Carvacrol, a Food-Additive, Provides Neuroprotection on Focal Cerebral Ischemia/Reperfusion Injury in Mice

Carvacrol (CAR), a naturally occurring monoterpenic phenol and food additive, has been shown to have antimicrobials, antitumor, and antidepressant-like activities. A previous study demonstrated that CAR has the ability to protect liver against ischemia/reperfusion injury in rats. In this study, we investigated the protective effects of CAR on cerebral ischemia/reperfusion injury in a middle cerebral artery occlusion mouse model. We found that CAR (50 mg/kg) significantly reduced infarct volume and improved neurological deficits after 75 min of ischemia and 24 h of reperfusion. This neuroprotection was in a dose-dependent manner. Post-treatment with CAR still provided protection on infarct volume when it was administered intraperitoneally at 2 h after reperfusion; however, intracerebroventricular post-treatment reduced infarct volume even when the mice were treated with CAR at 6 h after reperfusion. These findings indicated that CAR has an extended therapeutic window, but delivery strategies may affect the protective effects of CAR. Further, we found that CAR significantly decreased the level of cleaved caspase-3, a marker of apoptosis, suggesting the anti-apoptotic activity of CAR. Finally, our data indicated that CAR treatment increased the level of phosphorylated Akt and the neuroprotection of CAR was reversed by a PI3K inhibitor LY-294002, demonstrating the involvement of the PI3K/Akt pathway in the anti-apoptotic mechanisms of CAR. Due to its safety and wide use in the food industry, CAR is a promising agent to be translated into clinical trials

Antioxidant Activity of 7,8-Dihydroxyflavone Provides Neuroprotection Against Glutamate-Induced Toxicity

Glutamate, an excitatory neurotransmitter in the central nervous system, plays an important role in neurological disorders. Previous studies have shown that excess glutamate can cause oxidative stress in a hippocampal HT-22 cell line. 7,8-Dihydroxyflavone (7,8-DHF), a member of the flavonoid family, is a selective tyrosine kinase receptor B (TrkB) agonist that has neurotrophic effects in various neurological diseases such as stroke and Parkinson\u27s disease. In this study, we found that there is no TrkB receptor in HT-22 cells. Despite this, our data demonstrate that 7,8-DHF still protects against glutamate-induced toxicity in HT-22 cells in a concentration-dependent manner, indicating that 7,8-DHF prevents cell death through other mechanisms rather than TrkB receptors in this cell model. We further show that 7,8-DHF increases cellular glutathione levels and reduces reactive oxygen species (ROS) production caused by glutamate in HT-22 cells. Finally, our data demonstrate that 7,8-DHF protects against hydrogen peroxide and menadione-induced cell death, suggesting that 7,8-DHF has an antioxidant effect. In summary, although 7,8-DHF is considered as a selective TrkB agonist, our results demonstrate that 7,8-DHF can still confer neuroprotection against glutamate-induced toxicity in HT-22 cells via its antioxidant activity

The protection of CAR on infarct volume was in a dose-dependent manner.

<p>Mice were administered (i.p.) with CAR at doses of 5, 25, and 50 mg/kg at 2 h before ischemia. Cerebral infarct volume was determined by TTC staining after 75 min of ischemia and 24 h of reperfusion. Bars represent mean ± SEM of 6 brains. *, <i>P</i><0.05 versus vehicle-treated I/R group.</p

CAR treatment reduced cleaved caspase-3 level after cerebral I/R injury.

<p>Mice were treated with CAR (50 mg/kg, i.p.) or saline 2 h before ischemia. Cleaved caspase-3 level was determined by Western blot after 75 min of ischemia and 24 h of reperfusion. (A)The representative photographs show the levels of cleaved caspase-3 and beta-tubulin. Beta-tubulin was used as a loading control. (B)The quantitative analysis indicated that CAR treatment decreased ratio of cleaved caspase-3 to beta-tubulin. Bars represent mean ± SEM for samples from 4 brains in each group. *, <i>P</i><0.05 versus vehicle-treated I/R group.</p

A PI3K inhibitor LY-294002 blocked the neuroprotection of CAR on cerebral I/R injury.

<p>Mice were treated with CAR (50 mg/kg, i.p.) or saline 2 h before ischemia and 5 µl LY-294002 (10 mM) was administered (i.c.v.) at 15 min before ischemia. (A) After 30 min of ischemia and 6 h reperfusion, brain tissues were collected and p-Akt and t-Akt levels were determined by Western blot. The photographs show that LY-294002 treatment inhibited the activation of Akt by CAR. (B) After 75 min of ischemia and 24 h of reperfusion, cerebral infarct volume was determined by TTC staining. The representative TTC-stained coronal sections demonstrate that LY-294002 treatment abolished the protection of CAR on infarct volume. (C) Statistical analysis of cerebral infarct volume shows that PI3K inhibitor LY-294002 blocked the protective effects of CAR. I/R+CAR+LY: CAR and LY-294002-treated I/R group. Bars represent mean ± SEM of 6–9 brains. *, <i>P</i><0.05 versus vehicle-treated I/R group.</p

CAR treatment increased the activation of Akt after MCAO.

<p>Mice were treated with CAR (50 mg/kg, i.p.) or saline 2 h before ischemia. After 30 min of ischemia and 6 h reperfusion, brain tissues were collected and protein levels were determined by Western blot. (A)The representative photographs show levels of p-Akt, t-Akt, and beta-tubulin. Beta-tubulin was used as a loading control. (B) Quantitative analysis of the ratio of p-Akt to t-Akt was performed. Bars represent mean ± SEM for samples from 4 brains in each group. *, <i>P</i><0.05 versus sham group; #, <i>P</i><0.05 I/R group versus I/R+CAR group.</p

CAR pre-treatment reduced infarct volume and improved neurological deficits after MCAO.

<p>Mice were administered (i.p.) with CAR or saline at 2 h before cerebral ischemia. TTC staining and neurological scores were examined after 75 min of ischemia and 24 h of reperfusion. (A)The molecular structure of CAR is shown. (B) The representative TTC-stained coronal sections in vehicle-treated mice and CAR-treated mice (50 mg/kg,i.p.) are shown. (C)Statistical analysis of cerebral infarct volume was performed in I/R group and I/R+CAR group; CAR reduced infarct volume after cerebral I/R injury. (D)Neurological scores were evaluated according to a 5-points scale system. CAR treatment significantly decreased neurological deficits. Bars represent mean ± SEM of 6 brains. *, <i>P</i><0.05 versus vehicle-treated I/R group.</p

CAR post-treatment reduced infarct volume after cerebral I/R injury.

<p>CAR was administered (i.p. and i.c.v.) at different reperfusion times after 75 min of ischemia. TTC staining for cerebral infarct volume was performed after 24 h reperfusion. (A) Mice were administered CAR (50 mg/kg, i.p.) and saline at 0 h, 2 h, and 4 h after reperfusion. CAR had no protection on infarct volume when it was administered at 4 h after reperfusion. (B) Mice were administered with 10 µg CAR (i.c.v.) or saline at 2 h, 4 h, 6 h, and 7 h after reperfusion. CAR still had protective effect when mice treated with CAR after 6 h of reperfusion. Bars represent mean ± SEM of 6 brains. *, <i>P</i><0.05 versus vehicle-treated I/R group.</p