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Original Article  原创文章

Exploring the anti-inflammatory effects of Radix Curcumae essential oil in pulmonary sarcoidosis via the TLR4/MyD88/NF-кB pathway
通过 TLR4/MyD88/NF-кB 通路探索姜黄精油在肺结节病中的抗炎作用

Zhiguo Mao a,b, a,b,  ^("a,b, "){ }^{\text {a,b, }}, Xiangke Lin a,b, a,b,  ^("a,b, "){ }^{\text {a,b, }}, Yilong Hu a , b Hu a , b Hu^(a,b)\mathrm{Hu}^{\mathrm{a}, \mathrm{b}}, Ying Liu b , c b , c ^(b,c){ }^{\mathrm{b}, \mathrm{c}}, Shuaike Yu a , b Yu a , b Yu^(a,b)\mathrm{Yu}^{\mathrm{a}, \mathrm{b}}, Tianbao Zhou a,b a,b  ^("a,b "){ }^{\text {a,b }}, Jinying Zhang a,b a,b  ^("a,b "){ }^{\text {a,b }}, Xingyi Yang b,c b,c  ^("b,c "){ }^{\text {b,c }}, Shuo Tian a,b,c,*, a,b,c,*,  ^("a,b,c,*, "){ }^{\text {a,b,c,*, }} Mingsan Miao a,b,c,* a,b,c,*  ^("a,b,c,* "){ }^{\text {a,b,c,* }} (D)
毛治国 a,b, a,b,  ^("a,b, "){ }^{\text {a,b, }} ， 林祥科 a,b, a,b,  ^("a,b, "){ }^{\text {a,b, }} ， 一龙 Hu a , b Hu a , b Hu^(a,b)\mathrm{Hu}^{\mathrm{a}, \mathrm{b}} ， 刘 b , c b , c ^(b,c){ }^{\mathrm{b}, \mathrm{c}} 英 ， 帅科 Yu a , b Yu a , b Yu^(a,b)\mathrm{Yu}^{\mathrm{a}, \mathrm{b}} ， 周天宝 a,b a,b  ^("a,b "){ }^{\text {a,b }} ， 张 a,b a,b  ^("a,b "){ }^{\text {a,b }} 金英 ， 杨 b,c b,c  ^("b,c "){ }^{\text {b,c }} 兴义 ， 田 a,b,c,*, a,b,c,*,  ^("a,b,c,*, "){ }^{\text {a,b,c,*, }} 硕明三 a,b,c,* a,b,c,*  ^("a,b,c,* "){ }^{\text {a,b,c,* }} 苗 （D） a a  ^("a "){ }^{\text {a }} Department of Pharmacology, Henan University of Chinese Medicine, No. 156 Jinshui East Road, Zhengzhou, Henan 450046, China
a a  ^("a "){ }^{\text {a }} 河南中医药大学药理学系， 河南省郑州市金水东路 156 号， 河南450046， 中国 b b ^(b){ }^{\mathrm{b}} Collaborative Innovation Center of Research and Development on the Whole Industry Chain of Yu-Yao in Henan Province, Henan, China
b b ^(b){ }^{\mathrm{b}} 河南省余瑶全产业链研发协同创新中心 c c  ^("c "){ }^{\text {c }} Academy of Chinese Medicine Science, Henan University of Chinese Medicine, Zhengzhou 450046, China
c c  ^("c "){ }^{\text {c }} 河南中医药大学 中医科学院， 河南 郑州 450046

A R TICLE INFO  A r ticle 信息

Keywords:  关键字：

Pulmonary sarcoidosis  肺结节病
RCEO
TLR4/MyD88/NF-кB signalling pathway
TLR4/MyD88/NF-rkB 信号通路
Macrophages  巨噬细胞
CD4+T cells  CD4+T 细胞

Abstract  抽象

Background: Pulmonary sarcoidosis (PS) is an immune-mediated disorder characterised by a significant association with various immune cell types including macrophages, T-helper 1 (TH1) cells, and TH17 cells. Dysregulation of immune cell differentiation leads to pronounced inflammatory responses in the pulmonary system, resulting in granuloma formation. Currently, the therapeutic approach for PS predominantly involves the use of immunosuppressive agents. However, the considerable adverse effects associated with these treatments underscore the urgent need to develop novel anti-PS pharmacological interventions. Purpose: To investigate whether essential oil from Radix Curcumae (RCEO) improves pulmonary function in PS mice by inhibiting inflammation mediated by the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor kappa-B (NF-кB) (TLR4/MyD88/NF-кB) pathway. Methods: Bioinformatics and network pharmacology methodologies were used to predict potential therapeutic targets of RCEO in the context of PS. The primary components of RCEO were analysed using gas chromatography-mass spectrometry. A murine model of PS was established by induction with Propionibacterium acnes, and alterations in pulmonary function were evaluated using whole-body plethysmography. An enzymelinked immunosorbent assay was used to measure changes in inflammatory mediators in the blood and bronchoalveolar lavage fluid. Additionally, flow cytometry was performed to determine the differentiation of CD4+ T cells into TH1 and TH17 subsets in the lung tissues of mice in the model group. Immunofluorescence analysis was used to quantify the differentiation of macrophages into M1 and M2 phenotypes in the lung tissues. Polymerase chain reaction (PCR) and western blotting (WB) were used to assess alterations in the mRNAs expression levels and proteins associated with the TLR4/MyD88/NF-kB signalling pathway in pulmonary tissues. Results: Bioinformatics and network pharmacology predictions suggested that the therapeutic effects of RCEO on PS are linked to the modulation of inflammation via the TLR4/MyD88/NF-кB pathway. In a murine model of PS, RCEO significantly reduced pathological alterations in lung tissue and enhanced pulmonary function. Subsequent experimental analyses revealed that RCEO inhibited the differentiation of macrophages into M1-type macrophages and reduced the differentiation of CD4+ T cells into TH1 and TH17 cells, thereby preventing granuloma formation in the mouse pulmonary tissue. Further PCR and WB analyses indicated that RCEO modulated immune cell differentiation by inhibiting inflammation mediated through the TLR4/MyD88/NF-kB signalling pathway. Immunofluorescence experiments demonstrated that RCEO conferred a protective effect against PS by attenuating inflammation in macrophages. Conclusion: RCEO inhibited the differentiation of macrophages into M1-type macrophages and suppressed the differentiation of CD4 +T cells into TH1 and TH17 cells by blocking the inflammatory response mediated by the
背景： 肺结节病 （PS） 是一种免疫介导的疾病，其特征是与各种免疫细胞类型显著相关，包括巨噬细胞、T 辅助细胞 1 （TH1） 细胞和 TH17 细胞。免疫细胞分化失调导致肺系统出现明显的炎症反应，导致肉芽肿形成。目前，PS 的治疗方法主要涉及使用免疫抑制剂。然而，与这些治疗相关的相当大的不良反应凸显了开发新型抗 PS 药物干预措施的迫切需要。目的：探讨姜黄根精油是否通过抑制 Toll 样受体 4 （TLR4）/骨髓分化因子 88 （MyD88）/核因子 κ-B （NF-кB） （TLR4/MyD88/NF-кB） 通路介导的炎症来改善 PS 小鼠的肺功能。方法： 生物信息学和网络药理学方法用于预测 PS 背景下 RCEO 的潜在治疗靶点。使用气相色谱-质谱法分析 RCEO 的主要成分。通过痤疮丙酸杆菌诱导建立 PS 小鼠模型，并使用全身体积描记法评估肺功能的改变。酶联免疫吸附测定法用于测量血液和支气管肺泡灌洗液中炎症介质的变化。此外，进行流式细胞术以确定模型组小鼠肺组织中 CD4 + T 细胞分化为 TH1 和 TH17 亚群。免疫荧光分析用于量化巨噬细胞在肺组织中向 M1 和 M2 表型的分化。 聚合酶链反应 （PCR） 和蛋白质印迹 （WB） 用于评估肺组织中与 TLR4/MyD88/NF-kB 信号通路相关的 mRNAs 表达水平和蛋白质的变化。结果： 生物信息学和网络药理学预测表明，RCEO 对 PS 的治疗作用与通过 TLR4/MyD88/NF-кB 通路调节炎症有关。在 PS 小鼠模型中，RCEO 显着减少了肺组织的病理改变并增强了肺功能。随后的实验分析表明，RCEO 抑制巨噬细胞分化为 M1 型巨噬细胞，减少 CD4+ T 细胞向 TH1 和 TH17 细胞的分化，从而防止小鼠肺组织中肉芽肿的形成。进一步的 PCR 和 WB 分析表明，RCEO 通过抑制通过 TLR4/MyD88/NF-kB 信号通路介导的炎症来调节免疫细胞分化。免疫荧光实验表明，RCEO 通过减轻巨噬细胞中的炎症赋予对 PS 的保护作用。结论：RCEO通过阻断 CD4 +T 细胞介导的炎症反应，抑制巨噬细胞向 M1 型巨噬细胞的分化，抑制 CD4 +T 细胞向 TH1 和 TH17 细胞的分化。

TLR4/MyD88/NF-kB pathway. This further reduced the formation of granulomas in lung tissue and improved lung function in PS mice
TLR4/MyD88/NF-kB 通路。这进一步减少了肺组织中肉芽肿的形成并改善了 PS 小鼠的肺功能

Introduction  介绍

Pulmonary sarcoidosis (PS) is a systemic disorder characterised by the formation of non-caseating granulomas in the lungs, and its pathogenesis remains largely undefined (Yao et al., 2024). The onset of PS is modulated by several environmental, genetic, and immunological factors. A critical aspect of PS pathogenesis is immune dysregulation, in which CD4+ T lymphocytes and macrophages play an integral role (Carow et al., 2023). Despite ongoing research, the exact pathogenic mechanisms of PS are not fully understood and there is a notable lack of targeted therapeutic agents. Current treatment strategies primarily include administration of glucocorticoids and immunosuppressive drugs (Nakamizo et al., 2023; Redl et al., 2024). Prolonged glucocorticoid administration is associated with significant adverse effects, and the condition frequently recurs upon treatment cessation. Additionally, although PS may occasionally resolve spontaneously, certain patients may develop chronic pulmonary conditions including pulmonary fibrosis, respiratory failure, and lung cancer (Cleven et al., 2024; van Stigt et al., 2024). Therefore, investigating the precise pathogenic mechanisms underlying PS and identifying efficacious therapeutic agents are essential.
肺结节病 （PS） 是一种全身性疾病，其特征是在肺部形成非干酪性肉芽肿，其发病机制在很大程度上仍未确定（Yao 等人，2024 年）。PS 的发病受多种环境、遗传和免疫因素的调节。PS 发病机制的一个关键方面是免疫失调，其中 CD4+ T 淋巴细胞和巨噬细胞起着不可或缺的作用（Carow等人，2023 年）。尽管正在进行研究，但 PS 的确切致病机制尚不完全清楚，并且明显缺乏靶向治疗剂。目前的治疗策略主要包括糖皮质激素和免疫抑制药物的给药（Nakamizo 等人，2023 年;Redl et al.， 2024）。长期服用糖皮质激素与显著的不良反应相关，并且病情在治疗停止后经常复发。此外，尽管 PS 偶尔会自发消退，但某些患者可能会发展为慢性肺部疾病，包括肺纤维化、呼吸衰竭和肺癌（Cleven 等人，2024 年;van Stigt 等人，2024 年）。因此，研究 PS 的确切致病机制并确定有效的治疗药物至关重要。
Macrophages, which are integral to the innate immune defence of the body, are pivotal in the pathogenesis of PS and form a structural foundation for granulomas (Hutton and Deshane, 2024). Macrophages can identify foreign pathogens and subsequently differentiate into M1 macrophages, which release a wide array of proinflammatory mediators, including interleukin-23 (IL-23) and tumour necrosis factor-alpha (TNF- α α alpha\alpha ), thereby initiating cellular inflammatory responses (Franken et al., 2022; Wei et al., 2024). In contrast, the activation of M2 macrophages results in the secretion of anti-inflammatory cytokines, such as interleukin-10 (IL-10), which supports tissue repair and remodelling processes (Yang et al., 2024a). Stimulation of the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor kappa-B (NF-кB) signalling pathway promotes the activation of M1 macrophages, thereby facilitating pulmonary inflammation (Liu et al., 2024a). Additionally, CD4+ T cells can differentiate into T helper 1 (TH1) and TH17 cells in PS, forming the peripheral component of granulomas (Miedema et al., 2023; Zhang et al., 2023). TH1 and TH17 cells secrete interferon-gamma (IFN- γ γ gamma\gamma ) and IL-17A, respectively, to initiate intracellular inflammatory cascade reactions and facilitate granuloma formation (Ma et al., 2024a; Rungelrath et al., 2024; Yang et al., 2024c). Nonetheless, additional research is required to explore whether modulation of the TLR4/MyD88/NF-кB signalling pathway in PS can effectively reduce TH1 and TH17 cell populations as well as M1 macrophages, thereby ameliorating pulmonary inflammation.
巨噬细胞是身体先天免疫防御不可或缺的一部分，在 PS 的发病机制中起着关键作用，并构成了肉芽肿的结构基础（Hutton 和 Deshane，2024 年）。巨噬细胞可以识别外来病原体，随后分化成 M1 巨噬细胞，巨噬细胞释放多种促炎介质，包括白细胞介素 23 （IL-23） 和肿瘤坏死因子-α （TNF- α α alpha\alpha ），从而引发细胞炎症反应（Franken等人，2022 年;Wei et al.， 2024）。相比之下，M2 巨噬细胞的激活导致分泌抗炎细胞因子，例如白细胞介素 10 （IL-10），它支持组织修复和重塑过程（Yang等人，2024a）。刺激 Toll 样受体 4 （TLR4）/骨髓分化因子 88 （MyD88）/核因子 kappa-B （NF-кB） 信号通路促进 M1 巨噬细胞的激活，从而促进肺部炎症（Liu et al.， 2024a）。此外，CD4 + T 细胞可以在 PS 中分化为辅助性 T 细胞 1 （TH1） 和 TH17 细胞，形成肉芽肿的外周成分（Miedema 等人，2023 年;Zhang et al.， 2023）。TH1 和 TH17 细胞分别分泌干扰素-γ （IFN- γ γ gamma\gamma ） 和 IL-17A，以启动细胞内炎症级联反应并促进肉芽肿形成（马等人，2024a;Rungelrath 等人，2024 年;Yang et al.， 2024c）。尽管如此，还需要更多的研究来探索 PS 中 TLR4/MyD88/NF-rkB 信号通路的调节是否可以有效减少 TH1 和 TH17 细胞群以及 M1 巨噬细胞，从而改善肺部炎症。
Traditional Chinese medicine has gathered substantial clinical experience in the management of chronic pulmonary diseases. Yu Jin (Radix Curcumae), obtained from the desiccated tuberous roots of Curcuma wenyujin, Curcuma phaeocaulis, Curcuma kwangsiensis and Curcuma longa, all from the Zingiberaceae family, is traditionally recognised for its ability to enhance blood circulation, alleviate pain, and modulate qi to alleviate stagnation. Contemporary pharmacological studies have demonstrated that Radix Curcumae possesses significant antibacterial, anti-inflammatory, and antioxidant properties (Lee et al., 2024; Zhao et al., 2024b). Volatile compounds, which constitute the primary active components, have been shown to effectively inhibit intracellular inflammatory signalling pathways and decrease the release of proinflammatory mediators. These components have demonstrated considerable efficacy in the treatment of pulmonary diseases including pulmonary fibrosis and lung cancer (Kaya et al., 2019; Wu et al., 2021). However, the therapeutic potential and underlying mechanisms of the
中医在慢性肺病的管理方面积累了丰富的临床经验。鱼锦（姜黄）取自姜黄、姜黄、光生姜和长姜黄的干燥块茎根，均来自姜科，传统上被认为具有增强血液循环、缓解疼痛和调节气以缓解停滞的能力。当代药理学研究表明，姜黄具有显着的抗菌、抗炎和抗氧化特性（Lee 等人，2024 年;Zhao et al.， 2024b）。构成主要活性成分的挥发性化合物已被证明可有效抑制细胞内炎症信号通路并减少促炎介质的释放。这些成分在治疗包括肺纤维化和肺癌在内的肺部疾病方面显示出相当大的疗效（Kaya等人，2019 年;Wu et al.， 2021）。然而，
essential oil from Radix Curcumae (RCEO) for PS remain poorly understood. Preliminary findings from this study indicate that RCEO significantly ameliorates PS and suppresses inflammatory mediators, such as IL-23 and TNF- α α alpha\alpha. Moreover, increased expression of TLR4, MyD88, and NF-кB was detected in lung tissue affected by PS. Therefore, we hypothesised that the beneficial effects of RCEO on PS may be related to the differentiation and regulation of macrophages and CD4+T cells, possibly involving the TLR4/MyD88/NF-кB pathway. This study aimed to investigate whether RCEO exerts its effects on PS through the TLR4/MyD88/NF-кB signalling pathway.
来自姜黄 （RCEO） 的 PS 精油仍然知之甚少。本研究的初步结果表明，RCEO 显着改善 PS 并抑制炎症介质，如 IL-23 和 TNF- α α alpha\alpha 。此外，在受 PS 影响的肺组织中检测到 TLR4 、 MyD88 和 NF-кB 的表达增加。因此，我们假设 RCEO 对 PS 的有益影响可能与巨噬细胞和 CD4+T 细胞的分化和调节有关，可能涉及 TLR4/MyD88/NF-rkB 通路。本研究旨在探讨 RCEO 是否通过 TLR4/MyD88/NF-rkB 信号通路对 PS 产生影响。

Materials and methods  材料和方法

Gene expression omnibus (GEO) analysis
基因表达综合 （GEO） 分析

We screened the PS cohort (GSE16538) from the public GEO database (https://www.ncbi.nlm.nih.gov/geo/) and obtained large amounts of RNA sequencing data from both normal and PS samples. For datasets that were not normalised, we uniformly applied log ⁡ 2 log ⁡ 2 log 2\log 2 transformation. We used the Limma package (version 3.40.2) in the R R RR software to investigate differential mRNA expression (Barrett et al., 2013; Sun et al., 2019; Zhang et al., 2019). Adjusted P P PP values were analysed to correct for false positives. We defined the threshold for screening differentially expressed mRNAs as "adjusted P < 0.05 P < 0.05 P < 0.05P<0.05 and log2 (fold change) > 1 > 1 > 1>1 or log ⁡ 2 < − 1 log ⁡ 2 < − 1 log 2 < -1\log 2<-1 ". To further understand the pathogenic role of the target genes, we used the ClusterProfiler package in R to analyse the Gene Ontology (GO) functions of potential mRNAs and enriched Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways. Statistical analyses were performed using the R software (version 4.0.3). The results were considered statistically significant at P < 0.05 P < 0.05 P < 0.05P<0.05.
我们从公共 GEO 数据库 （https://www.ncbi.nlm.nih.gov/geo/） 中筛选了 PS 队列 （GSE16538），并从正常样本和 PS 样本中获得了大量 RNA 测序数据。对于未标准化的数据集，我们统一应用 log ⁡ 2 log ⁡ 2 log 2\log 2 了转换。我们在 R R RR 软件中使用 Limma 包（版本 3.40.2）来研究差异 mRNA 表达（Barrett等人，2013 年;Sun等人，2019 年;Zhang et al.， 2019）。分析调整后 P P PP 的值以纠正假阳性。我们将筛选差异表达 mRNA 的阈值定义为 “调整 P < 0.05 P < 0.05 P < 0.05P<0.05 后和 log2 （倍数变化） > 1 > 1 > 1>1 或 log ⁡ 2 < − 1 log ⁡ 2 < − 1 log 2 < -1\log 2<-1 ”。为了进一步了解靶基因的致病作用，我们使用 R 中的 ClusterProfiler 软件包来分析潜在 mRNA 的基因本体论 （GO） 功能，并丰富了京都基因和基因组百科全书 （KEGG） 通路。使用 R 软件 （4.0.3 版） 进行统计分析。结果被认为具有统计学意义。 P < 0.05 P < 0.05 P < 0.05P<0.05

Preparation of RCEO  RCEO 的准备

Radix Curcumae was purchased from Tongrentang, Beijing, China. Approximately 500 g of Radix Curcumae was pulverised and volatile oil was extracted using a supercritical fluid extractor (Speed SFE-2, Agilent Technologies, USA). The resulting brown or dark purple viscous liquid was sealed and stored at − 20 ∘ C − 20 ∘ C -20^(@)C-20^{\circ} \mathrm{C}.
姜黄 （Radix Curcumae） 购自中国北京同仁堂。将大约 500 g 姜黄碱粉碎，并使用超临界流体萃取器（Speed SFE-2，Agilent Technologies，美国）提取挥发油。将所得棕色或深紫色粘稠液体密封并储存在 − 20 ∘ C − 20 ∘ C -20^(@)C-20^{\circ} \mathrm{C} 。

Gas chromatography-mass spectrometry (GC-MS) analysis
气相色谱-质谱 （GC-MS） 分析

The composition of RCEO was analysed as previously reported (Liu et al., 2011). A GC-MS analyser (Agilent, USA) equipped with a DB-5 quartz capillary column ( 30 m × 250 μ m × 0.1 μ m 30 m × 250 μ m × 0.1 μ m 30mxx250 mumxx0.1 mum30 \mathrm{~m} \times 250 \mu \mathrm{~m} \times 0.1 \mu \mathrm{~m} ) was used for the analysis of RCEO. The testing conditions were as follows: helium ( 99.999 % 99.999 % 99.999%99.999 \% ) was used as the carrier gas with a flow rate of 1 mL / min 1 mL / min 1mL//min1 \mathrm{~mL} / \mathrm{min}. The temperature of the split injector was set to 270 ∘ C 270 ∘ C 270^(@)C270^{\circ} \mathrm{C} and the split ratio was 10 : 1 10 : 1 10:110: 1. The injection volume was 1 μ L 1 μ L 1muL1 \mu \mathrm{~L}. The temperature program was initiated at 50 ∘ C 50 ∘ C 50^(@)C50{ }^{\circ} \mathrm{C} for 2 min , then increased to 90 ∘ C 90 ∘ C 90^(@)C90^{\circ} \mathrm{C} at a rate of 20 ∘ C / min ∘ C / min ^(@)C//min{ }^{\circ} \mathrm{C} / \mathrm{min}, followed by a programmed increase to 150 ∘ C 150 ∘ C 150^(@)C150^{\circ} \mathrm{C} at 2.5 ∘ C / min 2.5 ∘ C / min 2.5^(@)C//min2.5^{\circ} \mathrm{C} / \mathrm{min} and maintained for 5 min . The temperature was subsequently increased to 250 ∘ C 250 ∘ C 250^(@)C250^{\circ} \mathrm{C} at a rate of 10 ∘ C / min 10 ∘ C / min 10^(@)C//min10^{\circ} \mathrm{C} / \mathrm{min} and held for 7 min . The ionisation voltage of the electron impact ion source was 70 eV , with an ion source temperature of 200 ∘ C 200 ∘ C 200^(@)C200^{\circ} \mathrm{C} and transfer line temperature of 260 ∘ C 260 ∘ C 260^(@)C260^{\circ} \mathrm{C}. The scanning range was 35 − 550 amu 35 − 550 amu 35-550amu35-550 \mathrm{amu}. The components of RCEO were identified by comparing the recorded mass spectra with those documented in the NIST mass spectral library.
如前所述分析 RCEO 的组成 （Liu et al.， 2011）。配备 DB-5 石英毛细管柱 （ 30 m × 250 μ m × 0.1 μ m 30 m × 250 μ m × 0.1 μ m 30mxx250 mumxx0.1 mum30 \mathrm{~m} \times 250 \mu \mathrm{~m} \times 0.1 \mu \mathrm{~m} ） 的 GC-MS 分析仪（安捷伦，美国）用于分析 RCEO。测试条件如下：以氦 （ 99.999 % 99.999 % 99.999%99.999 \% ） 为载气，流速为 1 mL / min 1 mL / min 1mL//min1 \mathrm{~mL} / \mathrm{min} 。将分流进样器的温度设置为 270 ∘ C 270 ∘ C 270^(@)C270^{\circ} \mathrm{C} ，分流比为 10 : 1 10 : 1 10:110: 1 。进样量为 1 μ L 1 μ L 1muL1 \mu \mathrm{~L} 。温度程序在 2 分钟时启动 50 ∘ C 50 ∘ C 50^(@)C50{ }^{\circ} \mathrm{C} ，然后以 20 的速率升高 90 ∘ C 90 ∘ C 90^(@)C90^{\circ} \mathrm{C} ∘ C / min ∘ C / min ^(@)C//min{ }^{\circ} \mathrm{C} / \mathrm{min} ，然后程序性地增加到 150 ∘ C 150 ∘ C 150^(@)C150^{\circ} \mathrm{C} at 2.5 ∘ C / min 2.5 ∘ C / min 2.5^(@)C//min2.5^{\circ} \mathrm{C} / \mathrm{min} 并保持 5 分钟。随后将温度升高至 250 ∘ C 250 ∘ C 250^(@)C250^{\circ} \mathrm{C} 10 ∘ C / min 10 ∘ C / min 10^(@)C//min10^{\circ} \mathrm{C} / \mathrm{min} 并保持 7 分钟。电子碰撞离子源的电离电压为 70 eV，离子源温度为 ， 200 ∘ C 200 ∘ C 200^(@)C200^{\circ} \mathrm{C} 传输线温度为 260 ∘ C 260 ∘ C 260^(@)C260^{\circ} \mathrm{C} 。扫描范围为 35 − 550 amu 35 − 550 amu 35-550amu35-550 \mathrm{amu} 。通过将记录的质谱与 NIST 质谱库中记录的质谱进行比较来鉴定 RCEO 的组分。

Network pharmacology and molecular docking analysis
网络药理与分子对接分析

The GC-MS results were used with Swiss Target Prediction (http://
GC-MS 结果与 Swiss Target Prediction （http://
www.swisstargetprediction.ch/) and Super PRED (https://prediction. charite.de/subpages/target_prediction.php) to identify the potential targets. The GeneCards database (https://www.genecards.org) and OMIM database (https://omim.org/) were used to obtain PS-related targets. Wei Sheng Xin (https://www.bioinformatics.com.cn/) was used to screen the targets for the RCEO treatment of PS. The STRING database (https://cn.string-db.org) was used to generate a proteinprotein interaction (PPI) network. Cytoscape was used to visualise and analyse the interaction information. Based on the core targets, the David database (https://davidbioinformatics.nih.gov/) was used for GO enrichment and KEGG pathway analysis. Wei Sheng Xin visualised the results.
www.swisstargetprediction.ch/）和 Super PRED （https://prediction.charite.de/subpages/target_prediction.php）以确定潜在目标。使用 GeneCards 数据库 （https://www.genecards.org） 和 OMIM 数据库 （https://omim.org/） 获得 PS 相关靶标。采用 Wei Sheng Xin （https://www.bioinformatics.com.cn/） 筛选 PS 的 RCEO 治疗靶点。使用 STRING 数据库 （https://cn.string-db.org） 生成蛋白质蛋白质相互作用 （PPI） 网络。Cytoscape 用于可视化和分析相互作用信息。基于核心靶标，使用 David 数据库 （https://davidbioinformatics.nih.gov/） 进行 GO 富集和 KEGG 通路分析。Wei Sheng Xin 将结果可视化。
AutoDockVina 1.2.2, a computational protein-ligand docking software, was used. Molecular structures of the drug components were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). The 3D coordinates of KDR were downloaded from the PDB (http://www.rcsb. org/). We first prepared the protein and ligand files, converting all protein and molecule files to PDBQT format and adding polar hydrogen atoms. The grid box was centred to cover each protein domain and accommodate free molecular movement. Molecular docking studies were conducted using Autodock Vina 1.2.2 (http://autodock.scripps. edu/) for model visualisation.
使用计算蛋白质-配体对接软件 AutoDockVina 1.2.2。药物成分的分子结构从 PubChem （https://pubchem.ncbi.nlm.nih.gov/） 获得。KDR 的 3D 坐标是从 PDB （http://www.rcsb. org/） 下载的。我们首先准备蛋白质和配体文件，将所有蛋白质和分子文件转换为 PDBQT 格式并添加极性氢原子。网格框居中以覆盖每个蛋白质结构域并容纳分子的自由移动。使用 Autodock Vina 1.2.2 （http://autodock.scripps. edu/） 进行分子对接研究以进行模型可视化。

Cellular thermal shift assay (CETSA)
细胞热转移测定 （CETSA）

The CETSA protocol was performed as previously described (Liu et al., 2024b) with minor modifications. Briefly, mouse macrophages RAW264.7 were treated with either germacrone ( 100 μ M 100 μ M 100 muM100 \mu \mathrm{M} ) or dimethylsulfoxide for 12 h in a CO 2 CO 2 CO_(2)\mathrm{CO}_{2} incubator at 37 ∘ C 37 ∘ C 37^(@)C37^{\circ} \mathrm{C}, followed by collection for CETSA (Lin et al., 2024). Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors. The cell suspension was then divided into eight aliquots, gradually heated for 3 min ( 37 , 42 , 47 , 52 , 57 , 62 , 67 3 min ( 37 , 42 , 47 , 52 , 57 , 62 , 67 3min(37,42,47,52,57,62,673 \mathrm{~min}(37,42,47,52,57,62,67, and 72 ∘ C ∘ C ^(@)C{ }^{\circ} \mathrm{C} ), and subsequently subjected to three freeze-thaw cycles using liquid nitrogen. After centrifugation at 4 ∘ C 4 ∘ C 4^(@)C4{ }^{\circ} \mathrm{C} for 15 min , the supernatants were collected and equal amounts of protein were used for TLR4 level determination.
CETSA 方案如前所述执行 （Liuet al.， 2024b），并进行了细微的修改。简而言之，小鼠巨噬细胞 RAW264.7 在 CO 2 CO 2 CO_(2)\mathrm{CO}_{2} 培养箱中用 germacrone （ 100 μ M 100 μ M 100 muM100 \mu \mathrm{M} ） 或二甲基亚砜处理 12 小时 37 ∘ C 37 ∘ C 37^(@)C37^{\circ} \mathrm{C} ，然后收集 CETSA （Linet al.， 2024）。使用含有蛋白酶和磷酸酶抑制剂的放射免疫沉淀测定 （RIPA） 缓冲液裂解细胞。然后将细胞悬液分成 8 等分试样，逐渐加热 3 min ( 37 , 42 , 47 , 52 , 57 , 62 , 67 3 min ( 37 , 42 , 47 , 52 , 57 , 62 , 67 3min(37,42,47,52,57,62,673 \mathrm{~min}(37,42,47,52,57,62,67 和 72 ∘ C ∘ C ^(@)C{ }^{\circ} \mathrm{C} ），随后使用液氮进行 3 次冻融循环。离心 4 ∘ C 4 ∘ C 4^(@)C4{ }^{\circ} \mathrm{C} 15 分钟后，收集上清液，并使用等量的蛋白质测定 TLR4 水平。

Animals  动物

Six-to-eight-week-old female C57BL/6 mice (18.0-22.0 g, n = 140 n = 140 n=140n=140 ) were provided by the Jinan Pengyue Laboratory Animal Breeding Co., Ltd. (License No.: SCXK (Lu) 20220006). The experimental animals were housed in an environment with a temperature of 22 ∘ C 22 ∘ C 22^(@)C22^{\circ} \mathrm{C}, humidity of 55 % % %\%, and light/dark cycle of 12 h / 12 h 12 h / 12 h 12h//12h12 \mathrm{~h} / 12 \mathrm{~h}, with ad libitum food and water. Animal experiments were conducted with the approval of the Experimental Animal Ethics Committee of Henan University of Chinese Medicine (IACUC-202405027) and fully complied with the ethical guidelines outlined in the NIH Guide for the Care and Use of Laboratory Animals.
6-8周龄雌性C57BL/6小鼠（18.0-22.0 g， n = 140 n = 140 n=140n=140 ）由济南鹏越实验动物育种有限公司提供（许可证号：SCXK（鲁）20220006）。实验动物被饲养在温度为 22 ∘ C 22 ∘ C 22^(@)C22^{\circ} \mathrm{C} 、湿度为 55 % % %\% 、光/暗循环为 12 h / 12 h 12 h / 12 h 12h//12h12 \mathrm{~h} / 12 \mathrm{~h} 的环境中，随意进食和水。动物实验经河南中医药大学实验动物伦理委员会 （IACUC-202405027） 批准进行，并完全遵守 NIH 实验动物护理和使用指南中概述的伦理准则。

Propionibacterium acnes culture
痤疮丙酸杆菌培养

P. acnes was cultured as previously described (BNCC, China) (Werner et al., 2017). Lyophilised P. acne powder was dissolved in sterile water ( 0.5 mL ) under aseptic conditions. After thorough mixing, the bacterial solution was evenly spread on Columbia agar blood plates ( 200 μ L 200 μ L 200 muL200 \mu \mathrm{~L} per plate). The plates were then incubated under anaerobic conditions at 37 ∘ C ∘ C ^(@)C{ }^{\circ} \mathrm{C} for 3-4 days to obtain activated P P PP. acnes.
痤疮丙酸杆菌如前所述培养（BNCC，中国）（Werner等人，2017 年）。在无菌条件下将冻干的痤疮丙酸杆菌粉溶于无菌水 （0.5 mL） 中。充分混合后，将细菌溶液均匀涂抹在 Columbia 琼脂血平板上（ 200 μ L 200 μ L 200 muL200 \mu \mathrm{~L} 每板）。然后将板在 37 ∘ C ∘ C ^(@)C{ }^{\circ} \mathrm{C} 的厌氧条件下孵育 3-4 天以获得活化 P P PP 的 .痤疮。

Establishment of PS model and drug administration
建立 PS 模型和给药

The PS mouse model was established as previously described (Song et al., 2019). P. acnes was dissolved in phosphate-buffered saline (PBS) and inactivated at 85 ∘ C 85 ∘ C 85^(@)C85^{\circ} \mathrm{C} for 10 min to prepare the P P PP. acnes suspension. On the first day of modelling, the mice were intraperitoneally injected with 0.25 mL of P P PP. acnes ( 2 mg / mL 2 mg / mL 2mg//mL2 \mathrm{mg} / \mathrm{mL} ). On days 14,28 , and 42 , the mice
如前所述建立 PS 小鼠模型 （Songet al.， 2019）。将痤疮丙酸杆菌溶于磷酸盐缓冲盐水 （PBS） 中，并在 10 分钟后灭活 85 ∘ C 85 ∘ C 85^(@)C85^{\circ} \mathrm{C} 以制备 P P PP .痤疮悬浮液。在建模的第一天，小鼠腹膜内注射 0.25 mL . P P PP 痤疮 （ 2 mg / mL 2 mg / mL 2mg//mL2 \mathrm{mg} / \mathrm{mL} ）。在第 14、28 和 42 天，小鼠
were intratracheally injected with 0.05 mL of inactivated P P PP. acnes (10 mg / mL mg / mL mg//mL\mathrm{mg} / \mathrm{mL} ), whereas the normal control group received an equal volume of PBS. The intratracheal injection procedure was as follows: the model group mice were anaesthetised with pentobarbital sodium, placed on the operating table with their abdomens facing upward, their neck hair removed, and disinfected with povidone-iodine. A 1 cm longitudinal incision was made in the middle of the neck and the trachea was exposed using blunt dissection with forceps. After intratracheal injection of 0.05 mL of P P PP. acnes, the incision was sutured quickly. The mice were rotated left and right, and then suspended head-up and tail-down for 1 min to evenly distribute P P PP. acnes in the lung tissue.
气管内注射 0.05 mL 灭活 P P PP .痤疮 （10 mg / mL mg / mL mg//mL\mathrm{mg} / \mathrm{mL} ），而正常对照组接受等体积的 PBS。气管内注射程序如下：模型组小鼠用戊巴比妥钠麻醉，置于手术台上，腹部朝上，去除颈部毛发，并用聚维酮碘消毒。在颈部中间做一个 1 cm 的纵向切口，用镊子钝器解剖气管。气管内注射 0.05 mL 后。 P P PP 痤疮，切口被迅速缝合。小鼠左右旋转，然后头朝上和尾朝下悬浮 1 min，使其分布 P P PP 均匀。肺组织中的痤疮。
The mice were randomly divided into five groups: control group, model group, dexamethasone (DXMS, 5 mg / kg 5 mg / kg 5mg//kg5 \mathrm{mg} / \mathrm{kg} ) group (Zhang et al., 2024), high-dose RCEO (YJ-10, 0.1 ml / kg 0.1 ml / kg 0.1ml//kg0.1 \mathrm{ml} / \mathrm{kg} ) group, and low-dose RCEO (YJ-5, 0.05 mL / kg 0.05 mL / kg 0.05mL//kg0.05 \mathrm{~mL} / \mathrm{kg} ) group. Drug administration was initiated on day 42 of modelling. RCEO was administered via nasal inhalation once a day. DXMS was administered intraperitoneally twice a week. The model and control groups were administered olive oil via nasal inhalation once a day for 14 consecutive days.
将小鼠随机分为 5 组：对照组、模型组、地塞米松 （DXMS， 5 mg / kg 5 mg / kg 5mg//kg5 \mathrm{mg} / \mathrm{kg} ） 组 （Zhang et al.， 2024）、高剂量 RCEO （YJ-10， 0.1 ml / kg 0.1 ml / kg 0.1ml//kg0.1 \mathrm{ml} / \mathrm{kg} ） 组和低剂量 RCEO （YJ-5， 0.05 mL / kg 0.05 mL / kg 0.05mL//kg0.05 \mathrm{~mL} / \mathrm{kg} ） 组。在建模的第 42 天开始给药。RCEO 通过鼻吸入给药，每天一次。DXMS 每周腹膜内给药 2 次。模型组和对照组通过鼻吸入每天一次给予橄榄油，连续 14 天。

Pulmonary function evaluation
肺功能评估

Fourteen days after administration, whole-body plethysmography (EMKA, China) was used to measure the tidal volume, respiratory frequency, and peak expiratory flow in each group of mice to evaluate lung function.
给药后 14 d，采用全身体积描记法 （EMKA， China） 测量每组小鼠的潮气量、呼吸频率和呼气峰流量，以评估肺功能。

Haematoxylin and eosin (HE) staining
苏木精和伊红 （HE） 染色

The lung tissues of the mice were fixed in 10 % 10 % 10%10 \% neutral formalin, embedded in paraffin, and sectioned at 4 μ m 4 μ m 4mum4 \mu \mathrm{~m}. After dewaxing, sections were rehydrated and subjected to histological analysis. Pathological changes in the lungs were observed through HE staining and a pathological slide scanner (KFBIO, China).
将小鼠的肺组织固定在中性福尔马林中 10 % 10 % 10%10 \% ，包埋在石蜡中，并在 处切片 4 μ m 4 μ m 4mum4 \mu \mathrm{~m} 。脱蜡后，切片再水化并进行组织学分析。通过 HE 染色和病理载玻片扫描仪 （KFBIO， China） 观察肺部的病理变化。

Enzyme-linked immunosorbent assay (ELISA)
酶联免疫吸附测定 （ELISA）

Blood was collected from the abdominal aorta 14 days after administration. Serum was separated by centrifugation and stored at − 20 ∘ C − 20 ∘ C -20^(@)C-20^{\circ} \mathrm{C}. The levels of transforming growth factor-beta (TGF- β β beta\beta ), IL-23, IL-10, and IL-17A (mlbio, China) were measured according to the manufacturer’s instructions.
给药后 14 天从腹主动脉采集血液。离心分离血清并储存在 − 20 ∘ C − 20 ∘ C -20^(@)C-20^{\circ} \mathrm{C} 。根据制造商的说明测量转化生长因子-β （TGF- β β beta\beta ）、IL-23、IL-10 和 IL-17A （mlbio， China） 的水平。
To collect bronchoalveolar lavage fluid (BALF), a 22-gauge catheter was inserted into the trachea of mice through a small incision. The lungs were then perfused three times with 0.7 mL of cold PBS to collect BALF, yielding approximately 2.1 mL . The collected BALF was centrifuged at 500 g for 5 min at 4 ∘ C 4 ∘ C 4^(@)C4^{\circ} \mathrm{C}. The precipitated cells were resuspended in 0.2 mL PBS for flow cytometry analysis, and the supernatant was used for ELISA. The levels of TGF- β β beta\beta, IL-23, IL-10, IL-17A, IFN- γ γ gamma\gamma, TNF- α α alpha\alpha, and lactate dehydrogenase (LDH) (mlbio, China) in BALF were measured according to the manufacturer’s instructions, and the protein levels in BALF were determined using a Bradford protein assay kit (Solarbio, China) according to the manufacturer’s instructions.
为了收集支气管肺泡灌洗液 （BALF），通过一个小切口将一根 22 号导管插入小鼠的气管中。然后用 0.7 mL 冷 PBS 灌注肺部 3 次以收集 BALF，得到约 2.1 mL。将收集的 BALF 在 500 g 下 4 ∘ C 4 ∘ C 4^(@)C4^{\circ} \mathrm{C} 离心 5 min。将沉淀的细胞重悬于 0.2 mL PBS 中用于流式细胞术分析，上清液用于 ELISA。根据制造商的说明测量 BALF 中 TGF- β β beta\beta 、IL-23、IL-10、IL-17A、IFN- γ γ gamma\gamma 、TNF- α α alpha\alpha 和乳酸脱氢酶 （LDH） （mlbio，中国） 的水平，并使用 Bradford 蛋白测定试剂盒（Solarbio，中国）根据制造商的说明测定 BALF 中的蛋白质水平。

Quantitative real-time polymerase chain reaction (qRT-PCR)
定量实时聚合酶链反应 （qRT-PCR）

Total RNA was extracted from the samples using an Animal Tissue Total RNA Extraction Kit (Servicebio, China). Subsequently, 50 mg of the extracted RNA was reverse-transcribed into cDNA, following the protocol outlined in the SweScript All-in-One RT SuperMix for qPCR (Servicebio, China). For qRT-PCR, SYBR qPCR Master Mix (Servicebio, China) was used in combination with QuantStudio 6 FLEX (ABI, USA). qRT-PCR data were analysed using the relative quantification ( 2 − Δ Δ C T 2 − Δ Δ C T 2^(-Delta Delta CT)2^{-\Delta \Delta C T} ) method. The primer sequences are listed in Table 1.
使用动物组织总 RNA 提取试剂盒 （Servicebio， China） 从样品中提取总 RNA。随后，按照用于 qPCR 的 SweScript All-in-One RT SuperMix（Servicebio，中国）中概述的方案，将 50 mg 提取的 RNA 逆转录成 cDNA。对于 qRT-PCR，SYBR qPCR 预混液（Servicebio，中国）与 QuantStudio 6 FLEX（ABI，USA）结合使用。使用相对定量 （ 2 − Δ Δ C T 2 − Δ Δ C T 2^(-Delta Delta CT)2^{-\Delta \Delta C T} ） 方法分析 qRT-PCR 数据。引物序列列于表 1 中。
  1. Abbreviations: PS, pulmonary sarcoidosis; RCEO, essential oil from Radix Curcumae; GC-MS, gas chromatography-mass spectrometry; TH1 cells, T-helper 1 cells; P. acnes, Propionibacterium acnes; BALF, bronchoalveolar lavage fluid; TNF- α α alpha\alpha, tumour necrosis factor-alpha; TLR4, Toll-like receptor 4; MyD88, myeloid differentiation factor 88; NF-kB, nuclear factor kappa-B; DXMS, dexamethasone; IFN- γ γ gamma\gamma, interferon-gamma; TGF- β β beta\beta, transforming growth factor-beta.
    缩写：PS，肺结节病;RCEO，来自姜黄的精油;GC-MS，气相色谱-质谱法;TH1 细胞，T-helper 1 细胞;P. acnes， 痤疮丙酸杆菌;BALF，支气管肺泡灌洗液;TNF- α α alpha\alpha ，肿瘤坏死因子-α;TLR4，Toll 样受体 4;MyD88，髓系分化因子 88;NF-kB，核因子 κ-B;DXMS，地塞米松;IFN- γ γ gamma\gamma ，干扰素-γ;TGF- β β beta\beta ，转化生长因子-β。
    • Corresponding authors at: Henan University of Chinese Medicine, No. 156 Jinshui East Road, Zhengdong New District, Zhengzhou, Henan 450046, China.
      通讯作者：河南中医药大学，中国河南省郑州市郑东新区金水东路 156 号450046。
    E-mail addresses: tianshuo0416@163.com (S. Tian), miaomingsan@163.com (M. Miao).
    电子邮件地址：tianshuo0416@163.com （S. Tian）、miaomingsan@163.com （M. Miao）。
    1 1 ^(1){ }^{1} These authors contributed equally to this work and share first authorship.
    1 1 ^(1){ }^{1} 这些作者对这项工作做出了同等贡献，并分享了第一作者身份。