OBJECTIVE: The purpose of this study was to determine the impact of TPR on the NLRP3, NLRC4, and AIM2 inflammasomes and the underlying mechanisms. Additionally, the efficacy of TPR was analysed in the further course of methionine- and choline-deficient (MCD)-induced NASH and lipopolysaccharide (LPS)-induced sepsis models of mice.
METHODS: In vitro studies used bone marrow-derived macrophages to assess the anti-inflammatory activity of TPR, and the techniques included western blot, testing of intracellular K+ and Ca2+, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), co-immunoprecipitation, ASC oligomerization assay, surface plasmon resonance (SPR), and molecular docking. We used LPS-induced sepsis models and MCD-induced NASH models in vivo to evaluate the effectiveness of TPR in inhibiting inflammatory diseases.
RESULTS: Our observations suggested that TPR could inhibit NLRP3, NLRC4, and AIM2 inflammasome activation. As shown in a mouse model of inflammatory diseases caused by MCD-induced NASH and LPS-induced sepsis, TPR significantly alleviated the progression of diseases. TPR interrupted the interactions between ASC and NLRP3/NLRC4/AIM2 in the co-immunoprecipitation experiment, and stable binding of TPR to ASC was also evident in SPR experiments. The underlying mechanisms of anti-inflammatory activities of TPR might be associated with targeting ASC, in particular, PYD domain of ASC.
CONCLUSIONS: In general, the requirement for ASC in multiple inflammasome complexes makes TPR, as a novel broad-spectrum inflammasome inhibitor, potentially useful for treating a wide range of multifactorial inflammasome-related diseases.
目的:本研究的目的是确定TPR对NLRP3,NLRC4和AIM2炎性体的影响及其潜在机制。此外,在甲硫氨酸和胆碱缺乏(MCD)诱导的NASH和脂多糖(LPS)诱导的小鼠脓毒症模型的进一步过程中分析了TPR的功效.
方法:体外研究使用骨髓来源的巨噬细胞评估TPR的抗炎活性,技术包括蛋白质印迹,细胞内K+和Ca2+的测试,免疫荧光,酶联免疫吸附测定(ELISA),免疫共沉淀,ASC寡聚化试验,表面等离子体共振(SPR),和分子对接。我们在体内使用LPS诱导的脓毒症模型和MCD诱导的NASH模型来评估TPR抑制炎性疾病的有效性。
结果:我们的观察表明TPR可以抑制NLRP3、NLRC4和AIM2炎性体的激活。如MCD诱导的NASH和LPS诱导的脓毒症引起的炎性疾病的小鼠模型所示,TPR显著缓解了疾病的进展。在免疫共沉淀实验中,TPR中断了ASC与NLRP3/NLRC4/AIM2之间的相互作用,在SPR实验中TPR与ASC的稳定结合也是明显的。TPR抗炎活性的潜在机制可能与靶向ASC有关,特别是,ASC的PYD域。
结论:一般来说,多个炎性体复合物对ASC的要求使得TPR,作为一种新型的广谱炎性体抑制剂,可能用于治疗多种多因素炎性体相关疾病。