Archive > Volume 16 Issue 3 > 2024,16(3):422-434. DOI:https://doi.org/10.1016/j.chmed.2024.04.001 Prev Next

增强对急性肺损伤的保护作用：金银花-连翘药对靶向炎性体和cGAS-STING信号通路

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Amplifying protection against acute lung injury: Targeting both inflammasome and cGAS-STING pathway by Lonicerae Japonicae Flos-Forsythiae Fructus drug pair

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    摘要:
    目的：急性肺损伤(ALI)以炎症为特征，目前缺乏有效的药物干预。金银花和连翘的联合用药具有抗感染、抗炎和治疗作用，特别是缓解呼吸道症状。在以往的研究中，中药对脂多糖(LPS)诱导的急性肺损伤有很好的疗效。然而，目前对于金银花和连翘联合治疗肺损伤的报道比较少。本研究的目的是比较中药药对“银翘”与金银花或连翘单独治疗肺损伤的疗效，探讨金银花与连翘是否在联合治疗肺损伤上有更好的效果，并探讨其作用机制。 方法：将36只小鼠按假手术/LPS气管灌注后给药情况分为对照组、模型组、金银花组、连翘组、银翘组、地塞米松组。采用H&E染色及肺水肿指数评价肺损伤严重程度。以支气管肺泡灌洗液(BALF)细胞计数计数肺泡渗出细胞(AECs)，用流式细胞术测定BALF中中性粒细胞百分比。采用酶联免疫吸附法(ELISA)检测BALF中髓过氧化物酶活性，采用定量聚合酶链反应(qPCR)和ELISA检测肺组织中IL-1β、TNF-α和IL-6的分泌及IL-1β、TNF-α和IL-6的分泌水平。采用Western blot法观察不同炎性体激活条件下金银花、连翘和银翘对骨髓源性巨噬细胞(BMDMs)上清caspase-1和IL-1β蛋白表达的影响。此外，采用ELISA法分别测定金银花、连翘和银翘给药后BMDMs上清液中IL-1β浓度和乳酸脱氢酶(MPO)释放水平的变化。Western blot检测金银花、连翘和银翘对BMDMs中STING和IRF3磷酸化的影响，qPCR检测IFN-β、TNF-α、IL-6和CXCL10 mRNA的变化。 结果：金银花连翘联合使用能明显减轻LPS引起的肺泡结构损伤、肺出血和炎性细胞浸润。肺指数评分和肺重湿/干比的下降证明了这一点。银翘治疗可使中性粒细胞浸润总数减少75%，MPO活性减少88%。银翘降低肺组织炎症因子IL-1β、TNF-α、IL-6的效果超过金银花或连翘，接近地塞米松的效果。在BMDMs中，与单独处理相比，金银花和连翘共给药0.2 mg/mL对尼日利亚菌素刺激的caspase-1和IL-1β的表达以及LDH的释放水平具有更好的抑制作用。同样，0.5 mg/mL 金银花和连翘联合使用可以更好地抑制ISD刺激下STING和IRF3的磷酸化水平以及IFN-β、TNF-α、IL-6和CXCL10的产生。 结论：金银花与连翘联用可提高LPS诱导ALI的治疗效果，其机制可能与金银花与连翘联用抑制cGAS-STING及NLRP3炎性小体通路有关。我们的研究为临床治疗ALI提供了新的候选药物。

    Abstract:
    Objective: Acute lung injury (ALI) is characterized by inflammation and currently lacks an efficacious pharmacological intervention. The medicine combination of Lonicerae Japonicae Flos (LJF) and Forsythiae Fructus (FF) demonstrates combined properties in its anti infective, anti-inflammatory, and therapeutic effects, particularly in alleviating respiratory symptoms. In previous studies, Chinese medicine has shown promising efficacy in lipopolysaccharides (LPS)-induced ALI. However, there have been no reports of LJF and FF pairing for lung injury. The aim of this study is to compare the efficacy of herb pair Lonicerae Japonicae Flos-Forsythiae Fructus (LF) with LJF or FF alone in the treatment of ALI, and to explore whether LJF and FF have a combined effect in the treatment of lung injury, along with the underlying mechanism involved. Methods: A total of 36 mice were divided into six groups (control, model, LJF, FF, LF, dexamethasone) based on the treatments they received after undergoing sham-operation/LPS tracheal instillation. H&E staining and pulmonary edema indexes were used to evaluate lung injury severity. Alveolar exudate cells (AECs) were counted based on cell count in bronchoalveolar lavage fluid (BALF), and neutrophil percentage in BALF was measured using flow cytometry. Myeloperoxidase (MPO) activity in BALF was measured using enzyme-linked immunosorbent assay (ELISA), while the production of IL-1b, TNF-a, and IL-6 in the lung and secretion level of them in BALF were detected by quantitative polymerase chain reaction (qPCR) and ELISA. The effect of LJF, FF, and LF on the expression of caspase-1 and IL-1b proteins in bone marrow derived macrophages (BMDMs) supernatant was assessed using Western blot method under various inflammasome activation conditions. In addition, the concentration of IL-1b and changes in lactatedehydrogenase (LDH) release levels in BMDMs supernatant after LJF, FF, and LF administration, respectively, were measured using ELISA. Furthermore, the effects of LJF, FF and LF on STING and IRF3 phosphorylation in BMDMs were detected by Western blot, and the mRNA changes of IFN-b, TNF-a, IL-6 and CXCL10 in BMDMs were detected by qPCR. Results: LF significantly attenuated the damage to alveolar structures, pulmonary hemorrhage, and infiltration of inflammatory cells induced by LPS. This was evidenced by a decrease in lung index score and wet/dry weight ratio. Treatment with LF significantly reduced the total number of neutrophil infiltration by 75 % as well as MPO activity by 88 %. The efficacy of LF in reducing inflammatory factors IL-1b, TNF-a, and IL-6 in the lungs surpasses that of LJF or FF, approaching the effectiveness of dexamethasone. In BMDMs, the co-administration of 0.2 mg/mL of LJF and FF demonstrated superior inhibitory effects on the expression of nigericin-stimulated caspase-1 and IL-1b, as well as the release levels of LDH, compared to individual treatments. Similarly, the combination of 0.5 mg/mL LJF and FF could better inhibit the phosphorylation levels of STING and IRF3 and the production of IFN-b, TNF-a, IL-6, and CXCL10 in response to ISD stimulation. Conclusion: The combination of LJF and FF increases the therapeutic effect on LPS-induced ALI, which may be mechanistically related to the combined effect inhibition of cyclic-GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) and NOD-like receptor family protein 3 (NLRP3) inflammasomes pathways by LJF and FF. Our study provides new medicine candidates for the clinical treatment of ALI.

    关键词:
    急性肺损伤;骨髓原代细胞;炎性小体;金银花-连翘药对;干扰素刺激基因

    Keywords:


    Project Supported:
    This study was granted by the Natural Science Foundation of Beijing, China (Grant No. 7232321), Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine (No: ZYYCXTD-C-202005), National Natural Science Foundation of China (Grant No. 81721002).

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