Conclusion
The FAK inhibitor PF-562271 alleviates endothelial cell damage induced by LPS and aging platelets by lowering cellular oxidative stress levels and reducing inflammatory responses. 目的: 探究FAK抑制剂对老化血小板诱导的人脐静脉内皮细胞（HUVEC）损伤的保护作用。 方法: 实验分为空白对照组、脂多糖（LPS）组、造模组（LPS+Plt）和FAK抑制剂PF-562271组（LPS+Plt+PF-562271）。通过Western blot和免疫荧光检测FAK、pFAK和PECAM-1的蛋白表达。流式细胞术检测HUVEC活性氧（ROS）含量。细胞通透性和跨内皮细胞电阻实验，检测HUVEC屏障功能的变化。RT-qPCR检测炎性因子mRNA表达，酶联免疫吸附测定法（ELISA）检测细胞上清液中，炎性因子的分泌情况。免疫荧光检测加入ROS抑制剂维生素C（Vit.C）后，PECAM-1的表达。 结果: 脂多糖和老化血小板处理后，FAK、pFAK和PECAM-1的蛋白表达增高，加入PF-562271后，FAK、pFAK和PECAM-1的蛋白表达降低（P＜0.05，P＜0.01）。流式细胞术结果显示，脂多糖和老化血小板可促进ROS的释放，而加入PF-562271后，ROS释放减少（P＜0.001）。脂多糖和老化血小板导致内皮细胞屏障受损，PF-562271可缓解内皮细胞屏障功能的损伤（P＜0.01）。脂多糖和老化血小板促进内皮细胞炎症因子TNF-α、IL-6、IL-8的表达，PF-562271可降低炎症因子的表达（P＜0.05）。加入维生素C后，PECAM-1蛋白表达降低（P＜0.01）。 结论: FAK抑制剂PF-562271可通过改善氧化应激水平和降低炎症反应，缓解脂多糖和老化血小板诱导的内皮细胞损伤。
Methods
Cultured HUVECs were treated with vehicle, lipopolysaccharide (LPS), LPS+aging platelets, or LPS+aging platelets+PF-562271. The changes in protein expressions of FAK, pFAK and PECAM-1 in the treated cells were detected using Western blotting and immunofluorescence assay, and the level of reactive oxygen species (ROS) was detected with flow cytometry. The changes of barrier function of the cells were assessed with cell permeability test and transendothelial cell resistance test. RT-qPCR was used to analyze mRNA expressions of inflammatory factors, and pro-inflammatory cytokine levels in the culture supernatants was determined with enzyme-linked immunosorbent assay. Immunofluorescence assay was used to examine the effect of the ROS inhibitor vitamin C on PECAM-1 expression in the cells with different treatments.
Objective
To investigate the protective effect of PF-562271, a FAK inhibitor, against aging platelet-induced injury in human umbilical vein endothelial cells (HUVECs).
Results
Treatment of HUVECs with LPS and aging platelets significantly increased cellular protein expressions of FAK, pFAK and PECAM-1, which were effectively lowered by addition of PF-562271 (P < 0.05). LPS and aged platelets obviously enhanced ROS production in the cells, which was inhibited by the addition of PF-562271 (P < 0.001). PF-562271 significantly alleviated the damage of endothelial cell barrier function of the cells caused by LPS and aging platelets (P < 0.01). The expressions of TNF-α, IL-6 and IL-8 in HUVECs increased significantly after exposure to LPS and aging platelets, and were obviously lowered after treatment with PF-562271 (P < 0.05). Treatment with vitamin C significantly decreased the expression of PECAM-1 protein in the cells (P < 0.01).