Lig4-4 selectively inhibits TREK-1 and plays potent neuroprotective roles in vitro and in rat MCAO model.

Two-pore domain potassium channel TREK-1 was known to play an important role in neuroprotection, especially in acute cerebral ischemia. In the present study we found that 3-(nitromethyl) isobenzofuran-1(3H)-one (lig4-4) could robustly inhibit TREK-1 currents with an IC of 2.06 μM. However, the IC of lig4-4 for other ion channel subtypes such as Kv2.1, Kv1.5, Kv3.1, hERG and neuronal Na and Ca channels were more than 30 μM, suggesting a specific inhibition of TREK-1 channel. MTT assay showed that lig4-4 significantly enhanced cell viability of cultured neurons under the condition of oxygen and glucose deprivation followed by reoxygenation (OGD/R). Annexin V/Propidium Iodide apoptosis assay also demonstrated that lig4-4 obviously reduced cell apoptosis in OGD/R-injured neurons. Western blotting results indicated that OGD/R-induced up-regulation of cleaved-caspase-3 expression and down-regulation of Bcl-2 could be notably reversed by lig4-4. The in vivo study showed that after oral administration of lig4-4 at 50 mg/kg, the infarct volume in middle cerebral artery occlusion (MCAO) rat model reduced from a vehicle control of 38.8% to 28%. Both in vitro and in vivo studies suggested that inhibition of TREK-1 with lig4-4 might produce a neuroprotective effect against cerebral ischemia. In conclusion, we demonstrated that lig4-4 selectively inhibited TREK-1 and protected brain from cerebral ischemic injury. The mechanisms might relate to block TREK-1 and inhibit neuronal apoptosis by modulating the expressions of Bcl-2 and cleaved-caspase-3.