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

doi:10.3969/j.issn.1674-8115.2025.02.004

Abstract

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

CLC Number:

R563.1

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.

Figures/Tables 5

TrendMD

References 26

1	SOHRABI C, ALSAFI Z, O'NEILL N, et al. World Health Organization declares global emergency: a review of the 2019 novel coronavirus (COVID-19)[J]. Int J Surg, 2020, 76: 71-76.
2	刘茜, 王荣帅, 屈国强, 等. 新型冠状病毒肺炎死亡尸体系统解剖大体观察报告[J]. 法医学杂志, 2020, 36(1): 21-23.
	LIU Q, WANG R S, QU G Q, et al. Gross examination report of a COVID-19 death autopsy[J]. Journal of Forensic Medicine, 2020, 36(1): 21-23.
3	XU Z, SHI L, WANG Y J, et al. Pathological findings of COVID-19 associated with acute respiratory distress syndrome[J]. Lancet Respir Med, 2020, 8(4): 420-422.
4	PEDERSEN S F, HO Y C. SARS-CoV-2: a storm is raging[J]. J Clin Invest, 2020, 130(5): 2202-2205.
5	BAIN C C, MACDONALD A S. The impact of the lung environment on macrophage development, activation and function: diversity in the face of adversity[J]. Mucosal Immunol, 2022, 15(2): 223-234.
6	BISWAS S K, CHITTEZHATH M, SHALOVA I N, et al. Macrophage polarization and plasticity in health and disease[J]. Immunol Res, 2012, 53(1): 11-24.
7	GANTER M T, ROUX J, MIYAZAWA B, et al. Interleukin-1β causes acute lung injury via αvβ5 and αvβ6 integrin-dependent mechanisms[J]. Circ Res, 2008, 102(7): 804-812.
8	ZHUGE F, NI Y H, NAGASHIMADA M, et al. DPP-4 inhibition by linagliptin attenuates obesity-related inflammation and insulin resistance by regulating M1/M2 macrophage polarization[J]. Diabetes, 2016, 65(10): 2966-2979.
9	SICA A, MANTOVANI A. Macrophage plasticity and polarization: in vivo veritas[J]. J Clin Invest, 2012, 122(3): 787-795.
10	WU Q, MA Y N, LIU Y, et al. CB2R agonist JWH-133 attenuates chronic inflammation by restraining M1 macrophage polarization via Nrf2/HO-1 pathway in diet-induced obese mice[J]. Life Sci, 2020, 260: 118424.
11	TORTORA C, PAOLA A D, ARGENZIANO M, et al. Effects of CB2 receptor modulation on macrophage polarization in pediatric celiac disease[J]. Biomedicines, 2022, 10(4): 874.
12	MAI P, YANG L, TIAN L, et al. Endocannabinoid system contributes to liver injury and inflammation by activation of bone marrow-derived monocytes/macrophages in a CB1-dependent manner[J]. J Immunol, 2015, 195(7): 3390-3401.
13	YE R S, LIU Z W. ACE2 exhibits protective effects against LPS-induced acute lung injury in mice by inhibiting the LPS-TLR4 pathway[J]. Exp Mol Pathol, 2020, 113: 104350.
14	LIAO M F, LIU Y, YUAN J, et al. Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19[J]. Nat Med, 2020, 26(6): 842-844.
15	王正, 张倩月, 宋怀东. 轻重症及转归阶段的COVID-19患者免疫细胞的单细胞转录谱分析[J]. 复旦学报(医学版), 2022, 49(2): 213-225.
	WANG Z, ZHANG Q Y, SONG H D. Immune cell profiling of patients with COVID-19 with mild, severe and prognosis states revealed by single-cell RNA sequencing[J]. Fudan University Journal of Medical Sciences, 2022, 49(2): 213-225.
16	LEE J S, PARK S, JEONG H W, et al. Immunophenotyping of COVID-19 and influenza highlights the role of type I interferons in development of severe COVID-19[J]. Sci Immunol, 2020, 5(49): eabd1554.
17	AEGERTER H, LAMBRECHT B N, JAKUBZICK C V. Biology of lung macrophages in health and disease[J]. Immunity, 2022, 55(9): 1564-1580.
18	CHEN X X, TANG J, SHUAI W Z, et al. Macrophage polarization and its role in the pathogenesis of acute lung injury/acute respiratory distress syndrome[J]. Inflamm Res, 2020, 69(9): 883-895.
19	CHEN Q, SHAO X B, HE Y Y, et al. Norisoboldine attenuates sepsis-induced acute lung injury by modulating macrophage polarization via PKM2/HIF-1α/PGC-1α pathway[J]. Biol Pharm Bull, 2021, 44(10): 1536-1547.
20	LI W F, ZHAO R Q, WANG X M, et al. Nobiletin-ameliorated lipopolysaccharide-induced inflammation in acute lung injury by suppression of NF-κB pathway in vivo and vitro[J]. Inflammation, 2018, 41(3): 996-1007.
21	SIMARD M, RAKOTOARIVELO V, MARZO V D, et al. Expression and functions of the CB2 receptor in human leukocytes[J]. Front Pharmacol, 2022, 13: 826400.
22	KIENZL M, KARGL J, SCHICHO R. The immune endocannabinoid system of the tumor microenvironment[J]. Int J Mol Sci, 2020, 21(23): 8929.
23	TIAN L, LI W Y, YANG L, et al. Cannabinoid receptor 1 participates in liver inflammation by promoting M1 macrophage polarization via RhoA/NF-κB p65 and ERK1/2 pathways, respectively, in mouse liver fibrogenesis[J]. Front Immunol, 2017, 8: 1214.
24	LOUVET A, TEIXEIRA-CLERC F, CHOBERT M N, et al. Cannabinoid CB2 receptors protect against alcoholic liver disease by regulating Kupffer cell polarization in mice[J]. Hepatology, 2011, 54(4): 1217-1226.
25	CHHATRIWALA M K, BETTS L, WORTHYLAKE D K, et al. The DH and PH domains of Trio coordinately engage Rho GTPases for their efficient activation[J]. J Mol Biol, 2007, 368(5): 1307-1320.
26	SIT S T, MANSER E. Rho GTPases and their role in organizing the actin cytoskeleton[J]. J Cell Sci, 2011, 124(Pt 5): 679-683.

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

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

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