Baicalin confers neuroprotection in animal models of stroke through its antioxidant and anti-apoptotic effects

Abstract

Importance: Ischemic stroke leads to neuronal cell death due to a lack of oxygen and glucose. Baicalin is a flavonoid that has antioxidant and anti-inflammatory properties.

Objective: The aim of this study is to elucidate the anti-oxidant and anti-apoptotic effects of baicalin in animal models of stroke.

Methods: Vehicle or baicalin (100 mg/kg) was administered intraperitoneally immediately after the middle cerebral artery occlusion (MCAO) surgery. Neurobehavioral tests were conducted 24 h post-MCAO and brain tissue was isolated to assess histopathological changes and apoptosis-associated protein expression. Additionally, reactive oxygen species (ROS) and lipid peroxidation (LPO) assays were performed to evaluate oxidative stress.

Results: MCAO animals exhibited severe neurological deficits, which were significantly alleviated by baicalin treatment. Baicalin mitigated the up-regulation in ROS and LPO levels induced by surgery. MCAO damage led to severe histopathological lesions and an increase in terminal deoxynucleotidyl transferase dUTP nick end labeling-positive reactions, these alterations were alleviated by baicalin treatment. MCAO damage decreases the expression of Bcl-2 and increases the expression of Bax, baicalin alleviates these changes. Baicalin also attenuated the upregulation of caspase-3 expression caused by MCAO injury.

Conclusions and relevance: These results can suggest evidence that baicalin exerts neuroprotective effects by preventing apoptosis during cerebral ischemia. In conclusion, baicalin acts as a potent neuroprotective agent through its antioxidant and anti-apoptotic effects on neuronal cell damage.

Keywords: Baicalin; cerebral ischemia; neuroprotection; stroke.

Fig. 1. Baicalin attenuates neurobehavioral deficits in MCAO animals. The chemical structure and molecular formula of baicalin (A), neurological deficit scoring test (B), corner test (C), grip strength assessment test (D), and vibrissae-evoked forelimb placing test (E) in the vehicle + sham, baicalin + sham, vehicle + MCAO, and baicalin + MCAO animals. Baicalin improved neurobehavioral disorder caused by MCAO damage. Data (n = 10) are shown as means ± standard error of means.

MCAO, middle cerebral artery occlusion. ^*p < 0.01, ^**p < 0.05 vs. vehicle + sham animals; ^#p < 0.05 vs. vehicle + MCAO animals.

Fig. 2. Baicalin exerts a neuroprotective effect in MCAO animals. ROS (A) and LPO (B) assays, representative microphotograph of triphenyltetrazolium chloride staining (C and D) of the brain in the vehicle + sham, baicalin + sham, vehicle + MCAO, and baicalin + MCAO animals. DCF and MDA levels were increased in vehicle + MCAO animals, while baicalin treatment attenuated these increases. Intact areas were stained red color, while ischemic areas were remained white color. Baicalin improved the infarct volume increased by MCAO damage. Data (n = 4) are shown as means ± standard error of means.

ROS, reactive oxygen species; LPO, lipid peroxidation; MCAO, middle cerebral artery occlusion; DCF, 2′,7′-dichlorodihydrofluorescein; MDA, malondialdehyde. ^*p < 0.01, ^**p < 0.05 vs. vehicle + sham animals; ^#p < 0.05 vs. vehicle + MCAO animals.

Fig. 3. Baicalin attenuates histopathological changes in MCAO animals. Representative microphotograph of (A) H&E staining and (B) TUNEL staining of the cerebral cortex in vehicle + sham, baicalin + sham, vehicle + MCAO, and baicalin + MCAO animals. (C) The number of damaged cells and TUNEL-positive cells increased in vehicle-treated + MCAO animals, whereas baicalin treatment attenuated these increases. Arrows indicate damaged cells with condensed and shrunken nuclei, swollen shape, and vacuolated forms (A). Arrows indicate the TUNEL-positive cells with dark brown (B). Data (n = 4) are presented as mean ± standard error of means. Scale bar: 100 μm.

H&E, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; MCAO, middle cerebral artery occlusion. ^*p < 0.01, ^**p < 0.05 vs. vehicle + sham animals; ^#p < 0.05 vs. vehicle + MCAO animals.

Fig. 4. Baicalin regulates Bcl-2 and Bax expressions in MCAO animals. (A-C) Western blot analysis and (D-F) immunofluorescence staining of Bcl-2 and Bax in the cerebral cortex of vehicle + sham, baicalin + sham animal, vehicle + MCAO, and baicalin + MCAO animals. Western blot analysis is presented as the ratio of each protein density to β-actin density. Results of immunostaining were expressed as a ratio of the number of each positive cell (green color) to that of DAPI-positive cells (blue color). The result for vehicle + sham animals was set to 1.0. Data (n = 4) are represented as mean ± standard error of means. Scale bar = 75 µm.

MCAO, middle cerebral artery occlusion; DAPI, 4′,6-diamidino-2-phenylindole. ^*p < 0.01, ^**p < 0.05 vs. vehicle + sham animals, ^#p < 0.05 vs. vehicle + MCAO animals.

Fig. 5. Western blot analysis and immunofluorescence staining of caspase-3 in the cerebral cortex of vehicle + sham, baicalin + sham animal, vehicle + MCAO, and baicalin + MCAO animals. Western blot analysis are presented as the ratio of each protein density to β-actin density (A and B). Results of immunostaining were expressed as a ratio of the number of caspase-3 positive cells (green color) to that of DAPI positive cells (blue color) (C and D). The result for vehicle + sham animals was set to 1.0. Data (n = 4) are represented as mean ± standard error of means. Scale bar = 75 µm.

MCAO, middle cerebral artery occlusion; DAPI, 4′,6-diamidino-2-phenylindole. ^*p < 0.01, ^**p < 0.05 vs. vehicle + sham animals; ^#p < 0.05 vs. vehicle + MCAO animals.

Fig. 6. A schematic of the neuroprotective effects of baicalin in focal cerebral ischemia. MCAO damage induced morphological changes, cerebral edema, infarction, and neurological disorders, and baicalin attenuated these changes through antioxidant and anti-apoptotic effects. MCAO damage decreased the expression of Bcl-2 and increased the expression of Bax and caspase-2, whereas baicalin alleviated these changes.

MCAO, middle cerebral artery occlusion.