Perampanel, an AMPAR antagonist, alleviates experimental intracerebral hemorrhage‑induced brain injury via necroptosis and neuroinflammation

Abstract

Spontaneous intracerebral hemorrhage (ICH) is a subtype of stroke with high mortality and morbidity due to the lack of effective therapies. The alpha‑amino‑3‑hydroxy‑5‑methyl‑4‑isoxazolepropionic acid receptor antagonist perampanel has been reported to alleviate early brain injury following subarachnoid hemorrhage and traumatic brain injury by reducing reactive oxygen species, apoptosis, autophagy, and necroptosis. Necroptosis is a caspase‑independent programmed cell death mechanism that serves a vital role in neuronal cell death following ICH. However, the precise role of necroptosis in perampanel‑mediated neuroprotection following ICH has not been confirmed. The present study aimed to investigate the neuroprotective effects and potential molecular mechanisms of perampanel in ICH‑induced early brain injury by regulating neural necroptosis in C57BL/6 mice and in a hemin‑induced neuron damage cell culture model. Mortality, neurological score, brain water content, and neuronal death were evaluated. The results demonstrated that perampanel treatment increased the survival rate and neurological score, and increased neuron survival. In addition, perampanel treatment downregulated the protein expression levels of receptor interacting serine/threonine kinase (RIP) 1, RIP3, and mixed lineage kinase domain like pseudokinase, and of the cytokines IL‑1β, IL‑6, TNF‑α, and NF‑κB. These results indicated that perampanel‑mediated inhibition of necroptosis and neuroinflammation ameliorated neuronal death in vitro and in vivo following ICH. The neuroprotective capacity of perampanel was partly dependent on the PTEN pathway. Taken together, the results of the present study demonstrated that perampanel improved neurological outcomes in mice and reduced neuronal death by protecting against neural necroptosis and neuroinflammation.

Keywords: early brain injury; intracerebral hemorrhage; necroptosis; neuroinflammation; perampanel.

Figure 1.

Perampanel alleviates early brain injury following ICH in vivo. (A) Diagram of the experimental design. (B) Neurological scores of the three experimental groups at 72 h after ICH (n=10). (C) Brain water content at 72 h after ICH (n=5). (D) Representative images of TUNEL assay staining in hippocampal sections at 72 h after ICH (n=5). Scale bar, 20 µm. Error bars indicate SEM. *P<0.01 vs. sham group; ^$P<0.05 vs. ICH group. ICH, intracerebral hemorrhage; Per, perampanel; SAH, subarachnoid hemorrhage.

Figure 2.

Perampanel relieves hemin-induced HT22 neuronal injury in vitro. (A) MTT assay of HT22 cells treated with 0–140 µM hemin. 100 µM hemin was used as the test dosage. (B) MTT assay of HT22 cells treated with hemin alone or hemin and perampanel (n=5). (C) Representative images of live (green)/dead (red) staining assay of HT-22 neurons exposed to hemin (100 µM, 72 h) in the presence and absence of perampanel (n=5). Scale bar=100 µm. Error bars indicate SEM. ^##P<0.01 vs. vehicle group; ^&&P<0.01 vs. hemin group. Per, perampanel.

Figure 3.

Perampanel relieves neuroinflammation following ICH. (A) Level of IL-1β, (B) IL-6, (C) TNF-α, and (D) NF-κB were measured after ICH in ipsilateral cortex tissue by ELISA. (E) Representative blots and (F) quantification of protein expression levels of TNF-α and NF-κB after hemin-induced ICH in vitro by western blotting. Error bars indicate SEM. **P<0.01 vs. sham group; ^$P<0.05 vs. ICH group; ^##P<0.01 vs. vehicle group; ^&P<0.05 vs. hemin group. ICH, intracerebral hemorrhage; Per, perampanel; Veh, vehicle.

Figure 4.

Perampanel suppresses necroptosis following ICH. (A) mRNA expression levels of RIP1, (B) RIP3, and (C) MLKL were determined after ICH by reverse transcription-quantitative PCR analysis. (D) Representative blots and (E-G) quantification of RIP1, RIP3, and MLKL protein expression levels after ICH, as determined by western blotting. (H) MTT assay of HT22 cells treated in vitro with hemin in the absence or presence of inhibitors of caspase-dependent apoptosis (z-VAD-fmk), autophagy (3-MA), or necroptosis (Nec-1). Error bars indicate SEM (n=5). *P<0.01 vs. sham group; ^$P<0.05 and ^$$P<0.01 vs. ICH group. ICH, intracerebral hemorrhage; RIP, receptor interacting serine/threonine kinase; MLKL, mixed lineage kinase domain like pseudokinase; Nec-1, necrostatin-1.

Figure 5.

Antinecroptosis effects of perampanel are mediated via the PTEN signaling pathway. (A) mRNA expression levels of PTEN in HT22 cells treated with hemin in the absence or presence of perampanel. (B) Representative blots and (C) quantification of protein expression levels of PTEN. (D) MTT assay of HT22 cells treated with hemin, perampanel and/or si-PTEN. (E) Representative images of live (green)/dead (red) staining assays of HT22 cells treated with hemin, perampanel and/or si-PTEN. Scale bar=100 µm. Error bars indicate SEM (n=5). ^#P<0.05 vs. vehicle group; ^&P<0.05 and ^&&P<0.01 vs. hemin group; ^£P<0.05 vs. hemin +Per group. si, small interfering; Per, perampanel; Veh, vehicle.

Figure 6.

Perampanel alleviates PTEN-dependent brain injury in vivo. si-PTEN was infused in the brain of mice 48 h prior to ICH establishment and permpanel treatment. (A) Neurological scores at 72 h after ICH. (B) Brain water content. (C) Representative blots and (D and E) quantification of RIP1 and RIP3 protein expression levels after ICH, as determined by western blotting. (F) Infusion with si-PTEN increased neuronal death after ICH, as determined by TUNEL staining. Scale bar=100 µm. Error bars indicate SEM (n=5). **P<0.01 vs. sham group; ^$P<0.05 and ^$$P<0.01 vs. ICH group; ^@@P<0.01 vs. ICH +Veh group; ^&&P<0.01 vs. ICH+si-Con+Per group. si, small interfering; ICH, intracerebral hemorrhage; RIP, receptor interacting serine/threonine kinase; Con, control; Per, perampanel; Veh, vehicle.