南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (9): 1476-1484.doi: 10.12122/j.issn.1673-4254.2023.09.04

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川藏香茶菜丙素抑制NLRP3炎症小体活化并缓解小鼠脓毒性休克

曹海若，张 玮，李明远，杨燕青，李玉云   

  1. 蚌埠医学院肿瘤基础研究与临床检验诊断重点实验室，安徽 蚌埠 233030；蚌埠医学院第一附属医院 检验科，安徽 蚌埠 233004；蚌埠医学院慢性疾病免疫学基础与临床安徽省重点实验室，安徽 蚌埠 233030
  • 出版日期:2023-09-20 发布日期:2023-09-28

Isodopharicin C inhibits NLRP3 inflammasome activation and alleviates septic shock in mice

CAO Hairuo, ZHANG Wei, LI Mingyuan, YANG Yanqing, LI Yuyun   

  1. Anhui Provincial Key Laboratory of Cancer Research and Clinical Laboratory Diagnosis, school of laboratory Medicine, Bengbu Medical College, Bengbu 233030, China; Clinical Laboratory, First Affiliated Hospital of Bengbu Medical College, Bengbu 233004, China; Anhui Provincial Key Laboratory of Immunology in Chronic Disease, Bengbu Medical College, Bengbu 233030, China
  • Online:2023-09-20 Published:2023-09-28

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摘要： 目的 探究中草药提取物川藏香茶菜丙素（Iso C）对NLRP3炎症小体活化的影响以及对脂多糖（LPS）诱导的小鼠脓毒性休克是否具有缓解作用。方法 体外实验：利用LPS预刺激小鼠骨髓来源巨噬细胞（BMDM）以及人急性单核细胞白血病（THP-1）细胞系。通过加入多种NLRP3炎症小体激动剂活化经典NLRP3炎症小体。利用胞内转染LPS活化非经典NLRP3炎症小体。利用转染聚脱氧腺苷酸（poly A：T）活化AIM2炎症小体。通过蛋白质印迹法（Western blot）检测NLRP3炎症小体活化产物caspase-1的剪切，酶联免疫吸附测定法（ELISA）检测上清中NLRP3依赖与非依赖的多种促炎细胞因子分泌情况。利用电感耦合等离子体发射光谱仪检测细胞内钾离子含量。体内实验：挑选SPF级C57BL/6J小鼠随机分为空白对照组（Control组），脓毒性休克组（LPS组），Iso C治疗组（LPS+Iso C组）。ELISA分析小鼠血清和腹腔灌洗液中促炎细胞因子白细胞介素 1β（1L-1β）、肿瘤坏死因子α（TNF-α）、白细胞介素 6（IL-6）的分泌情况，并观察注射LPS后小鼠48 h内生存时间，绘制小鼠生存曲线。结果 体外实验表明，在BMDM细胞中Iso C 以剂量依赖的方式抑制多种激动剂引起的经典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），Iso C对NLRP3炎症小体非依赖的促炎细胞因子TNF-α和IL-6的分泌无显著影响（P>0.05）。Iso C对AIM2炎症小体的活化无显著影响（P>0.05）。Iso C不影响NLRP3炎症小体活化上游信号钾离子外流（P>0.05）。在人THP-1细胞中Iso C抑制NLRP3炎症小体活化（P<0.05）。体内实验结果表明，与脓毒性休克组相比，Iso C治疗组小鼠血清和腹腔灌洗液中IL-1β的水平显著下降（P<0.05）且生存时间更长（P<0.05）。结论 中草药来源的Iso C可以特异性抑制NLRP3炎症小体的活化从而缓解小鼠脓毒性休克，是潜在的治疗炎症性疾病的小分子化合物。

关键词: 川藏香茶菜丙素；NLRP3炎症小体；脓毒性休克；川藏香茶菜

Abstract: Objective To investigate the effect of Isodopharicin C (Iso C), a traditional Chinese herbal medicine extract, on NLRP3 inflammasome activation and lipopolysaccharide (LPS)-induced septic shock in mice. Methods Murine bone marrow-derived macrophages (BMDM) and human monocytic THP-1 cells were stimulated with LPS before treatment with different NLRP3 inflammasome agonists to activate canonical NLRP3 inflammasomes. The non-canonical NLRP3 inflammasomes were activated by intracellular LPS transfection, and AIM2 inflammasomes were activated with poly A:T. The cleavage of caspase-1 induced by NLRP3 activation was measured using Western blotting. The levels of NLRP3-dependent and -independent pro-inflammatory cytokines in the cell culture supernatant were detected using ELISA, and the intracellular potassium ion concentration was measured using ICP-OES. In the animal experiment, C57BL/6J mouse models of septic shock (induced by intraperitoneal LPS injection) were treated with Iso C, and the levels of IL-1β, TNF-α and IL-6 in the serum and peritoneal lavage fluid were detected using ELISA. The survival time of the mice was observed within 48 h after LPS injection and a survival curve was plotted. Results In BMDM cells, Iso C dose-dependently inhibited the activation of canonical NLRP3 inflammasomes and non-canonical NLRP3 inflammasomes (P<0.05) without obviously affecting the secretion levels of TNF-α and IL-6 (P>0.05), the activation of AIM2 inflammasomes (P>0.05), or K+ efflux, the upstream signaling of NLRP3 activation (P>0.05). Iso C inhibited the activation of canonical NLRP3 inflammasomes in human THP-1 cells. In septic C57BL/6J mice, Iso C treatment significantly reduced IL-1β levels in the serum and peritoneal lavage fluid, and prolonged the survival time of the mice (P<0.05). Conclusion Iso C specifically inhibits NLRP3 inflammasome activation and alleviates septic shock in mice, and can serve as a potential small molecule compound for treatment of inflammatory diseases.

Key words: isodopharicin C; NLRP3 inflammasome; septic shock; Isodon pharicus