| Objectives:1. To detect the change of AMP-activated protein kinase (AMPK) activity in ischemic brain tissue and to investigate the significance of regulation of AMPK activity in cerebral ischemia reperfusion injury.2. To observe the change of astrocytes and microglias in ischemic brain tissue, to explore the impact of inhibition of AMPK activity on them and to examine the influence of AMPK activity change for nerve inflammation and upstream NF kappa B signaling pathway after cerebral ischemia reperfusion injury.3. To observe neuronal apoptosis in ischemic brain tissue after cerebral ischemia reperfusion injury and to investigate the impact of inhibiting AMPK activity on neuronal apoptosis and the related mechanism.Methods:Adult healthy male Kunming mice were randomly divided into three groups: sham group, saline control group (MCAO group) and Compound C intervention group (MCAO +CC group). Mice underwent 60min middle cerebral artery occlusion followed by a 24 hour reperfusion (MCAO/R), receiving either saline (MCAO group) or Compound C (20mg/kg ip) treatment(MCAO +CC group) immediately when the line was inserted. No drug was given to the sham group.1. Western Blot method was applied to detect pAMPK protein expression in ischemic brain tissue. Neural function defect scale was used to evaluate nerve function defect symptoms in mice. TTC staining was adopted to determine the volume of cerebral infarction.2. The expressions of astrocyte's specific marker GFAP and microglia's specific marker Ibal were characterized by immunohistochemical staining (IHC). RT-PCR method was adopted to detect Cx43 mRNA expression in ischemic brain tissue. The contents of TNFa and IL-1? were evaluated by ELISA method. Western Blot method was used to detect iNOS and NF?B/p65 protein expressions.3. TUNEL staining was used to observe neuronal apoptosis in ischemic brain tissue. The expression of AIF was characterized by IHC. Western Blot method was used to detect Cyt. C protein expression in the cytoplasm.Results:1. There was slight pAMPK protein expression in brain tissue of sham group mice. Compared with sham group, MCAO group mice had obvious nerve function defect symptoms (P<0.01) and focal cerebral infarcts (P<0.01). PAMPK protein expression significantly increased (P<0.01) in ischemic brain tissue of MCAO group mice. After Compound C was administrated, MCAO +CC group mice had significantly reduced nerve function score (P<0.05) and significantly decreased cerebral infarction volume (P<0.01). Meanwhile, pAMPK protein expression was significantly inhibited (cortex:P<0.01; hippocampus:P<0.05) in ischemic brain tissue of MCAO+CC group mice.2. After cerebral ischemia-reperfusion injury, immunohistochemical results showed that the numbers of GFAP-and Ibal-positive cells dramatically increased in the ischemic hippocampus (P<0.01) and cortex (P<0.05) of MCAO group mice, respectively. In ischemic brain tissue, Cx43 mRNA expression was significantly reduced (P<0.05), while TNF-? (P<0.01), IL-1? (P<0.01) and iNOS (P<0.05) expressions were significantly increased. At the same time, NF?B/p65 protein expression was significantly increased (P<0.01) in the nucleus of ischemic brain tissue. Compound C treatment caused that:?the numbers of GFAP- and Ibal-positive cells dramatically reduced in the ischemic hippocampus (P<0.05) and cortex (P<0.05) of MCAO+CC group mice, respectively. ?in ischemic brain tissue, Cx43 mRNA expression was significantly increased (P<0.05), while TNF-? (P<0.01), IL-1? (P<0.01) and iNOS (P<0.05) expressions were significantly decreased. ?in the nucleus of ischemic brain tissue, NF?B/p65 protein expression was significantly decreased (cortex:P<0.01, hippocampus:P<0.05).3. After cerebral ischemia-reperfusion injury, TUNEL positive cells dramatically increased (P<0.05) in ischemic brain tissue of MCAO group mice. Immunohistochemical results showed that the numbers of AIF-positive cells dramatically increased (P<0.05) in the ischemic brain tissue. Also, Cyt.C protein expression was significantly increased (P<0.01) in the cytoplasm of ischemic brain tissue. However, inhibiting AMPK activity caused some changes. TUNEL positive cells dramatically decreased (P<0.05) in ischemic brain tissue of MCAO+CC group mice. AIF-positive cells dramatically decreased (P<0.05) in the ischemic brain tissue. Meanwhile, Cyt.C protein expression was also significantly decreased (cortex:P<0.05, hippocampus:P<0.01) in the cytoplasm of ischemic brain tissue.Conclusion:1. After mice were subject to cerebral ischemia-reperfusion injury, AMPK was excessively activated in the ischemic brain tissue. Mice had obvious neurologic deficit symptoms and focal cerebral infarction. Inhibition of AMPK activity can greatly improve mice's neurologic deficit symptoms and reduce mice's cerebral infarction volume. The above shows that inhibiting AMPK activity may play certain nerve protective effect to cerebral ischemia reperfusion injury.2. Inhibiting AMPK activity can suppress the excessive activation of astrocytes and microglias, stabilize the protection effect of astrocytes and alleviate the glial cells-mediated neuroinflammation injury. Further, the mechanism of anti-inflammation of inhibiting AMPK activity may be related to inhibiting inflammatory upstream NF?B signaling pathways.3. After mice were subject to cerebral ischemia-reperfusion injury, neurons apoptosis was obvious in the ischemic brain tissue. At the same time, AIF and Cyt. C releases were increased. After inhibiting AMPK activity, neuron apoptosis was decreased, while AIF and Cyt C releases were also reduced. The results show that inhibiting AMPK activity can inhibit the neuron apoptosis. Its anti-apoptotic effect may be related to suppressing mitochondria-mediated apoptosis pathway. |
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