4-((5-(Tert-butyl)-3-chloro-2-hydroxybenzyl) amino)-2-hydroxybenzoic acid protects against oxygen-glucose deprivation/reperfusion injury

Aims:Oxidative stress is one of the most important pathological mechanisms which could aggravate ischemic stroke injury. In order to seek for better treatment therapies to alleviate stroke injury, novel chemicals have been synthetized. In the present study, a new compound 4-((5-(tert-butyl)-3-chloro-2-hydroxybenzyl) amino)-2- hydroxybenzoic acid, named LX009, was used to determine whether it could reduce the oxidative stress caused by oxygen-glucose deprivation (OGD)/reperfusion (RP) and exert neuroprotective effect both in mouse Neuro 2A (N2A) neuroblastoma cells and mouse primary cortical neurons.

Main methods:OGD/RP was performed as an in vitro model to mimic the pathologic process of ischemic stroke. We explored the anti-apoptosis effect of LX009 through CCK8 assay, calcein acetoxymethylester/propidium iodide (calcein-AM/PI) staining, Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit, caspase-3 activity assay. Besides, the anti-oxidative stress effect of the drug was determined by intracellular reactive oxygen species (ROS) detection, nitrite analysis, measurement of mitochondrial membrane potential (MMP), intracellular catalase (CAT) and Mn-superoxide dismutase (Mn-SOD) activity.

Key findings:Our results indicated that LX009 could alleviate OGD/RP-induced cell apoptosis. Furthermore, OGD/RP induced oxidative stress could be reserved by LX009, including measurements of intracellular ROS production, MMP, CAT and Mn-SOD activity. Mechanistically, the phosphorylation level of Akt, as well as the expression of nuclear factor-E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were elevated after LX009 treatment.

Significance:Our present study indicated that LX009 might have the potential to be an anti-oxidative stress agent in the future.

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