冰片配伍黄芪甲苷和三七总皂苷促进脑缺血再灌注后神经修复作用的研究

Study on borneol combined with astragaloside IV and panax notoginseng saponins promoting nerve repair after cerebral ischemia-reperfusion

目的:探讨冰片、黄芪甲苷(ASTⅣ)和三七总皂苷(PNS)配伍促进脑缺血再灌注后神经修复的作用和Wnt/β-catenin信号机制。方法:采用大鼠局灶性脑缺血再灌注模型,灌胃给药冰片、ASTⅣ、PNS及其配伍药物,神经功能评分和病理形态评价药物对脑组织损伤的影响,免疫组织化学方法检测脑组织巢蛋白(Nestin)、神经元核抗原(NeuN)反映神经发生,Western blot法检测Wnt/β-catenin信号通路相关蛋白表达。结果:与模型组比较,ASTⅣ+PNS配伍组、冰片+ASTⅣ+PNS配伍高、低剂量组可显著降低神经功能评分(P<0.01),冰片+ASTⅣ+PNS配伍低剂量组降低的效应均强于各药物单用(P<0.01)。病理学检测表明,各用药组细胞损伤明显减轻,ASTⅣ+PNS配伍组效应显著优于单用ASTⅣ组、单用PNS组,冰片+ASTⅣ+PNS配伍低剂量组效应优于各药物单用及ASTⅣ+PNS配伍组(P<0.01,P<0.05)。ASTⅣ+PNS配伍组、冰片+ASTⅣ+PNS配伍低剂量组使Nestin表达增加,冰片+ASTⅣ+PNS配伍低剂量组的作用显著强于各药物单用及ASTⅣ+PNS配伍组(P<0.01,P<0.05)。A S TⅣ+P N S配伍组、冰片+A S TⅣ+P N S配伍低剂量组使N e u N阳性细胞表达增多,药物配伍的作用显著强于单用A S TⅣ与单用冰片组。A S TⅣ+P N S配伍组、冰片+A S TⅣ+P N S配伍低剂量组使Wnt3a、Wnt7b、β-catenin蛋白表达显著增加,糖原合成酶激酶3β(GSK3β)表达下调,冰片+ASTⅣ+PNS配伍低剂量组升高Wnt3a、Wnt7b蛋白表达的效应强于各药物单用及ASTⅣ+PNS配伍组(P<0.01,P<0.05),ASTⅣ+PNS配伍组和冰片+ASTⅣ+PNS配伍低剂量组降低GSK3β表达和增强β-catenin蛋白表达的作用强于各药物单用(P<0.01)。结论:冰片、ASTⅣ、PNS配伍能够通过促进脑内神经细胞的增殖,修复受损的神经细胞,增强抗脑缺血再灌注损伤的作用,其作用与调控Wnt/β-catenin信号通路有关。

Objective:To research the effects of borneol combined with astragalosidesⅣ(ASTⅣ)and panax notoginseng saponins(PNS)promoting neural repair and the mechanism of Wnt/β-catenin signaling pathway.Methods:Focal cerebral ischemia-reperfusion model of middle cerebral artery in rats was established,borneol,ASTⅣ,PNS and the combinations were administered by gavage,the nerve function score and the neuropathological evaluation were used to assess the influences of the combination on brain injury,the expression of Nestin and neuronal nuclear antigen(NeuN)were used to reflect the neurogenesis via the immunohistochemistry detection,Western-blot method was used to measure the Wnt/β-catenin signaling pathway.Results:Compared with model group,the nerve function score was significantly decreased in ASTⅣ+PNS group and the high,low does combinations of borneol+ASTⅣ+PNS groups(P<0.01),and the effect of the low dose combinations of borneol+ASTⅣ+PNS group was higher than that of the single drug(P<0.01).The neuropathological change showed cell injury in the treatment groups was significantly relieved,and the effect in ASTⅣ+PNS group was significantly better than that in ASTⅣgroup and PNS group,the effect of low dose combination of borneol+ASTⅣ+PNS group was better than that of single alone and ASTⅣ+PNS group(P<0.01,P<0.05).Nestin positive cells was increased in ASTⅣ+PNS group and the low dose combination of borneol+ASTⅣ+PNS group,and the increase of Nestin positive cells in low dose combination of borneol+ASTⅣ+PNS group was significantly greater than that of alone group and ASTⅣ+PNS group(P<0.01,P<0.05).In ASTⅣ+PNS group and the low dose combination groups of borneol+ASTⅣ+PNS,NeuN positive cells was markedly increased,and the effect of increasing NeuN positive cells in combination of borneol+ASTⅣ+PNS groups was respectively stronger than that of ASTⅣand borneol alone.The proteins of Wnt3 a,Wnt7 b andβ-catenin were increased as well as the content of GSK3βprotein was decreased in ASTⅣ+PNS group and the low dose combination of borneol+ASTⅣ+PNS group,moreover,the effects of low dose combination group on up-regulating Wnt3 a,Wnt7 b proteins were stronger those of single group and ASTⅣ+PNS group(P<0.01,P<0.05),the increase ofβ-catenin and the decrease of GSK3βin ASTⅣ+PNS group and low dose combination of borneol+ASTⅣ+PNS group were better than those in single group(P<0.01).Conclusion:The combination of borneol,ASTⅣand PNS can boost the proliferation of neurons,repair nerve cells damaged,enhance the effect of anti-cerebral ischemia reperfusion injury,which may be relevant with Wnt/β-catenin signaling pathway.