Abstract：
BACKGROUND: Several factors contribute to ischemia/reperfusion injury (IRI), including activation of the NLRP3 inflammasome and its byproducts, such as interleukin-1β (IL-1β) and caspase-1. However, NLRP3 may paradoxically exhibit cardioprotective properties. This study aimed to assess the protective effects of the novel NLRP3 inhibitor, INF195, both in vitro and ex vivo.
METHODS: To investigate the relationship between NLRP3 and myocardial IRI, we synthetized a series of novel NLRP3 inhibitors, and investigated their putative binding mode via docking studies. Through in vitro studies we identified INF195 as optimal for NLRP3 inhibition. We measured infarct-size in isolated mouse hearts subjected to 30-min global ischemia/one-hour reperfusion in the presence of three different doses of INF195 (5, 10, or 20-μM). We analyzed caspase-1 and IL-1β concentration in cardiac tissue homogenates by ELISA. Statistical significance was determined using one-way ANOVA followed by Tukey\'s test.
CONCLUSIONS: INF195 reduces NLRP3-induced pyroptosis in human macrophages. Heart pre-treatment with 5 and 10-μM INF195 significantly reduces both infarct size and IL-1β levels. Data suggest that intracardiac NLRP3 activation contributes to IRI and that low doses of INF195 exert cardioprotective effects by reducing infarct size. However, at 20-μM, INF195 efficacy declines, leading to a lack of cardioprotection. Research is required to determine if high doses of INF195 have off-target effects or dual roles, potentially eliminating both harmful and cardioprotective functions of NLRP3. Our findings highlight the potential of a new chemical scaffold, amenable to further optimization, to provide NLRP3 inhibition and cardioprotection in the ischemia/reperfusion setting.
METHODS: To investigate the relationship between NLRP3 and myocardial IRI, we synthetized a series of novel NLRP3 inhibitors, and investigated their putative binding mode via docking studies. Through in vitro studies we identified INF195 as optimal for NLRP3 inhibition. We measured infarct-size in isolated mouse hearts subjected to 30-min global ischemia/one-hour reperfusion in the presence of three different doses of INF195 (5, 10, or 20-μM). We analyzed caspase-1 and IL-1β concentration in cardiac tissue homogenates by ELISA. Statistical significance was determined using one-way ANOVA followed by Tukey\'s test.
CONCLUSIONS: INF195 reduces NLRP3-induced pyroptosis in human macrophages. Heart pre-treatment with 5 and 10-μM INF195 significantly reduces both infarct size and IL-1β levels. Data suggest that intracardiac NLRP3 activation contributes to IRI and that low doses of INF195 exert cardioprotective effects by reducing infarct size. However, at 20-μM, INF195 efficacy declines, leading to a lack of cardioprotection. Research is required to determine if high doses of INF195 have off-target effects or dual roles, potentially eliminating both harmful and cardioprotective functions of NLRP3. Our findings highlight the potential of a new chemical scaffold, amenable to further optimization, to provide NLRP3 inhibition and cardioprotection in the ischemia/reperfusion setting.
摘要：
背景：几个因素有助于缺血/再灌注损伤（IRI），包括激活NLRP3炎性体及其副产物，如白细胞介素-1β（IL-1β）和caspase-1。然而,NLRP3可能矛盾地表现出心脏保护特性。本研究旨在评估新型NLRP3抑制剂的保护作用，INF195，体外和离体。
方法：为了研究NLRP3与心肌IRI的关系，我们合成了一系列新型的NLRP3抑制剂，并通过对接研究研究了它们的推定结合模式。通过体外研究,我们确定INF195对于NLRP3抑制是最佳的。我们在三种不同剂量的INF195（5、10或20μM）的存在下，测量了经过30分钟全身缺血/1小时再灌注的离体小鼠心脏的梗死面积。我们通过ELISA分析了心脏组织匀浆中的caspase-1和IL-1β浓度。使用单向ANOVA和随后的Tukey检验确定统计学显著性。
结论：INF195降低NLRP3诱导的人巨噬细胞的焦亡。使用5和10μMINF195的心脏预处理显着降低了梗死面积和IL-1β水平。数据表明，心内NLRP3激活有助于IRI，低剂量的INF195通过减少梗死面积发挥心脏保护作用。然而,在20μM，INF195疗效下降，导致缺乏心脏保护。需要研究以确定高剂量的INF195是否具有脱靶效应或双重作用，可能消除NLRP3的有害和心脏保护功能。我们的发现强调了一种新的化学支架的潜力，适合进一步优化，在缺血/再灌注环境中提供NLRP3抑制和心脏保护。
方法：为了研究NLRP3与心肌IRI的关系，我们合成了一系列新型的NLRP3抑制剂，并通过对接研究研究了它们的推定结合模式。通过体外研究,我们确定INF195对于NLRP3抑制是最佳的。我们在三种不同剂量的INF195（5、10或20μM）的存在下，测量了经过30分钟全身缺血/1小时再灌注的离体小鼠心脏的梗死面积。我们通过ELISA分析了心脏组织匀浆中的caspase-1和IL-1β浓度。使用单向ANOVA和随后的Tukey检验确定统计学显著性。
结论：INF195降低NLRP3诱导的人巨噬细胞的焦亡。使用5和10μMINF195的心脏预处理显着降低了梗死面积和IL-1β水平。数据表明，心内NLRP3激活有助于IRI，低剂量的INF195通过减少梗死面积发挥心脏保护作用。然而,在20μM，INF195疗效下降，导致缺乏心脏保护。需要研究以确定高剂量的INF195是否具有脱靶效应或双重作用，可能消除NLRP3的有害和心脏保护功能。我们的发现强调了一种新的化学支架的潜力，适合进一步优化，在缺血/再灌注环境中提供NLRP3抑制和心脏保护。
