AP-1及γ-GCS在大鼠呼吸机所致肺损伤中的作用及其防治研究
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摘要
目的通过检测不同潮气量机械通气大鼠肺组织激活蛋白-1(AP-1)和γ-谷氨酰半胱氨酸合成酶(γ-GCS) mRNA及其蛋白表达水平和大剂量氨溴索对大潮气量机械通气大鼠肺组织AP-1和γ-GCS表达的影响,探讨氧化／抗氧化系统失衡在呼吸机所致肺损伤(VILI)发病中的作用,以及大剂量氨溴索对VILI的干预作用。
方法本实验分为两部分
实验一：24只雄性健康Wistar大鼠随机分为3组,即对照组、小潮气量(L-VT)组和大潮气量(H-VT)组。采用硫代巴比妥酸比色法测定大鼠血浆及肺组织中丙二醛(MDA)含量；采用原位分子杂交技术和免疫组织化学染色法(SABC法)检测肺组织AP-1mRNA和γ-GCS mRNA及其蛋白表达水平。
实验二：32只雄性Wistar大鼠随机分为对照组、机械通气(MV)组、常规剂量氨溴索(CDAMB)组和大剂量氨溴索(HDAMB)组。在光镜下观察各组大鼠肺组织的病理学改变；采用原位分子杂交技术和免疫组织化学染色法检测肺组织AP-1mRNA和γ-GCS mRNA及其蛋白表达水平。
结果
实验一：
1.H-VT组大鼠肺组织和血浆中MDA含量明显高于对照组和L-VT组(均P<0.01)；L-VT组与对照组比较差异无统计学意义(P>0.05)。
2.H-VT组肺组织γ-GCSmRNA及其蛋白表达水平明显低于对照组和L-VT组(均P<0.01),而AP-1mRNA及其蛋白表达水平明显高于对照组和L-VT组(均P<0.01)；L-VT组与对照组比较差异均无统计学意义(P>0.05)。
实验二：
1.病理学改变：除对照组外,各实验组均有不同程度的肺组织损伤,表现为肺间质水肿和炎性细胞浸润,肺泡破裂融合,其严重程度从高到低依次为MV组、CDAMB组、HDAMB组和对照组。
2.MV组和CDAMB组大鼠肺组织AP-1mRNA及其蛋白表达水平明显高于对照组,而γ-GCS mRNA及其蛋白表达水平则明显低于对照组(均P<0.01)。
3.HDAMB组AP-1mRNA及其蛋白表达水平明显低于MV组和CDAMB组,而γ-GCS mRNA及其蛋白表达水平高于MV组和CDAMB组(均P<0.01)；MV组和CDAMB组各
项指标比较差异无统计学意义(P>0.05)。
结论1.大潮气量机械通气可通过诱导肺组织AP-1mRNA及其蛋白高表达,使γ-GCS表达降低,进一步导致谷胱甘肽合成减少,局部肺组织氧化／抗氧化系统失衡,是VILI发生的重要因素之一。
2.小潮气量机械通气对肺内AP-1、γ-GCS表达影响较小,对正常肺组织无明显损伤作用。
3.大剂量氨溴索可通过抑制机械通气大鼠肺组织AP-1的表达和上调γ-GCS的表达而发挥抗氧化作用,对VILI有一定保护作用。
Objective:
To explore the effect of the oxidant/antioxidant imbalance in ventilator induced lung injury (VILI) and the prevention of high dose ambroxol, by observing the expression of active protein-1 (AP-1),γ-glutamylcysteine synthetase(γ-GCS) mRNA and protein on different tidal volumes in rats and observing the effect of high dose ambroxol on the expression of AP-1 andγ-GCS in lung of rats in high tidal volume mechanical ventilation.
Methods:
1 Twenty-four male Wistar rats were divided into three groups randomly: control group, low tidal volume (L-VT) group and high tidal volume (H-VT) group. The levels of MDA in lung and blood were measured by Thiobarbituric acid method. Immunohistochemical staining and molecular hybridization in situ were employed to determine the expression level of AP-1 andγ-GCS mRNA and protein in pulmonary tissues.
2 Thirty-two male Wistar rats were divided into four groups:control group, mechanical ventilation group (MV),conventional dose ambroxol pretreatment group (CDAMB) and high dose ambroxol pretreatment group (HDAMB) randomly. The pathologic alterations of the lungs were observed under light microscope in groups;Immunohistochemial staining and molecular hybridization in situ were employed to determine the expression level of AP-1 and y-GCS mRNA and protein in pulmonary tissues.
Results:
1 Comparing with control group and low tidal volume group, the levels of MDA in lung and blood were highly significant in high tidal volume group (P<0.01); There was no statistics meaning for the differences between low tidal volume group and control group (P>0.05).
2 Comparing with control group and low tidal volume group, the expression of AP-1 mRNA and protein were highly significant (P<0.01) in high tidal volume group, with the expression of y-GCS decreased (P<0.01); There was no statistics meaning for the differences between low tidal volume group and control group (P>0.05).
3 Apart from the control group,different levels of interstitial and alveolar edema,infiltration and activation of inflammation cells could be seen in experimental groups. There was more obvious pathologic alterations in the MV group.
4 The expression of AP-1 mRNA and protein in MV group and CDAMB group are obviously increased compared with control group, and the expression ofγ-GCS was decreased(P<0.01).
5 While the expression of AP-1 mRNA and protein in HDAMB group decreased significantly as compared to MV group and CDAMB group, but the expression of y-GCS mRNA and protein increased significantly(P<0.01). But no statistic differences existed between MV group and CDAMB group (P>0.05).
Conclusion:
1 The over expression of AP-1 mRNA and protein, low expression ofγ-GCS and reduction of GSH can be induced by high tidal volume ventilation, which can lead to the imbalance of oxidant/antioxidant system. It is one of the most important factors in VILI.
2 There is small effect on the expression of AP-1 and y-GCS in lung tissue in the L-VT group and no obvious lung injury.
3 High dose ambroxol may play a role in the antioxidant therapy of VILI through downregulating the expression of AP-1 mRNA and protein and upregulating y-GCS mRNA and protein.
方法本实验分为两部分
实验一：24只雄性健康Wistar大鼠随机分为3组,即对照组、小潮气量(L-VT)组和大潮气量(H-VT)组。采用硫代巴比妥酸比色法测定大鼠血浆及肺组织中丙二醛(MDA)含量；采用原位分子杂交技术和免疫组织化学染色法(SABC法)检测肺组织AP-1mRNA和γ-GCS mRNA及其蛋白表达水平。
实验二：32只雄性Wistar大鼠随机分为对照组、机械通气(MV)组、常规剂量氨溴索(CDAMB)组和大剂量氨溴索(HDAMB)组。在光镜下观察各组大鼠肺组织的病理学改变；采用原位分子杂交技术和免疫组织化学染色法检测肺组织AP-1mRNA和γ-GCS mRNA及其蛋白表达水平。
结果
实验一：
1.H-VT组大鼠肺组织和血浆中MDA含量明显高于对照组和L-VT组(均P<0.01)；L-VT组与对照组比较差异无统计学意义(P>0.05)。
2.H-VT组肺组织γ-GCSmRNA及其蛋白表达水平明显低于对照组和L-VT组(均P<0.01),而AP-1mRNA及其蛋白表达水平明显高于对照组和L-VT组(均P<0.01)；L-VT组与对照组比较差异均无统计学意义(P>0.05)。
实验二：
1.病理学改变：除对照组外,各实验组均有不同程度的肺组织损伤,表现为肺间质水肿和炎性细胞浸润,肺泡破裂融合,其严重程度从高到低依次为MV组、CDAMB组、HDAMB组和对照组。
2.MV组和CDAMB组大鼠肺组织AP-1mRNA及其蛋白表达水平明显高于对照组,而γ-GCS mRNA及其蛋白表达水平则明显低于对照组(均P<0.01)。
3.HDAMB组AP-1mRNA及其蛋白表达水平明显低于MV组和CDAMB组,而γ-GCS mRNA及其蛋白表达水平高于MV组和CDAMB组(均P<0.01)；MV组和CDAMB组各
项指标比较差异无统计学意义(P>0.05)。
结论1.大潮气量机械通气可通过诱导肺组织AP-1mRNA及其蛋白高表达,使γ-GCS表达降低,进一步导致谷胱甘肽合成减少,局部肺组织氧化／抗氧化系统失衡,是VILI发生的重要因素之一。
2.小潮气量机械通气对肺内AP-1、γ-GCS表达影响较小,对正常肺组织无明显损伤作用。
3.大剂量氨溴索可通过抑制机械通气大鼠肺组织AP-1的表达和上调γ-GCS的表达而发挥抗氧化作用,对VILI有一定保护作用。
Objective:
To explore the effect of the oxidant/antioxidant imbalance in ventilator induced lung injury (VILI) and the prevention of high dose ambroxol, by observing the expression of active protein-1 (AP-1),γ-glutamylcysteine synthetase(γ-GCS) mRNA and protein on different tidal volumes in rats and observing the effect of high dose ambroxol on the expression of AP-1 andγ-GCS in lung of rats in high tidal volume mechanical ventilation.
Methods:
1 Twenty-four male Wistar rats were divided into three groups randomly: control group, low tidal volume (L-VT) group and high tidal volume (H-VT) group. The levels of MDA in lung and blood were measured by Thiobarbituric acid method. Immunohistochemical staining and molecular hybridization in situ were employed to determine the expression level of AP-1 andγ-GCS mRNA and protein in pulmonary tissues.
2 Thirty-two male Wistar rats were divided into four groups:control group, mechanical ventilation group (MV),conventional dose ambroxol pretreatment group (CDAMB) and high dose ambroxol pretreatment group (HDAMB) randomly. The pathologic alterations of the lungs were observed under light microscope in groups;Immunohistochemial staining and molecular hybridization in situ were employed to determine the expression level of AP-1 and y-GCS mRNA and protein in pulmonary tissues.
Results:
1 Comparing with control group and low tidal volume group, the levels of MDA in lung and blood were highly significant in high tidal volume group (P<0.01); There was no statistics meaning for the differences between low tidal volume group and control group (P>0.05).
2 Comparing with control group and low tidal volume group, the expression of AP-1 mRNA and protein were highly significant (P<0.01) in high tidal volume group, with the expression of y-GCS decreased (P<0.01); There was no statistics meaning for the differences between low tidal volume group and control group (P>0.05).
3 Apart from the control group,different levels of interstitial and alveolar edema,infiltration and activation of inflammation cells could be seen in experimental groups. There was more obvious pathologic alterations in the MV group.
4 The expression of AP-1 mRNA and protein in MV group and CDAMB group are obviously increased compared with control group, and the expression ofγ-GCS was decreased(P<0.01).
5 While the expression of AP-1 mRNA and protein in HDAMB group decreased significantly as compared to MV group and CDAMB group, but the expression of y-GCS mRNA and protein increased significantly(P<0.01). But no statistic differences existed between MV group and CDAMB group (P>0.05).
Conclusion:
1 The over expression of AP-1 mRNA and protein, low expression ofγ-GCS and reduction of GSH can be induced by high tidal volume ventilation, which can lead to the imbalance of oxidant/antioxidant system. It is one of the most important factors in VILI.
2 There is small effect on the expression of AP-1 and y-GCS in lung tissue in the L-VT group and no obvious lung injury.
3 High dose ambroxol may play a role in the antioxidant therapy of VILI through downregulating the expression of AP-1 mRNA and protein and upregulating y-GCS mRNA and protein.
引文
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[2]赖荣德,梁子敬.急性肺损伤的氧化作用与抗氧化研究进展[J].国际呼吸杂志,2006,26(12)：924-931.
[3]Chow CW,Abreu MTH, Suzuki T, et al. Oxidative stress and acute lung injury. Am J Respir Cell Mol Biol,2003,29 (4):4272431.
[4]Bin OY, Xiangdong G, Olga S, et al. Oxidant injury mediates TGF-β1 up-regulation in ventilator induced lung injury[J]. 解放军医学杂志，2006, (1):18-21.
[5]Karim A,Claire MD,Thierry M,et al. Transforming Growth Factor-β1 is a Potent Inhibitor of GlutathioneSynthesis in the Lung Epithelial Cell Line A549:Transcriptional Effect on the GSH Rate-limiting Enzyme y-Glutamylcysteine Synthetase[J]. Am J Respir, 1997,11:599-607.
[6]Nowak D,Antczak A,Krol M,et al.Antioxidant properties of ambroxol[J].Free Rad Biol Med,1994,16:517-522·
[7]Jang YY,Song J H,Shin YK,et al. Depressant effects of ambroxol and erdosteine on cytokine synthesis ,granule enzyme release ,and free radical production in rat alveolar macrophages activated by lipopolysaccharide. Pharmacol Toxicol,2003,92 (4):173-179.
[8]Nowak D, Antczak A,Pietras T,et al.Protective effect of ambroxol against heat-and hydrogen peroxide-induced damage to lung lipids in mice.Eur Respir J,1994;7:1629-1634
[9]Lecuona E,Saldias F,Comellas A,et al. ventilator-associated lung injury decreases lung ability to clear edema in rats[J]. Am J Respir Crit Care Med,1999,159(2):603-609.
[10]Saldias FJ,Lecuona E,Comellas AP,et al. Beta-adrenergic stimulation restores rat lung ability to clear edema in ventilator-associated lung injury[J]. Am J Respir Crit Care Med,2000,162:282-287.
[11]武庆平,刘萍,王市李,等.不同潮气量通气对大鼠呼吸机相关性肺损伤模型的影响[J].华中科技大学学报：医学版,2006,35(4)：51Ⅰ—515.
[12]Krieg H. Ambroxol, die Atelektasenbremse. Anaesthesist,1998,47 Suppl:11-12.
[13]Kim YK, Jang YY, Han ES, et al. Dep ressant effect of ambroxol onstimulated functional responses and cell death in rat alveolar macrophages exposed to silica in vitro [J]. Pharmacol Exp Ther,2002,300:629-637.
[14]GibbsBF, SchmutzlerW, Vollrath IB, et al. Ambroxol inhibits the release of histamine, leukotriences and cytokines from human leukocytes and mast cells [J]. Inflamm Res, 1999,48:86-93.
[15]胡乃浩,赵文静.机械通气致大鼠急性肺损伤时肺组织及血MDA、SOD的变化[J].徐州医学院学,2009,29(1)：1-3.
[16]Heping YANG,Jiaohong W,Huang ZZ,et al. Cloning and characterization of the flanking region of the rat glutamatecysteine ligase catalytic subunit[J]. Biochem J,2001,357(Pt2):447-455.
[17]Schreiber M,Wang ZQ,Jochum W,Fetka I,Elliott C,Wagner EF.Placental vascularisation requires the AP-1 component fral.[J]Development 2000; 127:4937-4948
[18]Ranieri VM,Suter PM,Tortorella C,et al. Effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome:a randomized controlled trial[J]. JAMA,1999,282(1):54-61.
[19]申严,戴爱国.γ-GCS的信号传导与慢性阻塞性肺疾病[J].国外医学内科学分册,2004,31(6)：260-263,267.
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[2]赖荣德,梁子敬.急性肺损伤的氧化作用与抗氧化研究进展[J].国际呼吸杂志,2006,26(12)：924-931.
[3]Chow CW,Abreu MTH, Suzuki T, et al. Oxidative stress and acute lung injury. Am J Respir Cell Mol Biol,2003,29 (4):4272431.
[4]Bin OY, Xiangdong G, Olga S, et al. Oxidant injury mediates TGF-β1 up-regulation in ventilator induced lung injury[J]. 解放军医学杂志，2006, (1):18-21.
[5]Karim A,Claire MD,Thierry M,et al. Transforming Growth Factor-β1 is a Potent Inhibitor of GlutathioneSynthesis in the Lung Epithelial Cell Line A549:Transcriptional Effect on the GSH Rate-limiting Enzyme y-Glutamylcysteine Synthetase[J]. Am J Respir, 1997,11:599-607.
[6]Nowak D,Antczak A,Krol M,et al.Antioxidant properties of ambroxol[J].Free Rad Biol Med,1994,16:517-522·
[7]Jang YY,Song J H,Shin YK,et al. Depressant effects of ambroxol and erdosteine on cytokine synthesis ,granule enzyme release ,and free radical production in rat alveolar macrophages activated by lipopolysaccharide. Pharmacol Toxicol,2003,92 (4):173-179.
[8]Nowak D, Antczak A,Pietras T,et al.Protective effect of ambroxol against heat-and hydrogen peroxide-induced damage to lung lipids in mice.Eur Respir J,1994;7:1629-1634
[9]Lecuona E,Saldias F,Comellas A,et al. ventilator-associated lung injury decreases lung ability to clear edema in rats[J]. Am J Respir Crit Care Med,1999,159(2):603-609.
[10]Saldias FJ,Lecuona E,Comellas AP,et al. Beta-adrenergic stimulation restores rat lung ability to clear edema in ventilator-associated lung injury[J]. Am J Respir Crit Care Med,2000,162:282-287.
[11]武庆平,刘萍,王市李,等.不同潮气量通气对大鼠呼吸机相关性肺损伤模型的影响[J].华中科技大学学报：医学版,2006,35(4)：51Ⅰ—515.
[12]Krieg H. Ambroxol, die Atelektasenbremse. Anaesthesist,1998,47 Suppl:11-12.
[13]Kim YK, Jang YY, Han ES, et al. Dep ressant effect of ambroxol onstimulated functional responses and cell death in rat alveolar macrophages exposed to silica in vitro [J]. Pharmacol Exp Ther,2002,300:629-637.
[14]GibbsBF, SchmutzlerW, Vollrath IB, et al. Ambroxol inhibits the release of histamine, leukotriences and cytokines from human leukocytes and mast cells [J]. Inflamm Res, 1999,48:86-93.
[15]胡乃浩,赵文静.机械通气致大鼠急性肺损伤时肺组织及血MDA、SOD的变化[J].徐州医学院学,2009,29(1)：1-3.
[16]Heping YANG,Jiaohong W,Huang ZZ,et al. Cloning and characterization of the flanking region of the rat glutamatecysteine ligase catalytic subunit[J]. Biochem J,2001,357(Pt2):447-455.
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