PI3K/Akt pathway contributes to neuroprotective effect of Tongxinluo against focal cerebral ischemia and reperfusion injury in rats.
Tongxinluo (TXL), a compound prescription, is formulated according to the collateral disease doctrine of traditional Chinese medicine, and is widely used for the treatment of cardio-cerebrovascular diseases in China.
We aimed to investigate the neuroprotective effect of TXL on focal cerebral ischemia and reperfusion injury in rats by attenuating its brain damage and neuronal apoptosis, and to assess the potential role of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in this protection.
Adult Male Sprague-Dawley rats (n=120) were randomly divided into 5 groups: sham, cerebral ischemia and reperfusion (I/R), cerebral ischemia and reperfusion plus TXL (1.6g/kg/day) (TXL1.6), TXL1.6 plus LY294002 and dimethyl sulfoxide (DMSO) (TXL1.6+LY294002), TXL1.6 plus DMSO (TXL1.6+vehicle). Prior to the grouping, TXL1.6 was selected to be the optimal dose of TXL by evaluating the neurological deficits score of five group rats (Sham, I/R, TXL0.4, TXL0.8 and TXL1.6, n=30) at 0, 1, 3, 5, and 7 days after reperfusion. Rats, being subjected to middle cerebral artery occlusion (MCAO) for 90min followed by 24h reperfusion, were the cerebral ischemia/reperfusion models. At 24h after reperfusion, cerebral infarct area was measured via tetrazolium staining and neuronal damage was showed by Nissl staining. The double staining of Terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) staining and immunofluorescence labeling with NeuN, was performed to evaluate neuronal apoptosis. Proteins involved in PI3K/Akt pathway were detected by Western blot.
The results showed that TXL markedly improved neurological function, reduced cerebral infarct area, decreased neuronal damage, and significantly attenuated neuronal apoptosis, while these effects were eliminated by inhibition of PI3K/Akt with LY294002. We also found that TXL up-regulated the expression levels of p-PDK1, p-Akt, p-c-Raf, p-BAD and down-regulated Cleaved caspase 3 expression notably, which were partially reversed by LY294002. Additionally, the increment of p-PTEN level on which LY294002 had little effect was also detected in response to TXL treatment.
These findings demonstrated that TXL provided neuroprotection against cerebral ischemia/reperfusion injury and neuronal apoptosis, and this effect was mediated partly by activation of the PI3K/Akt pathway.
Yu ZH
，Cai M
，Xiang J
，Zhang ZN
，Zhang JS
，Song XL
，Zhang W
，Bao J
，Li WW
，Cai DF
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Activation of Akt by SC79 decreased cerebral infarct in early cerebral ischemia-reperfusion despite increased BBB disruption.
Activation of Akt has been suggested to produce neuronal protection in cerebral ischemia. Decreasing blood-brain barrier (BBB) disruption has been associated with a better neuronal outcome in cerebral ischemia. We hypothesized that activation of Akt would decrease BBB disruption and contribute to decreasing the size of infarct in the early stage of cerebral ischemia-reperfusion within the therapeutic window. Transient middle cerebral artery occlusion (MCAO) was performed in rats under isoflurane anesthesia with controlled ventilation. Rats were treated with SC79 (a selective Akt activator which is cell and BBB permeable) 0.05 mg/kg × 3 i.p. or vehicle i.p. perioperatively. After one hour of MCAO and two hours of reperfusion, the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid (14C-AIB, molecular weight 104 Da) and the volume of 3H-dextran (molecular weight 70,000 Da) distribution were determined to measure the degree of BBB disruption. At the same time point, the size of infarction was determined using tetrazolium staining. In an additional group of rats, a higher dose of SC79 (0.5 mg/kg × 3) was administered to determine the size of infarct. Administration of SC79 increased the Ki in the ischemic-reperfused cortex (IR-C, +32%, p < 0.05) as well as in the contralateral cortex (CC, +35%, p < 0.05) when compared with the untreated animals with MCAO/reperfusion. The volume of dextran distribution was not significantly changed by SC79. SC79 treatment significantly produced a decrease in the percentage of cortical infarct out of total cortical area (12.7 ± 1.7% vs 6.9 ± 0.9%, p < 0.001). Increasing the dose of SC79 by ten times did not significantly affect the size of cortical infarct. Contrary to our hypothesis, our data demonstrated that SC79 decreased the size of the infarct in the ischemic-reperfused cortex despite an increase in BBB disruption. Our data suggest the importance of activation of Akt for neuronal survival in the early stage of cerebral ischemia-reperfusion within the therapeutic window and that the mechanism of neuroprotection may not be related to the BBB effects of SC79.
Liu X
，Kiss GK
，Mellender SJ
，Weiss HR
，Chi OZ
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Tamibarotene Improves Hippocampus Injury Induced by Focal Cerebral Ischemia-Reperfusion via Modulating PI3K/Akt Pathway in Rats.
The present study aimed to examine whether Am80 (tamibarotene) protects the hippocampus against cerebral ischemia-reperfusion (I/R) injury and whether phosphoinositide-3-kinase/Akt (PI3K/Akt) pathway mediates this effect.
Rats were subjected to 90 minutes of middle cerebral artery occlusion followed by 24 hours of reperfusion. The animals were randomly divided into 7 groups: sham-operated group; I/R group; groups pretreated with 2 mg/kg, 6 mg/kg, and 10 mg/kg of Am80; Am80 (6 mg/kg) combined with the selective PI3K inhibitor wortmannin (0.6 mg/kg), and wortmannin (0.6 mg/kg) only group. After 24 hours of reperfusion, neurological deficits and infarct volume were measured. Pathological changes in hippocampal neurons were analyzed by transmission electron microscopy. Neuronal survival was examined by TUNEL staining. The expression of Bcl-2, Bax, and Akt, and Akt phosphorylation (p-Akt) were measured by Western blotting and quantitative real-time polymerase chain reaction.
The pretreatment with Am80 improved the neurologic deficit score, reduced infarct volume, and decreased the number of TUNEL-positive cells in the hippocampus. Moreover, Am80 pretreatment downregulated the expression of Bax, upregulated the expression of Bcl-2, and increased the level of p-Akt. Wortmannin abolished in part the increase in p-Act and the neuroprotective effect exerted on the ischemic by Am80 pretreatment.
Our results documented that Am80 pretreatment protects ischemic hippocampus after cerebral I/R by regulating the expression of apoptosis-related proteins through the activation of the PI3K/Akt signaling pathway.
Tian X
，An R
，Luo Y
，Li M
，Xu L
，Dong Z
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