四逆散对脂多糖诱导的RAW264.7细胞极化的影响
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摘要
目的:探讨四逆散(Sinisan,SNS)对脂多糖(LPS)诱导的RAW264. 7巨噬细胞极化的调控作用。方法:RAW264. 7分为5组,分别为空白组,模型组,SNS低、中、高质量浓度组(10,20,40 mg·L~(-1));以LPS(100μg·L~(-1))刺激的RAW264. 7细胞为体外模型,使用不同质量浓度的SNS提前干预细胞,噻唑蓝(MTT)比色法检测不同质量浓度SNS对RAW264. 7细胞增殖的影响;光学显微镜下观察各组细胞分化程度;酶联免疫吸附测定(ELISA)检测细胞培养基上清中M1极化因子肿瘤坏死因子-α(TNF-α),白细胞介素-6(IL-6),白细胞介素-1β(IL-1β)以及M2极化因子白细胞介素-10(IL-10)的含量;实时荧光定量聚合酶链式反应(Real-time PCR)检测RAW264. 7细胞M1极化因子TNF-α,IL-6以及M2极化因子IL-10,精氨酸酶-1(Arg~(-1))的mRNA水平。结果:与空白组比较,模型组促进细胞增殖(P <0. 05),刺激M1极化因子TNF-α,IL-6,IL-1β的释放和上调TNF-α,IL-6的mRNA含量(P <0. 01),减少M2极化因子IL-10释放和IL-10,Arg~(-1)的mRNA水平(P <0. 05,P <0. 01)。与空白组比较,SNS对RAW264. 7细胞的活性没有影响。与模型组比较,SNS可抑制LPS诱导的细胞增殖(P <0. 05),减少LPS刺激的细胞分化,减少M1极化因子TNF-α,IL-6,IL-1β的释放和TNF-α,IL-6的mRNA含量(P <0. 05,P <0. 01),并增加M2极化因子IL-10释放和IL-10,Arg~(-1)的mRNA水平(P <0. 05,P <0. 01)。结论:SNS可抑制LPS诱导的RAW264. 7细胞炎症,其机制可能与调控巨噬细胞M1/M2表型极化平衡相关。
Objective: To investigate the regulatory effect of Sinisan( SNS) on the polarization of RAW264. 7 macrophages induced by lipopolysaccharide( LPS). Method: RAW264. 7 cells stimulated by LPS were used as the in vitro model. The cells were intervened with the different concentrations of SNS in advance. The effects of different concentrations of SNS on the proliferation of RAW264. 7 cells were detected by methyl thiazolyl tetrazolium( MTT) colorimetry. The degree of cell differentiation was detected by enzyme-linked immuno sorbent assay( ELISA) method. The contents of M1 polarization factors tumor necrosis factor-α( TNF-α),interleukin-6( IL-6),interleukin~(-1)β( IL-1β) and M2 polarization factors interleukin~(-1)0( IL-10) in cell culture supernatant were detected by ELISA method,mRNA levels of M1 polarization factors TNF-α,IL-6 and M2 polarization factors IL-10,arginase-1( Arg~(-1)) were detected by Real-time fluorescence quantitative polymerase chain reaction( Realtime PCR) method. Result: SNS had no effect on the cell viability of RAW264. 7 cells,inhibited LPS-induced cell proliferation,decreased LPS-stimulated cell differentiation,down-regulated M1 polarizing factors TNF-α,IL-6,IL-1β release and TNF-α,IL-6 mRNA levels,and increased the release of IL-10 and mRNA levels of IL-10 and Arg~(-1). Conclusion: SNS inhibits the inflammation of RAW264. 7 cells induced by LPS,and its mechanism may be related to the regulation of polarization balance of M1/M2 macrophages.
Objective: To investigate the regulatory effect of Sinisan( SNS) on the polarization of RAW264. 7 macrophages induced by lipopolysaccharide( LPS). Method: RAW264. 7 cells stimulated by LPS were used as the in vitro model. The cells were intervened with the different concentrations of SNS in advance. The effects of different concentrations of SNS on the proliferation of RAW264. 7 cells were detected by methyl thiazolyl tetrazolium( MTT) colorimetry. The degree of cell differentiation was detected by enzyme-linked immuno sorbent assay( ELISA) method. The contents of M1 polarization factors tumor necrosis factor-α( TNF-α),interleukin-6( IL-6),interleukin~(-1)β( IL-1β) and M2 polarization factors interleukin~(-1)0( IL-10) in cell culture supernatant were detected by ELISA method,mRNA levels of M1 polarization factors TNF-α,IL-6 and M2 polarization factors IL-10,arginase-1( Arg~(-1)) were detected by Real-time fluorescence quantitative polymerase chain reaction( Realtime PCR) method. Result: SNS had no effect on the cell viability of RAW264. 7 cells,inhibited LPS-induced cell proliferation,decreased LPS-stimulated cell differentiation,down-regulated M1 polarizing factors TNF-α,IL-6,IL-1β release and TNF-α,IL-6 mRNA levels,and increased the release of IL-10 and mRNA levels of IL-10 and Arg~(-1). Conclusion: SNS inhibits the inflammation of RAW264. 7 cells induced by LPS,and its mechanism may be related to the regulation of polarization balance of M1/M2 macrophages.
引文
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[3]陈伟伟,高润霖,刘力生,等.《中国心血管病报告2017》概要[J].中国循环杂志,2018,33(1):1-8.
[4] de Gaetano M,Crean D,Barry M,et al. M1-and M2-type macrophage responses are predictive of adverse outcomes in human atherosclerosis[J]. Front Immunol,2016,doi:10. 3389/fimmu. 2016. 00275.
[5] Moore K J,Sheedy F J,Fisher E A. Macrophages in atherosclerosis:a dynamic balance[J]. Nat Rev Immunol,2013,13(10):709-721.
[6] Shapouri-Moghaddam A,Mohammadian S,Vazini H,et al. Macrophage plasticity,polarization,and function in health and disease[J]. J Cell Physiol,2018,233(9):6425-6440.
[7] Sica A,Erreni M, Allavena P, et al. Macrophage polarization in pathology[J]. Cell Mol Life Sci,2015,72(21):4111-4126.
[8] FAN H J,XIE Z P,LU Z W,et al. Anti-inflammatory and immune response regulation of Si-Ni-San in 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin dysfunction[J]. J Ethnopharmacol,2018,222:1-10.
[9] JIANG J,ZHOU C,XU Q. Alleviating effects of si-nisan,a traditional Chinese prescription,on experimental liver injury and its mechanisms[J]. Biol Pharm Bull,2003,26(8):1089-1094.
[10] Ohta Y,Kobayashi T,Hayashi T,et al. Preventive effect of Shigyaku-san on progression of acute gastric mucosal lesions induced by compound 48/80,a mast cell degranulator,in rats[J]. Phytother Res,2006,20(4):256-262.
[11] Tanaka M,Satou T,Koike K. Anxiolytic-like effect of Shigyakusan extract with low side effects in mice[J]. J Nat Med,2013,67(4):862-866.
[12]张婧娴,芮俊乾,陈逸凡,等.四逆散有效成分对内脏高敏感大鼠5-HT信号通路的多靶点协同调控[J].中国实验方剂学杂志,2018,24(16):115-123.
[13] Lozano R,Naghavi M,Foreman K,et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010:a systematic analysis for the Global Burden of Disease Study 2010[J]. Lancet,2012,380(9859):2095-2128.
[14] YANG S,LI J, CHEN Y, et al. MicroRNA-216a promotes M1 macrophages polarization and atherosclerosis progression by activating telomerase via the Smad3/NF-kappaB pathway[J]. Biochim Biophys Acta Mol Basis Dis, 2018, doi:10. 1016/j.bbadis. 2018. 06. 016.
[15] WEI Y,ZHU M,Schober A. Macrophage MicroRNAs as therapeutic targets for atherosclerosis, metabolic syndrome,and cancer[J]. Int J Mol Sci,2018,doi:10. 3390/ijms19061756.
[16] Peled M,Fisher E A. Dynamic aspects of macrophage polarization during atherosclerosis progression and regression[J]. Front Immunol,2014,doi:10. 3389/fimmu. 2014. 00579.
[17]陈亚双,孙世伟.柴胡的化学成分及药理作用研究进展[J].黑龙江医药,2014,27(3):630-633.
[18]张霄潇,李正勇,马玉玲,等.中药枳实的研究进展[J].中国中药杂志,2015,40(2):185-190.
[19]吴修红,胡妮娜,李宝龙,等.赤芍与白芍脐中穴给药的药理作用比较研究[J].针灸临床杂志,2014,30(5):54-56.
[20]王颖,韩秀萍.甘草酸苷作用机制的研究进展[J].实用药物与临床,2018,21(1):109-113.