Chlorogenic acid alleviates the reduction of Akt and Bad phosphorylation and of phospho-Bad and 14-3-3 binding in an animal model of stroke

Abstract

Background: Stroke is caused by disruption of blood supply and results in permanent disabilities as well as death. Chlorogenic acid is a phenolic compound found in various fruits and coffee and exerts antioxidant, anti-inflammatory, and anti-apoptotic effects.

Objectives: The purpose of this study was to investigate whether chlorogenic acid regulates the PI3K-Akt-Bad signaling pathway in middle cerebral artery occlusion (MCAO)-induced damage.

Methods: Chlorogenic acid (30 mg/kg) or vehicle was administered peritoneally to adult male rats 2 h after MCAO surgery, and animals were sacrificed 24 h after MCAO surgery. Neurobehavioral tests were performed, and brain tissues were isolated. The cerebral cortex was collected for Western blot and immunoprecipitation analyses.

Results: MCAO damage caused severe neurobehavioral disorders and chlorogenic acid improved the neurological disorders. Chlorogenic acid alleviated the MCAO-induced histopathological changes and decreased the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells. Furthermore, MCAO-induced damage reduced the expression of phospho-PDK1, phospho-Akt, and phospho-Bad, which was alleviated with administration of chlorogenic acid. The interaction between phospho-Bad and 14-3-3 levels was reduced in MCAO animals, which was attenuated by chlorogenic acid treatment. In addition, chlorogenic acid alleviated the increase of cytochrome c and caspase-3 expression caused by MCAO damage.

Conclusions: The results of the present study showed that chlorogenic acid activates phospho-Akt and phospho-Bad and promotes the interaction between phospho-Bad and 14-3-3 during MCAO damage. In conclusion, chlorogenic acid exerts neuroprotective effects by activating the Akt-Bad signaling pathway and maintaining the interaction between phospho-Bad and 14-3-3 in ischemic stroke model.

Keywords: Chlorogenic acid; ischemic stroke; neuroprotection.

Fig. 1. Neurological deficits scoring test (A), corner test (B), adhesive removal test (C), and grip strength test (D) in vehicle + MCAO, CGA + MCAO, vehicle + sham, and CGA + sham animals. CGA improves neurological behavior deficits in ischemic brain injury. Data (n = 15) are shown as the mean ± standard error of mean.

MCAO, middle cerebral artery occlusion; CGA, chlorogenic acid. ^ap < 0.05.

Fig. 2. Representative photograph of hematoxylin and eosin staining (A-D) and TUNEL staining (E-H) of cerebral cortex tissue from vehicle + MCAO, CGA + MCAO, vehicle + sham, and CGA + sham animals. CGA migrated histopathological changes and attenuated increase in the number of TUNEL-positive cells caused by MCAO damage. Arrows indicate shrunken and condensed nuclei. The number of TUNEL-positive cells was markedly increased in vehicle + MCAO animals, while CGA alleviated this increase. Arrows indicate TUNEL-positive cells. Scale bar = 100 µm. Damaged cells represents the percentage of cells with histopathological lesions to total cells and apoptotic cells represents the percentage of TUNEL-positive cells to total cells (I). Data (n = 4) are shown as the mean ± standard error of mean.

TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; MCAO, middle cerebral artery occlusion; CGA, chlorogenic acid. ^ap < 0.05.

Fig. 3. Western blot analysis of phospho-PDK1, PDK1, phospho-Akt, Akt, phospho-Bad, and Bad in the cerebral cortex from vehicle + MCAO, CGA + MCAO, vehicle + sham, and CGA + sham animals. Each lane represents an individual experimental animal. Densitometric analysis is represented as a ratio of proteins intensity to actin intensity. Molecular weight (kDa) are depicted at right. Data (n = 5) are represented as mean ± standard error of mean.

PDK1, phosphoinositide-dependent kinase 1; MCAO, middle cerebral artery occlusion; CGA, chlorogenic acid. ^ap < 0.05.

Fig. 4. Immunofluorescence staining of phospho-Akt (A, C) and phospho-Bad (B, C) in the cerebral cortex from vehicle + MCAO, CGA + MCAO, vehicle + sham, and CGA + sham animals. CGA alleviated the decrease of phospho-Akt and phospho-Bad expression due to MCAO damage. The value of positive cells was expressed as a percentage of the number of positive cells to the number of total cells. Arrows indicate positive cells. Data (n = 5) are represented as mean ± standard error of mean. Scale bar = 100 µm.

MCAO, middle cerebral artery occlusion; CGA, chlorogenic acid. ^ap < 0.05.

Fig. 5. Immunoprecipitation analysis of phospho-Bad and 14-3-3 binding (A, B) and Western blot analysis of cytochrome c (C, D) in the cerebral cortex from vehicle + MCAO, CGA + MCAO, vehicle + sham, and CGA + sham animals. CGA attenuated the decrease of phospho-Bad and 14-3-3 interaction caused by MCAO damage. Each lane represents an individual experimental animal. Densitometric analysis is represented as a ratio of proteins intensity to IgG or β-actin intensity. Data (n = 5) are represented as mean ± standard error of mean.

MCAO, middle cerebral artery occlusion; CGA, chlorogenic acid. ^ap < 0.05.

Fig. 6. Western blot analysis of caspase-3 and cleaved caspase-3 in the cerebral cortex from vehicle + MCAO, CGA + MCAO, vehicle + sham, and CGA + sham animals. CGA attenuated the increase of caspase-3 and cleaved caspase-3 caused by MCAO damage. Each lane represents an individual experimental animal. Densitometric analysis is represented as a ratio of β-actin intensity. Data (n = 5) are represented as mean ± standard error of mean.

MCAO, middle cerebral artery occlusion; CGA, chlorogenic acid. ^ap < 0.05.