Ligustrazine Regulates Tight Junction Proteins In Vitro And In Vivo Models Via Alteration Of MiR-297c-5p

BackgroundBlood-brain barrier（BBB）,a highly specialized system for regulating substances passaging from peripheral fluids into brain,is composed of astrocytes,pericytes and endothelial cells in the central nervous system（CNS）.Endothelium,the first defense line between blood and brain,is maintained by tight junction proteins（TJPs）including Occludin,Claudin-5 and Zonula occludens-1（ZO-1）between adjacent endothelial cells.Caveolins,which are specific transport vesicles on the plasma membrane,perform transcytosis in the BBB under physiological condition.Endothelium barrier is a major determinant of BBB.Cerebral insult may influence TJPs expression and distribution as well as affect the expression and activation of Caveolins.Thus,exotics may pass through the BBB and provok severe neurotoxcity and impairment on neural functioning.Ligustrazine（LSZ）is an effective ingredient of Ligusticum chuanxiong Hort（chuanxiong）,which has been used as a traditional Chinese medicinal herb for thousands of years.Phosphatic or hydrochloridic LSZ is usually prescribed to patients with brain ischemia clinically.Scores of studies find that LSZ protects cerebral vasculature from cerebral ischemia by reducing oxidative stress,inhibiting the cascade reactions of apoptosis,promoting endothelial cell survival,and maintaining BBB stability.Increased TJPs serves as an important indicator of improved BBB integrity against ischemia injury.However,the protective effect of LSZ on the BBB and its underlying mechanism are barely elucidated.Micro RNAs（miRNAs）are small（20-22 nt）non-coding RNAs that play critical roles in post-transcriptional regulation of various cellular processes.MiRNA has gained increased attention owing to their important roles in the incidence and management of diseases.LSZ relieves lipopolysaccharide（LPS）-or hypoxia-induced cell injuries and neural apoptasis in injured spical cord via miRNA regulation.Therefore,we speculate that miRNAs may participate in LSZ treatment via regulation of TJPs.AimsBased on the protective effect of LSZ in vivo models of ischemia-reperfusion injury,the expression of TJPs was explored.High-througput miRNA sequencing combined with bioinformatics database analysis were used to find explicitly involved miRNAs after LSZ administration.The functional verification and dual-luciferase reporter test were applied to reveal the target of selected miRNA and clarify its possibility for BBB protection clinically.Methods1)OGD of b.End 3 cells was used as in vitro model.The optimal drug concentration was selected among gradient concentration by enzyme-linked immunosorbent assay（ELISA）.Western blot（WB）experiment verified LSZ regulating TJPs expression.2)High-throughput miRNA sequencing was used to find miRNAs involved in LSZ regulating BBB permeability.KEGG Pathway analysis was performed to find different geometric distributions based on the significant down-regulated miRNAs.Quantitative real-time PCR（q-PCR）experiment was used to verify the molecules with differently expression in the samples,and miRNA databases were applied to predict target genes.3)Middle cerebral artery occlusion（MCAO）was used to verify miR-297c-5p expression by q-PCR in model and LSZ-treated mice.4)Expression of TJPs and Caveolins was tested by WB experiment after miR-297c-5p agomir and antagomir transfection into b.End 3 cells.5)AAV9 packaged miR-297c-5p for overexpression was used to infect hippocampal CA1 area through stereotactic injection.Fluorescence microscopy was used to observe the efficiency of infection and WB experiment was performed to test Occludin expression.6)Dual-luciferase reporter test was used to reveal the interaction between miR-297c-5p and Ocln m RNA.7)MiR-297c-5p RNA oligo injection through implanted cannula in hippocampal CA1area combined with LSZ intraperitoneal administration were used to detect the changes of behavior and Occludin expression in MCAO mice.Results1)ELISA result indicated that LSZ（0.1μM and 1μM）increased Occludin expression in b.End 3 cells compared with OGD group.WB experiment revealed LSZ（0.1μM）significantly restored decreased expression of Occludin and ZO-1 induced by 3 h of OGD injury.2)High-throughput miRNA sequencing found 10 significantly reduced miRNAs compared with control mice under the condition of|log₂FC|≥1.0 and Q value≤0.05.KEGG Pathway was performed to analyse enriched pathways of differently expressed miRNAs.The pathways included tight junctions,adherens junctions,gap junctions,endocytosis,etc.Furthermore,q-PCR verified miRNAs expressions in MCAO and LSZ-treated mice.MiR-297c-5p was screened to find predictively corresponding target proteins including Occludin,ZO-1 and Caveolins.3)Neurologic deficits score and TTC staining indicated successful construction of MCAO mice model.Q-PCR showed that miR-297c-5p was up-regulated in MCAO mice and then down-regulated by LSZ administration.4)Transfection into b.End 3 cells with chemically synthesized RNA oligo showed miR-297c-5p agomir down-regulated Occludin,while antagomir up-regulated Occludin and ZO-1.On the contrary,agomir increased Caveolins expressions,while antagomir decreased their expressions.5)Stereotaxic injection of AAV9-packed miR-297c-5p indicated that AAV-miR-297c-5p reduced Occludin in hippocampus CA1 area.6)Dual-luciferase gene report showed that co-transfection of miR-297c-5p mimics and Ocln WT 3’UTR recombinant plasmid sgnificantly reduced the luciferase activity compared with co-transfection of mimics and Ocln mutated plasmid after 48 h incubation,which indicated Occludin as a direct target of miR-297c-5p.7)With pretreatment of RNA oligo and LSZ in MCAO mice,neurologic deficits test detected miR-297c-5p antagomir decreased neurologic deficits scores in MCAO mice.WB experiment indicated the expression of Occludin in hippocampus.The results showed the expression of Occludin in MCAO mice was lower than that in the sham group,MCAO+LSZ+Scramble group was higher than MCAO group,and lowered by miR-297c-5p agomir treatment;compared with MCAO mice,MCAO+miR-297c-5p antagomir increased Occludin expression.ConclusionsLSZ protected b.End 3 cells against OGD injury by increasing Occludin and ZO-1 levels.High-throughput miRNA sequencing relvealed differently expressed miRNAs after LSZ treatment.The alteration of miRNAs was verified by q-PCR.Bioinformatics analysis predicted target genes of miRNA and signaling pathways.Functional verification of miR-297c-5p by in vitro transfection and in vivo AAV injection was in line with dual-luciferase reporter test,which concluded Occludin was a direct target of miR-297c-5p.RNA oligo and LSZ treatment were used in MCAO animal model to investigate the role of miR-297c-5p in the regulation of LSZ on Occludin expression against ischemic injury.This study provided a theoretical basis for developing new drugs to protect cerebral blood vessels and blood-brain barrier.