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

doi:https://doi.org/10.1016/j.chmed.2024.04.001

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

增強對急性肺損傷的保護作用：金銀花-連翹藥對靶向炎性體和cGAS-STING信號通路

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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：急性肺損傷(ALI)以炎癥為特征，目前缺乏有效的藥物干預(yù)。金銀花和連翹的聯(lián)合用藥具有抗感染、抗炎和治療作用，特別是緩解呼吸道癥狀。在以往的研究中，中藥對脂多糖(LPS)誘導(dǎo)的急性肺損傷有很好的療效。然而，目前對于金銀花和連翹聯(lián)合治療肺損傷的報道比較少。本研究的目的是比較中藥藥對“銀翹”與金銀花或連翹單獨治療肺損傷的療效，探討金銀花與連翹是否在聯(lián)合治療肺損傷上有更好的效果，并探討其作用機制。方法：將36只小鼠按假手術(shù)/LPS氣管灌注后給藥情況分為對照組、模型組、金銀花組、連翹組、銀翹組、地塞米松組。采用H&E染色及肺水腫指數(shù)評價肺損傷嚴(yán)重程度。以支氣管肺泡灌洗液(BALF)細(xì)胞計數(shù)計數(shù)肺泡滲出細(xì)胞(AECs)，用流式細(xì)胞術(shù)測定BALF中中性粒細(xì)胞百分比。采用酶聯(lián)免疫吸附法(ELISA)檢測BALF中髓過氧化物酶活性，采用定量聚合酶鏈反應(yīng)(qPCR)和ELISA檢測肺組織中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法觀察不同炎性體激活條件下金銀花、連翹和銀翹對骨髓源性巨噬細(xì)胞(BMDMs)上清caspase-1和IL-1β蛋白表達(dá)的影響。此外，采用ELISA法分別測定金銀花、連翹和銀翹給藥后BMDMs上清液中IL-1β濃度和乳酸脫氫酶(MPO)釋放水平的變化。Western blot檢測金銀花、連翹和銀翹對BMDMs中STING和IRF3磷酸化的影響，qPCR檢測IFN-β、TNF-α、IL-6和CXCL10 mRNA的變化。結(jié)果：金銀花連翹聯(lián)合使用能明顯減輕LPS引起的肺泡結(jié)構(gòu)損傷、肺出血和炎性細(xì)胞浸潤。肺指數(shù)評分和肺重濕/干比的下降證明了這一點。銀翹治療可使中性粒細(xì)胞浸潤總數(shù)減少75%，MPO活性減少88%。銀翹降低肺組織炎癥因子IL-1β、TNF-α、IL-6的效果超過金銀花或連翹，接近地塞米松的效果。在BMDMs中，與單獨處理相比，金銀花和連翹共給藥0.2 mg/mL對尼日利亞菌素刺激的caspase-1和IL-1β的表達(dá)以及LDH的釋放水平具有更好的抑制作用。同樣，0.5 mg/mL 金銀花和連翹聯(lián)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的產(chǎn)生。結(jié)論：金銀花與連翹聯(lián)用可提高LPS誘導(dǎo)ALI的治療效果，其機制可能與金銀花與連翹聯(lián)用抑制cGAS-STING及NLRP3炎性小體通路有關(guān)。我們的研究為臨床治療ALI提供了新的候選藥物。

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.

關(guān)鍵詞:

急性肺損傷;骨髓原代細(xì)胞;炎性小體;金銀花-連翹藥對;干擾素刺激基因

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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