Wang Qiang, Yang Jing, Lei Yan, Xiu Chengkui, Huo Yanming, Shi Hang
Department of Cardiology, Wangjing Hospital, China Academy of Chinese Medical Sciences, Beijing100102, China.
Experimental Research Center, China Academy of Chinese Medical Sciences, Beijing 100700, China.
J Tradit Chin Med. 2020 Jun;40(3):376-385. doi: 10.19852/j.cnki.jtcm.2020.03.003.
To investigate the effects of extracts from Renshen (Radix Ginseng), Sanqi (Radix Notoginseng), and Chuanxiong (Rhizoma Chuanxiong) on the endothelial actin cytoskeleton in senescent human cardiac microvascular endothelial cells (HCMECs), and to propose the possible mechanism underlying the actions.
Lentiviral mediated RNA interference was applied to a replicative senescent HCMEC model by knocking down heat shock protein 27 (HSP27) gene. Cells were treated with extracts from Renshen (Radix Ginseng), Sanqi (Radix Notoginseng), and Chuanxiong (Rhizoma Chuanxiong) at final concentrations of 50, 100, and 200 mg/L, respectively and with 10 μM resveratrol for 48 h. Untreated cells were used as controls. Senescence was detected by senescence β-galactosidase staining and cell proliferation was analyzed by cell counting kit-8 assays. Secreted nitric oxide levels were detected by nitrate reductase. Morphological changes of F-actin and G-actin were observed by laser scanning confocal microscopy. Protein and gene expression of F- actin and HSP27 was detected by western blotting.
Compared with the control group, the proportion of senescent HSP27 shRNA cells treated with the extracts was decreased and their proliferation was increased. In the extract intervention group, F-actin around the cell periphery became irregular and jagged fractures formed gradually and then dissipated. Moreover, some dynamic actin stress fiber filaments appeared. The G-actin stretched to the cell periphery and punctate staining was scattered in the cytoplasm. In addition, the mean optical density value of F/G-actin was decreased significantly and the protein expression of F-actin was downregulated.
The extracts delayed microvascular endothelial cell senescence by downregulating the expression of F-actin through HSP27.
研究人参、三七和川芎提取物对衰老的人心脏微血管内皮细胞（HCMECs）内皮肌动蛋白细胞骨架的影响，并探讨其作用的可能机制。
采用慢病毒介导的RNA干扰技术，通过敲低热休克蛋白27（HSP27）基因，建立复制性衰老的HCMEC模型。分别用终浓度为50、100和200mg/L的人参、三七和川芎提取物以及10μM白藜芦醇处理细胞48小时。未处理的细胞作为对照。通过衰老β-半乳糖苷酶染色检测细胞衰老，通过细胞计数试剂盒-8法分析细胞增殖。用硝酸还原酶检测分泌型一氧化氮水平。通过激光扫描共聚焦显微镜观察F-肌动蛋白和G-肌动蛋白的形态变化。通过蛋白质印迹法检测F-肌动蛋白和HSP27的蛋白质和基因表达。
与对照组相比，用提取物处理的衰老HSP27 shRNA细胞比例降低，增殖增加。在提取物干预组中，细胞周边的F-肌动蛋白变得不规则，逐渐形成锯齿状断裂，然后消散。此外，出现了一些动态的肌动蛋白应力纤维丝。G-肌动蛋白伸展到细胞周边，点状染色分散在细胞质中。此外，F/G-肌动蛋白的平均光密度值显著降低，F-肌动蛋白的蛋白质表达下调。
提取物通过HSP27下调F-肌动蛋白的表达，延缓微血管内皮细胞衰老。
