Study On The Mechanism Of α-asarone Regulation Blood-brain Barrier And Biological Safety In Zebrafish

To study the influence of drugs α-asarone on zebrafish brain barrier tight junction structure and the expression of gene protein family Claudin, occludin, ZO, JAM, In order to study the mechanism of α-asarone open the blood brain barrier. While taking advantage of the zebrafish evaluation the development and reproductive toxicity of α- asarone on embryonic.Methods by taking Heart injection to help macromolecular fluorescent substance Dextran Texas Red injected to juvenile zebrafish after administrating α-asarone solution, observed α-asarone can help macromolecular fluorescent substance permeability of blood-brain barrier. By electron microscrope to observe structure of blood-brain barrier affects after α-asarone on the ultrastructure of the zebrafish brain barrier, affecting zebrafish brain tissue slices observed structure; using real-time quantitative PCR(Real-time quantitative,RT-qPCR)studies in zebrafish α-asarone closely connected family Claudin, affect ZO, occludin, JAM gene expression. α-asarone solution take 3 hpf(Hours post-ferilization,hpf) zebrafish embryos exposed to different concentrations at 24 hpf, 48 hpf, 72 hpf, 96 hpf morphological observation of embryonic development, zebrafish embryos independent statistical spontaneous movements, hatching rate, heart rate, embry deformity rate and mortality and other morphology, such as yolk sac edema, pericardial edema phenomenon; the expression of sepn1 was determined with real-time quantitative PCR technique at 96 hpf. The use of zebrafish behavioral system Noldus to evaluate the impact of α-asarone juvenile zebrafish movement and physiology. The use of zebrafish to study the determination of gonad index and the detection of sperm plasma membrane integrity on male zebrafish.Results are as follows by zebrafish heart injecting a fluorescent macromolecules, After administrating α-asarone 30 min, observed vascular vague, surrounded by a small amount of a fluorescent substance has exudation. After administrating α-asarone 60 min, observed zebrafish vascular more vague, description has a large number of fluorescent substances leaking from the vessel, while the control group did not see a fluorescent substance exudation. By electron microscopy showed that after administrating α-asarone, mild shrinkage of endothelial cells, the basement membrane rupture appeared swollen, the structure of tight junctions appear loose while the endothelial cells appeared crimple. But control group did not appear, tight junctions compact, the structure of basement membrane complete continuous(With the results of previous studies by our group is consistent). HE staining pathology results are consistent with the results of BBB ultrastructure. Intraperitoneal injection of α-asarone role 1h, the use of RT-qPCR to technique Claudin, ZO, occludin, JAM gene expression affect. The majority of claudin family genes as Claudin-19,-k,-j,-5a,-7a,-7b,-a,-h,-11 a expression down-regulated significantly. Of which Claudin-5b,-11 b up-regulated. Of which Claudin-2 does not change. Other gene families such as JAM,-2A,-2B,-3A,-3B, occluding A,-B were down-regulated significantly, but not the same magnitude of reduction. After administrating α-asarone 1h, Transcript abundance of Claudin-5a down-regulated most significantly.and recovered to at 2h, and gradually recovered at 8h to the normal levels. The zebrafish embryos exposed to different concentrations of α-asarone solution, the spontaneous movements of 24 hpf significantly reduce; zebrafish heart rate of 48 hpf significantly lower; 72 hpf juvenile zebrafish appeared curvature of the spine, pericardial edema, yolk sac edema other symptoms; 96 hpf hatching mortality differences; the concentrations of α-asarone is increasing, the zebrafish behavioral is inhibited. During the concentrations of α-asarone increase, the gonad index and the detection of sperm plasma membrane integrity on male zebra fish decrease.Conclution are resuscitation-inducing aromatic herbs could make a decrease in the expression of Claudins,which loosened the tight junction.In addition,the basement membrane ruptured and the endothelial cell crimpled.These factors weakened the barrier function of BBB.Gene reversible downregulation demonstrated that the open of BBB was physiological and the change would not bring about brain impairment.From the clinical safe medication perspective,borneol and muscone at high concentrations had significant toxic effects on the development of zebrafish embryos. Be caution of α- asarone.