Supplementary Material for: Isoliquiritigenin Ameliorates Indomethacin-Induced Small Intestinal Damage by Inhibiting NOD-Like Receptor Family, Pyrin Domain-Containing 3 Inflammasome Activation

Activation of the NOD-Like Receptor Family, Pyrin Domain-Containing 3 (NLRP3) inflammasome, which consists of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1, triggers pro-caspase-1 cleavage promoting the processing of pro-interleukin (IL)-1β into mature IL-1β, which is critical for the development of non-steroidal anti-inflammatory drug (NSAID)-induced enteropathy. We investigated the effects of isoliquiritigenin, a flavonoid derived from the roots of Glycyrrhiza species, on NSAID-induced small intestinal damage and the inflammasome activation. To induce enteropathy, mice were administered indomethacin by gavage with or without isoliquiritigenin pretreatment. Some mice received an intraperitoneal injection of recombinant murine IL-1β in addition to isoliquiritigenin and indomethacin. Indomethacin induced small intestinal damage and increased protein levels of cleaved caspase-1 and mature IL-1β in the small intestine. Treatment with 7.5 and 75 mg/kg isoliquiritigenin inhibited indomethacin-induced small intestinal damage by 40 and 56%, respectively. Isoliquiritigenin also inhibited the indomethacin-induced increase in cleaved caspase-1 and mature IL-1β protein levels, whereas it did not affect the mRNA expression of NLRP3, ASC, caspase-1, and IL-1β. Protection against intestinal damage in isoliquiritigenin-treated mice was completely abolished with exogenous IL-1β. NLRP3–/– and caspase-1–/– mice exhibited resistance to intestinal damage, and isoliquiritigenin treatment failed to inhibit the damage in NLRP3–/– and caspase-1–/– mice. Isoliquiritigenin prevents NSAID-induced small intestinal damage by inhibiting NLRP3 inflammasome activation.含吡啶结构域的NOD样受体家族3（NLRP3）炎症小体由NLRP3、含半胱天冬酶募集结构域的凋亡相关斑点样蛋白（ASC）以及半胱天冬酶-1前体（pro-caspase-1）组成，其活化可触发半胱天冬酶-1前体的剪切，促进白细胞介素-1β前体（pro-IL-1β）转化为成熟IL-1β，该过程与非甾体抗炎药（NSAID）诱导的肠病发生密切相关。本研究探讨了异甘草素（isoliquiritigenin）——一种从甘草属植物根部提取的黄酮类化合物——对NSAID诱导的小肠损伤及炎症小体活化的影响。为构建肠病模型，小鼠经口灌胃给予吲哚美辛，同时予以或不予异甘草素预处理；部分小鼠在给予异甘草素与吲哚美辛的基础上，额外腹腔注射重组小鼠IL-1β。实验结果显示，吲哚美辛可诱导小鼠小肠损伤，并提升小肠组织中剪切活化型caspase-1与成熟IL-1β的蛋白水平。分别以7.5 mg/kg与75 mg/kg剂量的异甘草素进行干预，可分别抑制40%与56%的吲哚美辛诱导的小肠损伤。此外，异甘草素还可阻断吲哚美辛诱导的剪切活化型caspase-1及成熟IL-1β蛋白水平升高，但对NLRP3、ASC、caspase-1及IL-1β的mRNA表达无显著影响。外源性给予IL-1β可完全抵消异甘草素对小鼠小肠损伤的保护作用。NLRP3基因敲除（NLRP3–/–）与半胱天冬酶-1基因敲除（caspase-1–/–）小鼠对小肠损伤表现出抗性，且异甘草素无法对这两类小鼠的小肠损伤产生抑制效果。综上，异甘草素可通过抑制NLRP3炎症小体活化，从而改善NSAID诱导的小鼠小肠损伤。