Suppression of cerebral ischemia injury induced blood brain barrier breakdown by dexmedetomidine via promoting CCN1

Abstract

Background: Blood-brain barrier (BBB) could aggravate cerebral ischemia injury. Dexmedetomidine (Dex) has been believed to play a protective role in cerebral ischemia injury-induced BBB injury.

Methods: Middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation (OGD) models were established to simulate cerebral ischemia injury. Animal experiments included 4 groups, Sham, MCAO, MCAO+Dex, MCAO+Dex+sh-CCN1. Generally applicable gene set enrichment analysis was performed to analyze gene expression difference. Total collagen content and Evans blue staining were performed to measure infarct ratio and BBB breakdown, respectively. The cell apoptosis, mRNA and protein expression were measured through flow cytometry, PCR, and western blotting, respectively. The levels of IL-1β, TNF-α, and IL-6 in serum were measured with commercial ELISA kits.

Results: Dex greatly promoted the expression level of CCN1. Dex suppressed cerebral ischemia injury, increased tight junction protein expression, improved the memory ability and neurological function of MCAO rats through targeting CCN1. The significant increase of inflammatory factors in the serum of MCAO rats were suppressed by Dex. Dex suppressed OGD induced increase of HRP permeability and promoting tight junction protein expression in vitro through regulating CCN1. The neurological function evaluation was performed with Neurological Severity Score (NSS) and Longa Score Scale.

Conclusions: Dex could remarkably alleviate cerebral ischemia injury by inhibiting BBB breakdown, inflammatory response, and promoting neurological function and tight junction protein expression via up-regulating CCN1. This study might provide a novel therapeutic target for the prevention and treatment of cerebral ischemia injury-induced BBB.

Keywords: CCN1; blood brain barrier; cerebral ischemia injury; dexmedetomidine; middle cerebral artery occlusion.

Figure 1

Dex greatly promoted the expression level of CCN1. (A) The level of CCN1 in HBMEC was measured with high throughput sequencing; (B) The level of CCN1 was measured with RT-PCR; (C) The level of CCN1 was measured with western blot. n = 3.

Figure 2

Dex suppressed cerebral ischemia injury and increased tight junction protein expression. (A) TTC staining was performed to study the infarct ratio; (B) Evans blue staining was conducted to investigate blood brain barrier injury; (C) The levels of IL-6, IL-1β, and TNF-α in serum were detected; (D) The expression levels of tight junction proteins were determined in brain tissues. ^*p <0.05. n = 5.

Figure 3

Dex remarkably improved the memory ability and neurological function of MCAO rats. (A) Morris water maze test was performed to evaluate the influence of Dex and sh-CCN1 on memory ability of rats (n = 5); (B) Escape latency was analyzed; (C) Swim distance was calculated; (D) The neurological function was evaluated with neurological severity score; (E) The neurological function was evaluated with Longa score. ^*p < 0.05. n = 5.

Figure 4

Gene differences analysis using brain tissues. (A) Gene differences between group Sham and MCAO were analyzed using volcano plot; (B) Gene differences between group Sham and MCAO+Dex were analyzed using volcano plot; (C) Gene differences were analyzed using Pheatmap analysis. n = 3.

Figure 5

Gene ontology enrichment analysis using brain tissues. (A) Gene ontology pathway enrichment analysis in the group MCAO; (B) Gene ontology pathway enrichment analysis in the group MCAO+Dex; (C) Differential gene GO annotation classification histogram. n = 3.

Figure 6

KEGG pathway enrichment analysis using brain tissues. (A) KEGG pathway enrichment analysis in the group MCAO; (B) KEGG pathway enrichment analysis in the group MCAO+Dex; (C) KEGG pathway classification was analyzed. n = 3.

Figure 7

Dex suppressed OGD induced increase of HRP permeability and promoting tight junction protein expression in vitro. (A) The expression levels of tight junction proteins in HBMEC were measured; (B) Blood brain barrier in vitro was evaluated with HRP permeability; (C) Cell apoptosis was detected with flow cytometry. ^*p < 0.05. n = 3.