| Part I Xiao-Xu-Ming decoction attenatues neurological injury and Brain-barrier disruption induced by cerebral ischemia and reperfusion in ratsObjective:In this part, we focused on identifying the effects of Xiao-Xu-Ming decoction (XXMD) on neurological injury and blood-brain barrier (BBB) disruption induced by focal cerebral ischemia and reperfusion.Methods:120 Male Sprague-Dawley rats were randomly divided into 5 groups:sham control(Sham), ischemia and reperfusion(IR), IR plus 15 g/kg/day XXMD(XXMD15), IR plus 30 g/kg/day XXMD(XXMD30), and IR plus 60 g/kg/day XXMD(XXMD60). The stroke model was induced by 90 min of middle cerebral artery occlusion followed by reperfusion. The brain lesion areas were evaluated by 2,3,5-triphenyltetrazolium chloride staining and pathological changes were estimated by hematoxylin-eosin (HE) staining at 24 h after reperfusion. Moreover, neurological deficits were observed at different time points after reperfusion. BBB dysfunction was evaluated by assessing brain water content and Evans blue content following 90 min of ischemia and 24 h of reperfusion. Pathological changes in BBB ultrastructure were observed with transmission electron microscopy. MMP-9, MMP-2 and VEGF expression levels were quantitatively determined by western blot and immunohistochemistry.Results:The results indicated that XXMD (30/60 g/kg/day) treatment significantly reduced cerebral infarct area, hemispheric swelling, ischemic injury and makedly improved behavioral function compared with IR group. Increasingly, we also found that XXMD (60 g/kg/day) treatment attenuated ultrastructure damage and permeability of BBB following ischemia and reperfusion. Moreover, XXMD downregulated the expression levels of MMP-9, MMP-2 and VEGF.Conclusions:These findings indicated that XXMD treatment (30/60 g/kg/day) could notably prevent brain damage following cerebral ischemia and reperfusion, and XXMD (60 g/kg/day) exert the maximal neuroprotective effects on cerebral ischemia. XXMD (60 g/kg/day) alleviated BBB disruption and cerebral ischemic injury, which may be achieved by inhibiting the expression levels of MMP-9, MMP-2, and VEGF.Part II PI3K/Akt pathway contributes to neurovascular unit protection of Xiao-Xu-Ming decoction against focal cerebral ischemia and reperfusion injury in ratsObjective:In this part, we focused on investigating the protective effects of Xiao-Xu-Ming Decoction (XXMD) on neurovascular unit and examining the role of PI3K (phosphatidylinositol 3-kinase)/Akt pathway in this protection.Methods:106 Male Sprague-Dawley rats were randomly divided into 5 groups:sham control (Sham), ischemia and reperfusion (I/R), I/R plus XXMD (60 g/kg/day)(XXMD60), I/R plus XXMD plus LY294002 (XXMD60+LY294002), and I/R plus XXMD plus Vehicle (XXMD60+Vehicle). The cerebral ischemia and reperfusion injury was induced by 90 min of middle cerebral artery occlusion. Cerebral infarct area was measured by tetrazolium staining and neurological function was observed at 24 h after reperfusion. Nissl staining was performed with toluidine blue to assess brain injury. DNA fragmentation assay combined with immunofluorescence were performed to evaluate apoptosis of neuron, astrocyte and vascular endothelial cell which constitute neurovascular unit. The expression levels of proteins involved in PI3K/Akt pathway were detected by western blot.Results:The results showed that XXMD (60 g/kg/day) improved neurological function, decreased cerebral infarct area and neuronal damage, and attenuated cellular apoptosis in neurovascular unit, while these effects were abolished by inhibition of PI3K/Akt with LY294002. We also found that XXMD up-regulated p-PDK1(Ser241), p-Akt(Ser473), p-GSK3β(Ser9) expression levels, which were partly reversed by LY294002. In addition, the increases of p-PTEN(Ser380), p-c-Raf(Ser259) expression levels which LY294002 had no effect on were also observed in response to XXMD treatment.Conclusions:The data indicated that the protective effects of XXMD on neurovascular unit partly through the activation of PI3K/Akt pathway.Part III Xiao-Xu-Ming decoction preserves mitochondrial integrity and reduces apoptosis after focal cerebral ischemia and reperfusion via the mitochondrial p53 pathwayObjective:The purpose of this part was to investigate the effects of XXMD on mitochondrial damage and apoptosis after cerebral ischemia and reperfusion.Methods:85 Male Sprague-Dawley rats were randomly divided into 3 groups:Sham, cerebral ischemia and reperfusion (I/R), and cerebral ischemia and reperfusion plus XXMD (60 g/kg/day) (XXMD60). Focal cerebral ischemia and reperfusion models were induced by middle cerebral artery occlusion. After 24 h of reperfusion, the rats were sacrificed and the brain tissues were collected for the following investigations. Ultrastructural features of mitochondria in the penumbra of the ischemic cortex were analyzed by transmission electron microscopy. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) staining and cleaved caspase 3 immunohistochemistry. The mitochondrial and cytoplasmic fractions were obtained at 24 h of reperfusion following 90 min of middle cerebral artery occlusion, and then proteins in the mitochondrial p53 pathway were detected by western blot and immunofluorescence.Results:The results showed that XXMD treatment markedly preserved mitochondrial integrity, and significantly reduced apoptosis. In addition, we found that XXMD treatment reduced p53 and Bax levels and increased Bcl-2 levels in mitochondrial fractions. XXMD significantly blocked the release of cytochrome c and Smac/Diablo from mitochondria, and inhibited activation of caspase 9 and caspase 3 in cytoplasmic fractions. Increased expression of c-IAP1 was observed in the XXMD60 group.Conclusions:The findings demonstrated that XXMD protected mitochondria from ischemic injury and inhibited apoptosis. The mitochondrial p53 pathway could be partially involved in the protective effects. |
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