Chlorogenic acid modulates the ubiquitin-proteasome system in stroke animal model

Abstract

Background: Chlorogenic acid, a phenolic compound, has potent antioxidant and neuroprotective properties. The ubiquitin-proteasome system is an important regulators of neurodevelopment and modulators of neuronal function. This system is associated with neurodevelopment and neurotransmission through degradation and removal of damaged proteins. Activation of the ubiquitin-proteasome system is a critical factor in preventing cell death. We have previously reported a decrease in the activity of the ubiquitin-proteasome system during cerebral ischemia. This study investigated whether chlorogenic acid regulates the ubiquitin-proteasome system in an animal stroke model. In adult rats, middle cerebral artery occlusion (MCAO) surgery was performed to induce focal cerebral ischemia. Chlorogenic acid (30 mg/kg) or normal saline was injected into the abdominal cavity 2 h after MCAO surgery, and cerebral cortex tissues were collected 24 h after MCAO damage.

Results: Chlorogenic acid attenuated neurobehavioral disorders and histopathological changes caused by MCAO damage. We identified the decreases in ubiquitin C-terminal hydrolase L1, ubiquitin thioesterase OTUB1, proteasome subunit α type 1, proteasome subunit α type 3, and proteasome subunit β type 4 expression using a proteomics approach in MCAO animals. The decrease in these proteins was alleviated by chlorogenic acid. In addition, the results of reverse transcription-polymerase chain reaction confirmed these changes. The identified proteins were markedly reduced in MCAO damage, while chlorogenic acid prevented these reductions induced by MCAO. The decrease of ubiquitin-proteasome system proteins in ischemic damage was associated with neuronal apoptosis.

Conclusions: Our results showed that chlorogenic acid regulates ubiquitin-proteasome system proteins and protects cortical neurons from neuronal damage. These results provide evidence that chlorogenic acid has neuroprotective effects and maintains the ubiquitin-proteasome system in ischemic brain injury.

Keywords: Cerebral ischemia; Chlorogenic acid; Neuroprotection; Ubiquitin–proteasome system.

Fig. 1

Neuroprotective effects of chlorogenic acid (CGA) in a middle cerebral artery occlusion (MCAO) animal model. Neurobehavioral corner test (A), vibrissae-evoked forelimb placement test (B), hematoxylin and eosin staining (C–E) in the vehicle + MCAO, CGA + MCAO, vehicle + sham, and CGA + sham animals. CGA attenuated MCAO-induced neurobehavioral deficits and histopathological changes. Square indicates the representative area of high magnification image. Damaged area was calculated as the ratio of infarct area to total brain area (D). Black arrows indicate condensed and shrunken nuclei, and white arrows indicate vacuolated and swollen forms (E). Scale bar = 100 μm. Data (A and B, n = 14; D and F n = 4) are represented as the mean ± S.E.M. * p < 0.05

Fig. 2

Chlorogenic acid (CGA) inhibits the decrease in ubiquitin–proteasome system proteins in the right ischemic cortex. Images of protein spots A of ubiquitin C-terminal hydrolase L1, ubiquitin thioesterase OTUB1, proteasome subunit α type 1, proteasome subunit α type 3, proteasome subunit β type 4, and hypoxanthine phosphoribosyltransferase in the cerebral cortex from vehicle + middle cerebral artery occlusion (MCAO), CGA + MCAO, vehicle + sham, and CGA + sham animals. MCAO significantly reduced the expression levels of the ubiquitin–proteasome system proteins and CGA treatment maintained the levels of these proteins. Each square indicates protein spots. Intensities of protein spots are represented as a ratio of intensity of each group to that of vehicle + sham group B. Data (n = 5 per group) are represented as the mean ± S.E.M. *p < 0.05, **p < 0.01

Fig. 3

Chlorogenic acid (CGA) maintains the mRNA levels of ubiquitin–proteasome system proteins in the right ischemic cortex. Image of reverse transcription-PCR products A of ubiquitin C-terminal hydrolase L1, ubiquitin thioesterase OTUB1, proteasome subunit α type 1, proteasome subunit α type 3, and proteasome subunit β type 4 in the cerebral cortex from vehicle + middle cerebral artery occlusion (MCAO), CGA + MCAO, vehicle + sham, and CGA + sham animals. MCAO induced cerebral ischemia markedly decreased the mRNA levels of the ubiquitin–proteasome system proteins while treatment with CGA inhibited the MCAO-induced decrease in the mRNA levels of these proteins. Intensities of PCR product are represented as a ratio of β-actin product intensity (B). Data (n = 5 per group) are represented as the mean ± S.E.M. *p < 0.05, **p < 0.01