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山东大学学报 (医学版) ›› 2021, Vol. 59 ›› Issue (1): 14-21.doi: 10.6040/j.issn.1671-7554.0.2020.1230

• 基础医学 • 上一篇    下一篇

柚皮素通过调控巨噬细胞NLRP3炎症小体活化对脓毒症致急性肺损伤的影响

江勇1,宋剑刚2,朱大侠1,刘礼剑1   

  1. 南华大学附属南华医院 1. 急诊科;2. 手足外科, 湖南 衡阳 421002
  • 发布日期:2021-01-09
  • 通讯作者: 宋剑刚. E-mail:549317378@qq.com

Effects of naringenin on acute lung injury induced by sepsis via regulating the activation of NLRP3 inflammasome in macrophages

JIANG Yong1, SONG Jiangang2, ZHU Daxia1, LIU Lijian1   

  1. 1. Emergency Department;
    2. Department of Hand and Foot Surgery, Nanhua Hospital Affiliated to Nanhua University, Hengyang 421002, Hunan, China
  • Published:2021-01-09

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摘要： 目的 探讨柚皮素(NAR)调控巨噬细胞NOD样受体蛋白3(NLRP3)炎症小体活化对脓毒症致急性肺损伤的影响。 方法 采用随机数字表法将60只雄性SD大鼠分为假手术组、模型组、柚皮素低剂量组(25 mg/kg)和柚皮素高剂量组(75 mg/kg),每组15只。采用盲肠结扎穿孔术(CLP)制备脓毒症大鼠模型,并于术后6、12、18 h腹腔注射不同浓度柚皮素进行干预治疗。术后24 h,颈内动脉插管采血测定氧合指数(OI);收集大鼠支气管肺泡灌洗液(BALF)并分离巨噬细胞,采用RT-PCR法和Western blotting法分别检测巨噬细胞中(NLRP3)、凋亡相关斑点样蛋白(ASC)和半胱天冬酶-1(caspase-1)mRNA及蛋白表达水平;取肺组织,测定肺组织湿/干比(W/D),采用苏木精-伊红染色法观察肺组织病理学改变并评分,采用免疫荧光染色法检测肺组织中巨噬细胞水平,采用ELISA法测定肺组织及肺泡灌洗液(BALF)中白介素1β(IL-1β)、白介素18(IL-18)水平。 结果 (1)与假手术组比较,模型组大鼠肺组织损伤严重,肺组织病理评分、W/D值及巨噬细胞水平升高(P<0.05),OI值降低(P<0.05),而肺组织和BALF中IL-1β、IL-18表达水平以及肺泡巨噬细胞中NLRP3 mRNA(1.027±0.064 vs 5.567±0.208)和蛋白(0.043±0.001 vs 1.242±0.065)、ASC mRNA(0.993±0.035 vs 5.000±0.200)和蛋白(0.018±0.001 vs 0.433±0.060)以及caspase-1 mRNA(0.973±0.038 vs 7.667±0.351)和蛋白(0.101±0.001 vs 0.959±0.078)表达水平均增加,差异有统计学意义(P<0.05);(2)与模型组比较,柚皮素高剂量组大鼠肺组织损伤程度得到改善,肺组织病理评分、W/D值及巨噬细胞水平显著降低(P<0.05),OI值上升(P<0.05),且肺组织和BALF中IL-1β、IL-18表达水平以及肺泡巨噬细胞中NLRP3 mRNA(5.567±0.208 vs 3.367±0.473)和蛋白(1.242±0.065 vs 0.172±0.023)、ASC mRNA(5.000±0.200 vs 3.433±0.404)和蛋白(0.433±0.060 vs 0.121±0.010)以及caspase-1 mRNA(7.667±0.351 vs 4.000±0.200)和蛋白(0.959±0.078 vs 1.020±0.088)表达水平均降低,差异有统计学意义(P<0.05)。而柚皮素低剂量组与模型组比较,相关指标差异均无统计学意义(P>0.05)。 结论 柚皮素可减轻脓毒症大鼠急性肺损伤,其机制可能与抑制肺泡巨噬细胞内NLRP3炎症小体的活化有关。

关键词: 柚皮素, 脓毒症, 急性肺损伤, 巨噬细胞, NOD样受体蛋白3炎症小体

Abstract: Objective To investigate the effects of naringenin(NAR)on sepsis-induced acute lung injury by regulating the activation of NOD-like receptor protein 3(NLRP3)inflammasomes in macrophages. Methods A total of 60 male SD rats were divided into sham operation group, model group, low-dose NAR group(L-NAR, 25 mg/kg)and high-dose NAR group(H-NAR, 75 mg/kg)by random digital table method, with 15 rats in each group. The rat models of sepsis were prepared by cecal ligation and puncture(CLP), and different concentrations of NAR were intraperitoneally injected at 6, 12, and 18 h after the operation. At 24 h after CLP, blood samples were taken with internal carotid artery cannula to measure oxygenation index(OI). The bronchoalveolar lavage fluid(BALF)was collected and macrophages were isolated. The mRNA and protein expressions of NLRP3, apoptosis associated speck like protein containing a CARD(ASC)and caspase-1 in the macrophages were detected with RT-PCR and Western blotting, respectively. The wet/dry ratio(W/D)of lung tissues was determined, and the histopathological changes were observed with HE staining. The level of macrophages in lung tissues was detected with immunofluorescence staining, and the levels of interleukin-1β(IL-1β)and interleukin-1β(IL-18)in lung tissues and BALF were determined with ELISA. Results (1) Compared with the sham group, the model group had severer lung tissue injury, elevated lung histopathological score, W/D and macrophage level(P<0.05), decreased OI(P<0.05), increased expressions of IL-1β and IL-18 in lung tissues and BALF, as well as increased NLRP3 mRNA(1.027±0.064 vs 5.567±0.208)and protein(0.043±0.001 vs 1.242±0.065), ASC mRNA(0.993±0.035 vs 5.000±0.200)and protein(0.018±0.001 vs 0.433±0.060), and caspase-1 mRNA(0.973±0.038 vs 7.667±0.351)and protein(0.101±0.001 vs 0.959±0.078)in macrophages(P<0.05). (2) Compared with the model group, the H-NAR group showed improved lung tissue injury, decreased lung histopathological score, W/D and macrophage level(P<0.05), increased OI(P<0.05), decreased expressions of IL-1β and IL-18 in lung tissues and BALF, as well as decreased NLRP3 mRNA(5.567±0.208 vs 3.367±0.473)and protein(1.242±0.065 vs 0.172±0.023), ASC mRNA(5.000±0.200 vs 3.433±0.404)and protein(0.433±0.060 vs 0.121±0.010)and caspase-1 mRNA(7.667±0.351 vs 4.000±0.200)and protein(0.959±0.078 vs 1.020±0.088)in macrophages(P<0.05). (3) There were no significant differences in the indicators between the L-NAR group and model group(P>0.05). Conclusion Naringenin can alleviate acute lung injury in sepsis rats, and the mechanism may be related to the inhibition of the activation of NLRP3 inflammasomes in alveolar macrophages.

Key words: Naringenin, Sepsis, Acute lung injury, Macrophages, NOD-like receptor protein 3

中图分类号: 

  • R574
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