Ligustrazine Alleviates Blood-Brain Barrier Damage by Downregulating Expression of miR-297c-5p

Abstract

Objective: Ligustrazine (LSZ), an ingredient of Ligusticum chuanxiong, has long been used to treat neurovascular diseases in China. This study investigates its protective effects for the impairment of the blood-brain barrier (BBB) and the underlying mechanisms.

Methods: In this study, the impacts of LSZ on the BBB function were firstly assessed in b. End3 cells in vitro. Oxygen-glucose deprivation (OGD) served as an injury factor and western blot (WB) analyzed the expressions of occludin and ZO-1, two tight junction proteins (TJs), essential for maintaining the integrity of the BBB. After bioinformatics analysis of the transcriptome in vivo, qRT-PCR of miR-297c-5p was conducted and a dual-luciferase reporter assay was used to verify the target protein, occludin, which was confirmed by hippocampal insertion using guide cannulas and microinfection of RNA oligos.

Results: A 3-h deprivation of OGD of b. End3 cells resulted in noticeable reductions in the level of occludin and ZO-1. However, administration of LSZ (0.1 μM) effectively restored these decreases. In normal mice, administration of LSZ (25 mg/kg, i.p., once daily for 9 days) resulted in a notable reduction in miR-297c-5p. In the middle cerebral artery occlusion (MCAO) mouse model, increased miR-297c-5p was also reversed by LSZ administration. Bioinformatics analysis revealed one of the targets of miR-297c-5p includes occludin. MiR-297c-5p was found to directly target occludin in the dual-luciferase reporter assay. Transfection of miR-297c-5p agomir into b. End3 cells resulted in a significant reduction in the level of occludin, while transfection of antagomir led to an increase in occludin. Besides, stereotaxic injection of AAV-miR-297c-5p into the hippocampus reduced occludin level in vivo. Ultimately, hippocampal microinfection of RNA oligos provided a confirmation that miR-297c-5p was downregulated by LSZ in MCAO mice with up-regulated occludin expression.

Conclusion: In conclusion, the present findings provide new insights into regulating occludin by LSZ through downregulation of miR-297c-5p.

Keywords: blood–brain barrier; ligustrazine; miR‐297c‐5p; tight junction.