双调蛋白对小鼠呼吸机相关性肺损伤的保护作用及机制研究
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摘要
目的研究上皮生长因子双调蛋白(Amphiregulin,Areg)对呼吸机相关性肺损伤(ventilator-associated lung injury,VALI)肺组织的保护作用,并探讨其作用机制。方法将C57BL/6小鼠根据随机数字表法随机分为8组:①对照组(Control组),每只腹腔注射磷酸盐缓冲液(PBS) 200μL,维持正常呼吸;②Areg组,腹腔注射重组小鼠Areg蛋白(rmAreg),维持正常呼吸;③PBS+VALI组,腹腔注射PBS,30min后行机械通气;④Areg+VALI组,腹腔注射rmAreg,30 min后行机械通气;⑤DMSO+VALI组;⑥DMSO+Areg+VALI组,每只腹腔注射二甲基亚砜(dimethylsulfoxide,DMSO)100μL,30 min后腹腔注射PBS或rmAreg,再经30 min后行机械通气;⑦AG1478+Areg+VALI组与⑧Perifosine+Areg+VALI组,每只腹腔注射AG1478 1 mg或Perifosine 1 mg,30 min后腹腔注射rmAreg,再经30 min后行机械通气。小鼠通过大潮气量机械通气(潮气量12 mL/kg,频率90次/分,通气4 h)制作VALI模型。机械通气24 h后行肺组织HE染色并观察病理学变化,检测支气管肺泡灌洗液(Bronchoalveolar lavage fluid,BALF)中总蛋白、免疫球蛋白M(Immunoglobulin M,IgM)、肿瘤坏死因子α-(Tumor necrosis factor,TNF-α)与白细胞介素-6(Interleukin-6,IL-6)的浓度;机械通气6 h后检测肺组织中上皮生长因子受体(Epidermal growth factor receptor,EGFR)与蛋白激酶B(AKT)的磷酸化水平。结果与0 h组[(62.0±19.4) pg/mL]相比,VALI小鼠在机械通气结束3、6、12、24 h后BALF中的Areg浓度有明显升高,其中在6 h升高最为显著[(231.8±58.7) pg/mL]。与Control组相比,PBS+VALI组小鼠的肺组织肺泡壁增厚,炎症反应显著增加,而在Areg+VALI组中这些病理损伤均有明显减轻。与Control组相比,PBS+VALI组小鼠BALF中总蛋白、IgM、TNF-α与IL-6均有显著升高,而相较于PBS+VALI组,Areg+VALI组中这些指标有明显降低。rmAreg显著提高了VALI小鼠肺组织中EGFR与AKT的磷酸化水平。AG1478与Perifosine均明显抑制了rmAreg对VALI肺组织的作用效果。结论小鼠发生VALI后肺组织中Areg的表达量显著增加。Areg通过EGFR-AKT信号通路减轻肺组织病理学变化、渗透性与炎症反应,因此在VALI中具有明显的保护作用。
Objective To study the protective effect of Amphiregulin(Areg) on ventilator-associated lung injury(VALI) in mice and to explore the potential mechanisms.Methods Male C57 BL/6 mice were randomly divided into eight groups according to the random number table:(1) control group,mice were injected with Phosphate buffer solution(PBS:200 μL) and treated with normal breathing;(2) Areg group,mice were injected with recombinant mouse Areg(rmAreg) and treated with normal breathing;(3) PBS+VALI group,mice were intraperitoneally administrated with PBS,and then were treated with high tidal volume mechanical ventilation 30 min later;(4) Areg+VALI group,mice were intraperitoneally injected with PBS,and then were subjected to high tidal volume mechanical ventilation 30 min later;(5) DMSO+VALI group and(6) DMSO+Areg+VALI group,mice were injected with DMSO(100 μL),followed by treatment with PBS or rmAreg 30 min later,and then were subjected to high tidal volume mechanical ventilation 30 min later;(7) AG1478+Areg+VALI group and(8) Perifosine+Areg+VALI group,mice were administered intraperitoneally with AG1478 or Perifosine,followed by injection with rmAreg 30 min later,and then were subjected to high tidal volume mechanical ventilation 30 min later. To establish VALI model,male C57 BL/6 mice were ventilated at high tidal volume(12 μL/kg) with 90 breaths/min for 4 hours after being anesthetized. At 24 hours after ventilation,mice lung tissues were stained with HE and examined at the light microscopy level,and the concentrations of total protein,Immunoglobulin M(IgM),Tumor necrosis factor(TNF-α) and Interleukin-6(IL-6) in Bronchoalveolar lavage fluid(BALF) were tested.At 6 hours after ventilation,the phosphorylation levels of epidermal growth factor receptor(EGFR) and protein kinase B(AKT) in lung tissues were detected by western blot.Results Compared with 0 h[(62.0±19.4) pg/mL] the concentrations of Areg in mice BALF were significantly increased at 3,6,12,24 h after high tidal volume ventilation,and the most significantly increased level of Areg was at 6 h after ventilation[(231.8±58.7) pg/mL].Compared with control group,the lung tissues of mice in PBS+VALI group had thicker alveolar wall and significantly increased inflammatory response.However,these pathological damages had been less observed in Areg +VALI group compared with PBS+VALI group.In addition,the BALF concentrations of total protein,IgM,TNF-α and IL-6 were all markedly increased in PBS+VALI group compared with control group,whereas the levels of these factors were all obviously decreased in Areg+VALI group compared with PBS+VALI group.Furthermore,treatment with rmAreg significantly increased the phosphorylation of EGFR and AKT in lung tissues of VALI mice.Both AG1478 and Perifosine obviously inhibited the effect of rmAreg on VALI lung tissues.Conclusion The expression level of Areg is significantly increased in lung tissue of mice after being subjected to high tidal volume ventilation.Areg can reduce the pathological damage,permeability and inflammatory response via EGFR-AKT signal transduction in lung tissue of mice suffering from VALI,which plays a significant protective role in VALI.
Objective To study the protective effect of Amphiregulin(Areg) on ventilator-associated lung injury(VALI) in mice and to explore the potential mechanisms.Methods Male C57 BL/6 mice were randomly divided into eight groups according to the random number table:(1) control group,mice were injected with Phosphate buffer solution(PBS:200 μL) and treated with normal breathing;(2) Areg group,mice were injected with recombinant mouse Areg(rmAreg) and treated with normal breathing;(3) PBS+VALI group,mice were intraperitoneally administrated with PBS,and then were treated with high tidal volume mechanical ventilation 30 min later;(4) Areg+VALI group,mice were intraperitoneally injected with PBS,and then were subjected to high tidal volume mechanical ventilation 30 min later;(5) DMSO+VALI group and(6) DMSO+Areg+VALI group,mice were injected with DMSO(100 μL),followed by treatment with PBS or rmAreg 30 min later,and then were subjected to high tidal volume mechanical ventilation 30 min later;(7) AG1478+Areg+VALI group and(8) Perifosine+Areg+VALI group,mice were administered intraperitoneally with AG1478 or Perifosine,followed by injection with rmAreg 30 min later,and then were subjected to high tidal volume mechanical ventilation 30 min later. To establish VALI model,male C57 BL/6 mice were ventilated at high tidal volume(12 μL/kg) with 90 breaths/min for 4 hours after being anesthetized. At 24 hours after ventilation,mice lung tissues were stained with HE and examined at the light microscopy level,and the concentrations of total protein,Immunoglobulin M(IgM),Tumor necrosis factor(TNF-α) and Interleukin-6(IL-6) in Bronchoalveolar lavage fluid(BALF) were tested.At 6 hours after ventilation,the phosphorylation levels of epidermal growth factor receptor(EGFR) and protein kinase B(AKT) in lung tissues were detected by western blot.Results Compared with 0 h[(62.0±19.4) pg/mL] the concentrations of Areg in mice BALF were significantly increased at 3,6,12,24 h after high tidal volume ventilation,and the most significantly increased level of Areg was at 6 h after ventilation[(231.8±58.7) pg/mL].Compared with control group,the lung tissues of mice in PBS+VALI group had thicker alveolar wall and significantly increased inflammatory response.However,these pathological damages had been less observed in Areg +VALI group compared with PBS+VALI group.In addition,the BALF concentrations of total protein,IgM,TNF-α and IL-6 were all markedly increased in PBS+VALI group compared with control group,whereas the levels of these factors were all obviously decreased in Areg+VALI group compared with PBS+VALI group.Furthermore,treatment with rmAreg significantly increased the phosphorylation of EGFR and AKT in lung tissues of VALI mice.Both AG1478 and Perifosine obviously inhibited the effect of rmAreg on VALI lung tissues.Conclusion The expression level of Areg is significantly increased in lung tissue of mice after being subjected to high tidal volume ventilation.Areg can reduce the pathological damage,permeability and inflammatory response via EGFR-AKT signal transduction in lung tissue of mice suffering from VALI,which plays a significant protective role in VALI.
引文
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[19] WESTPHALEN K,GUSAROVA GA,ISLAM MN,et al.Sessile alveolar macrophages communicate with alveolar epithelium to modulate immunity[J].Nature,2014,506(7489):503-506.
[20] 赵洁婷,钟文,徐建平.microRNA-21促进人肺腺癌细胞A549对顺铂耐药性的研究[J].安徽医药,2017,21(6):1082-1086.
[21] LU N,WANG LH,CAO HL,et al.Activation of the epidermal growth factor receptor in macrophages regulates cytokine production and experimental colitis[J].J Immunol,2014,192(3):1013-1023.
[22] LIN KK,ROSSI L,BOLES NC,et al.CD81 is essential for there-entry of hematopoietic stem cells to quiescence following stress-induced proliferation via deactivation of the akt pathway[J].PLoS Biol,2011,9(9):e1001148.DOI:10.1371/journal.pbio.1001148 DOI:10.1371/journal.pbio.1001148.
[2] SERPA NETO A,HEMMES SN,BARBAS CS,et al.Protective versus conventional ventilation for surgery:a systematic review and individual patient data meta-analysis[J].Anesthesiology,2015,123(1):66-78.
[3] SALMAN D,FINNEY SJ,GRIFFITHS MJ.Strategies to reduce ventilator-associated lung injury (VALI)[J].Burns,2013,39(2):200-211.
[4] BERASAIN C,AVILA MA.Amphiregulin[J].Semin Cell Dev Biol,2014,28:31-41.
[5] DOLINAY T,KAMINSKI N,FELGENDREHER M,et al.Gene expression profiling of target genes in ventilator-induced lung injury[J].Physiol Genomics,2006,26(1):68-75.
[6] STROSING KM,FALLER S,GYLLENRAM V,et al.Inhaled anesthetics exert different protective properties in a mouse model of ventilator-induced lung injury[J].Anesth Analg,2016,123(1):143-151.
[7] MATUTE-BELLO G,DOWNEY G,MOORE BB,et al.An official American Thoracic Society workshop report:features and measurements of experimental acute lung injury in animals[J].Am J Respir Cell Mol Biol,2011,44(5):725-738.
[8] MENG C,LIU GL,MU HL,et al.Amphiregulin may be a new biomarker of classically activated macrophages[J].Biochem Biophys Res Commun,2015,466(3):393-399.
[9] XU YN,MENG C,LIU GL,et al.Classically activated macrophages protect against lipopolysaccharide-induced acute lung injury by expressing amphiregulin in mice[J].Anesthesiology,2016,124(5):1086-1099.
[10] PREU? S,SCHEIERMANN J,STADELMANN S,et al.18:1/18:1-Dioleoyl-phosphatidylglycerol prevents alveolar epithelial apoptosis and profibrotic stimulus in a neonatal piglet model of acute respiratory distress syndrome[J].Pulm Pharmacol Ther,2014,28(1):25-34.
[11] SARGE T,TALMOR D.Targeting transpulmonary pressure to prevent ventilator induced lung injury[J].Minerva Anestesiol,2009,75(5):293-299.
[12] YERRAPUREDDY A,TOBIAS J,MARGULIES SS.Cyclic stretch magnitude and duration affect rat alveolar epithelial gene expression[J].Cell Physiol Biochem,2010,25(1):113-122.
[13] BRANDL K,SUN L,NEPPL C,et al.MyD88 signaling in nonhematopoietic cells protects mice against induced colitis by regulating specific EGF receptor ligands[J].Proc Natl Acad Sci USA,2010,107(46):19967-19972.
[14] PARDO-SAGANTA A,LATASA MU,CASTILLO J,et al.The epidermal growth factor receptor ligand amphiregulin is a negative regulator of hepatic acute-phase gene expression[J].J Hepatol,2009,51(6):1010-1020.
[15] BERASAIN C,GARCíA-TREVIJANO ER,CASTILLO J,et al.Amphiregulin:an early trigger of liver regeneration in mice[J].Gastroenterology,2005,128(2):424-432.
[16] LINGGI B,CARPENTER G.ErbB receptors:new insights on mechanisms and biology[J].Trends Cell Biol,2006,16(12):649-656.
[17] CITRI A,YARDEN Y.EGF-ERBB signalling:towards the systems level[J].Nat Rev Mol Cell Biol,2006,7(7):505-516.
[18] BERASAIN C,GARCíA-TREVIJANO ER,CASTILLO J,et al.Novel role for amphiregulin in protection from liver injury[J].J Biol Chem,2005,280(19):19012-19020.
[19] WESTPHALEN K,GUSAROVA GA,ISLAM MN,et al.Sessile alveolar macrophages communicate with alveolar epithelium to modulate immunity[J].Nature,2014,506(7489):503-506.
[20] 赵洁婷,钟文,徐建平.microRNA-21促进人肺腺癌细胞A549对顺铂耐药性的研究[J].安徽医药,2017,21(6):1082-1086.
[21] LU N,WANG LH,CAO HL,et al.Activation of the epidermal growth factor receptor in macrophages regulates cytokine production and experimental colitis[J].J Immunol,2014,192(3):1013-1023.
[22] LIN KK,ROSSI L,BOLES NC,et al.CD81 is essential for there-entry of hematopoietic stem cells to quiescence following stress-induced proliferation via deactivation of the akt pathway[J].PLoS Biol,2011,9(9):e1001148.DOI:10.1371/journal.pbio.1001148 DOI:10.1371/journal.pbio.1001148.