Preactivation of Notch1 in remote ischemic preconditioning reduces cerebral ischemia-reperfusion injury through crosstalk with the NF-κB pathway

Abstract

Background: Remote ischemic preconditioning (RIPC) initiates endogenous protective pathways in the brain from a distance and represents a new, promising paradigm in neuroprotection against cerebral ischemia-reperfusion (I/R) injury. However, the underlying mechanism of RIPC-mediated cerebral ischemia tolerance is complicated and not well understood. We reported previously that preactivation of Notch1 mediated the neuroprotective effects of cerebral ischemic preconditioning in rats subjected to cerebral I/R injury. The present study seeks to further explore the role of crosstalk between the Notch1 and NF-κB signaling pathways in the process of RIPC-induced neuroprotection.

Methods: Middle cerebral artery occlusion and reperfusion (MCAO/R) in adult male rats and oxygen-glucose deprivation and reoxygenation (OGD/R) in primary hippocampal neurons were used as models of I/R injury in vivo and in vitro, respectively. RIPC was induced by a 3-day procedure with 4 cycles of 5 min of left hind limb ischemia followed by 5 min of reperfusion each day before MCAO/R. Intracerebroventricular DAPT injection and sh-Notch1 lentivirus interference were used to inhibit the Notch1 signaling pathway in vivo and in vitro, respectively. After 24 h of reperfusion, neurological deficit scores, infarct volume, neuronal apoptosis, and cell viability were assessed. The protein expression levels of NICD, Hes1, Phospho-IKKα/β (p-IKK α/β), Phospho-NF-κB p65 (p-NF-κB p65), Bcl-2, and Bax were assessed by Western blotting.

Results: RIPC significantly improved neurological scores and reduced infarct volume and neuronal apoptosis in rats subjected to I/R injury. OGD preconditioning significantly reduced neuronal apoptosis and improved cell viability after I/R injury on days 3 and 7 after OGD/R. However, the neuroprotective effect was reversed by DAPT in vivo and attenuated by Notch1-RNAi in vitro. RIPC significantly upregulated the expression of proteins related to the Notch1 and NF-κB pathways. NF-κB signaling pathway activity was suppressed by a Notch1 signaling pathway inhibitor and Notch1-RNAi.

Conclusions: The neuroprotective effect of RIPC against cerebral I/R injury was associated with preactivation of the Notch1 and NF-κB pathways in neurons. The NF-κB pathway is a downstream target of the Notch1 pathway in RIPC and helps protect focal cerebral I/R injury.

Keywords: Cross reaction; Ischemia-reperfusion injury; NF-kappa B; Neuroprotection; Notch1 pathway; Remote ischemic preconditioning.

Fig. 1

Effect of RIPC on neurological deficit scores and infarct volume in rats after MCAO/R. The neurological deficit scores and brain infarction volume were evaluated 24 h after reperfusion in rats in the four groups. a Cerebral infarct images (white, infarct tissue; red, noninfarct tissue) stained using 2,3,5-triphenyltetrazolium chloride in coronal sections of rat brains. b Brain infarct volume presented as a percentage of the intact hemisphere. Data are presented as the means ± SEM. **P < 0.01 vs. the MCAO/R group. c Neurological scores are presented as the median (interquartile range). **P < 0.01 vs. the MCAO/R group

Fig. 2

RIPC activated the Notch and NF-κB signaling pathways in the ischemic penumbra after MCAO/R. a Protein bands of NICD, Hes1, IKKβ, NF-κB p65, and β-actin from Western blot analysis. b RIPC significantly upregulated the expression of proteins related to the Notch and NF-κB signaling pathways. Data are presented as the means ± SEM. *P < 0.05, **P < 0.01, vs. the MCAO/R group; ^#P < 0.05 vs. the sham group

Fig. 3

OGD preconditioning-induced neuroprotection was attenuated by Notch1-RNAi in vitro. a Hippocampal neurons were identified by Map-2 antibody labeling (red), and the purity was over 95%. b Screening results of Notch1 RNA interference fragments. **P < 0.01 vs. Notch1-NC group. c, d The expression of NICD was significantly reduced by Lv-sh-Notch1. **P < 0.01 vs. Lv-NC group. e, f OGD preconditioning significantly improved hippocampal neuronal proliferation rates and reduced neuronal apoptosis after OGD/R. The neuroprotective effect was attenuated by Notch1-RNAi. Data are presented as the means ± SEM, *P < 0.05, **P < 0.01 vs. the control cell group; ^#P < 0.05 vs. OGD/R group. ^&P < 0.05, ^&&P < 0.01 vs. OGD-Pre + OGD/R + Lv-NC group

Fig. 4

DAPT and Notch1-RNAi alleviated MCAO/R and OGD/R injury in vivo and in vitro, respectively. a, b Brain infarct volume presented as a percentage of the intact hemisphere. *P < 0.05 vs. the MCAO/R group. c Neurological scores are presented as the median (interquartile range), *P < 0.05 vs. the MCAO/R group. d, e Neuronal apoptosis assayed by TUNEL, NeuN, and DAPI immunofluorescence staining. The percent of TUNEL and NeuN double-positive cells. **P < 0.01 vs. the MCAO/R group. f Notch1-RNAi significantly improved hippocampal neuronal proliferation rates on day 3 and day 7 after OGD/R injury. **P < 0.01 vs. the OGD/R group

Fig. 5

In the hippocampal neuronal OGD/R model, OGD-preconditioning activated the Notch1 and NF-κB signaling pathways, and the NF-κB signaling pathway was suppressed by Notch1-RNAi. a Notch1-RNAi significantly reduced neuronal apoptosis on day 3 and day 7 following direct lethal OGD/R injury in vitro, *P < 0.05 vs. OGD/R group. b Protein bands of NICD, Hes1, p-IKK α/β, p-NF-κB p65, Bcl-2, Bax, and β-actin from Western blot analysis. c, d OGD-preconditioning significantly upregulated the expression of proteins related to the Notch1 and NF-κB signaling pathways after OGD/R. The expression of p-IKK α/β and p-NF-κB p65 was suppressed by Notch1-RNAi. Data are presented as the means ± SEM in c and d; *P < 0.05, **P < 0.01, ***P < 0.001 vs. the OGD/R group; ^##P < 0.01 vs. OGD-Pre + OGD/R + sh-NC group

Fig. 6

RIPC-induced neuroprotection was attenuated by a Notch1 signaling pathway inhibitor in vivo. The neurological deficit scores and brain infarction volume were evaluated 24 h after reperfusion in rats in the 4 groups. a Images of focal cerebral infarction staining by TTC in coronal brain slices (white, infarct tissue; red, noninfarct tissue). b Brain infarct volume presented as a percentage of the intact hemisphere. Data are presented as the means ± SEM. **P < 0.01 vs. MCAO/R group, ^##P < 0.01 vs. the RIPC + MCAO/R + DAPT group. c Neurological deficit scores are presented as the median with interquartile range. **P < 0.01 vs. MCAO/R group, ^##P < 0.01 vs. the RIPC + MCAO/R + DAPT group

Fig. 7

RIPC significantly reduced hippocampal neuronal cell apoptosis in rats after MCAO/R. However, the neuroprotective effects were attenuated by an inhibitor of the Notch1 signaling pathway. a Apoptosis was assayed by TUNEL, NeuN, and DAPI immunofluorescence staining. b The percent of TUNEL and NeuN double-positive cells. **P < 0.01 vs. the Sham group, ^#P < 0.05 vs. the MCAO/R group, ^&P < 0.05 vs. the RIPC + MCAO/R + DMSO group

Fig. 8

RIPC activated the Notch1 and NF-κB signaling pathways in rats after MCAO/R. NF-ΚB signaling pathway activity was suppressed by the Notch1 signaling pathway inhibitor DAPT. a, b DAPT significantly decreased the expression of NICD in rats after RIPC + MCAO/R. **P < 0.01 vs. DMSO group. c Protein bands of NICD, Hes1, p-IKK α/β, p-NF-κB p65, Bcl-2, Bax, and β-actin from Western blot analysis. d, e RIPC significantly upregulated the expression of proteins related to the Notch1 and NF-κB signaling pathways. The Notch1 signaling pathway inhibitor DAPT reduced the activity of the NF-κB signaling pathway. Data are presented as the means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001 vs. the RIPC + MCAO/R group, ^#P < 0.05, ^##P < 0.01 vs. the RIPC + MCAO/R + DMSO group