冰片配伍黄芪甲苷与三七总皂苷对脑缺血再灌注大鼠血脑屏障转运蛋白的影响

Effect of borneol combined with astragaloside Ⅳ and Panax notoginseng saponins on transporter proteins of blood-brain barrier after cerebral ischemia-reperfusion

目的 从血脑屏障(blood-brain barrier,BBB)转运蛋白角度探讨冰片配伍黄芪甲苷(astragalosides IV,AST IV)和三七总皂苷(Panax notoginseng saponins,PNS)在脑缺血再灌注状态下促进药物成分入脑的作用。方法 采用大鼠局灶性脑缺血再灌注模型,ig给予冰片、AST IV、PNS及其配伍药物,2,3,5-氯化三苯四氮唑(2,3,5-triphenyl tetrazolium chloride,TTC)染色法测定脑组织损伤,Western blotting法检测脑组织外排蛋白P-糖蛋白(P-glycoprotein,P-gp)和多药耐药相关蛋白1(multidrug resistance protein-1,MRP-1)、MRP-2、MRP-4、MRP-5及摄取蛋白有机阳离子转运体3(organic cation transporter3,OCT3)、有机阴离子转运多肽-2(organicaniontransportingpolypeptides-2,OATP-2)蛋白的表达,real-timePCR法检测脑组织多药耐药(multidrug resistance,mdr)基因mdr1a、mdr1b和mrp-1、mrp-2、mrp-4、mrp-5 mRNA表达。结果 TTC染色结果显示,脑缺血再灌注后,大鼠脑组织出现明显梗死灶。各药物能显著减少脑梗死体积,ASTIV+PNS的效应强于AST IV与PNS单用,冰片+AST IV+PNS的效应强于各药物单用及AST IV+PNS。外排蛋白和基因检测显示,脑缺血再灌注后,大鼠脑组织P-gp、MRP-1、MRP-2、MRP-4、MRP-5蛋白表达均显著增多。与模型组比较,冰片能显著下调大鼠脑组织P-gp、MRP-2、MRP-4蛋白表达水平,PNS能显著下调MRP-4、MRP-5蛋白表达水平,AST IV、AST IV+PNS与冰片+AST IV+PNS能显著下调P-gp、MRP-2、MRP-4、MRP-5蛋白表达水平,且冰片+AST IV+PNS的效应显著强于各药物单用及AST IV+PNS,AST IV+PNS的效应显著强于AST IV或PNS单用。基因表达结果与蛋白表达结果类似。摄取蛋白检测结果显示,与对照组比较,脑缺血再灌注后大鼠脑组织OCT-3蛋白表达在模型组及各给药组变化均不明显,而模型组OATP-2蛋白表达水平显著降低。PNS、AST IV+PNS及冰片+AST IV+PNS能显著上调OATP-2蛋白表达水平,且AST IV+PNS的效应显著强于AST IV、PNS单用,冰片+ASTIV+PNS的效应显著强于各药物单用及ASTIV+PNS。结论 脑缺血再灌注后,脑组织损伤,BBB的主要外排蛋白和基因表达均显著增强,而摄取蛋白OATP-2表达显著降低。冰片配伍AST IV及PNS后,能增强抗缺血性脑损伤的作用,其作用可能与下调BBB中外排蛋白P-gp、MRP-2、MRP-4、MRP-5和相应基因表达,同时上调摄取蛋白OATP-2表达,促使脑组织中冰片、AST IV及PNS有效成分的吸收与富集而发挥药理作用有关。

Objective To investigate the effects of borneol combined with astragaloside IV(AST IV)and Panax notoginseng saponins(PNS)on promoting the active components into the brain and anti-brain injury through the regulation of transporter proteins of blood-brain barrier(BBB)in the state of cerebral ischemia-reperfusion.Methods Focal cerebral ischemia-reperfusion model in rats was established,borneol,AST IV,PNS and the combination were administered by gavage,brain infarction rate was evaluated by 2,3,5-triphenyl tetrazolium chloride(TTC)staining,the expressions of efflux proteins such as p-glycoprotein(P-gp),multidrug resistance protein(MRP)-1,MRP-2,MRP-4,MRP-5 and uptake proteins such as organic cation transporter(OCT)-3,organicanion transporting polypep-tides(OATP)-2 in brain tissues were detected by Western blotting.The expressions of multidrug resistance(MDR)such as mdr1a,mdr1b and mrp-1,mrp-2,mrp-4,mrp-5 mRNA in brain tissues were determined by real-time PCR method.Results The results of TTC staining showed that brain infarct was found after cerebral ischemia-reperfusion.Each drug could significantly reduce brain infarction volume and decrease infarction rate,and the effect of AST IV+PNS was better than that of AST IV and PNS alone,the effect of borneol+AST IV+PNS was better than that of single drug and AST IV+PNS.The results of major efflux proteins and genes detection showed that the protein expressions of P-gp,MRP-1,MRP 2,MRP-4,and MRP-5 were significantly increased in rats after cerebral ischemia-reperfusion.Borneol could significantly down-regulate the expressions of P-gp,MRP-2,MRP-4 proteins,PNS could significantly down-regulate the levels of MRP-4,MRP-5 proteins;AST IV,AST IV+PNS and borneol+AST IV+PNS could significantly down-regulate P-gp,MRP-2,MRP-4,MRP-5 proteins,and the effects of borneol+AST IV+PNS were significantly better than those of single drug and AST IV+PNS;The effects of AST IV+PNS were significantly better than those of AST IV or PNS alone.The results of gene expressions were similar to those of protein expression.The results of major uptake proteins showed that the expression of OCT-3 protein did not change significantly in the model group and drug groups after cerebral ischemia-reperfusion;However,the expression of OATP-2 protein was significantly decreased in the model group.PNS,AST IV+PNS and borneol+AST IV+PNS could significantly up-regulate the expression of OATP-2 protein;Furthermore,the effect of borneol+AST IV+PNS was significantly greater than that of single drug and AST IV+PNS,and the effect of AST IV+PNS was significantly greater than that of AST IV and PNS alone.Conclusion After cerebral ischemia-reperfusion,brain tissues were damaged,the expressions of major efflux proteins and genes on BBB were significantly increased,while the expression of uptake protein such as OATP-2 was significantly decreased.Borneol combined with AST IV and PNS can enhance the effect of anti-ischemic brain injury,which may be related to the down-regulations of the expressions of efflux proteins such as P-gp,MRP-2,MRP-4,MRP-5 and corresponding genes in BBB,as well as the up-regulation of the expression of uptake proteins such as OATP-2,thus promoting the absorption and the enrichment of borneol,AST IV and the effective components of PNS in brain tissues,playing a better role in pharmacology.