Since several different pathways are involved in cerebral ischemia/reperfusion injury, combination therapy rather than monotherapy may be required for efficient neuroprotection. In this study, we examined the protective effects of an apoptosis inhibitor Gly 14 -humanin (HNG) and a necroptosis inhibitor necrostatin-1 (Nec-1) on hypoxia/ischemia/reperfusion injury. Cultured mouse primary cortical neurons were incubated with Nec-1, HNG or both in a hypoxia chamber for 60 min. Cell viability was determined by MTS assay at 24 h after oxygen-glucose deprivation (OGD) treatment. Mice underwent middle cerebral artery occlusion for 75 min followed by 24 h reperfusion. Mice were administered HNG and/or Nec-1 (i.c.v.) at 4 h after reperfusion. Neurological deficits were evaluated and the cerebral infarct volume was determined by TTC staining. Nec-1 or HNG alone had protective effects on OGD-induced cell death. Combined treatment with Nec-1 and HNG resulted in more neuroprotection than Nec-1 or HNG alone. Treatment with HNG or Nec-1 reduced cerebral infarct volume from 59.3 ± 2.6% to 47.0 ± 2.3% and 47.1 ± 1.5%, respectively. Combined treatment with HNG and Nec-1 improved neurological scores and decreased infarct volume to 38.6 ± 1.5%. In summary, we demonstrated that the combination treatment of HNG and Nec-1 conferred synergistic neuroprotection on hypoxia/ ischemia/reperfusion injury in vitro and in vivo. These findings provide a novel therapeutic strategy for the treatment of stroke by combining anti-apoptosis and anti-necroptosis therapy.
Humanin (HN) is an anti-apoptotic peptide that suppresses neuronal cell death induced by Alzheimer's disease, prion protein fragments, and serum deprivation. Recently, we demonstrated that Gly14-HN (HNG), a variant of HN in which the 14th amino acid serine is replaced with glycine, can decrease apoptotic neuronal death and reduce infarct volume in a focal cerebral ischemia/reperfusion mouse model. In this study, we postulate that the mechanism of HNG's neuroprotective effect is mediated by the PI3K/Akt pathway. Oxygen-glucose deprivation (OGD) was performed in cultured mouse primary cortical neurons for 60 min. The effect of HNG and PI3K/Akt inhibitors on OGD-induced cell death was examined at 24 h after reperfusion. HNG increased cell viability after OGD in primary cortical neurons, whereas the PI3K/Akt inhibitors wortmannin and Akti-1/2 attenuated the protective effect of HNG. HNG rapidly increased Akt phosphorylation, an effect that was inhibited by wortmannin and Akti-1/2. Mouse brains were injected intraventricularly with HNG before being subjected to middle cerebral artery occlusion (MCAO). HNG treatment significantly elevated p-Akt levels after cerebral I/R injury and decreased infarct volume. The protective effect of HNG on infarct size was attenuated by wortmannin and Akti-1/2. Taken as a whole, these results suggest that PI3K/Akt activation mediates HNG's protective effect against hypoxia/ischemia reperfusion injury.
Necrosis is generally believed to be a passive cellular response to external damage. To date, it has been difficult to investigate the potential mechanisms of necrosis. Recently, a new pathway to necrosis has been described by Yuan and colleagues. This pathway, called necroptosis, is a form of cell death that leads to necrosis. A chemical compound, necrostatin‐1 (Nec‐1), specifically inhibits this non‐apoptotic cell death. Iodoacetate (IAA) is an irreversible inhibitor of glycolysis and has been used as a tool for inducing chemical ischemia. We hypothesize that Nec‐1 can confer protection in this model. In this study, we explored the protective effects of Nec‐1 on IAA‐induced ischemia in rat myoblastic H9c2 cells. These cells were exposed to DMEM containing 7.5% dialyzed fetal bovine serum in the presence of 10–100 uM IAA for 2 h followed by incubation with regular medium overnight. Cell proliferation was measured by MTS assays. IAA was found to induce cell death in a dose‐dependent manner. At 50 uM IAA, cell proliferation was 30% as the control level. The addition of 25 uM Nec‐1 significantly attenuated IAA‐induced cell death. The protective effect of Nec‐1 was also dose‐dependent. These results demonstrate for the first time that necroptosis is involved in IAA‐induced cell death (Supported by American Heart Association Southeast Affiliate, Department of Veterans Affairs Merit Review and NIH HL‐087271).
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