通塞颗粒阻抑巨噬细胞炎症反应改善大鼠慢性阻塞性肺疾病急性加重

doi:10.12122/j.issn.1673-4254.2024.10.18

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (10): 1995-2003.doi: 10.12122/j.issn.1673-4254.2024.10.18

• • 上一篇

通塞颗粒阻抑巨噬细胞炎症反应改善大鼠慢性阻塞性肺疾病急性加重

程蒙蒙¹^,²^,³(), 刘新光¹^,²^,³, 魏焱鑫¹^,²^,³, 邢小香¹^,²^,³, 刘览¹^,²^,³, 辛楠¹^,²^,³, 赵鹏¹^,²^,³()

^1.河南中医药大学，呼吸疾病中医药防治省部共建协同创新中心//河南省中医药防治呼吸病重点实验室，河南郑州 450046
^2.河南中医药大学，中医药科学院，河南郑州 450046
^3.河南中医药大学第一附属医院呼吸科，河南郑州 450046

收稿日期:2024-08-09 出版日期:2024-10-20 发布日期:2024-10-31
通讯作者: 赵鹏 E-mail:mmxxnxfl@136.com;zhaopeng871@126.com
作者简介:程蒙蒙，硕士，E-mail: mmxxnxfl@136.com
基金资助:
河南省高校科技创新人才支持计划(24HASTIT073);河南省本科高校青年骨干教师培养计划(2023GGJS084);河南省高等学校重点科研项目(24A360013);郑州市科技协同创新专项(XTCX2021-05)

Tongsai Granules inhibit autophagy and macrophage-mediated inflammatory response to improve acute exacerbations of chronic obstructive pulmonary disease in rats

Mengmeng CHENG¹^,²^,³(), Xinguang LIU¹^,²^,³, Yanxin WEI¹^,²^,³, Xiaoxiang XING¹^,²^,³, Lan LIU¹^,²^,³, Nan XIN¹^,²^,³, Peng ZHAO¹^,²^,³()

^1.Collaborative Innovation Center for Chinese Medicine and Respiratory Diseases Co-constructed by Henan Province & Ministry of Education, Henan Key Laboratory of Chinese Medicine for Respiratory Disease, First Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou 450046, China
^2.Academy of Chinese Medical Sciences, Henan University of Chinese Medicine, Zhengzhou 450046, China, First Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou 450046, China
^3.Department of Respiratory Medicine, First Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou 450046, China

Received:2024-08-09 Online:2024-10-20 Published:2024-10-31
Contact: Peng ZHAO E-mail:mmxxnxfl@136.com;zhaopeng871@126.com

摘要/Abstract

摘要：

目的探讨通塞颗粒（TSG）阻抑巨噬细胞炎症反应改善慢性阻塞性肺疾病急性加重期（AECOPD）大鼠的作用机制。方法 24只SD大鼠随机分为对照（Control）组、模型（Model）组、TSG组和莫西沙星+沙丁胺醇（MXF+STL）组，6只/组。第1~8周建立慢性阻塞性肺疾病（COPD）大鼠模型，第9周第3天，经鼻滴入肺炎克雷伯杆菌建立AECOPD大鼠模型。第9周的第1~2天和第4~7天，Control组和Model组给予生理盐水灌胃，TSG组和MXF+STL组分别给予TSG和MXF+STL灌胃，灌胃结束后处死大鼠。HE染色观察大鼠肺组织病理变化；检测肺组织白细胞介素IL-1β、IL-6、肿瘤坏死因子TNF-α、基质金属蛋白酶MMP2和MMP9表达。采用脂多糖（LPS）刺激巨噬细胞MH-S，给予TSG含药血清及其代表成分干预，检测IL-1β、IL-6、TNF-α、环氧化酶COX-2及诱导型一氧化氮合酶（iNOS）mRNA水平，磷酸化P38（p-p38）、p-p62、微管相关蛋白1A/1B-轻链3（LC3）、Forkhead box O3（FoxO3a）及p-mTOR蛋白水平。结果 TSG显著改善AECOPD大鼠肺组织损伤与肺功能，包括降低支气管壁厚度、肺泡平均线性截距及上调平均肺泡数（P<0.05）；TSG明显抑制AECOPD大鼠肺组织炎症因子IL-1β、IL-6、TNF-α及基质金属蛋白酶MMP2和MMP9表达水平（P<0.01）。TSG显著抑制脂多糖诱导的巨噬细胞炎症因子IL-1β、IL-6、TNF-α及相关代谢酶COX-2、iNOS的表达（P<0.05）。血清药物化学联合网络药理鉴定了TSG含药血清10种化学成分，其对应466个靶点核心功能分析显示，TSG干预AECOPD可能与其主要成分木犀草素、槲皮素、贝母辛、贝母乙素等调控MAPK、mTOR、FoxO、自噬等有关。体外实验显示，木犀草素可以显著抑制LPS诱导巨噬细胞炎症反应及相关信号如p-p38，调控自噬相关信号FoxO3a、mTOR，以及抑制自噬相关蛋白p-p62、LC3的表达（P<0.05）。结论 TSG抑制巨噬细胞炎症反应缓解AECOPD，其机制可能与调控p38、mTOR、FoxO3a信号及抑制自噬有关。

关键词: 通塞颗粒, 慢性阻塞性肺疾病急性加重期, 巨噬细胞, 炎症反应

Abstract:

Objective To investigate the inhibitory effect of Tongsai Granules (TSG) on macrophage-mediated inflammatory response to alleviate acute exacerbation of chronic obstructive pulmonary disease (AECOPD) in rats and explore the underlying mechanism. Methods Twenty-four rats were divided into control group, AECOPD model group, TSG treatment group, and moxifloxacin+salbutamol (MXF+STL) treatment group. In the rat models of COPD, AECOPD was induced by nasal instillation of Klebsiella pneumoniae on day 3 of week 9 after modeling, and saline, TSG or MXF+STL were administered via gavage on days 1 and 2 and days 4 to 7 of week 9. After the treatments, lung tissues were collected for examining for pathologies and expressions of inflammatory markers, MMP2, and MMP9. In cultured macrophage MH-S cells with LPS stimulation, the effect of TSG-medicated serum on IL-1β, IL-6, TNF‑α, COX-2, and iNOS expressions and phosphorylation levels of p38, p-p62, LC3, FoxO3a, and mTOR were evaluated. Results TSG significantly improved lung pathologies and lung function in AECOPD rats by reducing bronchial wall thickness and mean alveolar linear intercept, increasing alveolar numbers, and reducing pulmonary expression of IL-1β, IL-6, TNF-α, MMP2 and MMP9. In MH-S cells, TSG significantly suppressed LPS-induced expressions of inflammatory cytokines, COX-2 and iNOS. Serum pharmacology coupled with network pharmacology identified 10 chemical components in TSG-medicated serum, and functional analysis of their 466 targets suggested that the therapeutic effect of TSG on AECOPD was mediated primarily by luteolin and quercetin, which regulate the MAPK, mTOR, FoxO, and autophagy pathways. In MH-S cells, luteolin significantly inhibited LPS-induced inflammatory responses and expressions of p-p38, FoxO3a, mTOR, p-p62 and LC3. Conclusion TSG reduces macrophage-mediated inflammatory responses to alleviate AECOPD in rats possibly by modulating p38, mTOR, and FoxO3a pathways and inhibiting autophagy.

Key words: Tongsai Granule, acute exacerbation of chronic obstructive pulmonary disease, macrophages, inflammatory response

程蒙蒙, 刘新光, 魏焱鑫, 邢小香, 刘览, 辛楠, 赵鹏. 通塞颗粒阻抑巨噬细胞炎症反应改善大鼠慢性阻塞性肺疾病急性加重[J]. 南方医科大学学报, 2024, 44(10): 1995-2003.

Mengmeng CHENG, Xinguang LIU, Yanxin WEI, Xiaoxiang XING, Lan LIU, Nan XIN, Peng ZHAO. Tongsai Granules inhibit autophagy and macrophage-mediated inflammatory response to improve acute exacerbations of chronic obstructive pulmonary disease in rats[J]. Journal of Southern Medical University, 2024, 44(10): 1995-2003.

图/表 5

图1 TSG改善AECOPD大鼠肺病理

Fig.1 Tongsai Granules (TSG) alleviates lung pathologies in AECOPD rats. A: Flow chart of animal experiments. B: Histopathological changes of the lung tissues detected in week 10 (HE staining, original magnification: ×200). C: Lung injury scores (BWT, S/Pi, MLI, and MAN). *P<0.05, **P<0.01 vs Control; #P<0.05, ##P<0.01 vs Model. MXF+STL: Moxifloxacin+Salbutamol group. CT: Control group. M: Model group.

图2 TSG降低AECOPD大鼠肺组织炎症因子表达

Fig.2 TSG inhibits expressions of inflammatory cytokines in the lung tissues of AECOPD rats. A: Expression levels of IL-6, IL-1β and TNF‑α in the lung tissue of the rats analyzed with immunohistochemistry (×200). B: Integral optical density (IOD) of the proteins (Mean±SD). **P<0.01 vs Control, ##P<0.01 vs Model.

图3 TSG降低AECOPD大鼠肺组织MMP表达

Fig.3 TSG inhibits expressions of MMPs in the lung tissues of AECOPD rats. A: Expressions of MMP2 and MMP9 in lung tissue of the rats analyzed by immunohistochemistry (×200). B: IOD of the proteins in each group (Mean±SD). **P<0.01 vs Control, ##P<0.01 vs Model.

图4 TSG干预AECOPD的网络药理分析

Fig.4 Network pharmacological analysis of the mechanisms of TSG for treating AECOPD in rats. A: Serum from TSG-treated rats inhibits expressions of IL-1β, IL-6, TNF-α, COX2, and iNOS in LPS-induced macrophages. B: PPI of 466 targets constructed using STRING. C: Orange circles represent the top 18 nodes ranked by degree value. D: Top 18 nodes analyzed for KEGG pathways. E: Sankey diagram illustrating the representative components of TSG-medicated serum and its target pathways. **P<0.01 vs Control, ##P<0.01 vs Model.

图5 TSG 抑制自噬阻抑LPS诱导的巨噬细胞炎症反应

Fig.5 TSG inhibits LPS-induced macrophage inflammation and autophagy. A: Luteolin inhibits expressions of IL-1β, IL-6, TNF-α, COX2, and iNOS in LPS-induced macrophages. B, C: Western blotting of expression levels of p-p38, p38, LC3, p-p62, p-FoxO3a, and p-mTOR (Mean±SD). *P<0.05, **P<0.01 vs Control; #P<0.05, ##P<0.01 vs Model. DEX: Dexamethasone.

参考文献 42

1	Ferrera MC, Labaki WW, Han MK. Advances in chronic obstructive pulmonary disease[J]. Annu Rev Med, 2021, 72: 119-34.
2	Ritchie AI, Wedzicha JA. Definition, causes, pathogenesis, and consequences of chronic obstructive pulmonary disease exacerbations[J]. Clin Chest Med, 2020, 41(3): 421-38.
3	Barnes PJ. Alveolar macrophages as orchestrators of COPD[J]. COPD, 2004, 1(1): 59-70.
4	Barnes PJ. Inflammatory mechanisms in patients with chronic obstructive pulmonary disease[J]. J Allergy Clin Immunol, 2016, 138(1): 16-27.
5	Zheng JT, Shi Y, Xiong LX, et al. The expression of IL-6, TNF-α, and MCP-1 in respiratory viral infection in acute exacerbations of chronic obstructive pulmonary disease[J]. J Immunol Res, 2017, 2017: 8539294.
6	Russell RE, Thorley A, Culpitt SV, et al. Alveolar macrophage-mediated elastolysis: roles of matrix metalloproteinases, cysteine, and serine proteases[J]. Am J Physiol Lung Cell Mol Physiol, 2002, 283(4): L867-73.
7	余学庆, 李建生, 李力. 慢性阻塞性肺疾病(COPD)中医证候分布规律研究[J]. 河南中医学院学报, 2003, 18(4): 44-6.
8	李建生, 余学庆. 慢性阻塞性肺疾病中医分期分级防治策略[J]. 中医杂志, 2019, 60(22): 1895-9.
9	李建生, 李素云, 马利军, 等. 通塞颗粒治疗老年慢性阻塞性肺疾病(COPD)急性加重期的临床疗效评价[J]. 河南中医学院学报, 2003, 18(5): 35-8.
10	Li Y, Li SY, Li JS, et al. A rat model for stable chronic obstructive pulmonary disease induced by cigarette smoke inhalation and repetitive bacterial infection[J]. Biol Pharm Bull, 2012, 35(10): 1752-60.
11	Tao LY, Lu XF, Fu ZJ, et al. Tong Sai Granule improves AECOPD via regulation of MAPK-SIRT1-NF‑κB pathway and cellular senescence alleviation[J]. J Ethnopharmacol, 2023, 314: 116622.
12	魏焱鑫, 魏毓, 刘新光, 等. 通塞颗粒对AECOPD大鼠气道上皮屏障及EGFR/ERK通路的影响[J]. 中国病理生理杂志, 2023, 39(12): 2204-13.
13	Venkatesan P. GOLD COPD report: 2024 update[J]. Lancet Respir Med, 2024, 12(1): 15-6.
14	Soler-Cataluña JJ, Martínez-García MA, Román Sánchez P, et al. Severe acute exacerbations and mortality in patients with chronic obstructive pulmonary disease[J]. Thorax, 2005, 60(11): 925-31.
15	Mannino DM, Buist AS. Global burden of COPD: risk factors, prevalence, and future trends[J]. Lancet, 2007, 370(9589): 765-73.
16	Zhu BF, Wang YF, Ming J, et al. Disease burden of COPD in China: a systematic review[J]. Int J Chron Obstruct Pulmon Dis, 2018, 13: 1353-64.
17	许硕, 刘欣, 袁太文, 等. 布地奈德联合特布他林治疗慢性阻塞性肺疾病急性加重期的疗效观察[J]. 中国医院用药评价与分析, 2016, 16(8): 1033-4, 1035.
18	杨振英, 王亚坤, 周礼清, 等. 糖皮质激素类联合肾上腺素能β激动剂对慢性阻塞性肺疾病急性加重期患者的疗效分析[J]. 安徽医药, 2018, 22(1): 151-5.
19	Vermeersch K, Gabrovska M, Aumann J, et al. Azithromycin during acute chronic obstructive pulmonary disease exacerbations requiring hospitalization (BACE). A multicenter, randomized, double-blind, placebo-controlled trial[J]. Am J Respir Crit Care Med, 2019, 200(7): 857-68.
20	Vollenweider DJ, Jarrett H, Steurer-Stey CA, et al. Antibiotics for exacerbations of chronic obstructive pulmonary disease[J]. Cochrane Database Syst Rev, 2012, 12: CD010257.
21	Strzelak A, Ratajczak A, Adamiec A, et al. Tobacco smoke induces and alters immune responses in the lung triggering inflammation, allergy, asthma and other lung diseases: a mechanistic review[J]. Int J Environ Res Public Health, 2018, 15(5): 1033.
22	杨泽辉, 陈晓东, 张强. 慢性阻塞性肺疾病患者血清及肺泡巨噬细胞相关细胞因子水平变化[J]. 中国慢性病预防与控制, 2014, 22(2): 238-40.
23	Barnes PJ. Cellular and molecular mechanisms of asthma and COPD[J]. Clin Sci: Lond, 2017, 131(13): 1541-58.
24	赵建荣, 李晓玫. 中药复方物质基础研究的现状与进展[J]. 中草药, 2003, 34(11): 963-6, 1010.
25	郭立民, 王长云, 顾谦群, 等. 中药复方效应物质基础研究方法及其发展趋势[J]. 中成药, 2007, 29(1): 118-21.
26	张雯霞, 冯敏, 苗雨露, 等. 中药血清药物化学的应用现状概述[J]. 药物评价研究, 2019, 42(7): 1448-53.
27	Santos BF, Grenho I, Martel PJ, et al. FOXO family isoforms[J]. Cell Death Dis, 2023, 14(10): 702.
28	Zhang Y, Yu WP, Chang WG, et al. Light chain amyloidosis-induced autophagy is mediated by the Foxo3a/beclin-1 pathway in cardiomyocytes[J]. Lab Invest, 2023, 103(2): 100001.
29	Fitzwalter BE, Thorburn A. FOXO3 links autophagy to apoptosis[J]. Autophagy, 2018, 14(8): 1467-8.
30	Audesse AJ, Dhakal S, Hassell LA, et al. FOXO3 directly regulates an autophagy network to functionally regulate proteostasis in adult neural stem cells[J]. PLoS Genet, 2019, 15(4): e1008097.
31	Racanelli AC, Choi AMK, Choi ME. Autophagy in chronic lung disease[M]//Progress in Molecular Biology and Translational Science. Amsterdam: Elsevier, 2020: 135-56.
32	Lv XX, Li K, Hu ZW. Chronic obstructive pulmonary disease and autophagy[M]// Autophagy: Biology and Diseases. Singapore: Springer, 2020: 559-67.
33	Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism, and disease[J]. Cell, 2017, 169(2): 361-71.
34	Kim YC, Guan KL. mTOR: a pharmacologic target for autophagy regulation[J]. J Clin Invest, 2015, 125(1): 25-32.
35	Huang QS, Yang HJ, Zhang CT, et al. Xiaoqinglong decoction protects the lungs of AECOPD mice through the AMPK/mTOR signaling pathway[J]. Evid Based Complement Alternat Med, 2020, 2020: 9865290.
36	Kaminska B. MAPK signalling pathways as molecular targets for anti-inflammatory therapy: from molecular mechanisms to therapeutic benefits[J]. Biochim Biophys Acta, 2005, 1754(1/2): 253-62.
37	Park MY, Ha SE, Kim HH, et al. Scutellarein inhibits LPS-induced inflammation through NF‑κB/MAPKs signaling pathway in RAW_264.7 cells[J]. Molecules, 2022, 27(12): 3782.
38	Yoshizaki T, Kusunoki C, Kondo M, et al. Autophagy regulates inflammation in adipocytes[J]. Biochem Biophys Res Commun, 2012, 417(1): 352-7.
39	Levine B, Kroemer G. Autophagy in the pathogenesis of disease[J]. Cell, 2008, 132(1): 27-42.
40	朱紫亨, 郑佳昆, 冯淬灵. 自噬在肺部慢性炎症性疾病中的作用[J]. 西南医科大学学报, 2024, 47(2): 176-179.
41	Yang S, Li FY, Lu SY, et al. Ginseng root extract attenuates inflammation by inhibiting the MAPK/NF‑κB signaling pathway and activating autophagy and p62-Nrf2-Keap1 signaling in vitro and in vivo [J]. J Ethnopharmacol, 2022, 283: 114739.
42	Matsuzawa-Ishimoto Y, Hwang S, Cadwell K. Autophagy and inflammation[J]. Annu Rev Immunol, 2018, 36: 73-101.

[1]	左涵珺, 段兆达, 王朝, 郭涛, 石金沙, 石浩龙, 李娟娟. 天麻素经PI3K/AKT通路改善新生大鼠缺氧缺血性脑损伤后小胶质细胞介导的炎症反应[J]. 南方医科大学学报, 2024, 44(9): 1712-1719.
[2]	刘鹏程, 娄丽娟, 刘霞, 王建, 姜颖. M2巨噬细胞特征基因风险评分能准确预测HBV相关肝细胞癌患者的预后[J]. 南方医科大学学报, 2024, 44(5): 827-840.
[3]	曹家樊, 孙跃, 丁鑫, 李盛文, 陈博, 兰天. 熊果苷通过抑制巨噬细胞募集并调控Akt/NF-κB和Smad信号通路改善小鼠肝纤维化[J]. 南方医科大学学报, 2024, 44(4): 652-659.
[4]	何程, 陈炜, 张念志, 栾军, 王三凤, 张尤. 参七虫草方通过ASS1/src/STAT3信号通路改善肺纤维化大鼠的炎症反应[J]. 南方医科大学学报, 2024, 44(4): 644-651.
[5]	包汉生, 王苏童, 吕穆杰, 王永成, 姜萍, 李晓. 激活α7nAchR促进肥胖小鼠的脂肪稳态和米色脂肪生成及产热作用[J]. 南方医科大学学报, 2024, 44(3): 499-506.
[6]	范毅平, 罗梦琳, 黄东宗, 刘琳, 傅博, 王潇宇, 关淼升, 李鸿波. 缓释地塞米松改性丝胶蛋白水凝胶支架促进大鼠下颌骨缺损修复：基于调节巨噬细胞M2极化[J]. 南方医科大学学报, 2024, 44(3): 533-540.
[7]	孔祥, 张腾, 张妍, 高灵犀, 汪文, 汪梦燕, 王国栋, 吕坤. 过表达lncRNA HEM2M改善非酒精性脂肪肝病小鼠的肝脏损伤[J]. 南方医科大学学报, 2024, 44(1): 1-8.
[8]	徐梦歧, 石宇彤, 刘俊平, 吴敏敏, 张凤梅, 何志强, 唐敏. JAG1影响单核-巨噬细胞重塑三阴性乳腺癌转移前微环境：基于外泌体中的LncRNA MALAT1[J]. 南方医科大学学报, 2023, 43(9): 1525-1535.
[9]	徐龙飞, 韩晶, 杨喆, 杨燕平, 陈金慧, 吴西军, 王琪, 洪艳. LRG1抑制小鼠肝巨噬细胞活化从而改善代谢相关脂肪性肝病：基于增强TGF-β1信号通路[J]. 南方医科大学学报, 2023, 43(7): 1164-1171.
[10]	林嘉宜, 娄安妮, 李旭. 脂多糖刺激巨噬细胞分泌含miR-155-5p的外泌体促进肝星状细胞的活化及迁移[J]. 南方医科大学学报, 2023, 43(6): 994-1001.
[11]	李敬怡, 杨思圆, 韩振, 江天乐, 朱耀, 周子航, 周静萍. Akt2抑制剂促进大鼠根尖周炎症微环境中巨噬细胞的极化：基于降低miR-155-5p的表达[J]. 南方医科大学学报, 2023, 43(4): 568-576.
[12]	张梦莹, 李志, 裴纬亚, 李雪琴, 杨辉, 朱小龙, 吕坤. M2型巨噬细胞来源的外泌体lncRNA NR_028113.1通过激活JAK2/STAT3通路促进巨噬细胞的极化[J]. 南方医科大学学报, 2023, 43(3): 393-399.
[13]	李晒, 李丽, 闵思敏, 刘赛赛, 秦志文, 熊志尚, 徐建国, 王博文, 丁渡山, 赵士弟. 大豆异黄酮可减轻大鼠脑缺血/再灌注损伤：基于抑制铁死亡及炎症级联反应[J]. 南方医科大学学报, 2023, 43(2): 323-330.
[14]	曹天然, 刘青芳, 潘美民, 张雪红. LncRNA SNHG8通过抑制miR-494-3p表达减轻脑缺血再灌注损伤[J]. 南方医科大学学报, 2023, 43(12): 2015-2022.
[15]	黄永祺, 喻伟, 游月华. 槟榔碱通过促进巨噬细胞分泌含miR-155-5p外泌体诱导人口腔成纤维细胞的活化[J]. 南方医科大学学报, 2023, 43(1): 60-67.

通塞颗粒阻抑巨噬细胞炎症反应改善大鼠慢性阻塞性肺疾病急性加重

Tongsai Granules inhibit autophagy and macrophage-mediated inflammatory response to improve acute exacerbations of chronic obstructive pulmonary disease in rats

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 42

相关文章 15

编辑推荐

Metrics

本文评价