替格瑞洛体外抗炎活性及机制研究

Study on Anti-inflammatory Activity and Mechanism of Ticagrelor in vitro

探讨替格瑞洛对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)和小鼠骨髓来源巨噬细胞(bone marrow-derived macrophages,BMDMs)构建NLRP3炎症小体抗炎活性的影响。LPS诱导HUVECs体外炎症模型,ELISA试剂法检测替格瑞洛对炎症细胞因子TNF-α、IL-1β、IL-6和IL-10的表达情况;LPS+尼日利亚菌素(nigericin)诱导BMDMs NLRP3炎症小体,ELISA试剂法和Western Blot法检测炎症小体激活情况;诱导BMDMs NLRP3炎症小体前后分别给予不同浓度替格瑞洛干预,ELISA试剂法和Western Blot法检测炎症因子IL-1β、TNF-α、Caspase-1表达情况;LPS诱导小鼠脓毒血症模型,替格瑞洛干预治疗,ELISA试剂法检测IL-1β、IL-6、TNF-α炎症因子表达情况。结果:1.LPS诱导HUVECs炎症模型呈浓度依赖性,随浓度的增加,细胞活性逐渐降低;中间质量浓度100、200 ng/mL诱导效率最高,随诱导时间增加,细胞活性稍增加或基本不变。2.不同浓度的替格瑞洛作用于LPS诱导的HUVECs炎症细胞模型中,均能下调TNF-α、IL-6、IFN-1β炎症因子,上调IL-10炎症因子水平,且炎症因子水平变化均呈现剂量浓度依赖趋势。3.炎症小体激活前、后进行药物干预,均能抑制炎症因子Caspase-1和IL-1β的表达,并且其抑制程度呈剂量依赖性;而激活前药物干预,TNF-α的表达量下降;激活后药物干预,TNF-α的表达量则基本不变。4.LPS诱导小鼠脓毒血症,替格瑞洛治疗组炎症因子IL-1β、IL-6、TNF-α表达显著降低。替格瑞洛在体外HUVECs细胞试验中,具有明显的抗炎活性;通过构建小鼠BMDMs炎症小体,研究替格瑞洛在免疫细胞上对炎症的调控作用,发现其可以作用于NLRP3炎症小体活化的启动阶段,抑制NF-κB信号通路的激活,阻止炎症反应的继续。

The test aims to investigate the effects of ticagrelor on the anti-inflammatory activity of NLRP3 inflammasome constructed by human umbilical vein endothelial cells(HUVECs)and mouse bone marrow-derived macrophages(BMDMs).The inflammation model of HUVECs in vitro was induced by LPS,the effects of ticagrelor on the expression of TNF-α,IL-1β,IL-6 and Il-10 were detected by ELISA;NLRP3 inflammasome of BMDMs was induced by LPS+nigericin,and the activation of the inflammasome was detected by ELISA and Western Blot.Before and after induction of BMDMs NLRP3 inflammasome,different concentrations of ticagrelor were given respectively,the expression of IL-1β,TNF-αand Caspase-1 were detected by ELISA and Western blot.The sepsis model was induced by LPS and treated with ticagrelor,the expression of IL-1β,IL-6 and TNF-αwere detected by ELISA.Result showed:1.The inflammation model of HUVECs induced by LPS was concentration-dependent,and the cell activity decreased gradually with the increase of concentration;the induction efficiency was the highest at the intermediate concentration of 100 ng/mL and 200 ng/mL,and the cell activity increased slightly or remained unchanged with the induction time.2.In the LPS-induced HUVECs inflammatory cell model,different concentrations of ticagrelor can down-regulate the levels of TNF-α,IL-6 and IL-1βinflammatory factors,and up-regulate the levels of IL-10 inflammatory factors,and the changes of inflammatory factors were dose-dependent.3.The expression of Caspase-1 and IL-1βcould be inhibited by drug intervention before and after the activation of inflammasome,and the degree of inhibition was dose-dependent;however,the expression of TNF-αdecreased during pre-activation drug intervention,but remained unchanged after activation drug intervention.4.The expressions of inflammatory factors IL-1β,IL-6 and TNF-αwere significantly decreased in the LPS-induced sepsis group.Ticagrelor has obvious anti-inflammatory activity in HUVECs cell assay in vitro.By constructing mouse BMDMs inflammasome,the regulation of inflammation on immune cells was studied,and it was found that ticagrelor can act on the activation of NLRP3 inflammasome,inhibit the activation of NF-κB signaling pathway,and avoid the continuation of inflammatory response.