目的 探究藏药十八味党参丸(TEP)的提取物调控脂多糖(LPS)诱导小鼠巨噬细胞RAW 264.7的炎性反应及M1/M2极化分型的探究。方法 Alarmarblue法评价RAW 264.7活力;荧光酶标定量分析细胞活性氧(reactive oxygen species,ROS) 水平;Griess法检测上清液中细胞一氧化氮(NO)的分泌情况;流式细胞术检测表面标志物CD206和CCR7蛋白的表达;RT-qPCR法检测细胞白介素(IL)-1β、IL-6、趋化因子2(CCL2)、一氧化氮合酶(iNOS)、精氨酸酶(ARG)-1和肿瘤坏死因子α(TNF-α)基因表达的情况;Western blot法检测TEP调控细胞合成iNOS、蛋白激酶B(Akt)和p38丝裂原活化激酶(p38MAPK)信号通路相关蛋白的表达情况。结果 与模型组相比,TEP组一定程度上提高了RAW 264.7的细胞活性,同时抑制了LPS诱导的细胞内ROS水平的升高。经过LPS刺激后,模型组NO表达量显著升高,在24 h后表达量达到最高,而TEP组NO表达显著降低。模型组的CCR7表达升高,CD206表达下降,TEP干预后CCR7表达下降,CD206表达升高,且趋势呈现浓度依赖。与模型组相比, TEP 组的IL-1β、IL-6、CCL2、iNOS、ARG和TNF-α基因表达以及iNOS、Akt 和p38MAPK蛋白表达水平均显著降低。结论 TEP能够抑制细胞内ROS水平,降低NO分泌和炎性基因的表达水平,抑制细胞的M1表型,促进M2表型,其机制可能与细胞内iNOS、Akt和p38MAPK信号通路蛋白表达被抑制有关。
Abstract
OBJECTIVE To explore the effect and mechanism of Tibetan Eighteen flavor Dangshen Pills (TEP) in regulating lipopolysaccharide (LPS) induced inflammatory response in macrophages RAW264.7. METHODS Alarmarblue assay was used to evaluate the effect of TEP on the viability of RAW264.7. The reactive oxygen species (ROS) level in RAW264.7 induced by LPS was quantified by fluorescence microplate. Griess assay was used to detect the secretion of nitric oxide (NO), and the surface markers CCR7 and CD206 of M1/M2 phenotype were detected by flow cytometer. RT-qPCR method was used to detect gene expression of IL-1β, IL-6, CCL2, iNOS, ARG-1 and TNF-α. Western blot was used to detect the expression of iNOS, pAkt, p38MAPK and p65NF-κB signal pathway related protein. RESULTS The viability of RAW264.7 cells induced by two dose groups was significantly improved compared with the model group. Meanwhile, the intracellular ROS level induced by LPS IN RAW264.7 cells was inhibited via the addition of TEP. The secretion of NO in LPS group increased significantly, reaching the highest expression after 24 hours, but that in two dose groups decreased significantly. The surface marker CCR7 of M1 pro-inflammatory phenotype was increased in RAW264.7 induced by LPS, however the addition of TEP decreased the expression of CCR7, and increased the expression of surface marker CD206 of anti-inflammatory M2 phenotype. The gene expression of IL-1β, IL-6, CCL2, iNOS, ARG and TNF-α in two dose groups was significantly reduced. The protein expression levels of iNOS, pAkt and p38MAPK in two dose groups were significantly reduced, however the expression levels of p65NF-κB were significantly increased. CONCLUSION TEP can suppress the ROS expression level, reduce cellular NO secretion and inflammatory gene expression of RAW264.7 cells, and inhibit M1 pro-inflammatory phenotype and promote M2 anti-inflammatory phenotype of macrophages. The mechanism may be related to the inhibition of iNOS, pAkt and p38MAPK signal pathway protein expression.
关键词
藏药十八味党参丸 /
巨噬细胞 /
炎性反应 /
M1/M2极化 /
Akt/p38MAPK信号通路
{{custom_keyword}} /
Key words
Tibetan Eighteen flavor Dangshen Pill /
macrophage /
inflammation /
M1/M2 polarization /
pAkt/p38MAPK signal pathway
{{custom_keyword}} /
中图分类号：
R965
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] ZHU Y M, QI G, YANG C Y, et al. An experimental study on the apoptosis of synovial cells and the expression of inflammatory factor. Chin High Alt Med Biol (中国高原医学与生物学杂志), 2017, 38(4):273-279.
[2] LIN H, CAO Y L, DING J X. Effect of saposhnikovan sulphate on the recruitment and reeducation of macrophages by MCF-7 cells in vitro through CCL3. Chin Pharm J(中国药学杂志), 2018, 53(20):1755-1761.
[3] HUANG X, LI Y, LI X, et al. TRIM14 promotes endothelial activation via activating NF-κB signaling pathway. J Mol Cell Biol, 2020, 12(3):176-189.
[4] HUANG N Q, JIN H, SHI J S, et al. Research progress of resveratrol inhibiting microglial activation in neuroinflammation. Chin Pharm J(中国药学杂志), 2018, 53(2):85-91.
[5] Y L Y A B, M L A B, X C A B, et al. Myricitrin blocks activation of NF-κB and MAPK signaling pathways to protect nigrostriatum neuron in LPS-stimulated mice. J Neuroimmunol,2019, 337:577049.
[6] KATTOOR A J, POTHINENI N V K, PALAGIRI D, et al. Oxidative stress in atherosclerosis. Curr Atheroscler Rep, 2017, 19(11):42.
[7] SIES H. Glutathione and its role in cellular functions. Free Radic Biol Med, 1999, 27(9-10):916-921.
[8] CAI H H, YANG F, FENG X E, et al. Effect of an active polyphenol compound LM49 on polarization of RAW264.7 macrophages. Chin Pharm J(中国药学杂志), 2020, 55(6):432-437.
[9] GUO L, HAN C Y. Role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) in hepatoma cell-induced M2 polarization of macrophages. Chin J Microbiol Immunol(中华微生物学和免疫学杂志), 2020, 40(12):934-940.
[10] ZHANG L. Investigation on the role of CCL2 and its signaling pathway in the pathological mechanism of rheumatoid arthritis and periodontitis. Shandong Med J(山东医药), 2020, 1165(7):103-107.
[11] CLOOTS R H E, SANKARANARAYANAN S, POYNTER M E, et al. Arginase 1 deletion in myeloid cells affects the inflammatory response in allergic asthma, but not lung mechanics, in female mice. BMC Pulm Med, 2017, 17(1):158.
[12] LARSON-CASEY J L, DESHANE J S, RYAN A J,et al. Macrophage Akt1 kinase-mediated mitophagy modulates apoptosis resistance and pulmonary fibrosis. Immunity, 2016, 44(3):582-596.
[13] HADRUP N, SHARMA A K, POULSEN M, et al. Toxicological risk assessment of elemental gold following oral exposure to sheets and nanoparticles. Regul Toxicol Pharmacol, 2015, 72(2):216-221.
[14] HUANG J, ZHANG Y, YAN J, et al. Immunomodulatory effect of dendrobium officinale polysaccharide on macrophage RAW264.7 induced by LPS. Chin Pharm J(中国药学杂志), 2017, 52(7):548-552.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
国家重点研发计划项目资助(2016YFC1102000);四川省苗子工程项目资助(2019076)
{{custom_fund}}
PDF(1594 KB)
