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目的探讨黄芪皂甙Ⅳ联合骨髓间充质干细胞(BMSCs)移植对缺血再灌注大鼠海马神经细胞凋亡及Bcl-2、Bax、Caspase-3表达的影响。方法实验动物随机分为假手术组、模型组、BMSCs组、BMSCs+黄芪组。采用线栓法建立大鼠大脑中动脉缺血(MCAO)模型,BMSCs组、BMSCs+黄芪组分别于建模成功后3h经尾静脉注射已制备好的PKH26标记BMSCs悬液。另外BMSCs+黄芪组于缺血前5min、缺血后12h,24h腹腔注射黄芪皂甙Ⅳ。缺血再灌注72h后,各组进行神经功能评分,应用荧光显微镜观察海马PKH-26标记的移植BMSCs分布,采用免疫组化法和实时荧光定量PCR方法检测大鼠海马Bcl-2、Bax、caspase-3的表达,运用TUNEL方法检测神经元细胞凋亡指数。结果 PKH-26标记的BMSCs在海马有表达。与模型组比较,BMSCs组与BMSCs+黄芪组均能够使大鼠神经功能损害评分降低(P<0.01),BMSCs+黄芪组神经功能恢复最为明显(P<0.05)。模型组Bcl-2、Caspase-3、Bax蛋白表达及细胞凋亡与假手术组差异显著(P<0.01)。与模型组比较,BMSCs+黄芪组和BMSCs组均可下调Caspase-3、Bax蛋白表达和细胞凋亡指数(P<0.05),上调Bcl-2蛋白表达及Bcl-2/Bax比值(P<0.05),其中以BMSCs+黄芪组作用更为显著(P<0.05)。实时荧光定量PCR显示:与模型组和BMSCs组比较,BMSCs+黄芪组能显著下调Caspase-3、Baxm RNA相对表达(P<0.05),上调Bcl-2mRNA相对表达(P<0.05)。结论黄芪皂甙Ⅳ联合BMSCs移植对脑缺血再灌注神经元细胞凋亡有协同抑制作用,其作用机制可能与黄芪皂甙Ⅳ促进BMSCs抑制Bax、caspase-3表达,增强Bcl-2表达和Bcl-2/Bax比值有关。
Objective To investigate the effects of astragaloside IV combined with bone marrow mesenchymal stem cells (BMSCs) transplantation on the apoptosis and the expression of Bcl-2, Bax and Caspase-3 in hippocampus during ischemia-reperfusion in rats. Methods Experimental animals were randomly divided into sham operation group, model group, BMSCs group, BMSCs + Astragalus group. The rat middle cerebral artery occlusion (MCAO) model was established by thread occlusion method. BMSCs and BMSCs + Astragalus group were injected with the prepared PKH26-labeled BMSCs suspension via caudal vein 3 h after successful modeling. In addition, BMSCs + Astragalus group was given intraperitoneal injection of astragaloside IV 5 min before ischemia and 12 h after ischemia. After 72h of ischemia-reperfusion, neurological scores were assessed in each group. The distribution of PKH-26 labeled BMSCs in the hippocampus was observed by fluorescence microscopy. The expressions of Bcl-2, Bax and caspase in hippocampus were detected by immunohistochemistry and real-time fluorescence quantitative PCR -3 expression, the use of TUNEL method to detect neuronal apoptosis index. Results PKH-26-labeled BMSCs were expressed in the hippocampus. Compared with model group, BMSCs group and BMSCs + Astragalus group could reduce the score of neurological impairment (P <0.01), and the recovery of neurological function of BMSCs + Astragalus group was the most obvious (P <0.05). The expression of Bcl-2, Caspase-3, Bax and apoptosis in model group were significantly different from those in sham operation group (P <0.01). Compared with model group, BMSCs + Astragalus group and BMSCs group could downregulate Caspase-3, Bax protein expression and apoptosis index (P <0.05), Bcl-2 protein expression and Bcl-2 / , Of which BMSCs + Astragalus group was more significant (P <0.05). Real-time quantitative PCR showed that compared with model group and BMSCs group, BMSCs + Astragalus group could significantly down-regulate Caspase-3 and Baxm RNA relative expression (P <0.05) and up-regulate Bcl-2 mRNA expression (P <0.05). Conclusion Astragaloside IV combined with BMSCs transplantation can inhibit the apoptosis of cerebral ischemia-reperfusion neurons in a synergistic manner. The possible mechanism is that Astragaloside IV can promote BMSCs to inhibit the expression of Bax and caspase-3, enhance the expression of Bcl-2 and Bcl-2 / Bax ratio related.