大麻素受体1通过Gαi/o/RhoA信号通路促进急性肺损伤小鼠巨噬细胞M1极化

doi:10.3969/j.issn.1674-8115.2025.02.004

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (2): 161-168.doi: 10.3969/j.issn.1674-8115.2025.02.004

大麻素受体1通过Gα_i/o/RhoA信号通路促进急性肺损伤小鼠巨噬细胞M1极化

马秀珍¹^,², 周妮³, 郭思琪²^,⁴, 王源媛²^,⁵, 麦平¹^,²()

^1.兰州大学第一临床医学院，兰州 730099
^2.甘肃省人民医院消化内科，兰州 730030
^3.陕西省西安国际医学中心医院，西安 710100
^4.甘肃中医药大学第一临床医学院，兰州 730000
^5.江苏大学医学院，镇江 212013

收稿日期:2024-07-04 接受日期:2024-10-28 出版日期:2025-02-24 发布日期:2025-02-28
通讯作者: 麦平 E-mail:maiping_lz@163.com
作者简介:马秀珍（1996—），女，东乡族，硕士生；电子信箱：2060935938@qq.com
周妮（1997—），女，住院医师，硕士；电子信箱：z18355425311@163.com。为共同第一作者。
麦平，主任医师、教授，博士；电子信箱：maiping_lz@163.com。
基金资助:
甘肃省自然科学基金(21JR1RA014)

Cannabinoid receptor 1 promotes M1 polarization of macrophages through the Gα_i/o/RhoA signaling pathway in mice with acute lung injury

MA Xiuzhen¹^,², ZHOU Ni³, GUO Siqi²^,⁴, WANG Yuanyuan²^,⁵, MAI Ping¹^,²()

^1.The First School of Clinical Medicine, Lanzhou University, Lanzhou 730099, China
^2.Department of Gastroenterology, Gansu Provincial People's Hospital, Lanzhou 730030, China
^3.Xi'an International Medical Center Hospital, Shaanxi Province, Xi'an 710100, China
^4.The First Clinical Medical College of Gansu University of Traditional Chinese Medicine, Lanzhou 730000, China
^5.Jiangsu University School of Medicine, Zhenjiang 212013, China

Received:2024-07-04 Accepted:2024-10-28 Online:2025-02-24 Published:2025-02-28
Contact: MAI Ping E-mail:maiping_lz@163.com
Supported by:
Natural Science Foundation of Gansu Province(21JR1RA014)

摘要/Abstract

摘要：

目的·探讨阻断大麻素受体1（cannabinoid receptor 1，CB1）对小鼠急性肺损伤（acute lung injury，ALI）的影响及其潜在的分子机制。方法·将40只小鼠随机分为空白对照组、AM281（CB1拮抗剂）对照组、脂多糖（lipopolysaccharide，LPS）组、LPS＋AM281组，每组10只。利用LPS诱导建立小鼠ALI模型。经苏木精-伊红（hematoxylin and eosin，H-E）染色观察各组小鼠肺组织病理表现并计算炎症评分。通过反转录和实时荧光定量聚合酶链反应（real-time fluorescent quantitative polymerase chain reaction，qPCR）检测各组小鼠肺巨噬细胞M1型标志物［肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-1β（interleukin-1β，IL-1β）、IL-12］和M2型标志物［精氨酸酶（arginase，Arg）、甘露糖受体C型2（mannose receptor，C type 2，Mrc2）、巨噬细胞半乳糖型凝集素1（macrophage galactose-type lectin 1，Mgl1）］的mRNA水平。体外培养人髓系白血病单核细胞THP-1，通过免疫荧光技术检测THP-1细胞中CB1和CB2的表达，并在进一步阻断CB1及抑制G_αi/o/RhoA信号通路后，检测M1型标志物的mRNA水平。结果·LPS组小鼠肺组织损伤明显，炎症评分明显升高；阻断CB1后，与LPS组相比，LPS＋AM281组小鼠肺损伤得到改善，表现为肺泡壁毛细血管充血改善、肺间质及肺泡腔内炎症细胞浸润减少，炎症评分下降（P=0.007）。与对照组相比，LPS组小鼠肺组织中M1型标志物水平上调，而在阻断CB1后巨噬细胞极性发生改变，M1/M2比例发生反转（均P<0.05）。体外研究发现，巨噬细胞表达CB1和CB2，利用花生四烯基-2-氯乙酰胺（arachidonyl-2-chloroethylamide，ACEA）激活CB1可上调M1型标志物的表达，阻断CB1及选择性抑制G_αi/o/RhoA信号通路后，M1型标志物的表达均明显下调（均P<0.05）。结论·CB1在ALI中通过Gα_i/o/RhoA信号通路促进巨噬细胞向M1极化，阻断CB1可改善肺损伤。

关键词: 急性肺损伤, 大麻素受体1, 巨噬细胞, M1极化, Gα_i/o/RhoA信号通路

Abstract:

Objective ·To explore the effects and potential molecular mechanisms of blocking cannabinoid receptor 1 (CB1) in acute lung injury (ALI) in mice. Methods ·Forty mice were randomly divided into blank control group, AM281 (CB1 antagonist) control group, lipopolysaccharide (LPS) group, and LPS＋AM281 group, with ten mice in each group. ALI models were induced by LPS. The pathological manifestations of lung tissues were observed in each group of mice by hematoxylin and eosin (H-E) staining and the inflammation scores were calculated. The mRNA levels of M1 markers [tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-12] and M2 markers [arginase (Arg), mannose receptor, C type 2 (Mrc2), macrophage galactose-type lectin 1 (Mgl1)] in lung macrophages were measured by reverse transcription and real-time fluorescence quantitative polymerase chain reaction (qPCR). Human myeloid leukemia monocytes THP-1 cells were cultured in vitro, and the expression of CB1 and CB2 in THP-1 cells was detected by immunofluorescence. After further blocking CB1 and inhibiting the Gα_i/o/RhoA signaling pathway, the mRNA levels of M1 markers were assessed. Results ·The LPS group showed significant lung tissue damage and a significant increase in inflammation scores in mice. After blocking CB1, compared with the LPS group, the LPS＋AM281 group of mice showed improvements in lung injury, manifested as improved congestion of alveolar wall capillaries, reduced infiltration of inflammatory cells in the lung interstitium and alveolar cavity, and a decreased inflammation score (P=0.007). Compared with the control group, the levels of M1 marker in the lung tissue of the LPS group were upregulated, while the polarization of macrophages changed and the M1/M2 ratio was reversed after blocking CB1 (all P<0.05). In vitro studies found that macrophages expressed CB1 and CB2. Activation of CB1 by arachidonyl-2-chloroethylamide (ACEA) upregulated the expression of M1 markers. Blocking CB1 and selectively inhibiting Gα_i/o/RhoA signaling significantly downregulated M1 markers (all P<0.05). Conclusion ·CB1 promotes the polarization of macrophage towards the M1 phenotype through the Gα_i/o/RhoA signaling pathway in ALI, and blocking CB1 can improve lung injury.

Key words: acute lung injury (ALI), cannabinoid receptor 1 (CB1), macrophage, M1 type polarization, Gα_i/o/RhoA signaling pathway

中图分类号:

R563.1

马秀珍, 周妮, 郭思琪, 王源媛, 麦平. 大麻素受体1通过Gα_i/o/RhoA信号通路促进急性肺损伤小鼠巨噬细胞M1极化[J]. 上海交通大学学报（医学版）, 2025, 45(2): 161-168.

MA Xiuzhen, ZHOU Ni, GUO Siqi, WANG Yuanyuan, MAI Ping. Cannabinoid receptor 1 promotes M1 polarization of macrophages through the Gα_i/o/RhoA signaling pathway in mice with acute lung injury[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(2): 161-168.

图/表 5

表1 引物序列

Tab 1 Primer sequences for qPCR

Gene	Forward primer (5'→3')	Reverse primer (5'→3')
18S rRNA	GTAACCCGTTGAACCCCATT	CCATCCAATCGGTAGTAGCG
TNF-α	GGCAGGTTCTGTCCCTTTCA	CTGTGCTCATGGTGTCTTTTCTG
IL-1β	CTACAGGCTCCGAGATGAACAAC	TCCATTGAGGTGGAGAGCTTTC
IL-12	CCTGAAGTGTGAAGCACCAAATT	CTTCAAGTCCATGTTTCTTTGCA
Arg1	AGCCACAGCTCTTCCCTTGA	CCTACCCCCGGAGAGTCACT
Mrc2	GAGCGACGGTCTAGGGTTTTC	GCCTCGGATATCATCGTCATC
Mgl1	AGAACAACGGCTCGGAAGTG	CCAATAGCAGCTGCCTTCATG

图1 THP-1细胞表达CB1和CB2Note： Representative images of CB1 and CB2 (green) immunofluorescence staining in THP-1 cells (×100) with DAPI staining to visualize the nuclei.

Fig 1 THP-1 cells expressing CB1 and CB2

图2 CB1阻断剂AM281减轻ALI小鼠的肺损伤和炎症反应Note： A. H-E staining. B. Inflammation scores. F=129.3, P=0.002, compared among the four groups; ①P=0.019, compared with the control group; ②P=0.007, compared with the LPS group.

Fig 2 CB1 blocker AM281 attenuated lung injury and inflammatory response in ALI mice

图3 CB1阻断剂AM281逆转巨噬细胞M1/M2比例Note： TNF-α: F=113.6, P=0.005, compared among the four groups; ①P=0.015, compared with the LPS group. IL-1β: F=117.5, P=0.001, compared among the four groups; ②P=0.008, compared with the LPS group. IL-12: F=97.92, P=0.004, compared among the four groups; ③P=0.031, compared with the LPS group. Mrc2: F=120.3, P=0.004, compared among the four groups; ④P=0.002, compared with the LPS group. Mgl1: F=93.52, P=0.005, compared among the four groups; ⑤P=0.015, compared with the LPS group.

Fig 3 CB1 blocker AM281 reversed the ratio of macrophage M1/M2

图4 CB1促进巨噬细胞M1极化可被AM281、PTX和C3转移酶阻断Note： A. F=32.17, P=0.012, compared among the five groups; ①P=0.025, compared with the control group; ②P=0.003, ③P=0.001, ④P=0.011, compared with the ACEA group. B. F=55.41, P=0.009, compare among the five groups; ⑤P=0.013, compared with the control group; ⑥P=0.008, ⑦P=0.017, ⑧P=0.024, compared with the ACEA group. C. F=47.18, P=0.010, compare among the five groups; ⑨P=0.005, compared with the control group; ⑩P=0.022,⑪P=0.039, ⑫P=0.026, compared with the ACEA group.

Fig 4 CB1-mediated macrophage M1 polarization can be blocked by AM281, PTX and C3 transferase

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大麻素受体1通过Gα_i/o/RhoA信号通路促进急性肺损伤小鼠巨噬细胞M1极化

Cannabinoid receptor 1 promotes M1 polarization of macrophages through the Gα_i/o/RhoA signaling pathway in mice with acute lung injury

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大麻素受体1通过Gαi/o/RhoA信号通路促进急性肺损伤小鼠巨噬细胞M1极化

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大麻素受体1通过Gα_i/o/RhoA信号通路促进急性肺损伤小鼠巨噬细胞M1极化

Cannabinoid receptor 1 promotes M1 polarization of macrophages through the Gα_i/o/RhoA signaling pathway in mice with acute lung injury