Lina Yu

Both PI3K/Akt and ERK1/2 pathways participate in the protection by dexmedetomidine against transient focal cerebral ischemia/reperfusion injury in rats

Dexmedetomidine (Dex) has been demonstrated to provide neuroprotection against ischemia/reperfusion (I/R) injury. However, the exact mechanism of this protection remains unknown. Here, we explored the neuroprotective effect of Dex in rats exposed to cerebral I/R-induced by middle cerebral artery occlusion (MCAO) and the role of phosphatidylinositol 3-kinase (PI3K)/Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), and glycogen synthase kinase-3β (GSK-3β) in this protective action. Adult male Sprague-Dawley rats were subjected to MCAO for 90 min followed by reperfusion for 24h and Dex (15 μg/kg, i.v.) was administered immediately after the onset of MCAO. The neurological deficit score, cerebral infarct volume, brain edema, and neuron survival were evaluated at 24h of reperfusion. The effect of Dex on p-Akt, p-ERK1/2 and p-GSK-3β expression in the ischemic hemisphere was assayed by Western blot. Treatment of rats exposed to I/R with Dex caused not only marked reduction in the neurological deficit score, cerebral infarct volume, and brain edema (P <0.01 vs. I/R alone), but also a decrease in neuron death in hippocampal CA1 and cortex (P<0.01 vs. I/R alone). The Dex-induced increment of neuron survival in the ischemic CA1 and cortex was diminished by the PI3K inhibitor LY294002 and the MEK inhibitor U0126. The increasing expressions of p-Akt and p-ERK1/2 induced by Dex in the ischemic hemisphere were markedly inhibited by LY294002 (or wortmannin) and U0126 (or PD98059), respectively. The up-regulation of p-GSK-3β by Dex in the ischemic hemisphere was significantly decreased by both LY294002 (or wortmannin) and U0126 (or PD98059). Our data demonstrated that treatment with Dex reduced cerebral injury in rats exposed to transient focal I/R, and this was mediated by the activation of the PI3K/Akt and ERK1/2 pathways as well the phosphorylation of downstream GSK-3β.

Postconditioning with sevoflurane protects against focal cerebral ischemia and reperfusion injury via PI3K/Akt pathway

Emerging evidence has demonstrated that postconditioning with sevoflurane provided neuroprotection. In this study, we investigated the neuroprotective effect of different concentrations of sevoflurane in rats with middle cerebral artery occlusion (MCAO). Furthermore, we tested the hypothesis that the neuroprotective effect of postconditioning with sevoflurane is associated with inhibition of apoptosis and mediated by activation of the phosphoinositide-3-kinase/Akt (PI3K/Akt) pathway. Adult male Sprague-Dawley rats were subjected to MCAO for 90 min and then treated with sevoflurane at the beginning of reperfusion. The infarct volume, neurological deficit scores and brain edema were evaluated at 24 hours. Spatial learning and memory was examined by Morris water maze. Apoptosis and apoptosis-related proteins were studied by TUNEL, immunohistochemistry and western blot. The neuroprotective effect and the amount of p-Akt after sevoflurane administration with or without wortmannin were analyzed. Postconditioning with sevoflurane 1.0 minimum alveolar concentration (MAC) and 1.5 MAC significantly decreased neurological deficit scores, infarct volume and brain edema and markedly improved spatial learning and memory. Postconditioning also reduced apoptotic cells, upregulated Bcl-2 and downregulated P53 and Bax. Wortmannin abolished the neuroprotective effect and prevented the increasing of p-Akt. Our data suggest postconditioning with sevoflurane (1.0 MAC and 1.5 MAC) not only reduced infarct volume but also improved learning and memory. Our study further showed that this neuroprotective effect may be partly due to the activation of PI3K/Akt pathway and inhibiting neuronal apoptosis.

Gender-Related Difference of Sevoflurane Postconditioning in Isolated Rat Hearts: Focus on Phosphatidylinositol-3-Kinase/Akt Signaling

BACKGROUND Previous studies have reported that female gender confers cardioprotection against ischemia/reperfusion (I/R) injury, partly because estrogen activates phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway. We have previously proven that cardioprotection of sevoflurane postconditioning is mediated by PI3K/Akt pathway in male rats. The purpose of the present study was to determine whether the cardioprotection of sevoflurane postconditioning is influenced by gender, and the role of PI3K/Akt pathway in such gender difference. MATERIALS AND METHODS Isolated hearts from 2-mo-old male and female SD rats were subjected to ischemia for 40 min and reperfusion for 2 h in the Langendorff apparatus, and were randomly assigned to the following groups: no ischemia/reperfusion (CON), ischemia/reperfusion (I/R), I/R+sevoflurane postconditioning (I/R+SPC), I/R+100 nM wortmannin (I/R+WOR), and I/R+SPC+WOR. Postconditioning was performed with administration of 3.0% sevoflurane at the first 10 min of reperfusion. Left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), and myocardial lactate dehydrogenase (LDH) release were measured. Infarct size was detected by riphenyltetrazolium chloride staining. The protein expression of total Akt (t-Akt) and phosphorylated Akt (Ser(473)) (p-Akt) were determined by Western blot. RESULTS The I/R group showed lower LVDP and higher LVEDP than CON group in the same gender during reperfusion period. The LDH release and infarct size were smaller in the female I/R group (P < 0.05 versus male I/R group). Sevoflurane postconditioning markedly improved left ventricular function and decreased LDH, infarct size in the male I/R+SPC group (P < 0.05 versus male I/R group) but not in the female I/R+SPC group. Wortmannin abolished the cardioprotection of sevoflurane postconditioning in the male I/R+SPC+Wort group (P < 0.05 versus male I/R+SPC group), and markedly increased the infarct size and LVEDP and decreased LVDP in female rats. The t-Akt protein expression was no significant difference in all groups. The ratio of p-Akt/t-Akt expression in the male CON group was a little lower than that in the female CON group, but there was no statistical significance. In male rats, the ratio of p-Akt/t-Akt was no difference between CON and I/R group, but it was higher in I/R+SPC group than that in I/R group (P < 0.05). In female rats, the level of p-Akt was markedly increased by I/R, which was markedly higher than that in male I/R group (P < 0.05). However, p-Akt was not different between I/R and I/R+SPC groups. Wortmannin decreased the p-Akt expression in both male and female rats. CONCLUSIONS It is concluded that female rat hearts showed greater resistance to I/R injury, and sevoflurane postconditioning developed cardioprotection in male rats but not in female rats. The PI3K/Akt pathway may be involved in the cardioprotection by both sevoflurane postconditioning and gender.

Dexmedetomidine protects against oxygen–glucose deprivation-induced injury through the I2 imidazoline receptor-PI3K/AKT pathway in rat C6 glioma cells

Objectives  To explore the protection and the mechanism of dexmedetomidine on the oxygen–glucose deprivation (OGD) insults in rat C6 glioma cells.

Effects of dexmedetomidine on the release of glial cell line-derived neurotrophic factor from rat astrocyte cells

Dexmedetomidine (DEX) has been found to improve neuronal survival after transient global or focal cerebral ischemia in rats. Astrocyte cells may possess beneficial properties that promote neuronal recovery by secreting neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF). The purpose of this study was to investigate the effects of DEX on GDNF release from astrocytes and the possible mechanisms involved. Astrocyte cells were treated with DEX, and GDNF level in the conditioned media was determined by ELISA assay. The expression of CREB, p-CREB and PKCα was analyzed by Western blotting to explore the mechanisms involved in GDNF release. Our results showed that DEX stimulated GDNF release in a time- and dose-dependent manner; and this stimulation was blocked by the α2-adrenoreceptor antagonist yohimbine, but not by α1-adrenoreceptor antagonist prasozin, demonstrating that DEX induced GDNF release likely acts via activating the α2A adrenoreceptor. In addition, DEX-stimulated GDNF release was also blocked by the universal PKC inhibitor Ro-318220 and PKCα/β inhibitor Gö 6976, but not by PKCδ inhibitor rottlerin and PKCβ inhibitor LY333531. Interestingly, DEX also activated CREB phosphorylation, which was inhibited by Ro-318220, Gö 697 and ERK kinase inhibitor PD98059. Silencing CREB by siRNA decreased the DEX-stimulated GDNF release. In addition, the membrane translocation of PKCα was enhanced following DEX treatment. Furthermore, we found that DEX stimulated GDNF release rescued neurons against OGD-induced neurotoxicity; this effect was partly abolished by GDNF antibody. Thus, through α2A adrenergic receptors, DEX may activate astrocytes, and promote GDNF release to protect neurons after stroke, and this signaling is possibly dependent on PKCα and CREB activation.

CX3CL1/CX3CR1 regulates nerve injury-induced pain hypersensitivity through the ERK5 signaling pathway.

Peripheral nerve injury induces the cleavage of CX3CL1 from the membrane of neurons, where the soluble CX3CL1 subsequently plays an important role in the transmission of nociceptive signals between neurons and microglia. Here we investigated whether CX3CL1 regulates microglia activation through the phosphorylation of extracellular signal‐regulated protein kinase 5 (ERK5) in the spinal cord of rats with spinal nerve ligation (SNL). ERK5 and microglia were activated in the spinal cord after SNL. The knockdown of ERK5 by intrathecal injection of antisense oligonucleotides suppressed the hyperalgesia and nuclear impact of nuclear factor‐κB induced by SNL. The blockage of CX3CR1, the receptor of CX3CL1, significantly reduced the level of ERK5 activation following SNL. In addition, the antisense knockdown of ERK5 reversed the CX3CL1‐induced hyperalgesia and spinal microglia activation. Our study suggests that CX3CL1/CX3CR1 regulates nerve injury‐induced pain hypersensitivity through the ERK5 signaling pathway.

Hypercholesterolemia blocked sevoflurane-induced cardioprotection against ischemia–reperfusion injury by alteration of the MG53/RISK/GSK3β signaling

BACKGROUND Recent studies have demonstrated that volatile anesthetic preconditioning confers myocardial protection against ischemia-reperfusion (IR) injury through activation of the reperfusion injury salvage kinase (RISK) pathway. As RISK has been shown to be impaired in hypercholesterolemia, we investigate whether anesthetic-induced cardiac protection was maintained in hypercholesterolemic rats. METHODS Normocholesteolemic or hypercholesterolemic rat hearts were subjected to 30 min of ischemia and 2 h of reperfusion. Animals received 2.4% sevoflurane during three 5 min periods with and without PI3K antagonist wortmannin (10 μg/kg, Wort) or the ERK inhibitor PD 98059 (1 mg/kg, PD). The infarct size, apoptosis, p-Akt, p-ERK1/2, p-GSK3β were determined. RESULTS Two hundred and six rats were analyzed in the study. In the healthy rats, sevoflurane significantly reduced infarct size by 42%, a phenomenon completely reversed by wortmannin and PD98059 and increased the phosphorylation of Akt, ERK1/2 and their downstream target of GSK3β. In the hypercholesterolemic rats, sevoflurane failed to reduce infarct size and increase the phosphorylated Akt, ERK1/2 and GSK3β. In contrast, GSK inhibitor SB216763 conferred cardioprotection against IR injury in healthy and hypercholesterolemic hearts. CONCLUSIONS Hyperchoesterolemia abrogated sevoflurane-induced cardioprotection against IR injury by alteration of upstream signaling of GSK3β and acute GSK inhibition may provide a novel therapeutic strategy to protect hypercholesterolemic hearts against IR injury.

PI3K Contributed to Modulation of Spinal Nociceptive Information Related to ephrinBs/EphBs

There is accumulating evidence to implicate the importance of EphBs receptors and ephrinBs ligands were involved in modulation of spinal nociceptive information. However, the downstream mechanisms that control this process are not well understood. In the present study, we investigated whether phosphatidylinositol 3-kinase (PI3K), as the downstream effectors, participates in modulation of spinal nociceptive information related to ephrinBs/EphBs. Intrathecal injection of ephrinB1-Fc produced a dose- and time-dependent thermal and mechanical hyperalgesia, accompanied by the increase of spinal PI3K-p110γ, phosphorylation of AKT (p-AKT) and c-Fos expression. Pre-treatment with PI3K inhibitor wortmannin or LY294002 prevented activation of spinal AKT induced by ephrinB1-Fc. Inhibition of spinal PI3K signaling dose-dependently prevented and reversed pain behaviors and spinal c-Fos protein expression induced by intrathecal injection of ephrinB1-Fc. Inhibition of EphBs receptors by intrathecal injection of EphB1-Fc reduced formalin-induced inflammation and chronic constrictive injury-induced neuropathic pain behaviors accompanied by decreased expression of spinal PI3K,p-AKT and c-Fos protein. Furthermore, pre-treatment with PI3K inhibitor wortmannin or LY294002 prevented ephrinB1-Fc-induced ERK activation in spinal. These data demonstrated that PI3K and PI3K crosstalk to ERK signaling contributed to modulation of spinal nociceptive information related to ephrinBs/EphBs.

Sevoflurane postconditioning reduces myocardial reperfusion injury in rat isolated hearts via activation of PI3K/Akt signaling and modulation of Bcl-2 family proteins

Sevoflurane postconditioning reduces myocardial infarct size. The objective of this study was to examine the role of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway in anesthetic postconditioning and to determine whether PI3K/Akt signaling modulates the expression of pro- and antiapoptotic proteins in sevoflurane postconditioning. Isolated and perfused rat hearts were prepared first, and then randomly assigned to the following groups: Sham-operation (Sham), ischemia/reperfusion (Con), sevoflurane postconditioning (SPC), Sham plus 100 nmol/L wortmannin (Sham+Wort), Con+Wort, SPC+Wort, and Con+dimethylsulphoxide (DMSO). Sevoflurane postconditioning was induced by administration of sevoflurane (2.5%, v/v) for 10 min from the onset of reperfusion. Left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), maximum increase in rate of LVDP (+dP/dt), maximum decrease in rate of LVDP (−dP/dt), heart rate (HR), and coronary flow (CF) were measured at baseline, R30 min (30 min of reperfusion), R60 min, R90 min, and R120 min. Creatine kinase (CK) and lactate dehydrogenase (LDH) were measured after 5 min and 10 min reperfusion. Infarct size was determined by triphenyltetrazolium chloride staining at the end of reperfusion. Total Akt and phosphorylated Akt (phospho-Akt), Bax, Bcl-2, Bad, and phospho-Bad were determined by Western blot analysis. Analysis of variance (ANOVA) and Student-Newman-Keuls’ test were used to investigate the significance of differences between groups. The LVDP, ±dP/dt, and CF were higher and LVEDP was lower in the SPC group than in the Con group at all points of reperfusion (P<0.05). The SPC group had significantly reduced CK and LDH release and decreased infarct size compared with the Con group [(22.9±8)% vs. (42.4±9.4)%, respectively; P<0.05]. The SPC group also had increased the expression of phospho-Akt, Bcl-2, and phospho-Bad, and decreased the expression of Bax. Wortmannin abolished the cardioprotection of sevoflurane postconditioning. Sevoflurane postconditioning may protect the isolated rat heart. Activation of PI3K and modulation of the expression of pro- and antiapoptotic proteins may play an important role in sevoflurane-induced myocardial protection.

Increased methylation of the MOR gene proximal promoter in primary sensory neurons plays a crucial role in the decreased analgesic effect of opioids in neuropathic pain

BackgroundThe analgesic potency of opioids is reduced in neuropathic pain. However, the molecular mechanism is not well understood.ResultsThe present study demonstrated that increased methylation of the Mu opioid receptor (MOR) gene proximal promoter (PP) in dorsal root ganglion (DRG) plays a crucial role in the decreased morphine analgesia. Subcutaneous (s.c.), intrathecal (i.t.) and intraplantar (i.pl.), not intracerebroventricular (i.c.v.) injection of morphine, the potency of morphine analgesia was significantly reduced in nerve-injured mice compared with control sham-operated mice. After peripheral nerve injury, we observed a decreased expression of MOR protein and mRNA, accompanied by an increased methylation status of MOR gene PP, in DRG. However, peripheral nerve injury could not induce a decreased expression of MOR mRNA in the spinal cord. Treatment with 5-aza-2′-deoxycytidine (5-aza-dC), inhibited the increased methylation of MOR gene PP and prevented the decreased expression of MOR in DRG, thereby improved systemic, spinal and periphery morphine analgesia.ConclusionsAltogether, our results demonstrate that increased methylation of the MOR gene PP in DRG is required for the decreased morphine analgesia in neuropathic pain.