Ezetimibe Attenuates Oxidative Stress and Neuroinflammation via the AMPK/Nrf2/TXNIP Pathway after MCAO in Rats

Abstract

Oxidative stress and neuroinflammation play essential roles in ischemic stroke-induced brain injury. Previous studies have reported that Ezetimibe (Eze) exerts antioxidative stress and anti-inflammatory properties in hepatocytes. In the present study, we investigated the effects of Eze on oxidative stress and neuroinflammation in a rat middle cerebral artery occlusion (MCAO) model. One hundred and ninety-eight male Sprague-Dawley rats were used. Animals assigned to MCAO were given either Eze or its control. To explore the downstream signaling of Eze, the following interventions were given: AMPK inhibitor dorsomorphin and nuclear factor erythroid 2-related factor 2 (Nrf2) siRNA. Intranasal administration of Eze, 1 h post-MCAO, further increased the endogenous p-AMPK expression, reducing brain infarction, neurologic deficits, neutrophil infiltration, microglia/macrophage activation, number of dihydroethidium- (DHE-) positive cells, and malonaldehyde (MDA) levels. Specifically, treatment with Eze increased the expression of p-AMPK, Nrf2, and HO-1; Romo-1, thioredoxin-interacting protein (TXNIP), NOD-like receptor protein 3 (NLRP3), Cleaved Caspase-1, and IL-1β were reduced. Dorsomorphin and Nrf2 siRNA reversed the protective effects of Eze. In summary, Eze decreases oxidative stress and subsequent neuroinflammation via activation of the AMPK/Nrf2/TXNIP pathway after MCAO in rats. Therefore, Eze may be a potential therapeutic approach for ischemic stroke patients.

Figure 1

Experimental design and animal groups. DHE: dihydroethidium; DMSO: dimethyl sulfoxide; dorsomorphin: AMPK inhibitor; Eze: Ezetimibe; IHC: immunohistochemistry; MCAO: middle cerebral artery occlusion; MDA: malonaldehyde; Scr siRNA: scramble siRNA; TTC: 2,3,5-triphenyltetrazolium chloride; WB: western blot.

Figure 2

Expression of endogenous p-AMPK after MCAO. (a) Representative western blot bands and quantitative analysis of p-AMPK expression in the ipsilateral hemisphere after MCAO. (b) Representative western blot band and quantitative analysis of p-AMPK expression after treatment with Ezetimibe. The dose of Ezetimibe was 500 μg/kg. ^∗p < 0.05 vs. sham. n = 6 per group.

Figure 3

Eze reduced brain infraction and improved neurological outcomes at 24 h after MCAO. (a–c) Intranasal administration of Eze reduced infarction volume and improved the Garcia score at 24 h after MCAO. (d) There were no differences in beam walking scores between the MCAO groups. ^∗p < 0.05 vs. sham, ^#p < 0.05 vs. vehicle, and ^&p < 0.05 vs. Eze 250 μg/kg. n = 6 per group. Eze: Ezetimibe.

Figure 4

Eze inhibited microglia/macrophage activation and neutrophil infiltration at 24 h after MCAO. (a, c) Immunofluorescence revealed that treatment with Eze reduced the number of MPO-positive cells and Iba-1-positive cells in the ischemic penumbra region. (b, d) Representative western blot bands and quantitative analyses of MPO and Iba-1 protein levels at 24 h after MCAO. ^∗p < 0.05 vs. sham; ^#p < 0.05 vs. vehicle. n = 6 per group. Eze: Ezetimibe. Scale bar = 100 μm.

Figure 5

Eze reduced oxidative stress injury at 24 h after MCAO. (a) Representative microphotograph of DHE staining in the ischemic penumbra region. (b) Quantitative analysis of DHE-positive cells in the ischemic penumbra region. (c) The MDA level in the ischemic penumbra region. (d) Representative western blot band and quantitative analysis of Romo-1 expression. (e, f) Representative western blot bands and quantitative analyses of total-Nrf2 and nuclear-Nrf2 expression. ^∗p < 0.05 vs. sham; ^#p < 0.05 vs. vehicle. n = 6 per group. DHE: dihydroethidium; Eze: Ezetimibe; MDA: malonaldehyde. Scale bar = 100 μm.

Figure 6

Dorsomorphin and Nrf2 siRNA reversed the neuroprotective effects of Eze after MCAO. (a) Representative image of TTC-staining brain slices, (b) quantified infarction volumes, (c) modified Garcia scores, and (d) beam walking scores at 24 h after MCAO. ^∗p < 0.05 vs. sham, ^#p < 0.05 vs. vehicle, ^&p < 0.05 vs. Eze+DMSO, and ^@p < 0.05 vs. Eze+Scr siRNA. n = 6 per group. Dorso: dorsomorphin; Eze: Ezetimibe; Scr siRNA: scramble siRNA.

Figure 7

Eze attenuated oxidative stress and neuroinflammation via the AMPK/Nrf2/TXNIP pathway after MCAO. (a) Representative western blot bands. (b–h) Quantitative analyses of p-AMPK, Nrf2, HO-1, TXNIP, NLRP3, Cleaved Caspase-1, and IL-1β in the ipsilateral hemisphere at 24 h after MCAO. ^∗p < 0.05 vs. sham, ^#p < 0.05 vs. vehicle, ^&p < 0.05 vs. Eze+DMSO, and ^@p < 0.05 vs. Eze+Scr siRNA. n = 6 per group. Eze: Ezetimibe; Scr siRNA: scramble siRNA.

Figure 8

Proposed pathway for Eze and its downstream targets. Eze increases levels of phosphorylated AMPK and activates Nrf2, which translocates into the nucleus to increase the transcription of HO-1—a protein that inhibits ROS and inflammation.