Inhibitory Effect of Serotonin Antagonist on Leukocyte-Endothelial Interactions In Vivo and In Vitro

BackgroundAlthough 5-HT2A serotonergic antagonists have been used to treat vascular disease in patients with diabetes mellitus or obesity, their effects on leukocyte-endothelial interactions have not been fully investigated. In this study, we assessed the effects of sarpogrelate hydrochloride (SRPO), a 5-HT2A receptor inverse agonist, on leukocyte-endothelial cell interactions in obesity both in vivo and in vitro.Methods and FindingsIn the in vivo experiment, C57BL/6 mice were fed a high-fat high-fructose diet (HFFD), comprising 20% fat and 30% fructose, with or without intraperitoneal injection of 5 mg/kg/day SRPO for 4 weeks. The body weight, visceral fat weight, and serum monocyte chemoattractant protein-1 levels in the mice increased significantly with the HFFD, but these effects were prevented by chronic injections of SRPO. Intravital microscopy of the femoral artery detected significant leukocyte-endothelial interactions after treatment with HFFD, but these leukocyte-endothelial interactions were reduced in the mice injected with SRPO. In the in vitro experiment, pre-incubation of activated human umbilical vein endothelial cells (HUVECs) with platelet-rich plasma (PRP) induced THP-1 cell adhesion under physiological flow conditions, but the adhesion was reduced by pretreatment of PRP with SRPO. A fluorescent immunobinding assay showed that PRP induced significant upregulation of E-selectin in HUVECs, but this upregulation was reduced by pretreatment of PRP with SRPO. In other in vitro conditions, pre-incubation of THP-1 cells with phorbol 12-myristate 13-acetate increased the adhesion of THP-1 cells to activated HUVECs under rotational conditions, but this adhesion was reduced by pretreatment with SRPO. Western blotting analysis showed that protein kinase C α activation in THP-1 cells was inhibited by SRPO.ConclusionOur findings indicated that SRPO inhibits vascular inflammation in obesity via inactivation of platelets and leukocytes, and improvement of obese.背景：尽管5-羟色胺2A（5-HT2A）受体拮抗剂已被用于治疗糖尿病或肥胖患者的血管疾病，但其对白细胞-内皮细胞相互作用的影响尚未得到充分研究。 本研究旨在评估5-羟色胺2A受体反向激动剂盐酸沙格雷酯（sarpogrelate hydrochloride，SRPO）对肥胖状态下体内及体外白细胞-内皮细胞相互作用的影响。 方法与结果：体内实验中，将C57BL/6小鼠分为两组，分别饲喂含20%脂肪与30%果糖的高脂高果糖饮食（high-fat high-fructose diet，HFFD），并以或不以5 mg/kg/天的剂量腹腔注射SRPO，持续4周。 高脂高果糖饮食造模后，小鼠体重、内脏脂肪重量及血清单核细胞趋化蛋白-1水平均显著升高，而长期腹腔注射SRPO可阻断上述变化。 对股动脉进行活体显微成像观察发现，高脂高果糖饮食造模后小鼠出现显著的白细胞-内皮细胞相互作用，而SRPO给药组的该类相互作用显著降低。 体外实验中，在生理流条件下，将活化的人脐静脉内皮细胞（human umbilical vein endothelial cells，HUVECs）与富血小板血浆（platelet-rich plasma，PRP）共同预孵育后，可诱导THP-1细胞黏附，而用SRPO预处理富血小板血浆可降低该黏附效应。 荧光免疫结合实验结果显示，富血小板血浆可显著上调HUVECs表面E-选择素的表达，而SRPO预处理富血小板血浆可抑制该上调作用。 其他体外实验条件下，将THP-1细胞与佛波醇12-肉豆蔻酸酯13-乙酸酯（phorbol 12-myristate 13-acetate，PMA）共同预孵育后，在旋转培养条件下可增强THP-1细胞与活化HUVECs的黏附能力，而SRPO预处理可降低该黏附作用。 蛋白质免疫印迹分析结果显示，SRPO可抑制THP-1细胞内蛋白激酶Cα的活化。 结论：本研究结果表明，SRPO可通过灭活血小板与白细胞，改善肥胖相关血管炎症，从而发挥对肥胖状态下血管炎症的抑制作用。