Synergistic protective effects of humanin and necrostatin-1 on hypoxia and ischemia/reperfusion injury - PubMed (original) (raw)

Synergistic protective effects of humanin and necrostatin-1 on hypoxia and ischemia/reperfusion injury

Xingshun Xu et al. Brain Res. 2010.

Abstract

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 24h after oxygen-glucose deprivation (OGD) treatment. Mice underwent middle cerebral artery occlusion for 75 min followed by 24h reperfusion. Mice were administered HNG and/or Nec-1 (i.c.v.) at 4h 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.

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Figures

Figure 1

Protective effect of Nec-1 and HNG on oxygen-glucose deprivation (OGD)-induced cell death in primary cortical neurons. Primary cortical neurons were used to perform OGD at DIV 10. Neurons were washed with HBSS and incubated with vehicle, Nec-1 (25 μM), HNG (0.2 μM), or both in a hypoxia chamber for 60 min. Cell viability was determined by MTS assay 24 h after reperfusion. Control neurons were exposed to oxygenated HBSS containing 5.5 mM glucose in normoxic conditions during the same time as the OGD culture. Bars represent mean ± SEM of 8 samples. *P<0.01, versus OGD group; #, P<0.01, versus OGD+HNG+Nec-1 group.

Figure 2

Protective effects of HNG and Nec-1 on neurological scores after cerebral ischemia/reperfusion injury. Mice were treated with HNG (0.1 μg in 5 μl saline and 1 μl DMSO) and/or Nec-1 (2.6 μg in 1 μl DMSO and 5 μl saline) after 75 min of ischemia and 4 h of reperfusion. Neurological deficits of the mice in four groups were evaluated. Bars represent mean ± SEM of 6–7 samples. *P<0.05, versus HNG+Nec-1 group; #, P<0.05, versus vehicle (Veh) group.

Figure 3

Protective effects of HNG and Nec-1 on infarct volume. Mice were treated with HNG (0.1 μg in 5 μl saline and 1 μl DMSO) and/or Nec-1 (2.6 μg in 1 μl DMSO and 5 μl saline) after 75 min of ischemia and 4 h of reperfusion. Cerebral infarct volume was determined by TTC staining 24 h after reperfusion. (A) Representative pictures of different treatments. (B) Quantitative analysis of cerebral infarct volume in four groups was performed. Bars represent mean ± SEM of 6–7 samples. *P<0.05, versus HNG+Nec-1 group; #, P<0.05, versus vehicle (Veh) group.

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Synergistic protective effects of humanin and necrostatin-1 on hypoxia and ischemia/reperfusion injury - PubMed (original) (raw)