黄芪甲苷与三七有效成分配伍对小鼠脑缺血再灌注后神经细胞凋亡和内质网应激的影响

Effects of astragaloside Ⅳ combined with active components of Panax notoginseng on apoptosis and endoplasmic reticulum stress in brain tissues after cerebral ischemia-reperfusion in mice

目的从内质网应激角度探讨黄芪甲苷和三七主要有效成分配伍对小鼠脑缺血再灌注后神经细胞凋亡的影响和作用机制。方法将 C57BL/6 小鼠随机分为假手术组、模型组、黄芪甲苷(ASTIV,40 mg/kg)组、人参皂苷Rg1(Rg1,50 mg/kg))组、人参皂苷Rb1(Rb1,40 mg/kg)组、三七皂苷R1(R1,10 mg/kg)组、4 种有效成分配伍(ASTIV+Rg1+Rb1+R1)组、ASTIV+Rg1 组、ASTIV+Rb1 组、ASTIV+R1 组及阳性对照依达拉奉组。各组小鼠预先给药3 d 后,夹闭双侧颈总动脉造成脑缺血再灌注模型,再灌注24 h 后,TUNEL 法检测海马CA1 区神经细胞凋亡,计算凋亡率;Western-blotting 法测定脑组织半胱氨酸天冬氨酸特异性蛋白酶-3(Caspase-3)、葡萄糖调节蛋白78(GRP78)、Caspase-12 和磷酸化的c-jun 氨基末端激酶1/2(p-JNK1/2)蛋白表达。结果脑缺血 20 min 再灌注24 h 后,小鼠海马CA1 区神经细胞凋亡率和Caspase-3 蛋白表达量增加。各给药组均能降低海马CA1 区神经细胞凋亡率,抑制Caspase-3 蛋白的表达。且ASTIV+Rg1 与ASTIV+R1 降低凋亡率和Caspase-3 蛋白表达的效应分别强于各有效成分单用;4 种有效成分配伍降低细胞凋亡率的效应强于各有效成分单用及ASTIV+Rb1,抑制Caspase-3 蛋白表达的效应强于各有效成分单用及ASTIV+Rb1、ASTIV+R1。脑缺血再灌注后,小鼠脑组织GRP78和Caspase-12、p-JNK1/2 蛋白表达增多,ASTIV、Rg1、R1 及各配伍组均能进一步上调脑组织GRP78 蛋白表达,且各有效成分配伍的效应分别强于各有效成分单用,4 种有效成分配伍的效应强于ASTIV+Rb1 及ASTIV+R1。R1、4 种有效成分配伍、ASTIV+Rg1、ASTIV+R1 均能降低Caspase-12 蛋白的表达,4 种有效成分配伍的效应强于各有效成分单用及ASTIV+Rb1。ASTIV、Rg1、4 种有效成分配伍、ASTIV+Rg1、ASTIV+Rb1 组脑组织p-JNK1/2 蛋白表达显著减少,4 种有效成分配伍下调p-JNK1/2 的效应强于各有效成分单用及ASTIV+Rg1、ASTIV+R1。结论黄芪甲苷与三七的主要有效成分配伍能增强对脑缺血再灌注后神经细胞凋亡的抑制作用,其机制与通过不同环节减轻内质网应激有关。ASTIV+Rb1 可能主要作用于JNK 通路,ASTIV+R1 可能主要作用于Caspase-12 通路,而4 种有效成分配伍和ASTIV+Rg1 均能作用于Caspase-12 和JNK 通路。

Objective To probe the effects and mechanisms of astragaloside IV combined with the active components from Panax notoginseng on apoptosis of nerve cells through endoplasmic reticulum stress（ERS） after cerebral ischemia-reperfusion（I/R） in mice.Methods C57BL/6 mice were randomly divided into Sham,model,astragaloside IV（AST IV）,ginsenoside Rg1（Rg1）,ginsenoside Rb1（Rb1）,notoginsenoside R1（R1）,four active components combination,AST IV＋Rg_1,AST IV＋Rb1,AST IV＋R1 and Edaravone group,pretreated for 3 d.After 1 h of the last administration,the model of cerebral I/R injury was established by bilateral common carotid artery（CCA） ligation followed by reperfusion,then TUNEL method was used to detect the apoptosis in hippocampal CA1 and apoptosis rate was calculated;The expression of cysteine aspartic acid specific protease（Caspase-3）,glucose regulated protein 78（GRP78）,Caspase-12 and phosphorylated C-Jun amino terminal enzyme（p-JNK1/2） proteins in brain tissues was tested by Western-blotting at 24 h after reperfusion.Results After cerebral ischemia for 20 min followed by reperfusion 24 h,the apoptosis rate of nerve cell in hippocampal CA1 and the expression of Caspase-3 protein in brain tissues were increased.All drugs could decrease the apoptosis rate and inhibit Caspase-3 protein expression.Furthermore,the decreased effects of AST Ⅳ＋Rg1 and AST Ⅳ＋R1 on the apoptosis rate and Caspase-3 protein expression were better than those of the active components alone;In the four active components combination,the decrease of the apoptosis rate was stronger than that of the four active components alone and the inhibition of AST Ⅳ＋Rb1 on Caspase-3 was greater than that of the four active components alone as well as AST Ⅳ＋Rb1 and AST Ⅳ＋R1.After the cerebral I/R,the expression of GRP78 and Caspase-12,p-JNK1/2 proteins were up-regulated.AST Ⅳ,Rg1,R1,and the combinations could further increase GRP78 protein expression in brain tissues,and the effect of the combinations was better than that of the active components alone;The effect of the four active components combination was better than that of AST Ⅳ＋Rb1 and AST IV ＋ R1.R1,the four active components combination,AST IV ＋ Rg1,and AST IV ＋ R1 could down-regulate Caspase-12 protein,and the effect of the four active components combination was more obvious than that of the four active components alone and AST IV ＋ Rb1.The expression of p-JNK1/2 in AST IV,Rg1,the four active components combination,AST IV ＋ Rg1,and AST IV＋Rb1 was decreased,the decrease in the four active components combination was stronger than that in the four active components alone as well as AST IV＋Rg1 and AST IV＋R1.Conclusion AST IV combined with the effective components from P.notoginseng has the potentiation on the inhibition of apoptosis,and the mechanism underlying might be associated with relieving ERS via different links.AST IV＋Rb1 might affect JNK pathway and AST IV＋R1 might act on the Caspase-12 pathway;Moreover,the four active components combination and AST IV＋Rg1 could act on both Caspase-12 and JNK.