Zhang Meng, Xiao Min, Li Jing-Jing, Li Jiang-Feng, Fan Guang-Hui
Clinical College of Traditional Chinese Medicine,Hubei University of Chinese Medicine Wuhan 430065,China Central Hospital of Wuhan Affiliated to Tongji Medical College of Huazhong University of Science and Technology Wuhan 430014,China.
Clinical College of Traditional Chinese Medicine,Hubei University of Chinese Medicine Wuhan 430065,China.
Zhongguo Zhong Yao Za Zhi. 2025 May;50(10):2787-2797. doi: 10.19540/j.cnki.cjcmm.20250312.301.
This article investigated the effect and mechanism of salidroside(SAL) on the expression of adhesion molecules in oxidized low-density lipoprotein(oxLDL)-induced mouse aortic endothelial cell(MAEC). The oxLDL-induced endothelial cell injury model was constructed, and the safe concentration and action time of SAL were screened. The cells were divided into control group, oxLDL group, low and high concentration groups of SAL, and ferrostatin-1(Fer-1) group. The cell viability was detected by CCK-8 assay; lactate dehydrogenase(LDH) leakage was measured by colorimetry; the expression of intercellular adhesion molecule 1(ICAM-1) and recombinant vascular cell adhesion molecule 1(VCAM-1) were detected by immunofluorescence; Fe(2+),glutathione(GSH),malondialdehyde(MDA),and 4-hydroxynonenal(4-HNE) levels were detected by kit method; reactive oxygen species(ROS) was detected by DCFH-DA probe; the levels of glutathione peroxidase 4(GPX4),silent mating type information regulation 2 homolog 1(SIRT1), and nuclear factor erythroid 2-related factor 2(Nrf2) were determined by using Western blot. The inhibitors of Nrf2 and SIRT1 were used, and endothelial cell were divided into control group, oxLDL group, SAL group, ML385 group(Nrf2 inhibitor), and EX527 group(SIRT1 inhibitor). The ultrastructure of mitochondria was observed by electron microscope; mitochondrial membrane potential(MMP) was detected by flowcytometry; the expressions of SIRT1,Nrf2,solute carrier family 7 member 11(SLC7A11),GPX4,ferroportin 1(FPN1),ferritin heavy chain 1(FTH1),ICAM-1, and VCAM-1 were detected by Western blot. The results showed that similar to Fer-1,low and high concentrations of SAL could improve cell viability, inhibit LDH release and the expression of ICAM-1 and VCAM-1 in oxLDL-induced endothelial cells(P<0.05 or P<0.01). It was related to increase in GSH level, decrease in Fe(2+),ROS,MDA, and 4-HNE level, and up-regulation of SIRT1,Nrf2, and GPX4 expression to inhibit ferroptosis(P<0.05 or P<0.01). The intervention effect of high concentration SAL was the most significant. ML385 and EX527 could partially offset the protection of SAL on mitochondrial structure and MMP and reverse the ability of SAL to up-regulate the expression of SIRT1,Nrf2,SLC7A11,GPX4,FPN1, and FTH1 and down-regulate the expression of ICAM-1 and VCAM-1(P<0.05 or P<0.01).To sum up, SAL could reduce the expression of ICAM-1 and VCAM-1 in oxLDL-induced endothelial cell, which may relate to activation of SLC7A11/GPX4 antioxidant signaling pathway mediated by SITR1/Nrf2, up-regulation of FPN1 and FTH1 expression, and inhibition of ferroptosis.
本文研究了红景天苷（SAL）对氧化型低密度脂蛋白（oxLDL）诱导的小鼠主动脉内皮细胞（MAEC）中黏附分子表达的影响及其机制。构建oxLDL诱导的内皮细胞损伤模型，筛选SAL的安全浓度和作用时间。将细胞分为对照组、oxLDL组、SAL低浓度和高浓度组以及铁死亡抑制剂1（Fer-1）组。采用CCK-8法检测细胞活力；比色法检测乳酸脱氢酶（LDH）泄漏；免疫荧光法检测细胞间黏附分子1（ICAM-1）和重组血管细胞黏附分子1（VCAM-1）的表达；试剂盒法检测Fe²⁺、谷胱甘肽（GSH）、丙二醛（MDA）和4-羟基壬烯醛（4-HNE）水平；DCFH-DA探针检测活性氧（ROS）；蛋白质免疫印迹法检测谷胱甘肽过氧化物酶4（GPX4）、沉默信息调节因子2同源物1（SIRT1）和核因子红细胞2相关因子2（Nrf2）的水平。使用Nrf2和SIRT1抑制剂，将内皮细胞分为对照组、oxLDL组、SAL组、ML385组（Nrf2抑制剂）和EX527组（SIRT1抑制剂）。通过电子显微镜观察线粒体超微结构；流式细胞术检测线粒体膜电位（MMP）；蛋白质免疫印迹法检测SIRT1、Nrf2、溶质载体家族7成员11（SLC7A11）、GPX4、铁转运蛋白1（FPN1）、铁蛋白重链1（FTH1）、ICAM-1和VCAM-1的表达。结果表明，与Fer-1相似，低浓度和高浓度的SAL均可提高oxLDL诱导的内皮细胞活力，抑制LDH释放以及ICAM-1和VCAM-1的表达（P<0.05或P<0.01）。这与GSH水平升高、Fe²⁺、ROS、MDA和4-HNE水平降低以及SIRT1、Nrf2和GPX4表达上调以抑制铁死亡有关（P<0.05或P<0.01）。高浓度SAL的干预效果最为显著。ML385和EX527可部分抵消SAL对线粒体结构和MMP的保护作用，并逆转SAL上调SIRT1、Nrf2、SLC7A11、GPX4、FPN1和FTH1表达以及下调ICAM-1和VCAM-1表达的能力（P<0.05或P<0.01）。综上所述，SAL可降低oxLDL诱导的内皮细胞中ICAM-1和VCAM-1的表达，这可能与SITR1/Nrf2介导的SLC7A11/GPX4抗氧化信号通路激活、FPN1和FTH1表达上调以及铁死亡抑制有关。
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