目的:探讨通过组蛋白乙酰化调节基质金属蛋白酶9(MMP-9)/基质金属蛋白酶组织抑制剂(TIMPs)基因表达是否是电针(EA)保护大脑中动脉阻塞(MCAO)大鼠模型血脑屏障(BBB)完整性的可能机制。
方法:雄性SD大鼠分为4组:假手术组,MCAO集团,MCAO+EA(MEA)组,和MCAO+EA+HAT抑制剂(HATi)组。通过阻断大脑中动脉产生MCAO模型。EA应用于百惠(GV20)。再灌注后1或3d收集样品。神经功能评分和伊文思蓝外渗用于评估中风后损伤。通过实时荧光定量聚合酶链反应(RT-qPCR)和染色质免疫沉淀(ChIP)评估EA对MMP-9/TIMPs基因表达的影响。
结果:我们的结果显示EA治疗显著改善了神经功能和改善了BBB破坏。RT-qPCR结果显示电针降低MMP-9的表达,促进TIMP-2mRNA的表达,但HATI逆转了EA的这些影响。此外,ChIP结果表明,EA降低了MMP-9启动子处H3K9ace/H3K27ace的富集,并显着刺激了TIMP-2启动子处H3K9ace/H3K27ace的募集。
结论:电针治疗百会(GV20)在卒中急性期通过组蛋白乙酰化修饰调节MMP-9和TIMP-2的转录,保留了MCAO大鼠BBB的结构完整性。这些发现表明,组蛋白乙酰化介导的靶基因转录活性可能是EA治疗中风的关键机制。
OBJECTIVE: To explore whether the regulation of matrix metalloproteinase 9 (MMP-9)/ tissue inhibitors of MMPs (TIMPs) gene expression through histone acetylation is a possible mechanism by which electroacupuncture (EA) protects blood-brain barrier (BBB) integrity in a middle cerebral artery occlusion (MCAO) rat model.
METHODS: Male Sprague-Dawley rats were divided into four groups: the sham group, the MCAO group, the MCAO + EA (MEA) group, and the MCAO + EA + HAT inhibitor (HATi) group. The MCAO model was generated by blocking the middle cerebral artery. EA was applied to Baihui (GV20). Samples were collected 1 or 3 d after reperfusion. Neurological function scores and Evans blue extravasation were employed to evaluate the poststroke injury. The effect of EA on MMP-9/TIMPs gene expression was assessed by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and chromatin immunoprecipitation (ChIP).
RESULTS: Our results showed that EA treatment prominently improved neurological function and ameliorated BBB disruption. The RT-qPCR assay showed that EA reduced the expression of MMP-9 and promoted TIMP-2 mRNA expression, but HATi reversed these effects of EA. In addition, ChIP results revealed that EA decreased the enrichment of H3K9ace/H3K27ace at MMP-9 promoters and notably stimulated the recruitment of H3K9ace/H3K27ace at TIMP-2 promoter.
CONCLUSIONS: EA treatment at Baihui (GV20) regulates the transcription of MMP-9 and TIMP-2 through histone acetylation modification in the acute stage of stroke, which preserves the structural integrity of the BBB in MCAO rats. These findings suggested that the histone acetylation-mediated transcriptional activity of target genes may be a crucial mechanism of EA treatment in stroke.