Liu Yinan, Niu Zenghui, Wang Xue, Xiu Chengkui, Hu Yanhong, Wang Jiali, Lei Yan, Yang Jing
Beijing Key Laboratory of Traditional Chinese Medicine Basic Research on Prevention and Treatment for Major Diseases, Experimental Research Center, China Academy of Chinese Medical Sciences, Beijing, 100700, China.
Graduate School of China Academy of Chinese Medical Sciences, Beijing, 100700, China.
Chin Med. 2024 Oct 14;19(1):143. doi: 10.1186/s13020-024-01008-7.
During the aging process, the number and functional activity of endothelial progenitor cells (EPCs) are impaired, leading to the unsatisfactory efficacy of transplantation. Previous studies demonstrated that Yiqihuoxue decoction (Ginseng-Sanqi-Chuanxiong, GSC) exerts anti-vascular aging effects. The purpose of this study is to evaluated the effects of GSC on D-galactose (D-gal)induced senescence and the underlying mechanisms.
The levels of cellular senescence-related markers P16, P21, P53, AMPK and p-AMPK were detected by Western blot analysis (WB). SA-β-gal staining was used to evaluate cell senescence. EPCs function was measured by CCK-8, Transwell cell migration and cell adhesion assay. The morphological changes of mitochondria were detected by confocal microscopy. The protein and mRNA expression of mitochondrial fusion fission Drp1, Mff, Fis1, Mfn1, Mfn2 and Opa1 in mitochondria were detect using WB and RT-qPCR. Mitochondrial membrane potential, mtROS and ATP of EPCs were measured using IF. H&E staining was used to observe the pathological changes and IMT of the aorta. The expressions of AGEs, MMP-2 and VEGF in aorta were measured using Immunohistochemical (IHC). The levels of SOD, MDA, NO and ET-1 in serum were detected by SOD, MDA and NO kits.
In vitro, GSC ameliorated the senescence of EPCs induced by D-gal and reduced the expression of P16, P21 and P53. The mitochondrial morphology of EPCs was restored, the expression of mitochondrial Drp1, Mff and Fis1 protein was decreased, the levels of mtROS and ATP were decreased, and mitochondrial function was improved. Meanwhile, the expression of AMPK and p-AMPK increased. The improvement effects of GSC on aging and mitochondrial morphology and function were were hindered after adding AMPK inhibitor. In vivo, GSC improved EPCs efficiency, ameliorated aortic structural disorder and decreased IMT in aging mice. The serum SOD level increased and MDA level decreased, indicating the improvement of antioxidant capacity. Increased NO content and ET-1 content suggested improvement of vascular endothelial function. The changes observed in SOD and MMP-2 suggested a reduction in vascular stiffness and the degree of vascular damage. The decreased expression of P21 and P53 indicates the delay of vascular senescence.
在衰老过程中，内皮祖细胞（EPCs）的数量和功能活性受损，导致移植效果不理想。先前的研究表明，益气活血汤（人参-三七-川芎，GSC）具有抗血管衰老作用。本研究旨在评估GSC对D-半乳糖（D-gal）诱导的衰老的影响及其潜在机制。
通过蛋白质免疫印迹分析（WB）检测细胞衰老相关标志物P16、P21、P53、AMPK和p-AMPK的水平。采用SA-β-半乳糖染色评估细胞衰老。通过CCK-8、Transwell细胞迁移和细胞黏附试验检测EPCs功能。用共聚焦显微镜检测线粒体的形态变化。使用WB和RT-qPCR检测线粒体融合裂变相关蛋白Drp1、Mff、Fis1、Mfn1、Mfn2和Opa1在mRNA和蛋白水平的表达。使用免疫荧光法检测EPCs的线粒体膜电位、线粒体活性氧（mtROS）和三磷酸腺苷（ATP）。用苏木精-伊红（H&E）染色观察主动脉的病理变化和内膜中层厚度（IMT）。采用免疫组织化学（IHC）检测主动脉中晚期糖基化终末产物（AGEs）、基质金属蛋白酶-2（MMP-2）和血管内皮生长因子（VEGF）的表达。通过超氧化物歧化酶（SOD）、丙二醛（MDA）和一氧化氮（NO）试剂盒检测血清中SOD、MDA、NO和内皮素-1（ET-1）的水平。
在体外，GSC改善了D-gal诱导的EPCs衰老，降低了P16、P21和P53的表达。EPCs的线粒体形态得以恢复，线粒体Drp1、Mff和Fis1蛋白的表达降低，mtROS和ATP水平降低，线粒体功能得到改善。同时，AMPK和p-AMPK的表达增加。添加AMPK抑制剂后，GSC对衰老以及线粒体形态和功能的改善作用受到阻碍。在体内，GSC提高了衰老小鼠的EPCs效率，改善了主动脉结构紊乱并降低了IMT。血清SOD水平升高，MDA水平降低，表明抗氧化能力得到改善。NO含量增加和ET-含量增加表明血管内皮功能得到改善。SOD和MMP-2的变化表明血管硬度和血管损伤程度降低。P21和P53表达降低表明血管衰老延迟。
