zone in the intestine for vitexin and rhamnosylvitexin. Both of them are poorly absorpted in duodenum, jejunum, ileum, and colon. Vitexin concentrations do not obvious influence intestine absorption. Within the concentration range from 20～160μg/ml, the intestinal absorption of vitexin presents auto concentration inhibition.Results:1. Under the condition of the same concentration, vitexin was absorpted in the four intestinal sections and there were no significant differences in the effective permeability coefficients (Peff) of vitexin between the four intestine segments. Rhamnosylvitexin could be absorpted in the four intestinal sections and there were no significant differences in the Peff of vitexin between the four intestines. Moreover, the Peff in vitexin and rhamnosylvitexin at the same intestine had no statistical significant difference.2. Under the condition of different concentration of vitexin at 20,40,80,160μg /ml, there were no significant differences in the Peff of vitexin in the jejunum section.Conclusion:There is not a preferential absorption zone in the intestine for vitexin and rhamnosylvitexin. Both of them are poorly absorpted in duodenum, jejunum, ileum, and colon. Vitexin concentrations do not obvious influence intestine absorption. Within the concentration range from 20～160μg/ml, the intestinal absorption of vitexin presents auto concentration inhibition.Neuroprotective effect and mechanism of Vitexin on focal cerebral ischemia/reperfusion injury in miceObjective:To investigate the protective effect and the mechanism of neuron apoptosis and its related cellular signal transduction pathways of vitexin on focal cerebral ischemia/reperfusion (I/R) injury in mice, in order to provide evidence for clinical application of vitexin.Methods:Healthy male Kun-ming mice were randomly divided into 6 groups:sham-operated contral (sham), ischemia/reperfusion group (I/R), I/R mice treated with breviscapine injection at a dose of 15.00 mg/kg (BRE), and I/R mice treated with vitexin at low (3.25 mg/kg), medium (7.50 mg/kg) and high (15.00 mg/kg) doses. Cerebral ischemia/reperfusion model in mice were established with suture method. The cerebral I/R model was induced by middle cerebral artery occlusion for 2 h followed by reperfusion for 22 h. The neurological function and infarct volume were assessed by using Long’s five-point scale system and TTC staining technique, respectively. The morphology of neurons in the cortex and hippocampus sections was examined by HE staining. ERK1/2, JNK and p38 phosphorylation, and apoptosis related protein of Bcl-2 and Bax were measured via Western blot in cortex and hippocampus tissue.Results:1. Compared with sham group, the neurological deficit score and infarct volume of I/R group were significantly increased and the brain tissue present clear pale grey. Compared with I/R group, treatment with medium and high vitexin and breviscapine injection could significantly reduced the neurological deficit score, infarct volume, ameliorated the cerebral ischemia/reperfusion injury.2. HE staining results show that the cellular morphology exhibited normally in sham group. There were deformation and swelling of nerve cells, pyknosis and anachromasis of nucleus as well as shrunken cell bodies in I/R group. Compared with I/R group, the medium and high vitexin and breviscapine markedly attenuated the neuronal damages. The pathological change of hippocampus CA1 neurons was more severe than cortical area.3. Compared with I/R group, medium and high vitexin groups significantly increased cortical and hippocampal p-ERK production. I/R injury significantly increased the levels of p-JNK and p-P38 compared with sham group, which were attenuated by vitexin treatment. In cortical tissues, low dose of vitexin did not significantly affect p-ERK and p-JNK level. Also, Low and medium vitexin did not affect the p-P38 level. In hippocampal tissues, low dose of vitexin did not significantly affect p-ERK level. Also low and medium vitexin did not affect the p-JNK and p-P38 levels.4. Compared with I/R group, medium and high vitexin groups significantly increased the Bcl-2 protein level in cortex and hippocampus. Also, medium and high vitexin groups inhibited the increase in cortical Bax expression induced by I/R injury. Moreover, high vitexin group inhibited an increase in hippocampal Bax expression compared to I/R group.Conclusion:The significant protective effect against cerebral ischemia/reperfusion injury in mice which exerted by vitexin may occur through modulating MAPK pathway and inhibiting apoptosis by downregulating Bax and upregulating Bcl-2, as well as reduction in neurological deficit score, cerebral infarct volume and histopathological damage.