Inhibition of G protein-coupled receptor 81 (GPR81) protects against ischemic brain injury

Abstract

Aim: Lactates accumulate in ischemic brains. G protein-coupled receptor 81 (GPR81) is an endogenous receptor for lactate. We aimed to explore whether lactate is involved in ischemic injury via activating GPR81.

Methods: N2A cells were transfected with GFP-GPR81 plasmids 24 h previously, and then treated with GPR81 antagonist 3-hydroxy-butyrate (3-OBA) alone or cotreated with agonists lactate or 3, 5-dihydroxybenzoic acid (3, 5-DHBA) during 3 h of oxygen-glucose deprivation (OGD). Adult male C57BL/6J mice and primary cultured cortical neurons were treated with 3-OBA at the onset of middle cerebral artery occlusion (MCAO) or OGD, respectively.

Results: The GPR81 overexpression increased the cell vulnerability to ischemic injury. And GPR81 antagonism by 3-OBA significantly prevented cell death and brain injury after OGD and MCAO, respectively. Furthermore, inhibition of GPR81 reversed ischemia-induced apoptosis and extracellular signal-regulated kinase (ERK) signaling may be involved in the neuroprotection.

Conclusions: G protein-coupled receptor 81 (GPR81) inhibition attenuated ischemic neuronal death. Lactate may aggravate ischemic brain injury by activating GPR81. GPR81 antagonism might be a novel therapeutic strategy for the treatment of cerebral ischemia.

Keywords: Cerebral ischemia; G protein-coupled receptor 81; Lactates; Neuroprotection.

Figure 1

G protein‐coupled receptor 81 (GPR81) is involved in oxygen–glucose deprivation‐induced neuronal cell death. N2A cells were transfected with vector or GFP‐GPR81 plasmids 24 h previously. Cells were treated with the compounds at indicated concentrations for 3 h during the oxygen–glucose deprivation (OGD). The cell viability was determined after the OGD exposure. (A) Transfection effects were confirmed by Western blot detecting GFP and GPR81. (B) The effects of GPR81 antagonist 3‐hydroxy‐butyrate (3‐OBA) on cell viability were determined in GFP‐GPR81‐transfected cells. (C) The effects of GPR81 agonists, lactate (1 mmol/L) and 3, 5‐DHBA (0.2 mmol/L) on cell viability were determined in GFP‐GPR81‐transfected cells. (D) Cell death was determined by double‐staining with Hoechst 33342 (blue) and propidium iodide (PI, red) in GFP‐GPR81 transfected cells (green). The arrows showed the PI‐positive cells. Scale bars, 20 μm. Three independent experiments were included and conducted in triplicate wells. Statistical comparisons were performed with one‐way ANOVA followed by Dunnett's t‐test. ^& P < 0.05 versus nontransfected group. **P < 0.01 and ***P < 0.001 versus OGD treatment group. ^## P < 0.01 and ^### P < 0.001 versus 3‐OBA treatment group.

Figure 2

Inhibition of GPR81 protected against neuronal injury in vitro and in vivo. (A) Primary cultured rat cortical neurons were treated with 3‐OBA at the beginning of oxygen–glucose deprivation (OGD). The cell viability was assessed by the MTT assay after 4‐h OGD. (B and C) Mice were given an intracerebroventricular injection of 3‐OBA at the onset of middle cerebral artery occlusion (MCAO). Infarct volumes were quantified by TTC staining 24 h after MCAO. Data were expressed as mean ± SD n = 6 for each group. Statistical comparisons were performed with one‐way ANOVA followed by Dunnett's t‐test; **P < 0.01 versus indicated groups.

Figure 3

Inhibition of GPR81 reduced oxygen–glucose deprivation (OGD)‐induced apoptosis. (A) Primary cultured rat cortical neurons were treated with 3‐OBA at the beginning of OGD. After OGD, the apoptosis was determined using terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick end labeling assay (TUNEL) staining, while the cell population was visualized by DAPI staining. The TUNEL‐positive ratio in each section was calculated from nine random fields. Data are expressed as mean ± SD n = 3 for each group. GFP‐GPR81‐transfected N2A cells were subjected to OGD for 3 h in the presence of 3 mmol/L 3‐hydroxy‐butyrate (3‐OBA) alone or with lactate (1 mmol/L). (B) In GFP‐GPR81 transfected N2A cells, Bax, Bcl‐xl, cytosolic Cyt C, and caspase‐3 were detected by Western blot at the end of OGD. Semi‐quantitative analysis of indicated proteins normalized to GAPDH was shown in the bar chart (mean ± SD, n = 3). (C) GFP‐GPR81 transfected N2A cells were immunostained with Cyt C (green) and Tom20 (red). Nuclei were stained with DAPI. Scale bars, 10 μm. Columns represent the overlap coefficient. At least 20 cells from three independent experiments were included for each group. The data were expressed as mean ± SD. Statistical comparisons were performed with one‐way ANOVA followed by Dunnett's t‐test. *P < 0.05, **P < 0.01 and ***P < 0.001 versus OGD treatment group. ^# P < 0.05 and ^## P < 0.01 versus 3‐OBA treatment group.

Figure 4

The impact of GPR81 agonist and antagonist on ATP level. (A) GFP‐GPR81 transfected cells treated with indicated compounds during 3 h oxygen–glucose deprivation (OGD). (B) Primary cultured neurons treated with indicated compounds during 4‐h OGD. ATP level in cellular supernatant was determined at the end of OGD. The data were expressed as mean ± SD n = 3 for each groups. Statistical comparisons were performed with one‐way ANOVA followed by Dunnett's t‐test. *P < 0.05 and **P < 0.01 versus OGD alone group. N.S, not statistically significant.

Figure 5

Extracellular signal‐regulated kinase (ERK) signaling pathway involved in protection of GPR81 antagonism in vitro. Primary cultured neurons were treated with 3 mmol/L 3‐hydroxy‐butyrate (3‐OBA) alone or with U0216 (5 μmol/L) for 3 h during oxygen–glucose deprivation (OGD). (A) The levels of phosphorylation ERK, total ERK, and caspase‐3 were determined by Western blot and the semi‐quantitative analyses normalized by GADPH were shown below. (B) Cell viability was determined by MTT assay after the OGD exposure. The data were expressed as mean ± SD n = 3 for each group. Statistical comparisons were performed with one‐way ANOVA followed by Dunnett's t‐test. ^& P < 0.05 versus control group. *P < 0.05 and **P < 0.01 versus OGD treatment group. ^# P < 0.05 and ^## P < 0.01 versus 3‐OBA treatment group.