School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
School of Pharmacy, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
Free Radic Biol Med. 2013 Dec;65:997-1004. doi: 10.1016/j.freeradbiomed.2013.08.178. Epub 2013 Aug 29.
D-Galactosamine (GalN) and lipopolysaccharide (LPS) are commonly used to study mechanisms of hepatic malfunction that result in hepatic inflammation and subsequent fulminant hepatic failure. Inflammasomes are intracellular multiprotein complexes that in response to cellular danger signals trigger the biological maturation of proinflammatory cytokines. Heme oxygenase-1 (HO-1) is a cytoprotective enzyme that induces anti-inflammatory and antioxidant activity against oxidative cellular stress. This study examined activation of the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome in GalN/LPS-induced hepatic injury and the role of HO-1 in the signaling pathways of inflammasome. Mice (C57BL/6) were pretreated twice with hemin (HO-1 inducer, 30 mg/kg) and zinc protoporphyrin (ZnPP; HO-1 inhibitor, 10mg/kg) at 12 and 2h before GalN (800 mg/kg)/LPS (40 μg/kg) administration. HO-1 induction with hemin reversed the lethality induced by GalN/LPS administration, and ZnPP pretreatment blocked this change. Lipid peroxidation markedly increased after GalN/LPS treatment, whereas glutathione content decreased in the GalN/LPS group. These changes were attenuated by hemin, but ZnPP reversed the effects of hemin. Serum levels of tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β increased after GalN/LPS treatment; these increases were attenuated by hemin. Hepatic mRNA levels of TNF-α, IL-1β, and NLRP3 increased after GalN/LPS treatment, and hemin attenuated increases in TNF-α and IL-1β. After GalN/LPS treatment, the hepatic expression of NLRP3, ASC, and caspase-1 (p10) was increased. In immunoprecipitation studies, hemin attenuated the interaction of NLRP3 with ASC and caspase-1. GalN/LPS induced expression of the thioredoxin-interacting protein (TXNIP) gene and the interaction between NLRP3 and TXNIP; again, hemin attenuated these effects. The effects of hemin were reversed by ZnPP. Our findings suggest that activation of the NLRP3 inflammasome leads to a GalN/LPS-induced inflammatory response through TXNIP-NLRP3 interaction. Furthermore, HO-1 overexpression may protect the liver against GalN/LPS-induced inflammation through suppression of the NLRP3 signaling pathway.
半乳糖胺（GalN）和脂多糖（LPS）常用于研究导致肝炎症和随后暴发性肝衰竭的肝功能障碍的机制。炎性体是细胞内多蛋白复合物，可响应细胞危险信号触发前炎性细胞因子的生物学成熟。血红素加氧酶-1（HO-1）是一种细胞保护性酶，可诱导针对氧化细胞应激的抗炎和抗氧化活性。本研究探讨了 GalN/LPS 诱导的肝损伤中 NACHT、LRR 和 PYD 结构域蛋白 3（NLRP3）炎性体的激活以及 HO-1 在炎性体信号通路中的作用。用血红素（HO-1 诱导剂，30mg/kg）和锌原卟啉（HO-1 抑制剂，10mg/kg）预处理两次，在 GalN（800mg/kg）/LPS（40μg/kg）给药前 12 小时和 2 小时，预处理 C57BL/6 小鼠。血红素诱导的 HO-1 逆转了 GalN/LPS 给药引起的致死性，而 ZnPP 预处理阻断了这种变化。GalN/LPS 处理后脂质过氧化明显增加，而 GalN/LPS 组谷胱甘肽含量减少。血红素减弱了这些变化，但 ZnPP 逆转了血红素的作用。GalN/LPS 处理后血清肿瘤坏死因子-α（TNF-α）和白细胞介素（IL）-1β水平升高，血红素减弱了升高。肝 TNF-α、IL-1β 和 NLRP3 的 mRNA 水平在 GalN/LPS 处理后增加，血红素减弱了 TNF-α和 IL-1β的增加。GalN/LPS 处理后，肝 NLRP3、ASC 和半胱氨酸天冬氨酸蛋白酶-1（p10）的表达增加。在免疫沉淀研究中，血红素减弱了 NLRP3 与 ASC 和半胱氨酸天冬氨酸蛋白酶-1 的相互作用。GalN/LPS 诱导硫氧还蛋白相互作用蛋白（TXNIP）基因的表达和 NLRP3 与 TXNIP 的相互作用；再次，血红素减弱了这些作用。ZnPP 逆转了血红素的作用。我们的发现表明，NLRP3 炎性体的激活通过 TXNIP-NLRP3 相互作用导致 GalN/LPS 诱导的炎症反应。此外，HO-1 过表达可能通过抑制 NLRP3 信号通路来保护肝脏免受 GalN/LPS 诱导的炎症。
