Catalpol Provides A Protective Effect On Fibrillary A?_1-42-induced Barrier Disruption In An In Vitro Model Of The Blood-brain Barrier

Research purpose:Catalpol is an iridoid glucoside extracted from the traditional Chinese herbal medicine root of Rehmannia glutinosa Libosch.Our previous research showed that the drug-containing serum of the traditional Chinese compound Bushen Yijing Decoction,which has significant protection to neuronal cells and extracorporeal blood-brain barrier models.Further in vitro studies found that the Rehmannia glutinosa Libosch,the mainly compoment of Bushen Yijing Decoction also has significant neuroprotective effect.Therefore,based on the results of previous studies,this study aims to explore the effects of catalpol on fibrous A?1-42-induced blood-brain barrier dysfunction in vitro and the potential mechanisms of it.Research methods:1.We used 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)method to detect the viability of bEnd.3 cells,to explore the appropriate time and concentration of fibrous A?1-42 on damaging cells,and the optimal concentration of catalpol that can effectively exert its protective effect;2.The non-contact co-culture model of blood-brain barrier(BBB)in vitro,which was constructed with immortal mouse brain microvascular endothelial bEnd.3 cells and primary astrocytes(As),was used to detect the fluorescence intensity of sodium fluorescein that across the BBB.Then evaluating the effect of catalpol on the per-meability of the BBB in vitro;3.Flow cytometry(FCM)FITC-V/PI method was used to detect the effect of Catalpol on the apoptosis of bEnd.3 cells induced by fibrous A?1-42.4.The monoyer bEnd.3 cells model of BBB in vitro was used to explor the levels of soluble A? that across the BBB.Then evaluating the effect of catalpol on the clea-rece of A? in vitro;5.Western blot method was used to detect the expression levels of related apoptotic proteins,matrix metalloproteinases,tight junction proteins and transporters in order to explore the specific mechanism of catalpol on protecting the BBB.Research results:1.The results of MTT assay showed that:(1)The viability inhibitory rate of A?1-42 to bEnd.3 cells was close to 50%when fibrous A?1-42 with 20?mol·L-1 damaged bEnd.3 cells for 24h and 48h,respectively,(2)The catalpol with 50-500 ?mol·L-1 significantly inhibited the damage of fibrillar A?1-42 to bEnd.3 cells,significantly increased cell viability,and its protective effect was positively correlated with the concentration of catalpol.(3)Catalpol with a concentration in the range of 50-500 ?nol·L-1 had no significant damage to bEnd.3 cells.2.Fluorescein sodium across the blood-brain barrier model and Western blot results showed that:(1)Catalpol can significantly reduce BBB leakage induced by the fibrillar A?1-42.(2)The catalpol inhibited the mitochondria-dependent pathways and the death receptor apoptosis pathway activated by fibrillar A?1-42 significantly,and associated with relation proteins expression(Bax,Bcl-2,Cytochromes C,Cleaved Caspase-8,Cleaved Caspase-9,Cleaved Caspase-3);decreased the protein levels of matrix metalloproteinases(MMPs)MMP-2,MMP-9,and in turn increased the tight junction proteins between endothelial cells,which included ZO-1,Occludin and Claudin-5.3.In addition,we also found that catalpol can increase the clearance rate of soluble A?across BBB models in vitro,which may be related to the decreasing of advanced glycation end products(RAGE)receptors and increasing of low density lipoprotein receptor protein 1(LRP-1)and P-glycoprotein(P-gp).Research conclusions:1.catalpol can significantly inhibit the cell apoptosis of vascular endothelial cells bEnd.3 induced by fibrillar A?1-42.2.Catalpol can significantly reduce BBB leakage induced by the fibrillar A?1-42 in vitro.The mechanism underline it may be related to the fact that catalpol inhibited the apoptosis of bEnd.3 and increase the levels of tight junction proteins induced by A?1-42.3.Catalpol can increase the effective clearance rate of soluble A? in brain,and the mechanism may be related to the fact that catalpol can effectively regulate the related transporters responsible for transporting A? on vascular endothelial cells.