大黄素对肝细胞脂质堆积和炎症的影响
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摘要
研究大黄素对肝细胞脂质堆积与炎症的影响。将低、中、高剂量(10. 93,54. 09,267. 70μmol·L-1)大黄素刺激L02肝细胞24 h后,显微观察细胞形态,试剂盒检测LDH释放量,油红O染色观察胞内脂滴变化,试剂盒检测胞内TC和TG含量,并采用Western blot法检测肝细胞中FASN,SREBF2和APOB以及炎症因子IL-6和p-NF-κB的蛋白表达情况。结果显示,大黄素(10. 93,54. 09,267. 70μmol·L-1)给药组细胞皱缩,体积变小,数量随剂量的增加逐渐减少; LDH释放量均高于对照组,且呈剂量依赖性显著增加; L02细胞内均可见红色脂滴;胞内TC,TG含量均高于对照组,且随浓度依赖性增加,其中中、高剂量组具有显著性差异(P<0. 05); FASN,SREBF2及炎症因子IL-6和p-NF-κB的蛋白表达水平均显著高于对照组,APOB表达水平低于对照组,差异具有统计学意义(P<0. 05)。研究结果表明,大黄素可剂量依赖诱导肝细胞脂质堆积与炎症损伤,进而损伤细胞,该过程与上调FASN,SREBF2,IL-6,p-NF-κB以及下调APOB的蛋白表达有关。
The aim of this study was to explore the effect of emodin on lipid accumulation and inflammation in hepatocytes. The cell morphology was observed by microscopy. LDH release was detected by the kit. Levels of intracellular lipid droplets were observed by oil red O staining. The contents of TC and TG in cells were detected by the kit. Western blot was used to determine protein expressions of FASN,SREBF2,APOB,IL-6 and p-NF-κB in hepatocytes. The results showed that the levels of L02 cell LDH were significantly increased after being treated with emodin,and the cells showed shrinkage,volume reduction,decrease in quantity with the increase of dose. Red lipid droplets were observed in L02 hepatocytes. Intracellular TC and TG contents of L02 cell increased in a concentrationdependent manner,with significant differences between medium and high-dose groups( P < 0. 05). Protein expressions of FASN,SREBF2,IL-6 and p-NF-κB were significantly higher than those of the control group,and the expression level of APOB was significantly lower than that of the control group( P<0. 05). In conclusion,emodin could induce lipid accumulation and inflammatory damage in hepatocytes in a dose-dependent manner,which in turn could damage liver cells. This process was related to the up-regulation of FASN,SREBF2,IL-6,p-NF-κB,as well as the down-regulation of the protein expression of APOB.
The aim of this study was to explore the effect of emodin on lipid accumulation and inflammation in hepatocytes. The cell morphology was observed by microscopy. LDH release was detected by the kit. Levels of intracellular lipid droplets were observed by oil red O staining. The contents of TC and TG in cells were detected by the kit. Western blot was used to determine protein expressions of FASN,SREBF2,APOB,IL-6 and p-NF-κB in hepatocytes. The results showed that the levels of L02 cell LDH were significantly increased after being treated with emodin,and the cells showed shrinkage,volume reduction,decrease in quantity with the increase of dose. Red lipid droplets were observed in L02 hepatocytes. Intracellular TC and TG contents of L02 cell increased in a concentrationdependent manner,with significant differences between medium and high-dose groups( P < 0. 05). Protein expressions of FASN,SREBF2,IL-6 and p-NF-κB were significantly higher than those of the control group,and the expression level of APOB was significantly lower than that of the control group( P<0. 05). In conclusion,emodin could induce lipid accumulation and inflammatory damage in hepatocytes in a dose-dependent manner,which in turn could damage liver cells. This process was related to the up-regulation of FASN,SREBF2,IL-6,p-NF-κB,as well as the down-regulation of the protein expression of APOB.
引文
[1]赵凤娥,石振东.中药相关性肝损伤的临床研究进展[J].世界华人消化杂志,2017,25(30):2689.
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[4]李秀玉,李超群,张志敏,等.中药相关性肝损害研究——从理论到临床[J].转化医学杂志,2015,4(4):244.
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[7] Lee M H,Kao L,Lin C C. Comparison of the antioxidant and transmembrane permeative activities of the different Polygonum cuspidatum extracts in phospholipid-based microemulsions[J].J Agric Food Chem,2011,59(17):9135.
[8] Naqvi S,Ullah M F,Hadi S M. DNA degradation by aqueous extract of Aloe vera in the presence of copper ions[J]. Indian J Biochem Biophys,2010,47(3):161.
[9] Yang Y C,Lim M Y,Lee H S. Emodin isolated from Cassia obtusifolia(Leguminosae)seed shows larvicidal activity against three mosquito species[J]. J Agric Food Chem,2003,51(26):7629.
[10] National Toxicology Program. NTP toxicology and carcinogenesis studies of EMODIN(CAS NO:518-82-1)feed studies in F344/N rats and B6C3F1 mice[J]. Natl Toxical Program Tech Rep Ser,2001,493:1.
[11] Ma J,Zheng L,He Y,et al. Hepatotoxic assessment of Polygoni Multiflori radix extract and toxicokinetic study of stilbene glucoside and anthraquinones in rats[J]. J Ethnopharmacol,2015,162:61.
[12]李雨萌,李瑞煜,牛明,等.饮片规格对何首乌化学成分溶出和肝细胞毒性的影响[J].中国中药杂志,2016,41(6):1033.
[13] Jiang L L,Zhao D S,Fan Y X,et al. Detection of emodin derived glutathione adduct in normal rats administered with large dosage of Polygoni Multiflori Radix[J]. Front Pharmacol,2017,8:446.
[14]雷湘,陈刚,陈科力,等.大黄素对小鼠的急性毒性研究[J].中药药理与临床,2008,24(1):29.
[15] Cui Y T,Liu B,Xie J,et al. The effect of emodin on cytotoxicity,apoptosis and antioxidant capacity in the hepatic cells of grass carp(Ctenopharyngodon idellus)[J]. Fish Shellfish Immunol,2014,38(1):74.
[16] Li C L,Ma J,Zheng L,et al. Determination of emodin in L-02cells and cell culture media with liquid chromatography-mass spectrometry:application to a cellular toxicokinetic study[J].J Pharm Biomed Anal,2012,71(12):71.
[17] Yang X W,Zhang Y H,Liu Y,et al. Emodin induces liver injury by inhibiting the key enzymes of ADH/NADPH transport in rat liver[J]. Toxicol Res,2018,7(5):888.
[18]张斌,丁慎华,钱雪梅,等.大剂量大黄素致大鼠肝损伤及其机制的初步研究[J].中国现代医学杂志,2015,25(35):18.
[19]刘德明,周春燕,吴嘉思,等.大黄素通过线粒体通路诱导HepG2细胞凋亡[J].中国实验方剂学杂志,2018,24(3):104.
[20] Liu X Y,Liu Y Q,Qu Y,et al. Metabolomic profiling of emodin-induced cytotoxicity in human liver cells and mechanistic study[J]. Toxicol Res,2015,4(4):948.
[21]宋丽,张正,朱丽明.中性脂质及肿瘤坏死因子α对正常人肝细胞生物活性的影响[J].华西医学,2007,22(2):332.
[22]徐冬梅,陈勇,方朝晖,等.基于MAPK途径探讨丹蛭降糖胶囊改善高脂血症大鼠肝损伤的机制[J].中国中药杂志,2019,doi:10. 19540/j.cnki.cjcmm.20190220. 001.
[23]田琳.炎症促进四氯化碳诱导的小鼠脂肪变肝细胞脂质异常集聚的分子机制[D].重庆:重庆医科大学,2014.
[24]吕琼.AMPK在炎症诱导的肝脏脂质沉积中的作用[D].重庆:重庆医科大学,2015.
[25]周利玲,吴和平,李树平,等.大黄素对鹌鹑脂质代谢的影响研究[J].实用预防医学,2006(4):821.
[26]韩伟,李小燕,周利玲,等.大黄素的降脂与抗衰老作用[J].中国医学创新,2009,6(29):14.
[27]刘涛,徐秋玲,赵岩,等.大黄素对非酒精性脂肪肝大鼠脂质水平及肝脏脂质代谢基因表达的影响[J].中草药,2010,41(9):1516.
[28]张征波,薛博瑜.大黄素对非酒精性脂肪肝小鼠肝脂质沉积的作用及机制[J].中华中医药杂志,2012,27(9):2423.
[29]欧健.大黄素对脂肪变性L02肝细胞UCP2 mRNA表达的影响[D].广州:暨南大学,2009.
[30]董杰.大黄素对脂质诱导胰岛素抵抗骨骼肌脂质沉积及脂肪酸氧化代谢通路关键蛋白影响[D].重庆:重庆医科大学,2015.
[31]王少杰,李晓洁,徐志猛,等.大黄素对小鼠急性脂肪肝的改善作用[J].中国药科大学学报,2017,48(1):89.
[32]李妹娟,王和生,王通渤,等.制何首乌中大黄素对ApoE-/-小鼠动脉粥样硬化模型中JAK2/STAT3通路的影响[J].中国实验方剂学杂志,2018,24(18):101.
[33] Musso G,Gambino R,Cassader M. Recent insights into hepatic lipid metabolism in non-alcoholic fatty liver disease(NAFDL)[J].Prog Lipid Res,2009,48(1):1.
[34] Czaja A J. Hepatic inflammation and progressive liver fibrosis in chronic liver disease[J]. World J Gastroenterol,2014,20(10):2515.
[35]张聪,盛磊,杨恬,等.鞣花酸对AKT基因转染诱导小鼠脂肪性肝病中炎症及氧化应激的影响[J].中国中药杂志,2019,44(9):1869.
[36]杨家耀,时昭红,马威,等.附子理中汤通过激活AMPK通路及抑制NF-κBp65通路降低非酒精性脂肪肝大鼠肝脏损伤[J].中国中药杂志,2018,43(15):3176.
[37]赵爽,叶强,李涛,等.炎症应激通过激活m TOR通路诱导THP-1巨噬细胞泡沫化的实验研究[J].中国药理学通报,2016,32(8):1105.
[38] Ataie-Kachoie P,Pourgholami M H,Morris D L. Inhibition of the IL-6 signaling pathway:a strategy to combat chronic inflammatory diseases and cancer[J]. Cytokine Growth Factor Rev,2013,24(2):163.
[39]张婷,陆山红,杨兴鑫,等.砂仁水提物对5-FU致大鼠肠黏膜屏障损伤的保护作用[J].中国现代应用药学,2019,36(3):286.
[40]王君燕,童晔玲,赵文慧.獐牙菜苦苷的抗炎活性及其对NF-κB通路相关因子p65和IKK-α表达的影响[J].中国现代应用药学,2018,35(12):1817.
[41] Kraakman M J,Kammoun H L,Allen T L,et al. Blocking IL-6trans-signaling prevents high-fat diet-induced adipose tissue macrophage recruitment but does not improve insulin resistance[J].Cell Metab,2015,21(3):403.
[2] Chalasani N P,Hayashi P H,Bonkovsky H L,et al. Practice Parameters Committee of the American College of Gastroenterology:ACG clinical guideline:the diagnosis and management of idiosyncratic drug induced liver injury[J]. Am J Gastroenterol,2014,109(7):950.
[3]于乐成,茅益民,陈成伟.药物性肝损伤诊治指南[J].肝脏,2015,20(10):750.
[4]李秀玉,李超群,张志敏,等.中药相关性肝损害研究——从理论到临床[J].转化医学杂志,2015,4(4):244.
[5] Wang J B,Zhao H P,Zhao Y L,et al. Hepatotoxicity or hepatoprotection? Pattern recognition for thearadoxical effect of the Chinese herb Rheum palmatum L. in treating rat liver injury[J].PLoS ONE,2011,6(9):e24498.
[6] Wang M,Zhao R,Wang W,et al. Lipid regulation effects of Polygoni Multiflori Radix,its processed products and its major substances on steatosis human liver cell line L02[J]. J Ethnopharmacol,2012,139(1):287.
[7] Lee M H,Kao L,Lin C C. Comparison of the antioxidant and transmembrane permeative activities of the different Polygonum cuspidatum extracts in phospholipid-based microemulsions[J].J Agric Food Chem,2011,59(17):9135.
[8] Naqvi S,Ullah M F,Hadi S M. DNA degradation by aqueous extract of Aloe vera in the presence of copper ions[J]. Indian J Biochem Biophys,2010,47(3):161.
[9] Yang Y C,Lim M Y,Lee H S. Emodin isolated from Cassia obtusifolia(Leguminosae)seed shows larvicidal activity against three mosquito species[J]. J Agric Food Chem,2003,51(26):7629.
[10] National Toxicology Program. NTP toxicology and carcinogenesis studies of EMODIN(CAS NO:518-82-1)feed studies in F344/N rats and B6C3F1 mice[J]. Natl Toxical Program Tech Rep Ser,2001,493:1.
[11] Ma J,Zheng L,He Y,et al. Hepatotoxic assessment of Polygoni Multiflori radix extract and toxicokinetic study of stilbene glucoside and anthraquinones in rats[J]. J Ethnopharmacol,2015,162:61.
[12]李雨萌,李瑞煜,牛明,等.饮片规格对何首乌化学成分溶出和肝细胞毒性的影响[J].中国中药杂志,2016,41(6):1033.
[13] Jiang L L,Zhao D S,Fan Y X,et al. Detection of emodin derived glutathione adduct in normal rats administered with large dosage of Polygoni Multiflori Radix[J]. Front Pharmacol,2017,8:446.
[14]雷湘,陈刚,陈科力,等.大黄素对小鼠的急性毒性研究[J].中药药理与临床,2008,24(1):29.
[15] Cui Y T,Liu B,Xie J,et al. The effect of emodin on cytotoxicity,apoptosis and antioxidant capacity in the hepatic cells of grass carp(Ctenopharyngodon idellus)[J]. Fish Shellfish Immunol,2014,38(1):74.
[16] Li C L,Ma J,Zheng L,et al. Determination of emodin in L-02cells and cell culture media with liquid chromatography-mass spectrometry:application to a cellular toxicokinetic study[J].J Pharm Biomed Anal,2012,71(12):71.
[17] Yang X W,Zhang Y H,Liu Y,et al. Emodin induces liver injury by inhibiting the key enzymes of ADH/NADPH transport in rat liver[J]. Toxicol Res,2018,7(5):888.
[18]张斌,丁慎华,钱雪梅,等.大剂量大黄素致大鼠肝损伤及其机制的初步研究[J].中国现代医学杂志,2015,25(35):18.
[19]刘德明,周春燕,吴嘉思,等.大黄素通过线粒体通路诱导HepG2细胞凋亡[J].中国实验方剂学杂志,2018,24(3):104.
[20] Liu X Y,Liu Y Q,Qu Y,et al. Metabolomic profiling of emodin-induced cytotoxicity in human liver cells and mechanistic study[J]. Toxicol Res,2015,4(4):948.
[21]宋丽,张正,朱丽明.中性脂质及肿瘤坏死因子α对正常人肝细胞生物活性的影响[J].华西医学,2007,22(2):332.
[22]徐冬梅,陈勇,方朝晖,等.基于MAPK途径探讨丹蛭降糖胶囊改善高脂血症大鼠肝损伤的机制[J].中国中药杂志,2019,doi:10. 19540/j.cnki.cjcmm.20190220. 001.
[23]田琳.炎症促进四氯化碳诱导的小鼠脂肪变肝细胞脂质异常集聚的分子机制[D].重庆:重庆医科大学,2014.
[24]吕琼.AMPK在炎症诱导的肝脏脂质沉积中的作用[D].重庆:重庆医科大学,2015.
[25]周利玲,吴和平,李树平,等.大黄素对鹌鹑脂质代谢的影响研究[J].实用预防医学,2006(4):821.
[26]韩伟,李小燕,周利玲,等.大黄素的降脂与抗衰老作用[J].中国医学创新,2009,6(29):14.
[27]刘涛,徐秋玲,赵岩,等.大黄素对非酒精性脂肪肝大鼠脂质水平及肝脏脂质代谢基因表达的影响[J].中草药,2010,41(9):1516.
[28]张征波,薛博瑜.大黄素对非酒精性脂肪肝小鼠肝脂质沉积的作用及机制[J].中华中医药杂志,2012,27(9):2423.
[29]欧健.大黄素对脂肪变性L02肝细胞UCP2 mRNA表达的影响[D].广州:暨南大学,2009.
[30]董杰.大黄素对脂质诱导胰岛素抵抗骨骼肌脂质沉积及脂肪酸氧化代谢通路关键蛋白影响[D].重庆:重庆医科大学,2015.
[31]王少杰,李晓洁,徐志猛,等.大黄素对小鼠急性脂肪肝的改善作用[J].中国药科大学学报,2017,48(1):89.
[32]李妹娟,王和生,王通渤,等.制何首乌中大黄素对ApoE-/-小鼠动脉粥样硬化模型中JAK2/STAT3通路的影响[J].中国实验方剂学杂志,2018,24(18):101.
[33] Musso G,Gambino R,Cassader M. Recent insights into hepatic lipid metabolism in non-alcoholic fatty liver disease(NAFDL)[J].Prog Lipid Res,2009,48(1):1.
[34] Czaja A J. Hepatic inflammation and progressive liver fibrosis in chronic liver disease[J]. World J Gastroenterol,2014,20(10):2515.
[35]张聪,盛磊,杨恬,等.鞣花酸对AKT基因转染诱导小鼠脂肪性肝病中炎症及氧化应激的影响[J].中国中药杂志,2019,44(9):1869.
[36]杨家耀,时昭红,马威,等.附子理中汤通过激活AMPK通路及抑制NF-κBp65通路降低非酒精性脂肪肝大鼠肝脏损伤[J].中国中药杂志,2018,43(15):3176.
[37]赵爽,叶强,李涛,等.炎症应激通过激活m TOR通路诱导THP-1巨噬细胞泡沫化的实验研究[J].中国药理学通报,2016,32(8):1105.
[38] Ataie-Kachoie P,Pourgholami M H,Morris D L. Inhibition of the IL-6 signaling pathway:a strategy to combat chronic inflammatory diseases and cancer[J]. Cytokine Growth Factor Rev,2013,24(2):163.
[39]张婷,陆山红,杨兴鑫,等.砂仁水提物对5-FU致大鼠肠黏膜屏障损伤的保护作用[J].中国现代应用药学,2019,36(3):286.
[40]王君燕,童晔玲,赵文慧.獐牙菜苦苷的抗炎活性及其对NF-κB通路相关因子p65和IKK-α表达的影响[J].中国现代应用药学,2018,35(12):1817.
[41] Kraakman M J,Kammoun H L,Allen T L,et al. Blocking IL-6trans-signaling prevents high-fat diet-induced adipose tissue macrophage recruitment but does not improve insulin resistance[J].Cell Metab,2015,21(3):403.