γ-Glutamylcysteine Alleviates Ischemic Stroke-Induced Neuronal Apoptosis by Inhibiting ROS-Mediated Endoplasmic Reticulum Stress

Abstract

Ischemic stroke is a severe and acute neurological disorder with limited therapeutic strategies currently available. Oxidative stress is one of the critical pathological factors in ischemia/reperfusion injury, and high levels of reactive oxygen species (ROS) may drive neuronal apoptosis. Rescuing neurons in the penumbra is a potential way to recover from ischemic stroke. Endogenous levels of the potent ROS quencher glutathione (GSH) decrease significantly after cerebral ischemia. Here, we aimed to investigate the neuroprotective effects of γ-glutamylcysteine (γ-GC), an immediate precursor of GSH, on neuronal apoptosis and brain injury during ischemic stroke. Middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation/reoxygenation (OGD/R) were used to mimic cerebral ischemia in mice, neuronal cell lines, and primary neurons. Our data indicated that exogenous γ-GC treatment mitigated oxidative stress, as indicated by upregulated GSH and decreased ROS levels. In addition, γ-GC attenuated ischemia/reperfusion-induced neuronal apoptosis and brain injury in vivo and in vitro. Furthermore, transcriptomics approaches and subsequent validation studies revealed that γ-GC attenuated penumbra neuronal apoptosis by inhibiting the activation of protein kinase R-like endoplasmic reticulum kinase (PERK) and inositol-requiring enzyme 1α (IRE1α) in the endoplasmic reticulum (ER) stress signaling pathway in OGD/R-treated cells and ischemic brain tissues. To the best of our knowledge, this study is the first to report that γ-GC attenuates ischemia-induced neuronal apoptosis by suppressing ROS-mediated ER stress. γ-GC may be a promising therapeutic agent for ischemic stroke.

Figure 1

γ-GC reduced apoptosis and oxidative injury in HT22 cells induced by OGD/R. HT22 cells were pretreated with γ-GC for one hour prior to OGD and then exposed to OGD for 12 h, followed by 12 h of reperfusion. γ-GC was present throughout the entire process of OGD and reperfusion. (a) Cell viability was measured by the CCK-8 assay. (b) HT22 cell apoptosis was assessed by Annexin V/7-AAD staining. Annexin V⁺⁷-AAD⁻ and Annexin V⁺⁷-AAD⁺ cells were defined as apoptotic cells. (c) The ratios of Bax/Bcl-2 and cleaved caspase-3/caspase-3 were measured by western blot analysis after 6 h of reperfusion. (d, e) Quantification of the levels of Bax/Bcl-2 and cleaved caspase-3/caspase-3. In the following experiments, HT22 cells were subjected to 12 h of reperfusion. (f) Quantification of total GSH. (g, h) The level of intracellular ROS. Scale bar: 100 μm. All data are presented as the mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01 versus the control group. ^#p < 0.05, ^##p < 0.01 versus the OGD/R group.

Figure 2

The DEGs of HT22 cells exposed to 12 h of OGD and 3 h of reperfusion were analyzed by transcriptome sequencing. (a) Heatmap of DEGs between the control group, OGD/R group, and OGD/R+γ-GC group (4 mM). (b) List of enriched GO analyses of biological processes, cellular components, and molecular functions. (c) Scatter plot of KEGG pathway enrichment analysis of DEGs.

Figure 3

γ-GC protected HT22 cells against OGD/R insult by repressing ER stress-induced apoptosis. After HT22 cells were subjected to 12 h of OGD and 1 h of reperfusion, the following experiments were conducted. (a) Western blot analysis of ER stress-associated proteins, including GRP78, p-PERK, p-IRE1α, and p-eIF2α. (b–e) Quantification of the levels of GRP78, p-PERK/PERK, p-IRE1α/IRE1α, and p-eIF2α/eIF2α. (f) The interaction between IRE1α and TRAF2 was evaluated by immunoprecipitation. (g) CHOP and p-JNK, which are indicators of ER-induced stress, were analyzed by western blotting at 6 h postreperfusion. (h) Quantification of CHOP and p-JNK/JNK expression. All data are presented as the mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01 versus the control group. ^#p < 0.05, ^##p < 0.01 versus the OGD/R group.

Figure 4

γ-GC protected primary cortical neurons against apoptosis and oxidative damage induced by OGD/R. Primary cortical neurons were pretreated with γ-GC for 1 hour and then subjected to 40 min of OGD followed by 12 h of reperfusion. (a) CCK-8 assays revealed cell viability. (b) Primary cortical neuronal apoptosis was examined by calcein/PI double staining. Scale bar: 50 μm. The percentage of calcein-positive cells was calculated as calcein‐positive cells/(calcein‐positive cells + PI‐positive cells). Bax, Bcl-2, and cleaved caspase-3 were examined after 6 h of reperfusion. (c) Representative western blot images of Bax/Bcl-2 and cleaved caspase-3/caspase-3. (d, e) Quantification of Bax/Bcl-2 and cleaved caspase-3/caspase-3 levels. (f) The level of total GSH in primary cortical neurons. (g) The level of intracellular ROS was visualized and quantified as the mean fluorescence intensity. Scale bar: 100 μm. All data are presented as the mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01 versus the control group. ^#p < 0.05, ^##p < 0.01 versus the OGD/R group.

Figure 5

γ-GC exerted its neuroprotective effect by suppressing ER stress-induced apoptosis in primary neurons. (a) Western blot analysis of sensors of ER stress, including GRP78, p-PERK, p-IRE1α, and p-eIF2α, after primary cortical neurons were exposed to 1 h of reperfusion. (b–e) Quantification of GRP78, p-PERK/PERK, p-IRE1α/IRE1α, and p-eIF2α/eIF2α levels. (f) The levels of CHOP and p-JNK were measured by western blot analysis after 6 h of reperfusion. (g, h) Quantification of CHOP and p-JNK/JNK expression. All data are presented as the mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01 versus the control group. ^#p < 0.05, ^##p < 0.01 versus the OGD/R group.

Figure 6

γ-GC alleviated neurological deficits in mice subjected to MCAO. 24 h after MCAO, the mice were sacrificed for in vivo examination. (a, b) Cerebral blood flow was measured by LSCI. Behavioral tests, including (c) mNSS, (d) grip strength, and (e) rotarod test, were performed to evaluate neurological function (n = 10). (f) The infarct area was visualized by TTC staining (n = 8). (g) Quantification of infarct volume. All data are presented as the mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01 versus the sham group. ^#p < 0.05, ^##p < 0.01 versus the MCAO group.

Figure 7

γ-GC protected MCAO mice against focal cerebral ischemic injury. (a) Image showing the ischemic penumbra that was isolated 24 h after MCAO for subsequent experiments. (b) Western blot analysis of apoptosis-associated proteins, including Bax, Bcl-2, and cleaved caspase-3. (c, d) The levels of Bax/Bcl-2 and cleaved caspase-3/caspase-3 were quantified. (e, f) Apoptotic cells were examined by TUNEL assays. Scale bar: 50 μm. (g) Measurement of total GSH in the ischemic penumbra. (h) Evaluation of the MDA level in the ischemic penumbra. (i, j) Representative images and quantification of intracellular ROS levels in the ischemic penumbra. Scale bar: 50 μm. All data are presented as the mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01 versus the sham group. ^#p < 0.05, ^##p < 0.01 versus the MCAO group.

Figure 8

γ-GC exerted its neuroprotective effect in vivo by alleviating ER stress. (a) Western blot analysis of GRP78, p-PERK, p-IRE1α, and p-eIF2α in the sham, MCAO+solvent, and MCAO+γ-GC groups 24 h after modeling. (b) Quantification of GRP78, p-PERK/PERK, p-IRE1α/IRE1α, and p-eIF2α/eIF2α levels in the ischemic penumbra. (c, d) The levels of CHOP and p-JNK/JNK were determined by western blot analysis. (e) The changes in p-IRE1α and p-eIF2α were visualized by immunofluorescence staining. Scale bar: 50 μm. (f) Quantification of p-IRE1α/NeuN and p-eIF2α/NeuN. All data are presented as the mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01 versus the sham group. ^#p < 0.05, ^##p < 0.01 versus the MCAO group.

Figure 9

γ-GC protected against ischemia/reperfusion-induced neuronal apoptosis by inhibiting ROS-mediated ER stress.