A11, a novel diaryl acylhydrazone derivative, exerts neuroprotection against ischemic injury in vitro and in vivo

Abstract

There is an urgent need to develop effective therapies for ischemic stroke, but the complicated pathological processes after ischemia make doing so difficult. In the current study, we identified a novel diaryl acylhydrazone derivative, A11, which has multiple neuroprotective properties in ischemic stroke models. First, A11 was demonstrated to induce neuroprotection against ischemic injury in a dose-dependent manner (from 0.3 to 3 μM) in three in vitro experimental ischemic stroke models: oxygen glucose deprivation (OGD), hydrogen peroxide, and glutamate-stimulated neuronal cell injury models. Moreover, A11 was able to potently alleviate three critical pathological changes, apoptosis, oxidative stress, and mitochondrial dysfunction, following ischemic insult in neuronal cells. Further analysis revealed that A11 upregulated the phosphorylation levels of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) in OGD-exposed neuronal cells, suggesting joint activation of the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MEK)/ERK pathways. In rats with middle cerebral artery occlusion, single-dose administration of A11 (3 mg/kg per day, i.v.) at the onset of reperfusion significantly reduced the infarct volumes and ameliorated neurological deficits. Our study, for the first time, reports the anti-ischemic effect of diaryl acylhydrazone chemical entities, especially A11, which acts on multiple ischemia-associated pathological processes. Our results may provide new clues for the development of an effective therapeutic agent for ischemic stroke.

Keywords: diaryl acylhydrazone; extracellular signal-regulated kinase; ischemic stroke; middle cerebral artery occlusion; neuroprotection; oxygen glucose deprivation; protein kinase B.

Fig. 1

Chemical structure of A11

Fig. 2

A11 alleviated the SH-SY5Y cell damage induced by OGD exposure. a Phase-contrast micrographs (10×) of cells subjected to different treatments. Scale bar = 100 μm. b SH-SYSY cells were treated with 0.3–3 μM A11 immediately before exposure to OGD. c The LDH levels in the culture media after different treatments were assayed. The results represent the mean ± SEM of three independent experiments. ^###P < 0.001 vs. the control group, *P < 0.05, **P < 0.01, ***P < 0.001 vs. the OGD group

Fig. 3

A11 increased the viability of neuronal cells induced by H₂O₂ and glutamate. a SH-SYSY cells were pretreated with A11 for 2 h followed by H₂O₂ injury. b HT22 cells were pretreated with A11 for 2 h followed by glutamate injury. The results represent the mean ± SEM of three independent experiments. ^###P < 0.001 vs. the control group, *P < 0.05, **P < 0.01, ***P < 0.001 vs. the toxic stimuli groups

Fig. 4

A11 inhibited apoptosis induced by OGD. a Apoptotic cells were detected by flow cytometry. b The apoptotic rate is represented as a histogram. Western blot analysis of cleaved Caspase-3 (c) and p53 (d) in SH-SY5Y cells treated with 1 μM A11 followed by exposure to OGD and cultured for 24 h after reoxygenation (n = 4). The results represent the mean ± SEM of three to four independent experiments. ^##P < 0.01, ^###P < 0.001 vs. the control group, **P < 0.01, ***P < 0.001 vs. the OGD group

Fig. 5

A11 suppressed ROS production and elevated the ATP level in SH-SY5Y cells. a Changes in ROS production in different treatment groups were measured by assessing the changes in the DCFH-DA fluorescence intensity. b Changes of the intracellular ATP level in different treatment groups. The results represent the mean ± SEM of three independent experiments. ^###P < 0.001 vs. the control group, *P < 0.05, ***P < 0.001 vs. the OGD group

Fig. 6

A11 prevented repression of phosphorylation of ERK and enhanced AKT phosphorylation in OGD-exposed SH-SY5Y cells. SH-SY5Y cells were incubated with A11 followed by OGD treatment and cultured for 15 min after reoxygenation. a Expression of ERK phosphorylation in different treatment groups. b Expression of AKT phosphorylation in different treatment groups. The results represent the mean ± SEM of four independent experiments. ^#P < 0.05, ^###P < 0.001 vs. the control group, *P < 0.05 vs. the OGD group

Fig. 7

A11 protected SH-SY5Y cells against OGD insult by activating the MEK/ERK and PI3K/AKT pathways. a Western blot analysis of ERK and p-ERK in hypoxic cells after treatment with MEK inhibitor U0126. b AKT and p-AKT levels in OGD injured cells treated with A11 and PI3K inhibitor LY294002. Cell viability was measured by MTT when treated with A11 and U0126 (c) or LY294002 (d). The results represent the mean  ± SEM of four independent experiments. ^#P < 0.05, ^###P < 0.001 vs. the control group, *P < 0.05, **P < 0.01 ***P < 0.001 vs. the OGD group, ^$$P < 0.01, ^$$$P < 0.001 vs. the OGD + A11 group

Fig. 8

Intravenous administration of A11 (3 mg/kg) at the onset of reperfusion reduced the cerebral infarct volumes and ameliorated neurological deficits in MCAO rats. a Representative photographs of TTC-stained brain slices of rats. b Quantitative analysis of the cerebral infarct volume. c Quantitative analysis of neurological scores. The results represent the mean ± SEM. ^###P < 0.001 vs. the sham group, *P < 0.05, **P < 0.01 vs. the vehicle-treated MCAO group, n = 16–19