The aim of the present study was to determine the effects of ferulic acid (FerA) administered immediately following the onset of permanent middle cerebral artery occlusion (MCAo) and then 7 days of ischemia, and also to explore the involvement of protein kinase B (Akt)-induced signaling in the penumbral cortex. Immediately following the onset of MCAo, FerA was intravenously administered to rats at a dose of 60 mg/kg (FerA-60 mg), 80 mg/kg (FerA-80 mg), or 100 mg/kg (FerA-100 mg). FerA-80 mg and FerA-100 mg effectively ameliorated cerebral infarction and neurological deficits 7 days following permanent cerebral ischemia. FerA-80 mg and FerA-100 mg significantly upregulated the expression of phospho-Akt (p-Akt), phospho-mammalian target of rapamycin (p-mTOR), and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1), and the phospho-4E-BP1 (p-4E-BP1)/4E-BP1 and mitochondrial Bcl-2/Bax ratios, and markedly downregulated the levels of cytochrome
During cerebral ischemia, mitogen-activated protein kinases and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling cascades are key in the regulation of cell proliferation, survival and apoptosis, and in the translation of anti-apoptotic factors B-cell lymphoma-2 (Bcl-2) and Bcl-extra-large (Bcl-xL), leading to neuroprotection against ischemic injury (
The family of eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) comprises three members, 4E-BP1, 4E-BP2 and 4E-BP3. As another downstream target of mTOR, 4E-BP1 is a translational repressor that binds to eIF4E and thereby inhibits its interaction with eIF4G (
Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FerA) is a major active ingredient derived from
Therefore, the present study evaluated the effects of various doses of FerA administered 7 days following permanent MCAo and examined the involvement of Akt signaling cascades in the penumbral cortex.
A total of 106 male Sprague-Dawley (SD) rats (BioLASCO Taiwan Co., Ltd., Taipei, Taiwan), 8–9 weeks of age and weighing 300–350 g, were used in the present study. The rats were housed in standard plastic cages, fed a constant provision of solid food, provided with water
The permanent MCAo model was performed on adult SD rats as described previously with modifications (
The neurological status of each rat was examined 1, 3, and 7 days following cerebral ischemia using a modified neurological deficit score (NDS; score of 0–18) as described previously (
The rats were randomly divided into five groups: Sham, Vehicle, FerA-60 mg, FerA-80 mg, and FerA-100 mg groups (n=4-5). In the FerA-60 mg, FerA-80 mg, and FerA-100 mg groups, the rats underwent MCAo surgery and were simultaneously administered FerA intravenously (iv) at doses of 60, 80 and 100 mg/kg, respectively. Following 7 days of cerebral ischemia, the rats were examined for neurological functions and then sacrificed. In the Vehicle group, the rats underwent the same procedures as that of the FerA-100 mg group, but the rats were administered saline instead of FerA. In the Sham group, the rats underwent the same procedures as that of the Vehicle group; however, the rats did not undergo MCAo.
Following 7 days of cerebral ischemia, the rats were examined for their neurological status and subsequently sacrificed. Their brains were carefully removed from the skull and divided into six serial 2-mm thick coronal slices using a brain slicer matrix. The brain slices were placed in a constant temperature box and incubated with 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC; Merck KGaA, Darmstadt, Germany) for 5 min at 37°C. Following TTC staining, the cerebral infarct in the MCA territory turned pale white, whereas the healthy brain tissue turned dark red. The TTC stained brain sections were visualized and photographed using a digital camera (Nikon Coolpix 775; Nikon Corporation, Tokyo, Japan). The areas of infarction were evaluated using image analysis software (ImageJ version 1.46; National Institutes of Health, Bethesda, MD, USA), and the percentage of cerebral infarction was obtained by determining the ratio of the cerebral infarct area to the total brain area.
The rats were randomly divided into the following five groups: Sham, Vehicle, FerA-60 mg, FerA-80 mg, and FerA-100 mg groups (n=4-5). They underwent the same procedures as described in Experiment A.
Following 7 days of cerebral ischemia, the rats were sacrificed and their brains were carefully removed. The coronal sections of the brains obtained from 1.7 mm anterior to 4.3 mm posterior to the bregma were used for western blot analysis. The penumbral cortex was homogenized in cold Cytosol Extraction Buffer mix (BioVision, Inc., Milpitas, CA, USA) using a tissue homogenizer (Kinematia Polytron RT-3000). The samples were further divided into cytosolic and mitochondrial fractions using the Mitochondria/Cytosol Fractionation kit (cat. no. K256-100 BioVision, Inc.). The concentrations of the cytosolic and mitochondrial proteins were evaluated using a Bio-Rad protein assay. Equal quantities (15 µg/well) of protein were loaded into the gel wells and separated according to their molecular weight by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as described previously (
Following 7 days of ischemia, the rats were sacrificed (n=3-4). Their brains were carefully removed, postfixed in 4% paraformalaldehyde, and cut into 15-µm-thick sections, as described previously (
TUNEL staining was performed in accordance with the manufacturer's protocol (QIA33; Calbiochem; Merck KGaA). The brain slides of the adjacent sections used in IHC analysis were first incubated with 20 µg/ml proteinase K for 20 min at room temperature, as described previously (
The rats were randomly divided into four groups: DS + Sham, DS + Vehicle, DS + FerA-100 mg, and LY + FerA-100 mg groups (n=4-5). In the LY + FerA-100 mg group, the rats underwent the same surgical procedures as that of the FerA-100 mg group (Experiment A); however, the rats also received an intracerebroventricular (ICV) infusion of LY294002 (LY), a PI3K/Akt inhibitor, 30 min prior to MCAo. In the DS + FerA-100 mg group, the rats underwent the same surgical procedures as that of the LY + FerA-100 mg group, but the rats received an ICV infusion of 1% dimethyl sulfoxide (DMSO). In the DS + Vehicle group, the rats underwent the same surgical procedures as that of the Vehicle group (Experiment A), but the rats also received an ICV infusion of 1% DMSO 30 min prior to MCAo. In the DS + Sham group, the rats underwent the same surgical procedures as that of the DS + Vehicle group, but the rats did not undergo MCAo.
The rats were anesthetized with 5% isoflurane, and anesthesia was maintained via facemask inhalation using 2% isoflurane. The rats received an ICV infusion of 10 µl of a solution containing LY (10 mM in DMSO, cat. no. 1747-5, BioVision, Inc.) or 1% DMSO into their right hemisphere. The infusions were performed over a 6-min period using a 10 µl Hamilton microliter syringe (Hamilton Company, Reno, NV, USA). The infusion was administered into the right cerebral ventricle (0.8 mm posterior to the bregma, 1.5 mm lateral on the right side, and 3.5 mm below the skull surface).
Following 7 days of ischemia, the rats were sacrificed, and their brains were immediately removed for incubation with antibodies for western blot analysis of the expression of Akt, p-Akt, mTOR, p-mTOR, 4E-BP1, p-4E-BP1, eIF4E (rabbit, cat. no. 9742; Cell Signaling Technology, Inc., 1:1,000), Bcl-2 and Bax. The protocol used for analysis of the protein samples using western blotting was the same as that described for Experiment B.
The rats were randomly divided into the following four groups: DS + Sham, DS + Vehicle, DS + FerA-100 mg, and LY + FerA-100 mg groups (n=3-4). They underwent the same experimental protocols as described in Experiment C.
At 7 days post-ischemia, the rats were sacrificed. They underwent the same experimental protocols for cerebral infarct detection as described in Experiment A.
All data in the present study are expressed as the mean ± standard deviation. Comparisons were performed using one-way analysis of variance with a Scheffe post hoc test. P<0.05 was considered to indicate a statistically significant difference. All data were analyzed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA).
At 7 days post-ischemia, the percentage of the cerebral infarct area was significantly increased in the Vehicle group compared with that in the Sham group (P<0.05) and was significantly reduced in the FerA-80 mg and FerA-100 mg groups compared with that in the Vehicle group (both P<0.05;
At 1 day post-ischemia, the NDS of the Vehicle, FerA-60 mg, FerA-80 mg, and FerA-100 mg groups did not differ significantly (P>0.05;
The results of the western blot analysis showed that the ratios of cytosolic p-Akt/Akt and p-mTOR/mTOR expression in the penumbral cortex were significantly reduced in the Vehicle group (both 0.3-fold) compared with those in the Sham group (both P<0.05) and were significantly increased in the FerA-80 mg (3.4- and 5.1-fold, respectively) and FerA-100 mg (5.0- and 5.1-fold, respectively) groups compared with the Vehicle group (all P<0.05;
The ratio of mitochondrial Bcl-2/Bax expression in the penumbral cortex was markedly reduced in the Vehicle group (0.2-fold) compared with that in the Sham group (P<0.05) and was markedly increased in the FerA-80 mg (10.5-fold) and FerA-100 mg (11.6-fold) groups compared with that in the Vehicle group (both P<0.05;
The numbers of cytochrome
The ratios of cytosolic p-Akt/Akt, p-mTOR/mTOR, 4E-BP1/actin, and eIF4E/actin expression in the penumbral cortex were significantly reduced in the DS + Vehicle group (0.4-, 0.3-, 0.4-, and 0.2-fold, respectively) compared with those in the DS + Sham group (all P<0.05) and were significantly increased in the DS + FerA-100 mg group (3.2-, 3.7-, 3.5-, and 3.3-fold, respectively) compared with those in the DS + Vehicle group (all P<0.05
The ratio of mitochondrial Bcl-2/Bax expression in the penumbral cortex was markedly reduced in the DS + Vehicle group (0.2-fold) compared with that in the DS + Sham group (P<0.05) and was markedly increased in the DS + FerA-100 mg group (3.5-fold) compared with that in the DS + Vehicle group (P<0.05;
The percentage cerebral infarct area was significantly increased in the DS + Vehicle group compared with that in the DS + Sham group (P<0.05) and was significantly reduced in the DS + FerA-100 mg group compared with that in the DS + Vehicle group (P<0.05;
In permanent focal cerebral ischemia, the infarct area rapidly increases within 1 day following the onset of ischemia and then undergoes delayed infarct expansion between 1 and 7 days post-ischemia (
Increasing evidence has revealed that apoptotic cells present in the ischemic penumbra contribute to infarct expansion during permanent (
Activated Akt can rapidly phosphorylate certain downstream proteins, including mTOR, and the activity of mTOR is regulated through the phosphorylation of its serine 2448 (
Accumulating evidence indicates that eIF4E is critical for translational control and is inactivated by stress, including ischemic stress, and activated by survival factors. In addition, the overexpression of eIF4E exerts beneficial effects on cytochrome
To determine the precise mechanism underlying the involvement of Akt-mediated anti-apoptotic signaling in FerA treatment, another experiment was performed in the FerA-100 mg group as the representative group of FerA treatment to evaluate the action of LY, a selective inhibitor of PI3K/Akt signaling, 7 days following cerebral ischemia. It was found that 1% DMSO (vehicle control) pretreatment (DS + Sham, DS + Vehicle, and DS + FerA-100 mg) did not alter the expression of p-Akt, p-mTOR or p-4E-BP1. Furthermore, the expression of eIF4E was markedly decreased in the DS + Vehicle group and the reduced expression of eIF4E was effectively restored in the DS + FerA-100 mg group. However, LY pretreatment (LY + FerA-100 mg) effectively abrogated the upregulating effects of FerA-100 mg treatment on the expression of p-Akt. LY + FerA-100 mg treatment consequently suppressed mTOR/4E-BP1/eIF4E signaling and activated the mitochondrial Bax-related apoptotic signaling cascade in the penumbral cortex, resulting in the exacerbation of the cerebral infarct size 7 days following permanent cerebral ischemia. On the basis of these results, it was hypothesized that FerA treatment exerts beneficial effects on cerebral infarction by activating Akt signaling, and that the downregulating effects of FerA treatment on mitochondrial Bax-induced apoptosis are attributed to the upregulation of the Akt/mTOR/4E-BP1/eIF4E/Bcl-2 anti-apoptotic signaling in the penumbral cortex 7 days following cerebral ischemia. To the best of our knowledge, the present study is the first to show that FerA treatment exerts neuroprotective effects against Bax-induced apoptosis by upregulating Akt/mTOR/4E-BP1-mediated, but not Akt/mTOR/p70S6K-mediated Bcl-2 anti-apoptotic signaling in the subacute phase of permanent MCAo.
Compelling evidence shows that Akt-mediated signaling downregulates the expression of inducible nitric oxide synthase, which elicits nitric oxide (NO)-induced apoptosis in the ischemic region following transient MCAo (
In conclusion, the findings of the present study suggest that FerA administered at a dose of 80 or 100 mg/kg immediately following the onset of cerebral ischemia effectively reduces cerebral infarction and improves neurological functions 7 days following cerebral ischemia, and that the anti-infarction effects of FerA treatment are associated with the activation of Akt-mediated anti-apoptotic signaling in the penumbral cortex. The effects of FerA treatment on mitochondrial Bax-induced apoptosis can be further attributed to the upregulation of Akt/mTOR/4E-BP1/Bcl-2 anti-apoptotic signaling, which inhibits the cytochrome
Not applicable.
The present study was supported by grants from China Medical University (grant no. CMU105-S-40) and China Medical University Hospital (grant nos. DMR-105-007 and DMR-107-165), Taichung, Taiwan.
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
CYC and YCL designed experiments. CYC performed the experiments, analyzed the data and wrote the manuscript. STK participated in the conception and design of the study, and helped to draft the manuscript. All authors have read and approved the final manuscript.
All study procedures were approved by the Institutional Animal Care and Use Committee of China Medical University (Taichung, Taiwan; permit no. 2016-321).
Not applicable.
The authors declare that they have no competing interests.
Cerebral infarct areas (S1-S6) in the experimental groups at 7 days following cerebral ischemia. In 2,3,5-triphenyltetrazolium chloride-stained sections, infarction is represented by a pale white color and non-infarction is represented by a dark red color. Scale bar=1 cm. FerA, ferulic acid.
Effects of FerA treatment on cerebral infarct area and neurological deficits 7 days following cerebral ischemia. (A) Percentages of cerebral infarct areas in the Sham, Vehicle, FerA-60 mg, FerA-80 mg, and FerA-100 mg groups were evaluated at 7 days post-ischemia (n=4-5). (B) NDS of the Sham, Vehicle, FerA-60 mg, FerA-80 mg, and FerA-100 mg groups were evaluated at 1, 3, and 7 days post-ischemia. *P<0.05 vs. the Sham group; #P<0.05 vs. the Vehicle group. FerA, ferulic acid; NDS, neurological deficit score.
Effects of FerA treatment on the cytosolic expression of p-Akt, Akt, p-mTOR, mTOR, p-4E-BP1, 4E-BP1, p-p70S6K, and p70S6K in the penumbral cortex. (A) Representative western blot images revealed the cytosolic expression of p-Akt, Akt, p-mTOR, mTOR, p-4E-BP1, 4E-BP1, p-p70S6K, and p70S6K in the penumbral cortex of the Sham, Vehicle, FerA-60 mg, FerA-80 mg, and FerA-100 mg groups at 7 days post-ischemia. Actin was used as an internal control for cytosolic extracts. The ratios of (B) p-Akt/Akt, (C) p-mTOR/mTOR, (D) 4E-BP1/actin, (E) p-4E-BP1/4E-BP1, (F) p70S6K/actin, and (G) p-p70S6K/p70S6K expression were measured in the penumbral cortex in the Sham, Vehicle, FerA-60 mg, FerA-80 mg, and FerA-100 mg groups (n=4-5). *P<0.05 vs. the Sham group; #P<0.05 vs. the Vehicle group. FerA, ferulic acid; cyto, cytosolic fraction; mTOR, mammalian target of rapamycin; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; p-, phosphorylated.
Effects of FerA treatment on the mitochondrial expression of Bcl-2, Bcl-xL, and Bax in the penumbral cortex. (A) Representative western blot images revealed the mitochondrial expression of Bcl-2, Bcl-xL and Bax in the penumbral cortex in the Sham, Vehicle, FerA-60 mg, FerA-80 mg, and FerA-100 mg groups at 7 days post-ischemia. HSP60 was used as an internal control for mitochondrial extracts. The ratios of (B) mitochondrial Bcl-2/Bax and (C) mitochondrial Bcl-xL/Bax expression were measured in the penumbral cortex in the Sham, Vehicle, FerA-60 mg, FerA-80 mg, and FerA-100 mg groups (n=4-5). *P<0.05 vs. the Sham group; #P<0.05 vs. the Vehicle group. FerA, ferulic acid; mito, mitochondrial fraction; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated X protein; Bcl-xL, Bcl-extra-large.
Effects of FerA treatment on the expression of cytochrome
Effects of DS + FerA-100 mg and LY + FerA-100 mg on the cytosolic expression of p-Akt, Akt, p-mTOR, mTOR, p-4E-BP1, 4E-BP1 and eIF4E in the penumbral cortex. (A) Representative images show cytosolic expression of p-Akt, Akt, p-mTOR, mTOR, p-4E-BP1, 4E-BP1, and eIF4E in the penumbral cortex in the DS + Sham, DS + Vehicle, DS + FerA-100 mg, and LY + FerA-100 mg groups at 7 days post-ischemia. The ratios of (B) p-Akt/Akt (C) p-mTOR/mTOR (D) 4E-BP1/actin (E) eIF4E/actin, and (F) p-4E-BP1/actin expression were measured in the penumbral cortex in the DS + Sham, DS + Vehicle, DS + FerA-100 mg, and LY+FerA-100 mg groups (n=4-5). *P<0.05 vs. the DS + Sham group; #P<0.05 vs. the DS + Vehicle group. FerA, ferulic acid; DS, dimethyl sulfoxide; LY, LY294002; cyto, cytosolic fraction; mTOR, mammalian target of rapamycin; eIF4E, eukaryotic initiation factor 4E; 4E-BP1, eIF4E-binding protein 1; p-, phosphorylated.
Effects of DS + FerA-100 mg and LY + FerA-100 mg on the mitochondrial expression of Bcl-2 and Bax in the penumbral cortex. (A) Representative images show the mitochondrial expression of Bcl-2 and Bax in the penumbral cortex in the DS + Sham, DS + Vehicle, DS + FerA-100 mg, and LY + FerA-100 mg groups at 7 days post-ischemia. The ratio of (B) mitochondrial Bcl-2/Bax expression was measured in the penumbral cortex in the DS + Sham, DS + Vehicle, DS + FerA-100 mg, and LY + FerA-100 mg groups (n=4-5). *P<0.05 vs. the DS + Sham group; #P<0.05 vs. the DS + Vehicle group. FerA, ferulic acid; DS, dimethyl sulfoxide; LY, LY294002; Mito, mitochondrial fraction; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-associated X protein.
Effects of DS + FerA-100 mg and LY + FerA-100 mg on cerebral infarction. (A) TTC staining reveals normal brain tissues (dark red color) and infarcted tissues (pale-white color) between S1 and S6 (scale bar=1 cm). (B) Percentages of cerebral infarct areas in the DS + Sham, DS + Vehicle, DS + FerA-100 mg, and LY + FerA-100 mg groups were evaluated at 7 days post-ischemia (n=3). *P<0.05 vs. the DS + Sham group; #P<0.05 vs. the DS + Vehicle group. FerA, ferulic acid; DS, dimethyl sulfoxide; LY, LY294002.
Expression of cytochrome
Factor | Sham | Vehicle | FerA-60 mg | FerA-80 mg | FerA-100 mg |
---|---|---|---|---|---|
Cytochrome |
0±0 | 322±26 |
272±28 | 129±21 |
95±15 |
Cleaved caspase-3 | 0±0 | 258±55 |
316±50 | 112±5 |
83±24 |
TUNEL | 0±0 | 818±110 |
699±107 | 366±65 |
291±42 |
P<0.05 vs. the Sham group
P<0.05 vs. the Vehicle group. FerA, ferulic acid; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling.