参芪调肾方减轻慢阻肺肺肾气虚证大鼠气道炎症的机制：基于铁死亡途径

doi:10.12122/j.issn.1673-4254.2024.10.12

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (10): 1937-1946.doi: 10.12122/j.issn.1673-4254.2024.10.12

• • 上一篇

参芪调肾方减轻慢阻肺肺肾气虚证大鼠气道炎症的机制：基于铁死亡途径

杨勤军¹^,²^,³(), 王卉¹, 徐淑钰¹, 杨程⁴, 丁焕章¹, 吴迪¹, 朱洁¹, 童佳兵¹^,², 李泽庚²^,³^,⁴()

^1.安徽中医药大学中医学院，安徽合肥 230038
^2.中医药防治肺系重大疾病应用转化安徽省重点实验室，安徽合肥 230031
^3.新安医学教育部重点实验室，安徽合肥 230038
^4.安徽中医药大学第一附属医院，安徽合肥 230031

收稿日期:2024-09-02 出版日期:2024-10-20 发布日期:2024-10-31
通讯作者: 李泽庚 E-mail:yangqjahtcm@163.com;li6609@126.com
作者简介:杨勤军，副教授，E-mail: yangqjahtcm@163.com
基金资助:
国家自然科学基金区域联合重点项目(U20A20398);国家自然科学基金(82374399);合肥综合性国家科学中心大健康研究院新安医学与中医药现代化研究所“揭榜挂帅”重大突破项目(2023CXMMTCM005);安徽省中医药传承创新科研项目(2024CCCX077)

Shenqi Tiaoshen Formula alleviates airway inflammation in rats with chronic obstructive pulmonary disease and kidney qi deficiency syndrome by inhibiting ferroptosis via regulating the Nrf2/SLC7A11/GPX4 signaling pathway

Qinjun YANG¹^,²^,³(), Hui WANG¹, Shuyu XU¹, Cheng YANG⁴, Huanzhang DING¹, Di WU¹, Jie ZHU¹, Jiabing TONG¹^,², Zegeng LI²^,³^,⁴()

^1.School of Traditional Chinese Medicine, Anhui University of Chinese Medicine, Hefei 230038, China
^2.Anhui Province Key Laboratory of the Application and Transformation of Traditional Chinese Medicine in the Prevention and Treatment of Major Pulmonary Diseases, Hefei 230031, China
^3.Key Laboratory of Xin'an Medical Science, Ministry of Education, Hefei 230038, China
^4.The First Affiliated Hospital of Anhui University of Chinese Medicine, Hefei 230031, China

Received:2024-09-02 Online:2024-10-20 Published:2024-10-31
Contact: Zegeng LI E-mail:yangqjahtcm@163.com;li6609@126.com
Supported by:
National Natural Science Foundation of China(82374399)

摘要/Abstract

摘要：

目的基于核因子E2相关因子2（Nrf2）/溶质载体家族7成员11（SLC7A11）/谷胱甘肽过氧化物酶4（GPX4）途径探讨参芪调肾方（SQTSF）调控铁死亡减轻COPD肺肾气虚证病证结合大鼠气道炎症的作用。方法 48只SD大鼠随机分为对照组（CON）、模型组（MOD）、SQTSF低、中、高剂量组（SQTSF-L、SQTSF-M、SQTSF-H）和氨茶碱组（APL），n=8。除对照组外，其余大鼠构建COPD肺肾气虚证模型，并从第30天开始灌胃SQTSF和APL。观察各组大鼠的体质量、抓力变化、肺功能、肺组织病理、肺泡灌洗液（BALF）炎性因子、肺组织氧化应激水平、铁离子代谢情况、肺组织细胞和线粒体超微结构以及Nrf2/SLC7A11/GPX4信号通路和铁死亡相关基因和蛋白的表达。结果与CON组比较，MOD组大鼠表现出皮毛枯黄脱落、对刺激反应迟钝，精神萎顿、嗜睡喜聚成堆闭目静卧等明显的气虚神疲表现；体质量和抓力明显下降（P<0.05）；第0.1秒用力呼气量（FEV_0.1）、用力肺活量（FVC）、FEV_0.1/FVC、Cdyn明显降低（P<0.05）；肺组织表现出明显的炎性浸润和肺气肿，支气管、血管周围和肺泡炎症评分、肺组织平均内衬间隔（MLI）、肺泡破坏指数（DI）明显上升，平均肺泡数（MAN）明显降低（P<0.05）；BALF中IL-1β、TNF-α、IL-6、IL-13明显上升（P<0.05）；肺组织ROS、MDA明显上升，GSH明显下降（P<0.05）；肺组织Fe²⁺和总铁离子明显上升（P<0.05）；透射电镜（TEM）观察发现MOD组大鼠肺组织细胞表现出线粒体外膜破裂，线粒体嵴消失等铁死亡特征性改变；肺组织Nrf2、GPX4、SLC7A11明显下降，酰基辅酶A合成酶长链家族成员4（ACSL4）明显上升（P<0.05）；与MOD组比较，SQTSF能明显改善各项指标的病理改变，且作用效果优于APL组（P<0.05）。结论 SQTSF能有效改善COPD肺肾气虚病证结合模型大鼠气道炎症和氧化应激，其机制可能与调控Nrf2/SLC7A11/GPX4信号通路抑制铁死亡有关。

关键词: 慢性阻塞性肺疾病, 参芪调肾方, Nrf2/SLC7A11/GPX4, 铁死亡, 气道炎症

Abstract:

Objective To investigate the effects of Shenqi Tiaoshen Formula (SQTSF) for alleviating airway inflammation in rats with both chronic obstructive pulmonary disease (COPD) and lung-kidney qi deficiency syndrome and explore its therapeutic mechanism. Methods Forty-eight SD rats were randomly divided into control group, model group, low-, medium-, and high-dose SQTSF groups, and aminophylline (APL) group. In all but the control group, rat models of COPD with lung-kidney qi deficiency syndrome were established and treated with saline, SQTSF or APL via daily gavage as indicated (starting from day 30). The rats were observed for changes in body weight, grip strength, lung function, lung pathology, inflammatory cytokines in bronchoalveolar lavage fluid (BALF), oxidative stress levels, iron ion metabolism, cellular and mitochondrial ultrastructural changes in the lung tissue, and expressions of Nrf2/SLC7A11/GPX4 signaling pathway and ferroptosis-related proteins. Results The rats in the model group exhibited obvious symptoms of lung-kidney qi deficiency syndrome with significantly decreased body weight, grip strength, and lung function parameters. Examination of the lung tissue revealed showed significant inflammatory cell infiltration and emphysema with obvious bronchial, perivascular, and alveolar inflammation and alveolar destruction, significantly increased IL-1β, TNF-α, IL-6, and IL-13 levels in BALF, and elevated pulmonary oxidative stress levels and Fe²⁺ and total iron ion concentrations. The rat models also showed characteristic ultrastructural changes of ferroptosis in the lung tissue cells under transmission electron microscope and significantly decreased Nrf2, GPX4, and SLC7A11 and increased ACSL4 expressions in the lung tissue. Treatment with SQTSF significantly improved these pathological changes in the rat models with a better effect than APL. Conclusion SQTSF can effectively improve airway inflammation and oxidative stress in COPD rats with lung-kidney qi deficiency possibly by inhibiting ferroptosis via regulating the Nrf2/SLC7A11/GPX4 signaling pathway.

Key words: chronic obstructive pulmonary disease, Shenqi Tiaoshen Formula, Nrf2/SLC7A11/GPX4, ferroptosis, airway inflammation

杨勤军, 王卉, 徐淑钰, 杨程, 丁焕章, 吴迪, 朱洁, 童佳兵, 李泽庚. 参芪调肾方减轻慢阻肺肺肾气虚证大鼠气道炎症的机制：基于铁死亡途径[J]. 南方医科大学学报, 2024, 44(10): 1937-1946.

Qinjun YANG, Hui WANG, Shuyu XU, Cheng YANG, Huanzhang DING, Di WU, Jie ZHU, Jiabing TONG, Zegeng LI. Shenqi Tiaoshen Formula alleviates airway inflammation in rats with chronic obstructive pulmonary disease and kidney qi deficiency syndrome by inhibiting ferroptosis via regulating the Nrf2/SLC7A11/GPX4 signaling pathway[J]. Journal of Southern Medical University, 2024, 44(10): 1937-1946.

图/表 9

图1 COPD肺肾气虚证大鼠模型建立与给药示意图

Fig.1 Schematic diagram of establishment and treatment of COPD rat models of lung-kidney qi deficiency syndrome.

图2 各组大鼠大鼠体质量、体质量系数和抓力比较

Fig.2 Comparison of body mass, body mass coefficient (A) and grip strength (B) of the rats among the groups. *P<0.05 vs control group, #P<0.05 vs model group.

图3 各组大鼠肺功能指标比较

Fig.3 Comparison of pulmonary function indexes among the groups. A: FEV0.1. B: FVC. C: FEV0.1/FVC. D: Cdyn. *P<0.05 vs control group, #P<0.05 vs model group.

图4 各组大鼠肺组织病理学形态和肺组织病理学评分比较

Fig.4 Comparison of lung histopathological morphology and lung histopathological scores among the groups (n=6, Mean±SD). A: HE staining of rat lung tissue (scale bar=40 μm). B: Airway and vascular inflammation score. C: Alveolar inflammation score. D: MAN. E: MLI. F: DI. *P<0.05 vs control group, #P<0.05 vs model group.

图5 各组大鼠BALF炎症因子比较

Fig.5 Comparison of BALF inflammatory factors among the groups. A: IL-1β. B: TNF-α. C: IL-6. D: IL-13. *P<0.05 vs control group, #P<0.05 vs model group.

图6 各组大鼠肺组织氧化应激指标比较

Fig.6 Comparison of oxidative stress indexes in lung tissues among the groups. A: Flow cytometry for detecting ROS in the lung tissue. B: MDA. C: GSH. D: MFI of ROS. *P<0.05 vs control group, #P<0.05 vs model group.

图7 各组大鼠肺组织离子代谢比较

Fig.7 Comparison of ion metabolism in the lung tissues among the groups. A: Fe2+. B:Total iron ion.*P<0.05 vs control group, #P<0.05 vs model group.

图8 TEM观察各组大鼠肺组织超微结构

Fig.8 Ultrastructure of lung tissue of rats in each group observed by TEM (Original magnification: ×10000, × 25000).

图9 各组大鼠铁死亡相关蛋白的表达

Fig.9 Expression of ferroptosis-related proteins in each group. A: Western blotting for detecting expressions of Nrf2, SLC7A11, GPX4, and ACSL4 in the lung tissue. B-E: Relative protein expression levels of Nrf2, SLC7A11, GPX4 and ACSL4. *P<0.05 vs control group, #P<0.05 vs model group.

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参芪调肾方减轻慢阻肺肺肾气虚证大鼠气道炎症的机制：基于铁死亡途径

Shenqi Tiaoshen Formula alleviates airway inflammation in rats with chronic obstructive pulmonary disease and kidney qi deficiency syndrome by inhibiting ferroptosis via regulating the Nrf2/SLC7A11/GPX4 signaling pathway

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