孙亚磊,郭宇,王欣雨,张雅素,程雪,朱珂,陈立典,冯晓东
SUN Ya-lei,GUO Yu,WANG Xin-yu,ZHANG Ya-su,CHENG Xue,ZHU Ke,CHEN Li-dian,FENG Xiao-dong

• 1:河南中医药大学康复医学院

摘要(Abstract)：

旨在探讨异鼠李素(Isor)对急性肺损伤(ALI)的干预作用及对NOD样受体热蛋白结构域3(NLRP3)/凋亡相关斑点样蛋白(ASC)/半胱氨酸蛋白酶-1(caspase-1)轴介导的细胞焦亡机制的调控效应。在体内实验中，将60只BALB/c小鼠分为5组，除对照组外，其他组小鼠灌胃给药1 h后气管滴注LPS造模，12 h后取材。体外实验中，RAW264.7细胞分为5组，除对照组外，其他组细胞药物预处理2 h后进行造模、指标检测。苏木素-伊红(HE)染色法观察肺组织的病理改变，同时检测肺肿胀程度、肺泡灌洗液(BALF)中蛋白水平、肺组织髓过氧化物酶(MPO)水平。细胞计数试剂8(CCK-8)方法进行细胞增殖毒性及细胞活力测定实验。酶联免疫吸附测定(ELISA)检测白细胞介素(IL)-1β、IL-6、IL-18、肿瘤坏死因子-α(TNF-α)水平，免疫组化、免疫荧光、免疫印迹等方法检测NLRP3、ASC、剪切的半胱氨酸蛋白酶-1(cleaved caspase-1)、气孔蛋白D的N端片段(GSDMD-N)等蛋白水平。结果显示，在体内实验中，Isor能显著改善小鼠肺组织的病理损伤状态，降低肺肿胀程度、BALF中蛋白水平、肺组织MPO水平、炎症因子IL-1β、IL-6、IL-18、TNF-α等的水平，抑制NLRP3/ASC/caspase-1轴及焦亡核心基因GSDMD-N的高表达。在体外实验中，通过细胞增殖毒性实验确定了Isor的安全剂量。Isor能减少细胞死亡，抑制NLRP3/ASC/caspase-1轴、GSDMD-N及炎症因子的表达水平。综上所述，Isor可能通过调控NLRP3/ASC/caspase-1轴介导的细胞焦亡，从而发挥减轻ALI的效应。
This study aims to explore the intervention effects of isorhamnetin(Isor) on acute lung injury(ALI) and its regulatory effects on pyroptosis mediated by the NOD-like receptor family pyrin domain containing 3(NLRP3)/apoptosis-associated speck-like protein containing a CARD(ASC)/cysteine aspartate-specific protease-1(caspase-1) axis. In the in vivo experiments, 60 BALB/c mice were divided into five groups. Except for the control group, the other groups were administered Isor by gavage 1 hour before intratracheal instillation of LPS to induce ALI, and tissues were collected after 12 hours. In the in vitro experiments, RAW264.7 cells were divided into five groups. Except for the control group, the other groups were pretreated with Isor for 2 hours before LPS stimulation and subsequent assessments. Hematoxylin-eosin(HE) staining was used to observe pathological changes in lung tissue, while lung swelling, protein levels in bronchoalveolar lavage fluid(BALF), and myeloperoxidase(MPO) levels in lung tissue were measured. Cell proliferation toxicity and viability were assessed using the cell counting kit-8(CCK-8) method. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of interleukin-1β(IL-1β), IL-6, IL-18, and tumor necrosis factor-α(TNF-α). Protein levels of NLRP3, ASC, cleaved caspase-1, and the N-terminal fragment of gasdermin D(GSDMD-N) were evaluated using immunohistochemistry, immunofluorescence, and Western blot. The results showed that in the in vivo experiments, Isor significantly improved pathological damage in lung tissue, reduced lung swelling, protein levels in BALF, MPO levels in lung tissue, and levels of inflammatory cytokines such as IL-1β, IL-6, IL-18, and TNF-α, and inhibited the high expression of the NLRP3/ASC/caspase-1 axis and the pyroptosis core gene GSDMD-N. In the in vitro experiments, the safe dose of Isor was determined through cell proliferation toxicity assays. Isor reduced cell death and inhibited the expression levels of the NLRP3/ASC/caspase-1 axis, GSDMD-N, and inflammatory cytokines. In conclusion, Isor may alleviate ALI by modulating pyroptosis mediated by the NLRP3/ASC/caspase-1 axis.

关键词(KeyWords)： 急性肺损伤;细胞焦亡;NLRP3/ASC/caspase-1轴;巨噬细胞;异鼠李素
acute lung injury;pyroptosis;NLRP3/ASC/caspase-1 axis;macrophage;isorhamnetin

Abstract:

Keywords:

基金项目(Foundation): 河南省重点研发与推广专项(科技攻关)(242102310508,252102310456);; 2023年度河南省“双一流”创建学科中医学科学研究专项(HSRP-DFCTCM-2023);; 河南中医药大学博士科研启动项目(00104311-2024-2-33)

作者(Author): 孙亚磊,郭宇,王欣雨,张雅素,程雪,朱珂,陈立典,冯晓东
SUN Ya-lei,GUO Yu,WANG Xin-yu,ZHANG Ya-su,CHENG Xue,ZHU Ke,CHEN Li-dian,FENG Xiao-dong

DOI: 10.19540/j.cnki.cjcmm.20250409.702

参考文献(References)：

• [1] WHEELER A P,BERNARD G R.Acute lung injury and the acute respiratory distress syndrome:a clinical review[J].Lancet,2007,369(9572):1553.
• [2] RUBENFELD G D,CALDWELL E,PEABODY E,et al.Incidence and outcomes of acute lung injury[J].N Engl J Med,2005,353(16):1685.
• [3] BELLANI G,LAFFEY J G,PHAM T,et al.Epidemiology,patterns of care,and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries[J].J Am Med Assoc,2016,315(8):788.
• [4] HUANG X,ZHANG R Y,FAN G H,et al.Incidence and outcomes of acute respiratory distress syndrome in intensive care units of China's mainland:a multicentre prospective longitudinal study[J].Crit Care,2020,24(1):515.
• [5] GU W J,ZENG Q,WANG X,et al.Acute lung injury and the NLRP3 inflammasome[J].J Inflamm Res,2024,17:3801.
• [6] ZHOU S J,YANG X,MO K L,et al.Pyroptosis and polarization of macrophages in septic acute lung injury induced by lipopolysaccharide in mice[J].Immun Inflamm Dis,2024,12(3):e1197.
• [7] GONG G,GUAN Y Y,ZHANG Z L,et al.Isorhamnetin:a review of pharmacological effects[J].Biomed Pharmacother,2020,128:110301.
• [8] LI Y,CHI G F,SHEN B Y,et al.Isorhamnetin ameliorates LPS-induced inflammatory response through downregulation of NF-κB signaling[J].Inflammation,2016,39(4):1291.
• [9] CHI G F,ZHONG W T,LIU Y,et al.Isorhamnetin protects mice from lipopolysaccharide-induced acute lung injury via the inhibition of inflammatory responses[J].Inflamm Res,2016,65(1):33.
• [10] 蔡楠.基于SIRT1-NLRP3信号通路探究姜黄素调节细胞焦亡改善急性肺损伤的作用机制[D].南京：南京中医药大学，2022.
• [11] DECHERT R E,HAAS C F,OSTWANI W.Current knowledge of acute lung injury and acute respiratory distress syndrome[J].Crit Care Nurs Clin North Am,2012,24(3):377.
• [12] FAN E,BRODIE D,SLUTSKY A S.Acute respiratory distress syndrome:advances in diagnosis and treatment[J].J Am Med Assoc,2018,319(7):698.
• [13] YANG B,MA L,WEI Y L,et al.Isorhamnetin alleviates lipopolysaccharide-induced acute lung injury by inhibiting mTOR signaling pathway[J].Immunopharmacol Immunotoxicol,2022,44(3):387.
• [14] ZOU Y N,WANG H T,FANG J,et al.Isorhamnetin as a novel inhibitor of pneumolysin against Streptococcus pneumoniae infection in vivo/in vitro[J].Microb Pathog,2023,185:106382.
• [15] SHEN Y,HE Y Y,PAN Y,et al.Role and mechanisms of autophagy,ferroptosis,and pyroptosis in sepsis-induced acute lung injury[J].Front Pharmacol,2024,15:1415145.
• [16] JIN X Y,MA Y C,LIU D D,et al.Role of pyroptosis in the pathogenesis and treatment of diseases[J].Med Comm,2023,4(3):e249.
• [17] DAI Z,LIU W C,CHEN X Y,et al.Gasdermin D-mediated pyroptosis:mechanisms,diseases,and inhibitors[J].Front Immunol,2023,14:1178662.
• [18] WANG C L,RUAN J B.An ancient defense mechanism:conservation of gasdermin-mediated pyroptosis[J].PLoS Biol,2023,21(5):e3002103.
• [19] 孟娇娇，刘蕾，付玉琪，等.中药通过调节NLRP3炎症小体治疗急性肺损伤的研究进展[J].中国实验方剂学杂志，2025,31(6):292.
• [20] VASUDEVAN S O,BEHL B,RATHINAM V A.Pyroptosis-induced inflammation and tissue damage[J].Semin Immunol,2023,69:101781.
• [21] JIANG P,JIN Y,SUN M,et al.Extracellular histones aggravate inflammation in ARDS by promoting alveolar macrophage pyroptosis[J].Mol Immunol,2021,135:53.
• [22] 徐志伟，周小杰，黄筠皓，等.石菖蒲挥发油抑制NLRP3炎症体介导的小胶质细胞焦亡实验研究[J].中国中药杂志，2024,49(18):4986.
• [23] 史纪元，姬乐，武世勋，等.淫羊藿苷通过抑制NLRP3炎性小体和Caspase-1通路减轻骨关节炎[J].世界中医药，2021,16(18):2706.
• [24] SCHWARZER R,LAURIEN L,PASPARAKIS M.New insights into the regulation of apoptosis,necroptosis,and pyroptosis by receptor interacting protein kinase 1 and caspase-8[J].Curr Opin Cell Biol,2020,63:186.
• [25] 陈凤，段乃凡，张兴，等.基于NLRP3/caspase-1/GSDMD通路探讨扶正通络颗粒对肺纤维化大鼠模型巨噬细胞焦亡的影响[J].中国中药杂志，2024,49(23):6399.
• [26] 刘培培.间充质干细胞来源外泌体抑制巨噬细胞焦亡减轻急性肺损伤及参附注射液治疗肺纤维化的研究[D].北京：北京协和医学院，2022.
• [27] SHEN C S,ZHANG Z G,XIE T,et al.Jinxin oral liquid inhibits human respiratory syncytial virus-induced excessive inflammation associated with blockade of the NLRP3/ASC/caspase-1 pathway[J].Biomed Pharmacother,2018,103:1376.