刘晓丹 丁煌 李艳玲 陆展辉 杨芙蓉 黄小平 邓常清

〔摘要〕 目的 觀察黄芪甲苷（AST IV）配伍三七总皂苷（PNS）对氧糖剥夺后再复糖复氧（OGD/R）大鼠脑微血管内皮细胞（BMECs）增殖、凋亡及线粒体功能的影响。方法 差速密度梯度离心法提取BMECs，免疫荧光检测血管性血友病因子（vWF）表达对细胞进行鉴定，取第3代BMECs，采用AST IV与PNS高（100 μmol/L+60 μmol/L）、中（50 μmol/L+30 μmol/L）、低（25 μmol/L+15 μmol/L）剂量配伍预处理24 h，以OGD/R建立缺血再灌注损伤模型，同时设立正常组和模型组。CCK8法测定细胞增殖情况，LDH漏出率检测细胞损伤，AnnexinⅤ/PI双染检测细胞凋亡，TraKineTMPro活细胞线粒体染色试剂盒对线粒体进行染色，激光共聚焦观察线粒体结构，JC1染色测定线粒体膜电位变化情况。结果 成功培养分离BMECs，阳性表达vWF，与正常组比较，模型组细胞存活数量明显减少（P<0.05），LDH漏出率显著增加（P<0.01），细胞凋亡率显著增加（P<0.01）;与模型组比较，AST IV与PNS不同剂量配伍均能促进BMECs增殖、抑制LDH的释放和抑制细胞凋亡（P<0.05，P<0.01）。与正常组比较，模型组线粒体荧光强度明显降低，分布不均;与模型组比较，AST IV与PNS不同剂量配伍组深红色荧光强度有所增强。与正常组比较，模型组线粒体膜电位水平下降（P<0.01），与模型组比较，AST IV与PNS不同剂量配伍组线粒体膜电位明显增强（P<0.05，P<0.01）。结论 AST IV配伍PNS在体外能促进缺血再灌注模型BEMCs细胞增殖，抑制细胞凋亡，降低LDH漏出率，其机制与保护线粒体，增加线粒体膜电位有关。

〔关键词〕 脑微血管内皮细胞;脑缺血再灌注;黄芪甲苷;三七总皂苷;凋亡;增殖;线粒体

〔中图分类号〕R285.5       〔文献标志码〕A       〔文章编号〕doi：10.3969/j.issn.1674-070X.2021.04.002

〔Abstract〕 Objective To observe the effects of astragaloside IV （AST IV） combined with panax notoginseng saponins （PNS） on proliferation， apoptosis， and mitochondrial function of rats brain microvascular endothelial cells （BMECs）. Methods BMECs were extracted by differential density gradient centrifugation. The cells were identified by immunofluorescence detection of the expression of von willebrand factor （VWF）. The third generation of BMECs were pretreated with AST IV combined with PNS at high （100 μmol/L + 60 μmol/L）， medium （50 μmol/L + 30 μmol/L）， and low （25 μmol/L + 15 μmol/L） doses for 24 hours. OGD/R was used to establish ischemia reperfusion injury model， and control group and model group were established at the same time. CCK8 assay was used to detect cell proliferation， LDH leakage rate was used to detect cell injury， Annexin V/PI double staining was used to detect cell apoptosis， and TraKineTMPro living cell mitochondrion staining kit was used stain the mitochondria. The structure of mitochondria was observed by confocal laser scanning. The changes of mitochondrial membrane potential were measured by JC1 staining. Results BMECs were successfully cultured and separated， and with positive expression of vWF. Compared with the control group， the number of surviving cells in the model group was significantly reduced （P<0.05）， the leakage rate of LDH was significantly increased （P<0.01）， and the apoptosis rate was significantly increased （P<0.01）. Compared with the model group， the combination of IV and PNS at different doses could promote BMECs proliferation， inhibit the release of LDH and inhibit apoptosis （P<0.05， P<0.01）; compared with the control group， fluorescence intensity of mitochondria in the model group was significantly decreased and the distribution was uneven; compared with model group， the intensity of dark red fluorescence was enhanced in AST IV and PNS groups with different doses. Compared with the control group， the level of mitochondrial membrane potential in the model group was decreased （P<0.01）. Compared with the model group， the mitochondrial membrane potential of AST IV and PNS in different doses was significantly increased （P<0.05， P<0.01）. Conclusion AST IV combined with PNS can promote the proliferation of ischemia reperfusion model BMECs， inhibit cell apoptosis and reduce the leakage rate of LDH in vitro. The mechanism is related to the protection of mitochondria and the increase of mitochondrial membrane potential.

〔Keywords〕 brain microvascular endothelial cells; cerebral ischemia reperfusion injury; astragaloside IV; panax notoginseng  saponins; apoptosis; proliferation; mitochondria

脑缺血性疾病的病因及发病机制复杂，致残率、死亡率很高，再灌注治疗引起的脑微血管内皮细胞功能障碍是继发性神经功能受损的关键环节[1]。脑微血管内皮细胞是脑微循环的重要组成部分，也是血脑屏障的组成单位，在维持血管的完整、稳态平衡中发挥了重要的作用。因此，对脑微血管内皮细胞的保护成为脑缺血再灌注治疗的重要靶点。

黄芪总皂苷（astragalosides， AST）和三七总皂苷（Panax notoginseng saponins， PNS）为黄芪和三七发挥心脑血管效应的有效组分。课题组大量研究[2-11]证实黄芪甲苷（AST IV）和PNS配伍可多环节多靶点发挥对脑缺血再灌注损伤大鼠的神经保护作用，并且能够修复脑缺血再灌注损伤大鼠血脑屏障。……