大气细颗粒物对胶原结构样巨噬细胞受体基因敲除小鼠呼吸和循环系统的影响
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摘要
目的探讨大气细颗粒物(PM_(2.5))对胶原结构样巨噬细胞受体(MARCO)基因敲除小鼠呼吸和循环系统的作用。方法将12只野生型C57BL/6小鼠随机分为野生型生理盐水组和野生型PM_(2.5)组,12只MARCO~(-/-)型C57BL/6小鼠随机分为MARCO~(-/-)型生理盐水组和MARCO~(-/-)型PM_(2.5)组,每组6只。野生型PM_(2.5)组和MARCO~(-/-)型PM_(2.5)组小鼠气管滴注PM_(2.5)混悬液4 mg·kg~(-1),野生型生理盐水组和MARCO~(-/-)型生理盐水组小鼠给予等量无菌生理盐水,隔日1次,共6次。末次滴注24 h后检测各组小鼠支气管肺泡灌洗液(BALF)中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)、乳酸脱氢酶(LDH)及血清中C-反应蛋白(CRP)、TNF-α和LDH水平;免疫组织化学法检测各组小鼠肺组织中磷酸化表皮生长因子受体(EGFR)表达情况。结果野生型生理盐水组与MARCO~(-/-)型生理盐水组小鼠BALF中IL-6、TNF-α、LDH水平及血清中CRP、TNF-α、LDH水平比较差异均无统计学意义(tBALF=-0. 230、-0. 964、1. 263,t血清=-0. 944、-1. 908、-0. 392,P> 0. 05)。野生型PM_(2.5)组与野生型生理盐水组小鼠BALF中IL-6、TNF-α、LDH水平及血清中CRP、TNF-α、LDH水平比较差异无统计学意义(tBALF=-0. 583、-2. 130、-1. 016,t血清=-0. 890、-1. 649、-0. 885,P> 0. 05)。MARCO~(-/-)型PM_(2.5)组小鼠BALF中IL-6、TNF-α、LDH水平及血清中CRP、LDH水平显著高于MARCO~(-/-)型生理盐水组(tBALF=2. 469、3. 364、3. 000,t血清=2. 530、4. 605,P <0. 05),2组小鼠血清TNF-α水平比较差异无统计学意义(t=1. 911,P> 0. 05)。MARCO~(-/-)型PM_(2.5)组与野生型PM_(2.5)组小鼠BALF中IL-6、TNF-α、LDH水平及血清中CRP、TNF-α水平比较差异无统计学意义(tBALF=1. 723、1. 114、-0. 324,t血清=1. 643、1. 311,P> 0. 05);MARCO~(-/-)型PM_(2.5)组小鼠血清LDH水平显著高于野生型PM_(2.5)组(t=2. 378,P <0. 05)。免疫组织化学结果显示,野生型与MARCO~(-/-)型生理盐水组小鼠肺组织中均未见磷酸化EGFR表达,而野生型与MARCO~(-/-)型PM_(2.5)组小鼠肺组织均可见磷酸化EGFR表达,且MARCO~(-/-)型PM_(2.5)组小鼠肺组织中磷酸化EGFR水平高于野生型PM_(2.5)组。结论PM_(2.5)急性暴露可导致MARCO~(-/-)小鼠呼吸和循环系统发生炎性效应,MARCO可能在PM_(2.5)导致的小鼠急性呼吸和循环系统炎症中发挥重要作用。
Objective To investigate the effect of fine particulate matter( PM_(2.5)) on respiratory and cardiovascular systems in mice with macrophage receptor with collagenous structure( MARCO) gene knockout. Methods Twelve wild-type C57 BL/6 mice were randomly divided into wide-type normal saline group and wild-type PM_(2.5) group,with 6 mice in each group. Twelve MACRO~(-/-)C57 BL/6 mice were randomly divided into MARCO~(-/-)normal saline group and MARCO~(-/-)PM_(2.5) group,with 6 mice in each group. The mice in the wild-type PM_(2.5) group and MARCO~(-/-)PM_(2.5) group were treated with PM_(2.5) suspension( 4 mg·kg~(-1)) by intratracheal instillation,while the mice in the wide-type normal saline group and MARCO~(-/-)normal saline group were treated with equal volume of normal saline by intratracheal instillation,once every other day for six times. The levels of tumor necrosis factor-α( TNF-α),interleukin-6( IL-6),lactate dehydrogenase( LDH) in bronchoalveolar lavage fluid( BALF) and the levels of serum C-reactive protein( CRP),TNF-α,LDH were detected at 24 hours after the last infusion. The expression of phosphorylated epidermal growth factor receptor( EGFR) in lung tissues of mice in each group was determined by immunohistochemistry. Results There was no significant difference in the levels of IL-6,TNF-α,LDH in BALF and the levels of CRP,TNF-α,LDH in serum between the wild-type normal saline group and MARCO~(-/-)normal saline group( tBALF=-0. 230,-0. 964,1. 263; tserum=-0. 944,-1. 908,-0. 392; P > 0. 05). There was no significant difference in the levels of IL-6,TNF-α,LDH in BALF and the levels of CRP,TNF-α,LDH in serum between wide-type normal saline group and wild-type PM_(2.5) group( tBALF=-0. 583,-2. 130,-1. 016; tserum=-0. 890,-1. 649,-0. 885; P > 0. 05). The levels of IL-6,TNF-α,LDH in BALF and the levels of CRP,LDH in serum of mice in the MARCO~(-/-)PM_(2.5) group were significantly higher than those in the MARCO~(-/-)normal saline group( tBALF= 2. 469,3. 364,3. 000; tserum= 2. 530,4. 605; P < 0. 05).There was no significant difference in the level of serum TNF-α between the MARCO~(-/-)PM_(2.5) group and MARCO~(-/-)normal saline group( t = 1. 911,P > 0. 05). There was no significant difference in the levels of IL-6,TNF-α,LDH in BALF and the levels of CRP,TNF-α in serum between the MARCO~(-/-)PM_(2.5) group and wild-type PM_(2.5) group( tBALF= 1. 723,1. 114,-0. 324; tserum= 1. 643,1. 311; P > 0. 05). The level of serum LDH of mice in the MARCO~(-/-)PM_(2.5) group was significantly higher than that in the wild-type PM_(2.5) group( t = 2. 378,P < 0. 05). The immunohistochemical results showed that there was no the expression of phosphorylated EGFR in lung tissues of mice in the wide-type normal saline group and MARCO~(-/-)normal saline group,while the expression of phosphorylated EGFR was observed in lung tissues of mice in the wild type PM_(2.5) group and MARCO~(-/-)PM_(2.5) group,and the expression of phosphorylated EGFR in lung tissues of mice in the MARCO~(-/-)PM_(2.5) group was higher than that in the wild type PM_(2.5) group. Conclusion The inflammatory reaction of respiratory and cardiovascular systems caused by PM_(2.5) acute exposure in MARCO~(-/-)mice is more serious than that in wild type mice.MARCO may play an important role in the acute inflammatory reaction of respiratory and cardiovascular systems of mice caused by PM_(2.5).
Objective To investigate the effect of fine particulate matter( PM_(2.5)) on respiratory and cardiovascular systems in mice with macrophage receptor with collagenous structure( MARCO) gene knockout. Methods Twelve wild-type C57 BL/6 mice were randomly divided into wide-type normal saline group and wild-type PM_(2.5) group,with 6 mice in each group. Twelve MACRO~(-/-)C57 BL/6 mice were randomly divided into MARCO~(-/-)normal saline group and MARCO~(-/-)PM_(2.5) group,with 6 mice in each group. The mice in the wild-type PM_(2.5) group and MARCO~(-/-)PM_(2.5) group were treated with PM_(2.5) suspension( 4 mg·kg~(-1)) by intratracheal instillation,while the mice in the wide-type normal saline group and MARCO~(-/-)normal saline group were treated with equal volume of normal saline by intratracheal instillation,once every other day for six times. The levels of tumor necrosis factor-α( TNF-α),interleukin-6( IL-6),lactate dehydrogenase( LDH) in bronchoalveolar lavage fluid( BALF) and the levels of serum C-reactive protein( CRP),TNF-α,LDH were detected at 24 hours after the last infusion. The expression of phosphorylated epidermal growth factor receptor( EGFR) in lung tissues of mice in each group was determined by immunohistochemistry. Results There was no significant difference in the levels of IL-6,TNF-α,LDH in BALF and the levels of CRP,TNF-α,LDH in serum between the wild-type normal saline group and MARCO~(-/-)normal saline group( tBALF=-0. 230,-0. 964,1. 263; tserum=-0. 944,-1. 908,-0. 392; P > 0. 05). There was no significant difference in the levels of IL-6,TNF-α,LDH in BALF and the levels of CRP,TNF-α,LDH in serum between wide-type normal saline group and wild-type PM_(2.5) group( tBALF=-0. 583,-2. 130,-1. 016; tserum=-0. 890,-1. 649,-0. 885; P > 0. 05). The levels of IL-6,TNF-α,LDH in BALF and the levels of CRP,LDH in serum of mice in the MARCO~(-/-)PM_(2.5) group were significantly higher than those in the MARCO~(-/-)normal saline group( tBALF= 2. 469,3. 364,3. 000; tserum= 2. 530,4. 605; P < 0. 05).There was no significant difference in the level of serum TNF-α between the MARCO~(-/-)PM_(2.5) group and MARCO~(-/-)normal saline group( t = 1. 911,P > 0. 05). There was no significant difference in the levels of IL-6,TNF-α,LDH in BALF and the levels of CRP,TNF-α in serum between the MARCO~(-/-)PM_(2.5) group and wild-type PM_(2.5) group( tBALF= 1. 723,1. 114,-0. 324; tserum= 1. 643,1. 311; P > 0. 05). The level of serum LDH of mice in the MARCO~(-/-)PM_(2.5) group was significantly higher than that in the wild-type PM_(2.5) group( t = 2. 378,P < 0. 05). The immunohistochemical results showed that there was no the expression of phosphorylated EGFR in lung tissues of mice in the wide-type normal saline group and MARCO~(-/-)normal saline group,while the expression of phosphorylated EGFR was observed in lung tissues of mice in the wild type PM_(2.5) group and MARCO~(-/-)PM_(2.5) group,and the expression of phosphorylated EGFR in lung tissues of mice in the MARCO~(-/-)PM_(2.5) group was higher than that in the wild type PM_(2.5) group. Conclusion The inflammatory reaction of respiratory and cardiovascular systems caused by PM_(2.5) acute exposure in MARCO~(-/-)mice is more serious than that in wild type mice.MARCO may play an important role in the acute inflammatory reaction of respiratory and cardiovascular systems of mice caused by PM_(2.5).
引文
[1] ZHOU M,HE G,FAN M,et al. Smog episodes,fine particulate pollution and mortality in China[J]. Environ Res,2015,136:396-404.
[2] LU F,XU D,CHENG Y,et al. Systematic review and meta-analysis of the adverse health effects of ambient PM2. 5and PM10pollution in the Chinese population[J]. Environ Res,2015,136:196-204.
[3]熊琪,茹琴,陈琳,等. PM2. 5对肺泡巨噬细胞功能影响的研究进展[J].环境与健康杂志,2013,30(12):1128-1130.
[4] GILBERTI R M,KNECHT D A. Macrophages phagocytose nonopsonized silica particles using a unique microtubuledependent pathway[J]. Mol Biol Cell,2015,26(3):518-529.
[5] NOVAKOWSKI K E,HUYNH A,HAN S J,et al. A naturally occurring transcript variant of MARCO reveals the srcr domain is critical for function[J]. Immunol Cell Biol,2016,94(7):646-655.
[6] SOUKUP J M,BECKER S. Human alveolar macrophage responses to air pollution particulates are associated with insoluble components of coarse material,including particulate endotoxin[J].Toxicol Appl Pharmacol,2001,171(1):20-26.
[7] POZZI R,BERARDIS B D,PAOLETTI L,et al. Inflammatory mediators induced by coarse(PM2. 5-10)and fine(PM2. 5)urban air particles in RAW 264. 7 cells[J]. Toxicology,2003,183(1/2/3):243-254.
[8] BECKER S,SOUKUP J M,SIOUTAS C,et al. Response of human alveolar macrophages to ultrafine,fine,and coarse urban air pollution particles[J]. Exp Lung Res,2003,29(1):29-44.
[9]姜智海,宋伟民,周晓瑜,等. PM2. 5对小鼠肺急性损伤的试验研究[J].卫生研究,2004,33(3):264-266.
[10] YAN Z,JIN Y,AN Z,et al. Inflammatory cell signaling following exposures to particulate matter and ozone[J]. Biochim Biophys Acta,2016,1860(12):2826-2834.
[11] ARREDOUANI M S,YANG Z,IMRICH A,et al. The macrophage scavenger receptor SR-AI/II and lung defense against pneumococci and particles[J]. Am J Respir Cell Mol Biol,2012,35(4):474-478.
[12] JR H R,THAKUR S A,MAYFAIR J K,et al. MARCO mediates silica uptake and toxicity in alveolar macrophages from C57BL/6mice[J]. J Biol Chem,2006,281(45):34218-34226.
[13]晋乐飞,吴卫东,张巧,等.吸入式气管滴注法的建立[J].郑州大学学报(医学版),2015,50(1):75-78.
[14] JIN Y,WU W,ZHANG W,et al. Involvement of EGF receptor signaling and NLRP12 inflammasome in fine particulate matter‐induced lung inflammation in mice[J]. Environ Toxicol,2017,32(4):1121-1134.
[15]高丽云,张学军,汪涛,等.环境中细颗粒物PM2. 5对呼吸系统的作用机制[J].新乡医学院学报,2017,34(3):163-165.
[16]高丽云,聂林坤,李潇,等.大气细颗粒物PM2. 5对心血管疾病损害机制的研究进展[J].新乡医学院学报,2018,35(7):551-553.
[17] LI G,XUE M,ZENG Q,et al. Association between fine ambient particulate matter and daily total mortality:an analysis from 160communities of China[J]. Sci Total Environ,2017,599:108-113.
[18] YORIFUJI T,KASHIMA S,DOI H. Acute exposure to fine and coarse particulate matter and infant mortality in Tokyo,Japan(2002-2013)[J]. Sci Total Environt,2016,551:66-72.
[19] POPE C A. Epidemiology of fine particulate air pollution and human health:biologic mechanisms and who's at risk[J].Environ Health Perspect,2000,108(Suppl 4):713-723.
[20] SIBILLE Y,REYNOLDS H Y. Macrophages and polymorphonuclear neutrophils in lung defense and injury[J]. Am Rev Respir Dis,1990,141(2):471-501.
[21] GHIO A J,KENNEDY T P,SCHAPIRA R M,et al. Hypothesis:is lung disease after silicate inhalation caused by oxidant generation[J]. Lancet,1990,336(8721):967-969.
[22] ISHII H,FUJII T,HOGG J C,et al. Contribution of IL-1βand TNF-αto the initiation of the peripheral lung response to atmospheric particulates(PM10)[J]. Am J Physiol Lung Cell Mol Physiol,2004,287(1):L176-L183.
[23] JALAVA P I,SALONEN R O,PENNANEN A S,et al.Heterogeneities in inflammatory and cytotoxic responses of RAW264. 7 macrophage cell line to urban air coarse,fine,and ultrafine particles from six European sampling campaigns[J]. Inhal Toxicol,2007,19(3):213-225.
[24] HUANG N H,WANG Q,XU D Q,et al. Immunological effect of PM2. 5on cytokine production in female Wistar rats[J]. Biomed Environ Sci,2008,21(1):63-68.
[25] MIYATA R,VAN EEDEN S F. The innate and adaptive immune response induced by alveolar macrophages exposed to ambient particulate matter[J]. Toxicol Appl Pharmacol,2011,257(2):209-226.
[26] LI R,KOU X,XIE L,et al. Effects of ambient PM2. 5on pathological injury,inflammation,oxidative stress,metabolic enzyme activity,and expression of c-fos and c-jun in lungs of rats[J]. Environ Sci Pollut Res Int,2015,22(24):1-10.
[27] SHVEDOVA A A,KISIN E,MURRAY A R,et al. Inhalation vs.aspiration of single-walled carbon nanotubes in C57BL/6 mice:inflammation,fibrosis,oxidative stress,and mutagenesis[J]. Am J Physiol Lung Cell Mol Physiol,2008,295(4):L552-L565.
[28] BACKES J M,HOWARD P A,MORIARTY P M. Role of C-reactive protein in cardiovascular disease[J]. Rev Colomb De Cardio,2011,18(5):273-278.
[29] RD P C,HANSEN M L,LONG R W,et al. Ambient particulate air pollution,heart rate variability,and blood markers of inflammation in a panel of elderly subjects[J]. Environ Health Perspect,2004,112(3):339-345.
[30] HACKEL P O,ZWICK E,PRENZEL N,et al. Epidermal growth factor receptors:critical mediators of multiple receptor pathways[J]. Curr Opin Cell Biol,1999,11(2):184-189.
[31] OVREVIK J,REFSNES M,TOTLANDSDAL A I,et al. TACE/TGF-α/EGFR regulates CXCL8 in bronchial epithelial cells exposed to particulate matter components[J]. Eur Respir J,2011,38(5):1189-1199.
[32] WU W,SAMET J M,GHIO A J,et al. Activation of the EGF receptor signaling pathway in airway epithelial cells exposed to Utah Valley PM[J]. Am J Physiol Lung Cell Mol Physiol,2001,281(2):L483-L489.
[2] LU F,XU D,CHENG Y,et al. Systematic review and meta-analysis of the adverse health effects of ambient PM2. 5and PM10pollution in the Chinese population[J]. Environ Res,2015,136:196-204.
[3]熊琪,茹琴,陈琳,等. PM2. 5对肺泡巨噬细胞功能影响的研究进展[J].环境与健康杂志,2013,30(12):1128-1130.
[4] GILBERTI R M,KNECHT D A. Macrophages phagocytose nonopsonized silica particles using a unique microtubuledependent pathway[J]. Mol Biol Cell,2015,26(3):518-529.
[5] NOVAKOWSKI K E,HUYNH A,HAN S J,et al. A naturally occurring transcript variant of MARCO reveals the srcr domain is critical for function[J]. Immunol Cell Biol,2016,94(7):646-655.
[6] SOUKUP J M,BECKER S. Human alveolar macrophage responses to air pollution particulates are associated with insoluble components of coarse material,including particulate endotoxin[J].Toxicol Appl Pharmacol,2001,171(1):20-26.
[7] POZZI R,BERARDIS B D,PAOLETTI L,et al. Inflammatory mediators induced by coarse(PM2. 5-10)and fine(PM2. 5)urban air particles in RAW 264. 7 cells[J]. Toxicology,2003,183(1/2/3):243-254.
[8] BECKER S,SOUKUP J M,SIOUTAS C,et al. Response of human alveolar macrophages to ultrafine,fine,and coarse urban air pollution particles[J]. Exp Lung Res,2003,29(1):29-44.
[9]姜智海,宋伟民,周晓瑜,等. PM2. 5对小鼠肺急性损伤的试验研究[J].卫生研究,2004,33(3):264-266.
[10] YAN Z,JIN Y,AN Z,et al. Inflammatory cell signaling following exposures to particulate matter and ozone[J]. Biochim Biophys Acta,2016,1860(12):2826-2834.
[11] ARREDOUANI M S,YANG Z,IMRICH A,et al. The macrophage scavenger receptor SR-AI/II and lung defense against pneumococci and particles[J]. Am J Respir Cell Mol Biol,2012,35(4):474-478.
[12] JR H R,THAKUR S A,MAYFAIR J K,et al. MARCO mediates silica uptake and toxicity in alveolar macrophages from C57BL/6mice[J]. J Biol Chem,2006,281(45):34218-34226.
[13]晋乐飞,吴卫东,张巧,等.吸入式气管滴注法的建立[J].郑州大学学报(医学版),2015,50(1):75-78.
[14] JIN Y,WU W,ZHANG W,et al. Involvement of EGF receptor signaling and NLRP12 inflammasome in fine particulate matter‐induced lung inflammation in mice[J]. Environ Toxicol,2017,32(4):1121-1134.
[15]高丽云,张学军,汪涛,等.环境中细颗粒物PM2. 5对呼吸系统的作用机制[J].新乡医学院学报,2017,34(3):163-165.
[16]高丽云,聂林坤,李潇,等.大气细颗粒物PM2. 5对心血管疾病损害机制的研究进展[J].新乡医学院学报,2018,35(7):551-553.
[17] LI G,XUE M,ZENG Q,et al. Association between fine ambient particulate matter and daily total mortality:an analysis from 160communities of China[J]. Sci Total Environ,2017,599:108-113.
[18] YORIFUJI T,KASHIMA S,DOI H. Acute exposure to fine and coarse particulate matter and infant mortality in Tokyo,Japan(2002-2013)[J]. Sci Total Environt,2016,551:66-72.
[19] POPE C A. Epidemiology of fine particulate air pollution and human health:biologic mechanisms and who's at risk[J].Environ Health Perspect,2000,108(Suppl 4):713-723.
[20] SIBILLE Y,REYNOLDS H Y. Macrophages and polymorphonuclear neutrophils in lung defense and injury[J]. Am Rev Respir Dis,1990,141(2):471-501.
[21] GHIO A J,KENNEDY T P,SCHAPIRA R M,et al. Hypothesis:is lung disease after silicate inhalation caused by oxidant generation[J]. Lancet,1990,336(8721):967-969.
[22] ISHII H,FUJII T,HOGG J C,et al. Contribution of IL-1βand TNF-αto the initiation of the peripheral lung response to atmospheric particulates(PM10)[J]. Am J Physiol Lung Cell Mol Physiol,2004,287(1):L176-L183.
[23] JALAVA P I,SALONEN R O,PENNANEN A S,et al.Heterogeneities in inflammatory and cytotoxic responses of RAW264. 7 macrophage cell line to urban air coarse,fine,and ultrafine particles from six European sampling campaigns[J]. Inhal Toxicol,2007,19(3):213-225.
[24] HUANG N H,WANG Q,XU D Q,et al. Immunological effect of PM2. 5on cytokine production in female Wistar rats[J]. Biomed Environ Sci,2008,21(1):63-68.
[25] MIYATA R,VAN EEDEN S F. The innate and adaptive immune response induced by alveolar macrophages exposed to ambient particulate matter[J]. Toxicol Appl Pharmacol,2011,257(2):209-226.
[26] LI R,KOU X,XIE L,et al. Effects of ambient PM2. 5on pathological injury,inflammation,oxidative stress,metabolic enzyme activity,and expression of c-fos and c-jun in lungs of rats[J]. Environ Sci Pollut Res Int,2015,22(24):1-10.
[27] SHVEDOVA A A,KISIN E,MURRAY A R,et al. Inhalation vs.aspiration of single-walled carbon nanotubes in C57BL/6 mice:inflammation,fibrosis,oxidative stress,and mutagenesis[J]. Am J Physiol Lung Cell Mol Physiol,2008,295(4):L552-L565.
[28] BACKES J M,HOWARD P A,MORIARTY P M. Role of C-reactive protein in cardiovascular disease[J]. Rev Colomb De Cardio,2011,18(5):273-278.
[29] RD P C,HANSEN M L,LONG R W,et al. Ambient particulate air pollution,heart rate variability,and blood markers of inflammation in a panel of elderly subjects[J]. Environ Health Perspect,2004,112(3):339-345.
[30] HACKEL P O,ZWICK E,PRENZEL N,et al. Epidermal growth factor receptors:critical mediators of multiple receptor pathways[J]. Curr Opin Cell Biol,1999,11(2):184-189.
[31] OVREVIK J,REFSNES M,TOTLANDSDAL A I,et al. TACE/TGF-α/EGFR regulates CXCL8 in bronchial epithelial cells exposed to particulate matter components[J]. Eur Respir J,2011,38(5):1189-1199.
[32] WU W,SAMET J M,GHIO A J,et al. Activation of the EGF receptor signaling pathway in airway epithelial cells exposed to Utah Valley PM[J]. Am J Physiol Lung Cell Mol Physiol,2001,281(2):L483-L489.