Role of activated cannabinoid receptor 2 in lipopolysaccharide-induced secretion of RAW264.7 macrophage inflammatory cytokines and its possible mechanism

Q4 Nursing

中华急诊医学杂志 Pub Date : 2019-08-10 DOI:10.3760/CMA.J.ISSN.1671-0282.2019.08.012

Q. Yuan, Anpeng Liu, Qiangsheng Liu, Fei Zheng, Yanlin Wang, Zongze Zhang

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引用次数: 0

Abstract

Objective To investigate the role of activated cannabinoid receptor 2 (CB2R) in lipopolysaccharide (LPS)-induced secretion of RAW264.7 macrophage inflammatory cytokines and its possible mechanism. Methods Macrophages were seeded in 6-well plates (2 mL/well) at the density of 1×105 cells/mL and randomly divided into four groups (n=6 each group): control group (group C), LPS group (group LPS), LPS plus CB2R agonist HU308 group (group LPS+HU308), and LPS plus HU308 plus 3-Methyladenine group (group LPS+HU308+3-MA). LPS with the final concentration of 1 μg/mL were added in group LPS, group LPS+HU308 and group LPS+HU308+3-MA. After incubation for 15 min, 3-MA with a final concentration of 10 mmol/L was added into group LPS+HU308+3-MA . HU308 with the final concentration of 10 μmol/L was added in group LPS+HU308 and group LPS+HU308+3-MA at 15 min after 3-MA intervention, and the cells were then incubated for 24 h. The concentrations of TNF-α, IL-18 and IL-1β in supernatant serum of each group were determined by ELISA. The expressions of ICAM-1 and NLRP3 mRNA were detected by RT-PCR. The expressions of LC3 and Beclin1 were detected by Western blot, and the ratio of LC3-II/LC3-I was calculated. LSD-t test was used for sample pairwise comparison, and one way ANOVA for inter-group comparison. A P<0.05 was considered statistically significant. Results Compared with group C, the concentrations of TNF-α [(228.86±10.20) pg/mL vs (140.05±5.54) pg/mL], IL-1β [(363.62±8.14) pg/mL vs (244.82±9.11) pg/mL], and IL-18 [(293.28±13.57) pg/mL vs (202.84±9.54) pg/mL] in supernatant serum were increased (all P<0.05), the expressions of ICAM-1 [(5.88±0.32) vs (1.00±0.03)] and NLRP3 [(8.07±0.93) vs (1.01±0.05)] mRNA were increased (all P<0.05), the expressions of LC3-II/LC3-I ratio [(0.50±0.03) vs (0.40±0.06)] and Beclin1 [(0.51±0.04) vs (0.16±0.03)] were up-regulated in group LPS (all P<0.05). Compared with group LPS, the concentrations of TNF-α [(165.44±7.07) pg/mL], IL-1β [(272.09±3.35) pg/mL] and IL-18 [(220.41±6.01) pg/mL] in supernatant serum were significantly decreased (all P<0.05), the expressions of ICAM-1 [(3.21±0.35)] and NLRP3 [(1.54±0.30)] mRNA were decreased (all P<0.05), the expressions of LC3-II/LC3-I ratio [(0.71±0.03)] and Beclin1 [(0.71±0.02)] were up-regulated in group LPS+HU308 (all P<0.05). Compared with group LPS+HU308, the concentrations of TNF-α [(197.06±5.59) pg/mL], IL-1β [(318.98±11.54) pg/mL] and IL-18 [(243.33±8.71) pg/mL] in supernatant serum were significantly increased (all P<0.05), the expressions of ICAM-1 [(4.04±0.21)] and NLRP3 [(5.87±0.77)] mRNA were increased (all P<0.05), the expressions of LC3-II/LC3-I ratio [(0.44±0.08)] and Beclin1 [(0.32±0.03)] were down-regulated in group LPS+HU308+3-MA (all P<0.05). Conclusions Activation of cannabinoid receptor 2 can alleviate LPS-induced the secretion of RAW264.7 macrophage inflammatory cytokines, and its mechanism may be related to enhanced autophagy. Key words: Cannabinoid receptor 2; LPS; Inflammatory cytokines; Secretion; Immunoregulation; Raw264.7; Macrophage; Autophagy; NLRP3

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活化大麻素受体2在脂多糖诱导巨噬细胞RAW264.7炎症因子分泌中的作用及其可能机制

目的探讨活化大麻素受体2（CB2R）在脂多糖（LPS）诱导巨噬细胞分泌RAW264.7炎性细胞因子中的作用及其可能机制。方法将巨噬细胞以1×105细胞/mL的密度接种在6孔板（2mL/孔）中，随机分为4组（每组n=6）：对照组（C组）、LPS组（LPS组）、LPS+CB2R激动剂HU308组（LPS+HU308组）和LPS+HU308加3-甲基腺嘌呤组（LPP+HU308+3-MA组）。LPS组、LPS+HU308组和LPS+HU308+3-MA组分别加入终浓度为1μg/mL的LPS。孵育15分钟后，将最终浓度为10mmol/L的3-MA加入LPS+HU308+3-MA组。LPS+HU308组和LPS+HU308+3-MA组在3-MA干预后15min加入终浓度为10μmol/L的HU308，孵育24h。用ELISA法测定各组上清液中TNF-α、IL-18和IL-1β的浓度。RT-PCR检测ICAM-1和NLRP3mRNA的表达。用蛋白质印迹法检测LC3和Beclin1的表达，并计算LC3-II/LC3-I的比值。样本配对比较采用LSD-t检验，组间比较采用单向方差分析。P＜0.05被认为具有统计学意义。结果与C组相比，上清液中TNF-α[（228.86±10.20）pg/mLvs（140.05±5.54）pg/mL]、IL-1，LPS组ICAM-1[（5.88±0.32）vs（1.00±0.03）]和NLRP3[（8.07±0.93）vs（1.01±0.05）]mRNA表达增加（均P<0.05），LPS+HU308组上清液中IL-1β[（272.09±3.35）pg/mL]和IL-18[（220.41±6.01）pg/mL]显著降低（均P<0.05），ICAM-1[（3.21±0.35）]和NLRP3[（1.54±0.30）]mRNA表达降低（均<0.05），上清液中TNF-α[（197.06±5.59）pg/mL]、IL-1，结论激活大麻素受体2可减轻LPS诱导的巨噬细胞分泌RAW264.7炎性细胞因子，其机制可能与增强自噬有关。关键词：大麻素受体2；LPS；炎症细胞因子；分泌物；免疫调节；Raw264.7；巨噬细胞；自噬；NLRP3

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来源期刊

中华急诊医学杂志 Nursing-Emergency Nursing

CiteScore

0.10

自引率

0.00%

发文量

8629

期刊介绍： Chinese Journal of Emergency Medicine is the only national journal which represents the development of emergency medicine in China. The journal is supervised by China Association of Science and Technology, sponsored by Chinese Medical Association, and co-sponsored by Zhejiang University. The journal publishes original research articles dealing with all aspects of clinical practice and research in emergency medicine. The columns include Pre-Hospital Rescue, Emergency Care, Trauma, Resuscitation, Poisoning, Disaster Medicine, Continuing Education, etc. It has a wide coverage in China, and builds up communication with Hong Kong, Macao, Taiwan and international emergency medicine circles.