脂质过氧化炎症损伤模型中参莲提取物对巨噬细胞功能的药效与机制探讨
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摘要
目的:研究参莲(SL)提取物在脂质过氧化炎症损伤模型中,对巨噬细胞功能的作用和促进炎症消散的药效和药理机制。方法:利用氧化低密度脂蛋白(ox-LDL)体外诱导小鼠巨噬细胞RAW264. 7细胞和人单核细胞THP-1造成脂质过氧化损伤模型,酶联免疫吸附测定(ELISA)检测不同质量浓度的SL提取物(2. 5,5. 0,10. 0,20. 0 mg·L~(-1))对巨噬细胞肿瘤坏死因子-α(TNF-α),白细胞介素-1β(IL-1β),单核细胞趋化蛋白-1(MCP-1)的含量;流式细胞术检测巨噬细胞泡沫化形成;transwell实验检测SL提取物对THP-1趋化功能的影响;以及用蛋白免疫印迹法(Western blot)检测诱导型一氧化氮合酶(i NOS),炎症消散因子5脂氧合酶(ALOX5)以及对核转录因子-κB(NF-κB)信号通路中的磷酸化p65(p-p65)和磷酸化IκK(p-IκK)蛋白表达情况。结果:与正常组比较,ox-LDL提高M1型巨噬细胞标志物TNF-α,IL-1β,i NOS的表达(P <0. 01),抑制M2型标志物IL-10的表达,促进泡沫细胞形成(P <0. 01),诱导THP-1向ox-LDL趋化,提高MCP-1分泌量(P <0. 01),抑制ALOX5的表达。与模型组比较,SL提取物可以降低TNF-α,IL-1β,i NOS的表达(P <0. 01),提高IL-10的表达(P <0. 05);能显著降低巨噬细胞吞噬脂质后泡沫化的形成(P <0. 01);能降低THP-1细胞MCP-1的分泌(P <0. 01),减少趋化的细胞数目(P <0. 01);促进炎症消散因子ALOX5的升高(P <0. 05),同时抑制p65和IκK磷酸化表达(P <0. 01)。结论:在脂质过氧化炎症损伤模型中,SL提取物具有诱导巨噬细胞M2极化,抑制巨噬细胞对ox-LDL的趋化和泡沫化的形成,提高ALOX5表达,促进炎症消散,从而改善脂质过氧化炎症损伤状态,上述功能与抑制NF-κB信号通路中p65和IκK磷酸化水平有关。
Objective: To study the effect of Shenlian extract( SL extract) on macrophage function and inflammatory resolution in lipid peroxidation inflammatory injury models. Method: The effects of different concentrations of SL extract( 2. 5,5. 0,10. 0,20. 0 mg·L~(-1)) on the polarization type,foam formation and chemotactic function of macrophages were detected with RAW264. 7 cells induced by oxidized low-density lipoprotein( ox-LDL). Western blot was used to detect pro-inflammatory resolution factor arachidonate5-lipoxygenase( ALOX5) and inducible inducible nitric oxide synthase( i NOS), and phosphorylated p65( p-p65) and phosphorylated IκK( p-IκK) in nuclear factor( NF)-κB related signaling pathways. Result:Compared with the control group,ox-LDL enhanced the expressions of M1 macrophage markers TNF-α,IL-1β,i NOS( P < 0. 01),inhibited the expressions of IL-10 of M2 markers,promoted foam cell formation( P < 0. 01),induced THP-1 chemotaxis ability,increased the content of MCP-1( P < 0. 01),and inhibited the expression of ALOX5. Compared with the model group,SL extract reduced the expressions of TNF-α,IL-1β,and i NOS( P < 0. 01),increased the expression of IL-10( P < 0. 05),and down-regulated the formation of foam of macrophages( P < 0. 01). In transwell assay,SL extract obviously decreased the MCP-1 secretion and the number of chemotaxis cells( P < 0. 01),and promoted the increase of the inflammatory resolution factor ALOX5. The phosphorylation of p65 and IκK was inhibited significantly( P < 0. 01). Conclusion: In the inflammatory damage model of lipid peroxidation,SL extract can regulate the polarization of macrophage,inhibit the chemotaxis and foaming of ox-LDL,increase the inflammatory resolution molecular expression,and improve the state of lipid peroxidation,which may be related to the inhibition of NF-κB signaling pathway.
Objective: To study the effect of Shenlian extract( SL extract) on macrophage function and inflammatory resolution in lipid peroxidation inflammatory injury models. Method: The effects of different concentrations of SL extract( 2. 5,5. 0,10. 0,20. 0 mg·L~(-1)) on the polarization type,foam formation and chemotactic function of macrophages were detected with RAW264. 7 cells induced by oxidized low-density lipoprotein( ox-LDL). Western blot was used to detect pro-inflammatory resolution factor arachidonate5-lipoxygenase( ALOX5) and inducible inducible nitric oxide synthase( i NOS), and phosphorylated p65( p-p65) and phosphorylated IκK( p-IκK) in nuclear factor( NF)-κB related signaling pathways. Result:Compared with the control group,ox-LDL enhanced the expressions of M1 macrophage markers TNF-α,IL-1β,i NOS( P < 0. 01),inhibited the expressions of IL-10 of M2 markers,promoted foam cell formation( P < 0. 01),induced THP-1 chemotaxis ability,increased the content of MCP-1( P < 0. 01),and inhibited the expression of ALOX5. Compared with the model group,SL extract reduced the expressions of TNF-α,IL-1β,and i NOS( P < 0. 01),increased the expression of IL-10( P < 0. 05),and down-regulated the formation of foam of macrophages( P < 0. 01). In transwell assay,SL extract obviously decreased the MCP-1 secretion and the number of chemotaxis cells( P < 0. 01),and promoted the increase of the inflammatory resolution factor ALOX5. The phosphorylation of p65 and IκK was inhibited significantly( P < 0. 01). Conclusion: In the inflammatory damage model of lipid peroxidation,SL extract can regulate the polarization of macrophage,inhibit the chemotaxis and foaming of ox-LDL,increase the inflammatory resolution molecular expression,and improve the state of lipid peroxidation,which may be related to the inhibition of NF-κB signaling pathway.
引文
[1] Moss J W,Davies T S,Garaiova I,et al. A unique combination of nutritionally active ingredients can prevent several key processes associated with atherosclerosis in vitro[J]. PLo S One,2016,11(3):e0151057.
[2] Viola J,Soehnlein O. Atherosclerosis-a matter of unresolved inflammation[J]. Semin Immunol,2015,27(3):184-193.
[3] Fredman G,Tabas I. Boosting inflammation resolution in atherosclerosis:the next frontier for therapy[J]. Am J Pathol,2017,187(6):1211-1221.
[4] Motwani M P,Gilroy D W. Macrophage development and polarization in chronic inflammation[J]. Semin Immunol,2015,27(4):257-266.
[5] LIU Y C,ZOU X B,CHAI Y F,et al. Macrophage polarization in inflammatory diseases[J]. Int J Biol Sci,2014,10(5):520-529.
[6] Tabas I. Macrophage death and defective inflammation resolution in atherosclerosis[J]. Nat Rev Immunol,2010,10(1):36-46.
[7] Gimbrone M A, Garcia-Cardena G. Vascular endothelium,hemodynamics,and the pathobiology of atherosclerosis[J]. Cardiovasc Pathol,2013,22(1):9-15.
[8] Colin S,Chinetti-Gbaguidi G,Staels B. Macrophage phenotypes in atherosclerosis[J]. Immunol Rev,2014,262(1):153-166.
[9] Shaikh S,Brittenden J,Lahiri R,et al. Macrophage subtypes in symptomatic carotid artery and femoral artery plaques[J]. Eur J Vasc Endovasc Surg,2012,44(5):491-497.
[10] Hirata Y,Tabata M,Kurobe H,et al. Coronary atherosclerosis is associated with macrophage polarization in epicardial adipose tissue[J]. J Am Coll Cardiol,2011,58(3):248-255.
[11]陈丽娜,李玉洁,杨庆,等. SL方对大鼠动脉粥样硬化病变形成过程影响的动态观察[J].中国中药杂志,2011,36(1):66-71.
[12]郭琰.基于炎症消散的SL提取物抗动脉粥样硬化作用的机理研究[D].北京:中国中医科学院,2015.
[13]李玉洁,朱晓新,杨庆,等. SL提取物对动脉粥样硬化家兔血管病理形态和脂质代谢的影响[J].中草药,2011,42(4):760-764.
[14]李玉洁,杨庆,翁小刚,等. SL提取物抗炎药理活性评价[J].中药新药与临床药理,2011,22(1):65-69.
[15]刘思思,李琦,孙立东,等. SL提取物对LPS诱导的巨噬细胞炎症反应的影响[J].中国实验方剂学杂志,2017,23(10):85-91.
[16]周冰冰,李玉洁,李琦,等. SL提取物对M1巨噬细胞的影响[J].中国中药杂志,2014,39(11):2086-2090.
[17] Jongstra-Bilen J,ZHANG C X,Wisnicki T,et al.Oxidized low-density lipoprotein loading of macrophages downregulates TLR-induced proinflammatory responses in a gene-specific and temporal manner through transcriptional control[J]. J Immunol,2017,199(6):2149-2157.
[18] Clement C,Alma Z. Macrophages and immune cells in atherosclerosis:recent advances and novel concepts[J].Basic Res Cardiol,2015,110(4):34.
[19] Martin R B,Sanjay S,Gary K O. Vascular smooth muscle cells in atherosclerosis[J]. Circ Res,2016,118(4):692-702.
[20] Tabas I. Macrophage death and defective inflammation resolution in atherosclerosis[J]. Nat Rev Immunol,2010,10(1):36-46.
[21]王波,余珊,何江波,等.熊果酸对RAW264. 7巨噬细胞源性泡沫细胞胆固醇逆转运及对PPAR-γ的基因及蛋白表达的影响[J].中国实验方剂学杂志,2017,23(10):127-132.
[22] Ravandi A,Leibundgut G,Hung M Y,et al. Release and capture of bioactive oxidized phospholipids and oxidized cholesteryl esters during percutaneous coronary and peripheral arterial interventions in humans[J]. J Am Coll Cardiol,2014,63(19):1961-1971.
[23] Caligiuri G,Rudling M,Ollivier V,et al. Lesions and Th17 in interleukin-10 deficiency increases atherosclerosis,thrombosis,and low-density lipoproteins in apolipoprotein E-knockout mice[J]. Mol Med,2003,9(1/2):10-17.
[24] Getz G S,Reardon C A. Atherogenic lipids and macrophage subsets[J]. Curr Opin Lipidol,2015,26(5):357-361.
[25] WANG H H,Garruti G,LIU M,et al. Cholesterol and lipoprotein metabolism and atherosclerosis:recent advances in reverse cholesterol transport[J]. Ann Hepatol,2017,16(Suppl. 1:s3-105):s27-s42.
[26] Sergin I,Evans T D,Razani B. Degradation and beyond:the macrophage lysosome as a nexus for nutrient sensing and processing in atherosclerosis[J]. Curr Opin Lipidol,2015,26(5):394-404.
[27] Di Pietro N,Formoso G,Pandolfi A. Physiology and pathophysiology of ox LDL uptake by vascular wall cells in atherosclerosis[J]. Vascul Pharmacol,2016,84:1-7.
[28] Sugimoto M A,Ribeiro A L C,Costa B R C,et al.Plasmin and plasminogen induce macrophage reprogramming and regulate key steps of inflammation resolution via annexin A1[J]. Blood,2017,129(21):2896-2907.
[29]刘玉晖,侯贝贝,游宇,等.补阳还五汤稳定Apo E-/-小鼠动脉粥样硬化易损斑块的作用机制[J].中国实验方剂学杂志,2018,24(15):112-119.
[30]万强,刘中勇.定心方通过上调CD4+CD25+Foxp3+调节性T细胞表达对PM2. 5所致动脉粥样硬化的影响[J].中国实验方剂学杂志,2018,24(17):139-144.
[31] YUAN T,YANG T,CHEN H,et al. New insights into oxidative stress and inflammation during diabetes mellitus-accelerated atherosclerosis[J]. Redox Biol,2018,20:247-260.
[32] Gordon S,Pluddemann A,Martinez E F. Macrophage heterogeneity in tissues:phenotypic diversity and functions[J]. Immunol Rev,2014,262(1):36-55.
[2] Viola J,Soehnlein O. Atherosclerosis-a matter of unresolved inflammation[J]. Semin Immunol,2015,27(3):184-193.
[3] Fredman G,Tabas I. Boosting inflammation resolution in atherosclerosis:the next frontier for therapy[J]. Am J Pathol,2017,187(6):1211-1221.
[4] Motwani M P,Gilroy D W. Macrophage development and polarization in chronic inflammation[J]. Semin Immunol,2015,27(4):257-266.
[5] LIU Y C,ZOU X B,CHAI Y F,et al. Macrophage polarization in inflammatory diseases[J]. Int J Biol Sci,2014,10(5):520-529.
[6] Tabas I. Macrophage death and defective inflammation resolution in atherosclerosis[J]. Nat Rev Immunol,2010,10(1):36-46.
[7] Gimbrone M A, Garcia-Cardena G. Vascular endothelium,hemodynamics,and the pathobiology of atherosclerosis[J]. Cardiovasc Pathol,2013,22(1):9-15.
[8] Colin S,Chinetti-Gbaguidi G,Staels B. Macrophage phenotypes in atherosclerosis[J]. Immunol Rev,2014,262(1):153-166.
[9] Shaikh S,Brittenden J,Lahiri R,et al. Macrophage subtypes in symptomatic carotid artery and femoral artery plaques[J]. Eur J Vasc Endovasc Surg,2012,44(5):491-497.
[10] Hirata Y,Tabata M,Kurobe H,et al. Coronary atherosclerosis is associated with macrophage polarization in epicardial adipose tissue[J]. J Am Coll Cardiol,2011,58(3):248-255.
[11]陈丽娜,李玉洁,杨庆,等. SL方对大鼠动脉粥样硬化病变形成过程影响的动态观察[J].中国中药杂志,2011,36(1):66-71.
[12]郭琰.基于炎症消散的SL提取物抗动脉粥样硬化作用的机理研究[D].北京:中国中医科学院,2015.
[13]李玉洁,朱晓新,杨庆,等. SL提取物对动脉粥样硬化家兔血管病理形态和脂质代谢的影响[J].中草药,2011,42(4):760-764.
[14]李玉洁,杨庆,翁小刚,等. SL提取物抗炎药理活性评价[J].中药新药与临床药理,2011,22(1):65-69.
[15]刘思思,李琦,孙立东,等. SL提取物对LPS诱导的巨噬细胞炎症反应的影响[J].中国实验方剂学杂志,2017,23(10):85-91.
[16]周冰冰,李玉洁,李琦,等. SL提取物对M1巨噬细胞的影响[J].中国中药杂志,2014,39(11):2086-2090.
[17] Jongstra-Bilen J,ZHANG C X,Wisnicki T,et al.Oxidized low-density lipoprotein loading of macrophages downregulates TLR-induced proinflammatory responses in a gene-specific and temporal manner through transcriptional control[J]. J Immunol,2017,199(6):2149-2157.
[18] Clement C,Alma Z. Macrophages and immune cells in atherosclerosis:recent advances and novel concepts[J].Basic Res Cardiol,2015,110(4):34.
[19] Martin R B,Sanjay S,Gary K O. Vascular smooth muscle cells in atherosclerosis[J]. Circ Res,2016,118(4):692-702.
[20] Tabas I. Macrophage death and defective inflammation resolution in atherosclerosis[J]. Nat Rev Immunol,2010,10(1):36-46.
[21]王波,余珊,何江波,等.熊果酸对RAW264. 7巨噬细胞源性泡沫细胞胆固醇逆转运及对PPAR-γ的基因及蛋白表达的影响[J].中国实验方剂学杂志,2017,23(10):127-132.
[22] Ravandi A,Leibundgut G,Hung M Y,et al. Release and capture of bioactive oxidized phospholipids and oxidized cholesteryl esters during percutaneous coronary and peripheral arterial interventions in humans[J]. J Am Coll Cardiol,2014,63(19):1961-1971.
[23] Caligiuri G,Rudling M,Ollivier V,et al. Lesions and Th17 in interleukin-10 deficiency increases atherosclerosis,thrombosis,and low-density lipoproteins in apolipoprotein E-knockout mice[J]. Mol Med,2003,9(1/2):10-17.
[24] Getz G S,Reardon C A. Atherogenic lipids and macrophage subsets[J]. Curr Opin Lipidol,2015,26(5):357-361.
[25] WANG H H,Garruti G,LIU M,et al. Cholesterol and lipoprotein metabolism and atherosclerosis:recent advances in reverse cholesterol transport[J]. Ann Hepatol,2017,16(Suppl. 1:s3-105):s27-s42.
[26] Sergin I,Evans T D,Razani B. Degradation and beyond:the macrophage lysosome as a nexus for nutrient sensing and processing in atherosclerosis[J]. Curr Opin Lipidol,2015,26(5):394-404.
[27] Di Pietro N,Formoso G,Pandolfi A. Physiology and pathophysiology of ox LDL uptake by vascular wall cells in atherosclerosis[J]. Vascul Pharmacol,2016,84:1-7.
[28] Sugimoto M A,Ribeiro A L C,Costa B R C,et al.Plasmin and plasminogen induce macrophage reprogramming and regulate key steps of inflammation resolution via annexin A1[J]. Blood,2017,129(21):2896-2907.
[29]刘玉晖,侯贝贝,游宇,等.补阳还五汤稳定Apo E-/-小鼠动脉粥样硬化易损斑块的作用机制[J].中国实验方剂学杂志,2018,24(15):112-119.
[30]万强,刘中勇.定心方通过上调CD4+CD25+Foxp3+调节性T细胞表达对PM2. 5所致动脉粥样硬化的影响[J].中国实验方剂学杂志,2018,24(17):139-144.
[31] YUAN T,YANG T,CHEN H,et al. New insights into oxidative stress and inflammation during diabetes mellitus-accelerated atherosclerosis[J]. Redox Biol,2018,20:247-260.
[32] Gordon S,Pluddemann A,Martinez E F. Macrophage heterogeneity in tissues:phenotypic diversity and functions[J]. Immunol Rev,2014,262(1):36-55.
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