基于NF-κB通路的芒果苷干预脂多糖诱导的细胞炎症作用机制研究
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摘要
目的研究芒果苷对小鼠巨噬细胞RAW 264.7的保护作用,并探讨其可能的作用机制。方法采用脂多糖(LPS)诱导RAW 264.7细胞炎症模型。实验分四步:(1)细胞存活率实验分5组,即:空白组、LPS组、LPS+不同浓度(50、100、150μg/mL)芒果苷组。MTT法检测细胞存活率。(2)一氧化氮(NO)、白介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)及白介素6(IL-6)分泌实验分6组,即:空白组、LPS组、LPS和不同浓度芒果苷(50、100、150μg/mL)组、LPS+BAY 11-7082[核因子κB(NF-κB)抑制剂]组。Griess法检测NO分泌量,ELISA法检测IL-1β、TNF-α及IL-6分泌量。(3)诱导型一氧化氮合酶(iNOS)与环氧化酶-2(COX-2)mRNA及蛋白表达实验分组同细胞存活率实验。实时荧光定量PCR检测iNOS及COX-2 mRNA水平变化,Western blot检测iNOS及COX-2蛋白表达水平。(4)细胞质、细胞核中NF-κB表达实验分3组,即:空白组、LPS组、LPS+150μg/mL(终末浓度)芒果苷共处理组, Western blot检测细胞质、细胞核中NF-κB p65蛋白表达量。结果 (1)与空白组比较,LPS组显著降低RAW264.7细胞存活率(P<0.05);与LPS组比较,50～150μg/mL芒果苷预处理对RAW264.7细胞增殖无显著影响(P>0.05)。(2)与空白组比较,LPS组NO、IL-1β、TNF-α、IL-6分泌量显著升高(P<0.05);与LPS组比较,除50μg/mL芒果苷预处理组NO、TNF-α分泌量无显著变化外(P>0.05),其余各组NO、IL-1β、TNF-α、IL-6分泌量均显著降低(P<0.05)。(3)与空白组比较,LPS组iNOS、COX-2 mRNA及蛋白表达量均显著升高(P<0.05);与LPS组比较,50～150μg/mL芒果苷预处理组iNOS、COX-2 mRNA及蛋白表达显著下降(P<0.05)。(4)与空白组比较,LPS组细胞质中NF-κB p65蛋白量减少,细胞核中则增多(P<0.05);与LPS组比较,150μg/mL芒果苷预处理组细胞质中NF-κB p65蛋白量升高,细胞核中蛋白量减少(P<0.05)。结论芒果苷可能通过抑制NF-κB信号通路,而抑制iNOS、COX-2表达及相关炎症因子的分泌,干预LPS诱导的RAW264.7细胞炎症。
Objective To study the protective effect and mechanism of mangiferin on inflammation induced by lipopolysaccharide(LPS) in RAW264.7 cells. Methods RAW264.7 cell inflammation model was induced by LPS. The experiment was divided into four steps:(1) Cell viability experiment was conducted. RAW264.7 cells were divided into 5 groups, i.e., blank group, LPS group, LPS+different concentrations(50, 100, 150 μg/mL) mangiferin groups. The cell viability was measured by MTT assay.(2) Nitric oxide(NO), IL-1β, tumor necrosis factor alpha(TNF-α), and IL-6 secretion experiments were conducted. Cells were divided into 6 groups, i.e., blank group, LPS treatment group, LPS+different concentrations of mangiferin(50, 100, 150 μg/mL) groups, LPS+BAY 11-7082(NF-κB inhibitor) group. Detection of NO secretion was detected by Griess assay, and the secretions of IL-1β, TNF-α, and IL-6 were detected by ELISA.(3) Inducible nitric oxide synthase(iNOS) and cyclooxygenase-2(COX-2) mRNA and protein expression experiments were conducted. The groups were divided in the same way as that in the cell viability experiments. Real-time quantitative PCR was used to detect the changes of iNOS and COX-2 mRNA levels. And the expression levels of iNOS and COX-2 protein were detected by Western blot.(4) The expressions of nuclear factor-kappaB(NF-κB) in cytoplasm and nucleus were detected. The cells were divided into three groups, i.e., blank group, LPS treatment group, LPS+150 μg/mL(final concentration) mangiferin co-treatment group. The expressions of NF-κB p65 protein in cytoplasm and nucleus were detected by Western blot. Results(1) Compared with the blank group, the survival rate of RAW264.7 cells was significantly reduced in the LPS group(P<0.05). Compared with the LPS group, 50-150 μg/mL mangiferin pretreatment had no significant effect on the proliferation of RAW264.7 cells(P>0.05).(2) Compared with the blank group, the secretion levels of NO, IL-1β, TNF-α, and IL-6 were significantly increased in the LPS group(P<0.05). Compared with the LPS group, the secretion levels of NO and TNF-α in the 50 μg/mL mangiferin pretreatment group had no significant changes(P>0.05), but the secretion of NO, IL-1β, TNF-α, and IL-6 significantly decreased in the rest groups(P<0.05).(3) Compared with the blank group, the expression levels of iNOS and COX-2 mRNA and protein were significantly increased in the LPS group(P<0.05). Compared with the LPS group, pretreatment with 50-150 μg/mL mangiferin reduced iNOS and COX-2 mRNA and protein expression levels(P<0.05).(4) Compared with the blank group, the NF-κB p65 protein was significantly decreased in the cytoplasm and increased in the nucleus in the LPS group(P<0.05). Compared with the LPS group, 150 μg/mL mangiferin pretreatment increased the NF-κB p65 protein in the cytoplasm, and decreased it in the nucleus(P<0.05). Conclusion Mangiferin intervened LPS-induced RAW 264.7 cell inflammation possibly by inhibiting NF-κB signaling pathway, thus further inhibiting the expression levels of iNOS/COX-2 and the secretion of related inflammatory factors.
Objective To study the protective effect and mechanism of mangiferin on inflammation induced by lipopolysaccharide(LPS) in RAW264.7 cells. Methods RAW264.7 cell inflammation model was induced by LPS. The experiment was divided into four steps:(1) Cell viability experiment was conducted. RAW264.7 cells were divided into 5 groups, i.e., blank group, LPS group, LPS+different concentrations(50, 100, 150 μg/mL) mangiferin groups. The cell viability was measured by MTT assay.(2) Nitric oxide(NO), IL-1β, tumor necrosis factor alpha(TNF-α), and IL-6 secretion experiments were conducted. Cells were divided into 6 groups, i.e., blank group, LPS treatment group, LPS+different concentrations of mangiferin(50, 100, 150 μg/mL) groups, LPS+BAY 11-7082(NF-κB inhibitor) group. Detection of NO secretion was detected by Griess assay, and the secretions of IL-1β, TNF-α, and IL-6 were detected by ELISA.(3) Inducible nitric oxide synthase(iNOS) and cyclooxygenase-2(COX-2) mRNA and protein expression experiments were conducted. The groups were divided in the same way as that in the cell viability experiments. Real-time quantitative PCR was used to detect the changes of iNOS and COX-2 mRNA levels. And the expression levels of iNOS and COX-2 protein were detected by Western blot.(4) The expressions of nuclear factor-kappaB(NF-κB) in cytoplasm and nucleus were detected. The cells were divided into three groups, i.e., blank group, LPS treatment group, LPS+150 μg/mL(final concentration) mangiferin co-treatment group. The expressions of NF-κB p65 protein in cytoplasm and nucleus were detected by Western blot. Results(1) Compared with the blank group, the survival rate of RAW264.7 cells was significantly reduced in the LPS group(P<0.05). Compared with the LPS group, 50-150 μg/mL mangiferin pretreatment had no significant effect on the proliferation of RAW264.7 cells(P>0.05).(2) Compared with the blank group, the secretion levels of NO, IL-1β, TNF-α, and IL-6 were significantly increased in the LPS group(P<0.05). Compared with the LPS group, the secretion levels of NO and TNF-α in the 50 μg/mL mangiferin pretreatment group had no significant changes(P>0.05), but the secretion of NO, IL-1β, TNF-α, and IL-6 significantly decreased in the rest groups(P<0.05).(3) Compared with the blank group, the expression levels of iNOS and COX-2 mRNA and protein were significantly increased in the LPS group(P<0.05). Compared with the LPS group, pretreatment with 50-150 μg/mL mangiferin reduced iNOS and COX-2 mRNA and protein expression levels(P<0.05).(4) Compared with the blank group, the NF-κB p65 protein was significantly decreased in the cytoplasm and increased in the nucleus in the LPS group(P<0.05). Compared with the LPS group, 150 μg/mL mangiferin pretreatment increased the NF-κB p65 protein in the cytoplasm, and decreased it in the nucleus(P<0.05). Conclusion Mangiferin intervened LPS-induced RAW 264.7 cell inflammation possibly by inhibiting NF-κB signaling pathway, thus further inhibiting the expression levels of iNOS/COX-2 and the secretion of related inflammatory factors.
引文
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[13] Guo HW,Yun CX,Hou GH,et al.Mangiferin attenuates TH1/TH2 cytokine imbalance in an ovalbumin-induced asthmatic mouse model[J].PLoS One,2014,9(6):e100394.
[14] Prabhu S,Jainu M,Sabitha KE,et al.Effect of mangiferin on mitochondrial energy production in experimentally induced myocardial infarcted rats[J].Vascul Pharmacol,2006,44(6):519-525.
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[16] Natarajan K,Abraham P,Kota R,et al.NF-κB-iNOS-COX2-TNF-α inflammatory signaling pathway plays an important role in methotrexate induced small intestinal injury in rats[J].Food Chem Toxicol,2018,118(1):766-783.
[17] Sang W,Zhong Z,Linghu K,et al.Siegesbeckia pubescens Makino inhibits Pam3CSK4-induced inflammation in RAW 264.7 macrophages through suppressing TLR1/TLR2-mediated NF-κB activation[J].Chin Med,2018,13(37):1-10.
[18] 王晓晨,吉爱国.NF-κB信号通路与炎症反应[J].生理科学进展,2014,45(1):68-71.
[19] Kang SR,Han DY,Park KI,et al.Suppressive effect on lipopolysaccharide-induced proinflammatory mediators by Citrus aurantium L.in macrophage RAW 264.7 cells via NF-κB signal pathway[J].Evid Based Complement Alternat Med,2011,2011(1):1-12.
[20] Park KI,Kang SR,Park HS,et al.Regulation of proinflammatory mediators via NF-κB and p38 MAPK-dependent mechanisms in RAW 264.7 macrophages by polyphenol components isolated from Korea Lonicera japonica THUNB[J].Evid Based Complement Alternat Med,2012,2012(1):1-10.
[21] Lee MS,Kwon MS,Choi JW,et al.Anti-inflammatory activities of an ethanol extract of Ecklonia stolonifera in lipopolysaccharide-stimulated RAW 264.7 murine macrophage cells[J].J Agric Food Chem,2012,60(36):9120-9129.
[2] 黄云,胡文姬,李学坚,等.芒果苷近10年研究进展[J].中药药理与临床,2015,31 (1):269–274.
[3] 王志萍,邓家刚,谭珍媛.芒果苷片体外抑菌杀虫作用的实验研究[J].时珍国医国药,2009,20(9):2167-2168.
[4] 郭宏伟,邓家刚,运晨霞,等.芒果苷抑制哮喘小鼠气道炎症的机制[J].中国实验方剂学杂志,2012,18(9):187-190.
[5] 谭之强.芒果苷通过抑制氧化应激及抗凋亡作用保护大鼠颅脑损伤[J].神经损伤与功能重建,2018,13(1):26-28.
[6] 吴巍,滕厚雷.小檗碱与芒果苷及其配伍对L6 成纤维细胞葡萄糖摄取的影响及机制[J].中国药理学通报,2010,26(9):1259-1260.
[7] Dinh QN,Drummond GR,Sobey CG,et al.Roles of inflammation,oxidative stress,and vascular dysfunction in hypertension[J].Biomed Res Int,2014,2014(1):406960.
[8] Lontchi-Yimagou E,Sobngwi E,Matsha TE,et al.Diabetes mellitus and inflammation[J].Curr Diabetes Rep,2013,13(3):435-444.
[9] Hoenerhoff MJ.Inflammation and cancer:Partners in crime[J].Vet J,2015,206(1):1-2.
[10] Yang C,Liu P,Wang S,et al.Shikonin exerts anti-inflammatory effects in LPS-induced mastitis by inhibiting NF-κB signaling pathway[J].Biochem Biophys Res Commun,2018,505(1):1-6.
[11] Hou J,Gu Y,Zhao S,et al.Anti-inflammatory Effects of Aurantio-Obtusin from seed of Cassia obtusifolia L.through modulation of the NF-κB pathway[J].Molecules,2018,23(12):E3093.
[12] 黄云,胡文姬,李学坚,等.芒果苷近10年研究进展[J].中药药理与临床,2015,31(1):269-274.
[13] Guo HW,Yun CX,Hou GH,et al.Mangiferin attenuates TH1/TH2 cytokine imbalance in an ovalbumin-induced asthmatic mouse model[J].PLoS One,2014,9(6):e100394.
[14] Prabhu S,Jainu M,Sabitha KE,et al.Effect of mangiferin on mitochondrial energy production in experimentally induced myocardial infarcted rats[J].Vascul Pharmacol,2006,44(6):519-525.
[15] Morais TC,Arruda BR,de Sousa Magalh?es H,et al.Mangiferin ameliorates the intestinal inflammatory response and the impaired gastrointestinal motility in mouse model of postoperative ileus[J].Naunyn Schmiedebergs Arch Pharmacol,2015,388(5):531-538.
[16] Natarajan K,Abraham P,Kota R,et al.NF-κB-iNOS-COX2-TNF-α inflammatory signaling pathway plays an important role in methotrexate induced small intestinal injury in rats[J].Food Chem Toxicol,2018,118(1):766-783.
[17] Sang W,Zhong Z,Linghu K,et al.Siegesbeckia pubescens Makino inhibits Pam3CSK4-induced inflammation in RAW 264.7 macrophages through suppressing TLR1/TLR2-mediated NF-κB activation[J].Chin Med,2018,13(37):1-10.
[18] 王晓晨,吉爱国.NF-κB信号通路与炎症反应[J].生理科学进展,2014,45(1):68-71.
[19] Kang SR,Han DY,Park KI,et al.Suppressive effect on lipopolysaccharide-induced proinflammatory mediators by Citrus aurantium L.in macrophage RAW 264.7 cells via NF-κB signal pathway[J].Evid Based Complement Alternat Med,2011,2011(1):1-12.
[20] Park KI,Kang SR,Park HS,et al.Regulation of proinflammatory mediators via NF-κB and p38 MAPK-dependent mechanisms in RAW 264.7 macrophages by polyphenol components isolated from Korea Lonicera japonica THUNB[J].Evid Based Complement Alternat Med,2012,2012(1):1-10.
[21] Lee MS,Kwon MS,Choi JW,et al.Anti-inflammatory activities of an ethanol extract of Ecklonia stolonifera in lipopolysaccharide-stimulated RAW 264.7 murine macrophage cells[J].J Agric Food Chem,2012,60(36):9120-9129.