Sevoflurane Preconditioning Confers Delayed Cardioprotection by Upregulating AMP-Activated Protein Kinase Levels to Restore Autophagic Flux in Ischemia-Reperfusion Rat Hearts.

BACKGROUND Volatile anesthetic preconditioning confers delayed cardioprotection against ischemia/reperfusion injury (I/R). AMP-activated protein kinase (AMPK) takes part in autophagy activation. Furthermore, autophagic flux is thought to be impaired after I/R. We hypothesized that delayed cardioprotection can restore autophagic flux by activating AMPK. MATERIAL AND METHODS All male rat hearts underwent 30-min ischemia and 120-min reperfusion with or without sevoflurane exposure. AMPK inhibitor compound C (250 µg/kg, iv) was given at the reperfusion period. Autophagic flux blocker chloroquine (10 mg/kg, ip) was administrated 1 h before the experiment. Myocardial infarction, nicotinamide adenine dinucleotide (NAD⁺) content, and cytochrome c were measured. To evaluate autophagic flux, the markers of microtubule-associated protein 1 light chain 3 (LC3) I and II, P62 and Beclin 1, and lysosome-associated membrane protein-2 (LAMP 2) were analyzed. RESULTS The delayed cardioprotection enhanced post-ischemic AMPK activation, reduced infarction, CK-MB level, NAD⁺ content loss and cytochrome c release, and compound C blocked these effects. Sevoflurane restored impaired autophagic flux through a lower ratio of LC3II/LC3I, downregulation of P62 and Beclin 1, and higher expression in LAMP 2. Consistently, compound C inhibited these changes of autophagy flux. Moreover, chloroquine pretreatment abolished sevoflurane-induced infarct size reduction, CK-MB level, NAD⁺ content loss, and cytochrome c release, with concomitant increase the ratios of LC3II/LC3I and levels of P62 and Beclin 1, but p-AMPK expression was not downregulated by chloroquine. CONCLUSIONS Sevoflurane exerts a delayed cardioprotective effects against myocardial injury in rats by activation of AMPK and restoration of I/R-impaired autophagic flux.

Sevoflurane postconditioning protects against myocardial ischemia/reperfusion injury by restoring autophagic flux via an NO-dependent mechanism.

Volatile anesthetics improve postischemic cardiac function and reduce infarction even when administered for only a brief time at the onset of reperfusion. A recent study showed that sevoflurane postconditioning (SPC) attenuated myocardial reperfusion injury, but the underlying mechanisms remain unclear. In this study, we examined the effects of sevoflurane on nitric oxide (NO) release and autophagic flux during the myocardial ischemia/reperfusion (I/R) injury in rats in vivo and ex vivo. Male rats were subjected to 30 min ischemia and 2 h reperfusion in the presence or absence of sevoflurane (1.0 minimum alveolar concentration) during the first 15 min of reperfusion. We found that SPC significantly improved hemodynamic performance after reperfusion, alleviated postischemic myocardial infarction, reduced nicotinamide adenine dinucleotide content loss, and cytochrome c release in heart tissues. Furthermore, SPC significantly increased the phosphorylation of endothelial nitric oxide synthase (NOS) and neuronal nitric oxide synthase, and elevated myocardial NOS activity and NO production. All these effects were abolished by treatment with an NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg, i.v.). We also observed myocardial I/R-induced accumulation of autophagosomes in heart tissues, as evidenced by increased ratios of microtubule-associated protein 1 light chain 3 II/I, up-regulation of Beclin 1 and P62, and reduced lysosome-associated membrane protein-2 expression. SPC significantly attenuated I/R-impaired autophagic flux, which were blocked by L-NAME. Moreover, pretreatment with the autophagic flux blocker chloroquine (10 mg/kg, i.p.) increased autophagosome accumulation in SPC-treated heart following I/R and blocked SPC-induced cardioprotection. The same results were also observed in a rat model of myocardial I/R injury ex vivo, suggesting that SPC protects rat hearts against myocardial reperfusion injury by restoring I/R-impaired autophagic flux via an NO-dependent mechanism.

Neuroprotective Effects of DTIO, A Novel Analog of Nec-1, in Acute and Chronic Stages After Ischemic Stroke.

Receptor-interacting protein 1 kinase (RIP1K) plays a key role in necroptosis. Necrostatin-1 (Nec-1), a specific inhibitor of RIP1K, provides neuroprotection against ischemic brain injury, associating with inhibition of inflammation. Recently, our group synthesized a novel analog of Nec-1, 5-(3',5'-dimethoxybenzal)-2-thio-imidazole-4-ketone (DTIO). The present study investigated the effect of DTIO on ischemic stroke-induced brain injury in both acute and chronic phase and its underlying mechanism. In vivo, DTIO treatment reduced infarct volume and improved neurological deficits in the acute phase after permanent middle cerebral artery occlusion (pMCAO) and it also attenuated brain atrophy and promoted brain functional recovery in the chronic phase post-cerebral ischemia/reperfusion (I/R). In vitro, DTIO treatment decreased lactate dehydrogenase (LDH) leakage and necrotic cell death in the oxygen and glucose deprivation (OGD) or oxygen and glucose deprivation and reoxygenation (OGD/R)-induced neuronal or astrocytic cell injury. Simultaneously, DTIO suppressed the production and release of inflammatory cytokines, and reduced the formation of glial scar. Homology modeling analysis illustrated that DTIO had an ability of binding to RIP1K. Furthermore, immunoprecipitation analysis showed that DTIO inhibited the phosphorylation of RIP1K and decreased the interaction between the RIP1K and RIP3K. In addition, knockdown of RIP1K had neuroprotective effects and inhibited the release of proinflammatory cytokines, but didn't have a significant effect on DTIO-mediated neuroprotection. In conclusion, DTIO has protective effects on acute ischemic stroke and promotes functional recovery during chronic phase, associating with protecting ischemic neurons and astrocytes, inhibiting inflammation, and lessening the glial scar formation via inhibiting of the RIP1K.

RIP1K Contributes to Neuronal and Astrocytic Cell Death in Ischemic Stroke via Activating Autophagic-lysosomal Pathway.

Although the receptor-interacting protein 1 kinase (RIP1K)-regulated necroptosis can be evoked by cerebral ischemia, the effects of RIP1K in mediating neuronal and astrocytic cell death and the underlying mechanisms remain poorly understood. This study evaluates the contribution of RIP1K to ischemic stroke-induced neuronal and astrocytic cell death, and the activation of autophagic-lysosomal pathway. Using an in vitro oxygen and glucose deprivation (OGD) in primary cultured neurons or astrocytes and a permanent middle cerebral artery occlusion (pMCAO) model in rats or mice, we observed the role of RIP1K in the ischemic neuronal and astrocytic cell death and the underlying mechanisms by pharmacological or genetic inhibition of RIP1K. pMCAO or OGD condition led to an increase in RIP1K, RIP3K and RIP1K-RIP3K complex. RIP1K knockdown or necrostatin-1 (Nec-1, a specific inhibitor of RIP1K) treatment reduced infarct volume, improved neurological deficits, increased microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) levels, and attenuated neuronal or astrocytic necrotic cell death in the ischemic cortex. RIP1K knockdown decreased RIP1K-RIP3K complex formation, light chain 3 II (LC3II) and active cathepsin B levels and lysosomal membrane permeability (LMP). Furthermore, a combination of Nec-1 and an inhibitor of autophagy or cathepsin B produced an enhancement of protective effect on neuronal or astrocytic cell death. RIP1K-mediated necroptosis may play important roles in ischemia-induced neuronal and astrocytic cell death through the activation of autophagic-lysosomal pathway.

Sevoflurane postconditioning attenuates reactive astrogliosis and glial scar formation after ischemia-reperfusion brain injury.

Cerebral ischemia leads to astrocyte's activation and glial scar formation. Glial scar can inhibit axonal regeneration during the recovery phase. It has demonstrated that sevoflurane has neuroprotective effects against ischemic stroke, but its effects on ischemia-induced formation of astrogliosis and glial scar are unknown. This study was designed to investigate the effect of sevoflurane postconditioning on astrogliosis and glial scar formation in ischemic stroke model both in vivo and in vitro. The results showed that 2.5% of sevoflurane postconditioning could significantly reduce infarction volume and improve neurologic deficits. And it could also decrease the expression of the glial scar marker glial fibrillary acidic protein (GFAP), neurocan and phosphacan in the peri-infarct region and markedly reduce the thickness of glial scar after ischemia/reperfusion (I/R). Consistent with the in vivo data, in the oxygen and glucose deprivation/reoxygenation (OGD/Re) model, sevoflurane postconditioning could protect astrocyte against OGD/Re-induced injury, decrease the expression of GFAP, neurocan and phosphacan. Further studies demonstrated that sevoflurane postconditioning could down-regulate the expression of Lamp1 and active cathepsin B, and block I/R or OGD/Re-induced release of cathepsin B from the lysosomes into cytoplasm. In order to confirm whether inhibition of cathepsin B could attenuate the formation of glial scar, we used cathepsin B inhibitor CA-074Me as a positive control. The results showed that inhibition of cathepsin B could decrease the expression of GFAP, neurocan and phosphacan. Taken together, sevoflurane postconditioning can attenuate astrogliosis and glial scar formation after ischemic stroke, associating with inhibition of the activation and release of lysosomal cathepsin B.

Inhibition of autophagy blocks cathepsins-tBid-mitochondrial apoptotic signaling pathway via stabilization of lysosomal membrane in ischemic astrocytes.

Our previous study and others have demonstrated that autophagy is activated in ischemic astrocytes and contributes to astrocytic cell death. However, the mechanisms of ischemia-induced autophagy remain largely unknown. In this study, we established a rat's model of permanent middle cerebral artery occlusion (pMCAO) and an in vitro oxygen and glucose deprivation (OGD) model. Autophagy was inhibited by either pharmacological treatment with 3-methyladenine (3-MA) and wortmannin (Wort) or genetic treatment with knockdown of Atg5 in primary cultured astrocytes and knockout of Atg5 in mouse embryonic fibroblast (MEF) cells, respectively. We found that pharmacological or genetic inhibition of autophagy reversed pMCAO or OGD-induced increase in LC3-II, active cathepsin B and L, tBid, active caspase-3 and cytoplastic cytochrome c (Cyt-c), and suppressed the injury-induced reduction in mitochondrial Cyt-c in ischemic cortex, in injured astrocytes and MEF cells. Immunofluorescence analysis showed that 3-MA or Wort treatment reversed OGD-induced release of cathepsin B and L from the lysosome to the cytoplasm and activation of caspase-3 in the astrocytes. Furthermore, treatment of 3-MA or Wort decreased OGD-induced increase in lysosomal membrane permeability and enhanced OGD-induced upregulation of lysosomal heat shock protein 70.1B (Hsp70.1B) in astrocytes. Inhibition of autophagy by 3-MA or Wort reduced infarction volume in rats and protected OGD-induced astrocytic cell injury. A non-selective caspase inhibitor z-VAD-fmk or a specific caspase-3 inhibitor Q-DEVD-OPh also rescued OGD-induced astrocytic cell injury. In conclusion, our presenting data suggest that inhibition of autophagy blocks cathepsins-tBid-mitochondrial apoptotic signaling pathway via stabilization of lysosomal membranes, possibly due to upregulation of the lysosomal Hsp70.1B in ischemic astrocytes.