甲基巴多索龙通过抑制NLRP3炎症小体活化缓解小鼠急性肝损伤

doi:10.12122/j.issn.1673-4254.2024.09.05

摘要/Abstract

摘要：

目的探究小分子化合物甲基巴多索隆（CDDO-Me）抑制NLRP3炎症小体活化并缓解急性肝损伤的作用机制。方法体外实验：CDDO-Me预处理小鼠骨髓来源巨噬细胞（BMDM）或人急性单核细胞白血病（THP-1）细胞后，利用多种NLRP3炎症小体活化剂（Nigericin、ATP、MSU、胞内LPS转染）活化NLRP3炎症小体，使用聚脱氧腺苷酸（poly A∶T）活化AIM2炎症小体，通过Western blotting和ELISA检测细胞培养上清中caspase-1和白细胞介素1β（IL-1β）的分泌水平，确定CDDO-Me对NLRP3炎症小体的抑制效果及其特异性。体内实验：将SPF级雄性C57BL/6J小鼠随机分为4组，即空白对照组（Control组）、急性肝损伤组（APAP组）、CDDO-Me低剂量治疗组（APAP+CDDO-Me20 mg/kg组）和CDDO-Me高剂量治疗组（APAP+CDDO-Me40 m/kg组），ELISA检测小鼠血清中IL-1β、肿瘤坏死因子α（TNF-α）、谷草转氨酶（AST）、谷丙转氨酶（ALT）的表达水平，HE染色观察肝组织结构变化。结果 CDDO-Me可剂量依赖性的抑制多种激动剂诱导的NLRP3炎症小体活化以及胞内转染LPS诱导非经典NLRP3炎症小体的活化（P<0.05），但对NLRP3炎症小体非依赖的相关炎性因子白细胞介素6（IL-6）、TNF-α的分泌无显著影响（P>0.05）。此外，CDDO-Me对AIM2炎症小体的活化无显著影响（P>0.05）。动物实验结果表明，相较急性肝损伤组（APAP组），CDDO-Me治疗组（APAP+CDDO-Me20 mg/kg组和APAP+CDDO-Me40 mg/kg组）小鼠血清IL-1β、AST和ALT的表达水平显著降低，肝组织HE染色结果显示CDDO-Me能明显改善ALI小鼠损伤的肝组织结构，减少炎性细胞浸润，且呈剂量依赖性（P<0.05）。结论 CDDO-Me能够特异性抑制NLRP3炎症小体活化并缓解APAP诱导的小鼠急性肝损伤。

关键词: 甲基巴多索龙, NLRP3炎症小体, 炎症小体相关疾病, 急性肝损伤

Abstract:

Objective To investigate the inhibitory effect of bardoxolone methyl (CDDO-Me) on activation of NLRP3 inflammasome and its mechanism for alleviating acute liver injury (ALI). Methods Mouse bone marrow-derived macrophages (BMDM) and THP-1 cells were pre-treated with CDDO-Me followed by treatment with Nigericin, ATP, MSU, intracellular LPS transfection for activation of NLRP3 inflammasomes, or poly A:T for activation of AIM2 inflammasomes. The levels of caspase-1 and IL-1β in the cell culture supernatant was determined with Western blotting and ELISA to assess the inhibitory effect of CDDO-Me on NLRP3 inflammasomes and its specificity. In the animalexperiment, male C57BL/6J mouse models of acetaminophen-induced ALI were treated with low-dose (20 mg/kg) and high-dose (40 mg/kg) CDDO-Me, and the changes in serum levels of IL-1β, TNF-α, AST and ALT were measured by ELISA and liver tissue pathology was observed using HE staining. Results In mouse BMDM and THP-1 cells, CDDO-Me dose-dependently inhibited the activation of NLRP3 inflammasomes without significantly affecting the secretion of non-inflammasome-related inflammatory factors IL-6 and TNF-α or AIM2 inflammasome activation. In the mouse models of ALI, CDDO-Me treatment at both the low and high doses significantly reduced serum levels of IL-1β, AST and ALT, ameliorated histological changes and reduced inflammatory cell infiltration in the liver tissue, and the effects exhibited a distinct dose dependence. Conclusion CDDO-Me can specifically inhibit the activation of NLRP3 inflammasomes to alleviate acetaminophen-induced ALI in mice.

Key words: bardoxolone methyl, NLRP3 inflammasomes, inflammasome-related diseases, acute liver injury

李明远, 张玮, 华梦晴. 甲基巴多索龙通过抑制NLRP3炎症小体活化缓解小鼠急性肝损伤[J]. 南方医科大学学报, 2024, 44(9): 1662-1669.

Mingyuan LI, Wei ZHANG, Mengqing HUA. Bardoxolone methyl alleviates acute liver injury in mice by inhibiting NLRP3 inflammasome activation[J]. Journal of Southern Medical University, 2024, 44(9): 1662-1669.

图/表 6

图1 CDDO-Me抑制nigericin诱导的NLRP3炎症小体活化

Fig.1 CDDO-Me suppresses nigericin-induced NLRP3 inflammasome activation in mouse bone marrow-derived macrophages (BMDM). A: Western blotting of cleaved p20 in the culture supernatants (SN) and pro-caspase-1 (Pro-casp1) in the cell lysate (Input) of BMDM. B, C: Quantitative analysis of the expressions of p20 and Pro-casp1 in BMDM. D-F: ELISA of IL-1β, TNF-α and IL-6 in cell culture supernatants, respectively. *P<0.05, ***P<0.001.

图 2 CDDO-Me抑制ATP和MSU诱导的NLRP3炎症小体活化

Fig.2 CDDO-Me inhibits NLRP3 inflam-masome activation in BMDM induced by ATP, MSU and LPS. A, B: Western blotting of p20 in supernatants (SN) and Pro-casp1 in cell lysates (Input) of BMDM. C, D: ELISA of mature IL-1β in the culture supernatant of BMDM. *P<0.05, **P<0.01, ***P<0.001.

图 3 CDDO-Me抑制非经典NLRP3炎症小体活化

Fig.3 CDDO-Me inhibits non-canonical NLRP3 inflammasome activation in BMDM. A: Western blotting of p20 in culture supernatants (SN) and Pro-casp1 in lysates (Input) of BMDM. B, C: Quantitative analysis of the expressions of p20 and Pro-casp1 in BMDM. D: ELISA of mature IL-1β in the culture supernatant of BMDM. *P<0.05, **P<0.01, ***P<0.001.

图 4 CDDO-Me对AIM2炎症小体活化无影响

Fig.4 CDDO-Me does not affect AIM2 inflammasome activation in BMDM. A: Western blotting of p20 in culture supernatants (SN) and Pro-casp1 in lysates (Input) of BMDM. B, C: Quantitative analysis of the expressions of p20 and Pro-casp1 in BMDM. D: ELISA of matureIL-1β in the culture supernatant of BMDM. ***P<0.001.

图 5 CDDO-Me抑制人THP-1细胞中NLRP3炎症小体活化

Fig.5 CDDO-Me inhibits NLRP3 inflammasome activation in human THP-1 cells. A: Western blotting of p20 in culture supernatants (SN) and Pro-casp1 in lysates (Input) of human THP-1 cells. B, C: Quantitative analysis of the expressions of p20 and Pro-casp1 in human THP- 1 cells. D, E: ELISA of IL-1β (D) and TNF-α (E) in the culture supernatant of THP-1 cells. ***P<0.001.

图 6 CDDO-Me缓解对乙酰氨基酚(APPA)引起的急性肝损伤(AILI)

Fig.6 CDDO-Me relieves acetaminophen (APPA)-induced acute liver injury (ALI) in mice. A, B: ELISA of IL-1β (A) and TNF-α (B) in the serum from C57BL/6J mice. C-D: Levels of AST (C) and ALT (D) determined with ELISA in the serum from C57BL/6J mice in control, APAP and APAP plus CDDO-Me (20 and 40 mg/kg) groups. E: HE staining of liver sections from ALI mice in control, APAP and APAP plus CDDO-Me (20 and 40 mg/kg) groups (Original magnification: ×200). F: Quantification of necrotic areas by Image J software. ***P<0.001 vs control; ##P<0.01 vs APPA group, ###P<0.001 vs APPA group.

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