TRPM7 inhibitor carvacrol protects brain from neonatal hypoxic-ischemic injury

Abstract

Background: Our previous study found that suppression of TRPM7 reduced neuronal death in adult rat ischemic brain injury. It was reported that carvacrol blocked TRPM7 and attenuated brain injury in an adult rat MCAO model. The effects of carvacrol on neonatal stroke remain unknown. This study investigated the effects of carvacrol on neuronal injury and behavioral impairment after hypoxia-ischemia in neonatal mice and the potential signaling pathway underlying these effects.

Results: Carvacrol inhibited TRPM7 current in HEK293 cells over-expressing TRPM7 and TRPM7-like current in hippocampal neurons in a dose-dependent manner. Carvacrol (>200 μM) reduced OGD-induced neuronal injury in cortical neurons. 24 hours after HI, TRPM7 protein level in the ipsilateral hemisphere was significantly higher than in the contralateral hemisphere. Carvacrol (30 and 50 mg/kg) pre-treatment reduced brain infarct volume 24 hours after HI in a dose-dependent manner. Carvacrol pre-treatment also improved neurobehavioral outcomes. Furthermore, animals pre-treated with carvacrol had fewer TUNEL-positive cells in the brain compared to vehicle-treated animals 3 days after HI. Carvacrol pre-treatment also increased Bcl-2/Bax and p-Akt/t-Akt protein ratios and decreased cleaved caspase-3 protein expression 24 hours after HI.

Conclusions: Carvacrol pre-treatment protects against neonatal hypoxic-ischemic brain injury by reducing brain infarct volume, promoting pro-survival signaling and inhibiting pro-apoptotic signaling, as well as improving behavioral outcomes. The neuroprotective effect may be mediated by the inhibition of TRPM7 channel function. Carvacrol is a potential drug development target for the treatment of neonatal stroke.

Figure 1

Carvacrol (CAR) inhibits TRPM7 and TRPM7-like currents and protects cortical neurons from OGD-induced injury. A, HEK293 cells over-expressing TRPM7 were induced by tetracycline (1 μM) for 24 hours. TRPM7 current was recorded as described in methods section. Representative I-V curves are shown. Perfusion with carvacrol (500 μM and 1 mM) caused a dramatic decrease in the TRPM7 current in dose-dependent manner (n = 6 cells). B, TRPM7-like current in primary hippocampal neurons (HPC) was recorded as described in methods section. Perfusion with carvacrol (500 μM and 1 mM) dose-dependently blocked TRPM7-like current in HPC. Representative I-V curves are shown (n = 6 cells). C, cortical neurons were incubated with carvacrol or vehicle (0.1% DMSO) for 30 min and then treated with OGD for 1 hour and transferred to regular medium for 24 hours. Cells were then stained with PI and the fluorescent intensity was measured using Synergy HT Multi-Mode Micro plate Reader. Results demonstrated that carvacrol (200-800 μM) significantly protected neurons from OGD-induced injury (*, p < 0.05 compared with vehicle treated group, n = 5, One-way ANOVA followed by Newman-Keuls test). D and E, cortical neurons were treated with carvacrol (300 μM) for 30 min, and then OGD and PI staining were conducted as described above. Representative images were taken using a Zeiss LSM 710 Confocal Microscope. Scale bar = 10 μm. *, p < 0.05 compared with control group; #, p < 0.05 compared with OGD group, n = 4, One-way ANOVA followed by Newman-Keuls test.

Figure 2

Carvacrol (CAR) pre-treatment reduced infarct volume of neonatal hypoxic-ischemic brain injury in a dose-dependent manner. A, TTC staining was performed as described in methods section. The representative images of TTC-stained coronal brain slices are shown (Left panel). Infarct volume is smaller in carvacrol pre-treatment group than vehicle group (*, p < 0.05 compared with vehicle group. #, p < 0.05 compared with carvacrol 30 mg/kg group, One-way ANOVA followed by Newman-Keuls test). B, representative images of whole brain and Nissl staining for brain slices (sham, n = 7; vehicle group, n = 12; carvacrol pretreatment group, n = 15).

Figure 3

Carvacrol pretreatment improves neurobehavioral performance after cerebral HI. Neurobehavioral evaluation was performed as described in Material and Methods. Geotaxis reflex (A), cliff avoidance test (B) and grip test (C) of sham (n = 7), vehicle group (HI + vehicle, n = 12) and carvacrol (50 mg/kg) pretreatment group (HI + CAR, n = 15) were measured 1 day, 3 days and 7 days after HI (*, p < 0.05 versus sham group; #, p < 0.05 versus vehicle group, One-way ANOVA followed by Newman-Keuls test).

Figure 4

TRPM7 protein expression in the cortex of HI mice and TUNEL staining of mouse brain slices. A, western blotting results of TRPM7 protein expression in the injured hemisphere (ipsilateral) and uninjured contralateral hemisphere 24 hours after HI. TRPM7 protein expression level is higher in injured hemisphere than the contralateral hemisphere (*, p < 0.05, n = 4). B, TUNEL staining was carried out 3 days after HI and images were viewed using fluorescence microscopy. Left panel shows the representative images of TUNEL staining. Red fluorescence in TUNEL-positive cells indicates apoptosis. Blue fluorescence is DAPI staining for nucleus. Scale bar represents 10 μm. Right panel shows the mouse brain coronal sections. C, Quantitative analysis of TUNEL-positive cells showed that pre-treatment with carvacrol (50 mg/kg) reduced the number of TUNEL positive cells (*, p < 0.05 compared with sham group. #, p < 0.05 compared with vehicle group, n = 3, One-way ANOVA followed by Newman-Keuls test).

Figure 5

Immunofluorescence assay of TRPM7 and cleaved caspase-3 expression in brain sections of HI mice. Triple immunofluorescence staining was employed for TRPM7, cleaved caspase-3 and NeuN in the penumbra of the injured hemisphere (ipsilateral) and in a similar region of the uninjured contralateral hemisphere 24 hours after HI. Representative images are shown. Green, blue and red fluorescence indicates NeuN, TRPM7 and cleaved caspase-3, respectively. The white arrow indicates the co-localization of TRPM7 and activated caspase-3 with NeuN. N = 3, Scale bar in 20× and 40× magnifying images = 10 μm. Scale bar in 63× magnifying images = 20 μm.

Figure 6

Western blotting results. Experiments were carried out as described in the methods section. A, representative images of Bcl-2 (26 kDa), Bax (20 kDa), cleaved caspase-3 (19/17 kDa), p-Akt (Ser 473, 60 kDa) and t-Akt (60 kDa) protein expression. β-actin (42 kDa) was used as a loading control. Carvacrol (50 mg/kg) pretreatment increased the protein ratio of Bcl-2/Bax (B), decreased cleaved caspase-3 protein expression (C) and increased p-Akt/t-Akt protein ratio (D) 24 hours after HI (*, p < 0.05 versus sham group; #, p < 0.05 versus vehicle group, n = 3, One-way ANOVA followed by Newman-Keuls test). When primary mouse cortical neurons were exposed to OGD for 60 minutes and additional 18 hours in normal culture medium, Bcl-2/Bax ratio in the OGD group was lower than that of the control group, and significantly increased in the Carvacrol (300 μM) pre-treatment group (E and F, *, p < 0.05 versus the sham group; #, p < 0.05 versus the vehicle group, n = 3, One-way ANOVA followed by Newman-Keuls test).