Shexiang Baoxin Pill, a Proprietary Multi-Constituent Chinese Medicine, Prevents Locomotor and Cognitive Impairment Caused by Brain Ischemia and Reperfusion Injury in Rats: A Potential Therapy for Neuropsychiatric Sequelae of Stroke

Abstract

Ischemic stroke is a common type of cerebrovascular event and also the leading cause of disability. Post-stroke cognitive impairment occurs frequently in stroke survivors. Shexiang Baoxin Pill (SBP) is a proprietary Chinese medicine, initially used to treat cardiovascular diseases. Herein, we aim to explore the effects of SBP on oxygen glucose deprivation and reoxygenation (OGD/R) in neuronal cells (CATH.a) and cerebral ischemia/reperfusion injury induced post-stroke cognitive impairment in middle cerebral artery occlusion (MCAO) rat model. MCAO rats received two doses of oral SBP treatment (28 or 56 mg/kg) after 1 h of operation and once daily for 2 weeks continuously. Behavioral tests, immunoblotting, and immunofluorescence were examined after 14 days. Current data suggest that SBP enhanced cell viability and downregulated apoptosis via activating the PI3K/Akt signaling pathway in CATH. a cells. Furthermore, 14 days of SBP treatment promoted the recovery of learning and locomotor function in the MCAO rats. SBP up-regulated the expression of p-Akt, p-GSK3β, as well as the expression of NMDAR1, PSD-95, and AMPAR. Also, SBP down-regulated the expression of p-CaMKII. These results indicated that long-term SBP treatment might be a potential option for cognitive impairment induced by the ischemic stroke.

Keywords: ischemic stroke; locomotor ability; middle cerebral artery occlusion; post stroke cognitive impairment; shexiang baoxin pill.

FIGURE 1

SBP enhanced cell viability and downregulated apoptosis, and upregulated the expression of p-Akt/Akt and p-GSK3β/GSK3β in vitro. (A) CCK8 assay for cell viability in CATH. a cells under OGD/R conditions with or without SBP. (B, C) The apoptosis of CATH. a was assessed by flow cytometry, and the results showed that OGD/R can enhance apoptosis, while the apoptosis was downregulated upon SBP treatment. (D) The protein levels of p-Akt/Akt and p-GSK3β/GSK3 were upregulated and activated by SBP. All data are means ± SEM, n = 3; ^* p < 0.05, ^** p < 0.01, and ^*** p <.001 compared to OGD/R group.

FIGURE 2

SBP reduces cerebral infarction at the early stage of stroke in rats 24 h after ischemia/reperfusion injury. Data are expressed as mean ± SEM, n = 8; ^*** p < 0.001 compared to MCAO.

FIGURE 3

SBP Improved Behavioural Effects on Cognitive Performance in Rats 14 days after Ischemia-Reperfusion Brain Injury. Effects of SBP in MCAO model rats in the Morris water maze test. (A) Schematic representation of the experimental design. (B) representative swimming path. (C) Escape latency to the hidden platform in training trials. (D) time stay in the target quadrant. (E) total distance move. Data are expressed as mean ± SEM, n = 9; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 compared to MCAO.

FIGURE 4

SBP Improved Behavioural Effects on Locomotor and Anxiety in Rats 14 days after Ischemia-Reperfusion Brain Injury. (A) representative open field test path (B) Entries in the target quadrant during the probe test. (C) Time stay in target quadrant during open field test. (D) Recognition index from noval object recognition. (E) Time to stay at rotarod at day 7. (F) Time to stay at rotarod at day 14. Data are expressed as mean ± SEM, n = 9: ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 compared to MCAO.

FIGURE 5

(A) Western blot analysis of p-Akt, Akt, p-GSK3β, and GSK3 in the ipsilateral side of the brain of rats at 14 d after MCAO (or sham surgery) operation. (B)Western blot analysis of PSD-95, NMDAR1, AMPAR, p-CaMKII, and CaMKII in the ipsilateral hippocampus of rats at 14 days after MCAO (or sham surgery) operation. Data are expressed as mean ± SEM, n = 5; ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.

FIGURE 6

Immunofluorescence images on PSD-95 (green) and NeuN (red) in the cornu ammonis 3 (CA3) and dentate gyrus (DG) of hippocampal subregions of rats at 14 days after MCAO (or sham surgery) operation. Data are expressed as mean ± SEM, n = 3; ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 compared to MCAO.