高迁移率族蛋白1在实验大鼠呼吸机所致肺损伤中的作用及血必净注射液的干预作用
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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高迁移率族蛋白1 (HMGB1) mRNA及其蛋白表达水平和血必净注射液对大潮气量机械通气大鼠肺组织HMGB1的影响,探讨HMGB1在呼吸机所致肺损伤(VILI)发病中的作用,以及血必净注射液对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。采用髓过氧化物酶检测试剂盒测定各组大鼠血浆和肺泡灌洗液(BALF)中髓过氧化物酶(MPO)活性,采用免疫组织化学法(SABC法)和原位分子杂交技术检测肺组织HMGB1表达水平。
实验二：24只雄性健康Wistar大鼠随机分为3组,即对照组、大潮气量组、血必净干预组。采用髓过氧化物酶检测试剂盒测定各组大鼠血浆和肺泡灌洗液中MPO活性,采用免疫组织化学法(SABC法)和原位分子杂交技术检测肺组织HMGB1表达水平。
结果：
实验一：
1、大潮气量组大鼠血浆及BALF中MPO活性明显高于对照组和小潮气量组(均P<0.01),对照组与小潮气量组比较差异无统计学意义(P>0.05)。
2、大潮气量组肺组织气道上皮细胞HMGB1mRNA及其蛋白表达水平明显高于对照组和小潮气量组(均P<0.01),小潮气量组与对照组比较差异均无统计学意义(均P>0.05)。
实验二：
1、大潮气量组和血必净干预组大鼠血浆及BALF中MPO活性明显高于对照组(均P<0.01)；大潮气量组大鼠血浆及BALF中MPO活性高于血必净干预组(均P<0.01)。
2、大潮气量组和血必净干预组大鼠肺组织气道上皮细胞HMGB1 mRNA及其蛋白表达水平明显高于对照组(均P<0.01)；大潮气量组大鼠肺组织气道上皮细胞HMGB1 mRNA及其蛋白表达水平高于血必净干预组(均P<0.01)。
结论：
1、大潮气量机械通气可诱导肺组织HMGB1mRNA及其蛋白高表达,HMGB1分泌增多导致肺组织炎症反应扩大,可能是呼吸机所致肺损伤(VILI)发生的重要因素之一。
2、小潮气量机械通气对肺组织HMGB1表达影响较小,对正常肺组织无明显损伤作用。
3、血必净注射液在一定程度上可抑制肺组织HMGB1mRNA及其蛋白的表达,对机械通气所致大鼠急性肺损伤具有一定防治作用。
Objective:
To explore the role of high mobility group box 1(HMGB1) in acute lung injury induced by different tidal volume of ventilation in rats and its prevention and treatment of XueBiJing injection in the ventilaor induced lung injury by detecting the expression of high mobility group box 1(HMGB1) in lung tissue.
Methods:
1 Twenty-four male Wistar rats were randomly divided into three groups:control group, low tidal volume group (L-VT), high tidal volume group (H-VT). The levels of myloperoxidase(MPO) activity in plasma and bronchoalveolar lavage fluid (BALF) were determined by biochemical methods respectively.Immunohistochemial staining and molecular hybridization in situ were employed to determine the expression level of HMGB1 mRNA and protein in pulmonary tissues.
2 Twenty-four male Wistar rats were divided into three groups randomly:control group, H-VT group and XueBiJing pretreatment group. The levels of myloperoxidase(MPO) activity in plasma and bronchoalveolar lavage fluid (BALF) were determined by biochemical methods respectively.Immunohistochemial staining and molecular hybridization in situ were employed to determine the expression level of HMGB1 mRNA and protein in pulmonary tissues.
Results:
1 Comparing with control group and low tidal volume group, the levels of MPO activity in plasma and BALF 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 HMGB1 mRNA and protein were highly significant (P<0.01) in high tidal volume group. There was no statistics meaning for the differences between low tidal volume group and control group (P>0.05).
3 Comparing with control group, the levels of MPO activity in plasma and BALF were highly significant in high tidal volume group and XueBiJing pretreatment group(P＜0.01); Comparing with high tidal volume group, the levels of MPO activity in plasma and BALF were decreased significant in XueBiJing pretreatment group(P<0.01).
4 Comparing with control group, the expression of HMGB1 mRNA and protein was highly significant in high tidal volume group and XueBiJing pretreatment group (P＜0.01); Comparing with high tidal volume group, the expression of HMGB1 mRNA and protein was decreased significant in XueBiJing pretreatment group(P<0.01).
Conclusion:
1 The over expression of HMGB1 mRNA and protein can be induced by high tidal volume ventilation, which can lead to increased inflammatory response in lung tissue to expand. It is one of the most important factors in VILI.
2 There is small effect on the expression of HMGB1 in lung tissue in the L-VT group and no obvious lung injury.
3 XueBiJing injection may play a role of prevention and treatment in the ventilator induced lung injury by inhibiting the expression of HMGB1.
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。采用髓过氧化物酶检测试剂盒测定各组大鼠血浆和肺泡灌洗液(BALF)中髓过氧化物酶(MPO)活性,采用免疫组织化学法(SABC法)和原位分子杂交技术检测肺组织HMGB1表达水平。
实验二：24只雄性健康Wistar大鼠随机分为3组,即对照组、大潮气量组、血必净干预组。采用髓过氧化物酶检测试剂盒测定各组大鼠血浆和肺泡灌洗液中MPO活性,采用免疫组织化学法(SABC法)和原位分子杂交技术检测肺组织HMGB1表达水平。
结果：
实验一：
1、大潮气量组大鼠血浆及BALF中MPO活性明显高于对照组和小潮气量组(均P<0.01),对照组与小潮气量组比较差异无统计学意义(P>0.05)。
2、大潮气量组肺组织气道上皮细胞HMGB1mRNA及其蛋白表达水平明显高于对照组和小潮气量组(均P<0.01),小潮气量组与对照组比较差异均无统计学意义(均P>0.05)。
实验二：
1、大潮气量组和血必净干预组大鼠血浆及BALF中MPO活性明显高于对照组(均P<0.01)；大潮气量组大鼠血浆及BALF中MPO活性高于血必净干预组(均P<0.01)。
2、大潮气量组和血必净干预组大鼠肺组织气道上皮细胞HMGB1 mRNA及其蛋白表达水平明显高于对照组(均P<0.01)；大潮气量组大鼠肺组织气道上皮细胞HMGB1 mRNA及其蛋白表达水平高于血必净干预组(均P<0.01)。
结论：
1、大潮气量机械通气可诱导肺组织HMGB1mRNA及其蛋白高表达,HMGB1分泌增多导致肺组织炎症反应扩大,可能是呼吸机所致肺损伤(VILI)发生的重要因素之一。
2、小潮气量机械通气对肺组织HMGB1表达影响较小,对正常肺组织无明显损伤作用。
3、血必净注射液在一定程度上可抑制肺组织HMGB1mRNA及其蛋白的表达,对机械通气所致大鼠急性肺损伤具有一定防治作用。
Objective:
To explore the role of high mobility group box 1(HMGB1) in acute lung injury induced by different tidal volume of ventilation in rats and its prevention and treatment of XueBiJing injection in the ventilaor induced lung injury by detecting the expression of high mobility group box 1(HMGB1) in lung tissue.
Methods:
1 Twenty-four male Wistar rats were randomly divided into three groups:control group, low tidal volume group (L-VT), high tidal volume group (H-VT). The levels of myloperoxidase(MPO) activity in plasma and bronchoalveolar lavage fluid (BALF) were determined by biochemical methods respectively.Immunohistochemial staining and molecular hybridization in situ were employed to determine the expression level of HMGB1 mRNA and protein in pulmonary tissues.
2 Twenty-four male Wistar rats were divided into three groups randomly:control group, H-VT group and XueBiJing pretreatment group. The levels of myloperoxidase(MPO) activity in plasma and bronchoalveolar lavage fluid (BALF) were determined by biochemical methods respectively.Immunohistochemial staining and molecular hybridization in situ were employed to determine the expression level of HMGB1 mRNA and protein in pulmonary tissues.
Results:
1 Comparing with control group and low tidal volume group, the levels of MPO activity in plasma and BALF 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 HMGB1 mRNA and protein were highly significant (P<0.01) in high tidal volume group. There was no statistics meaning for the differences between low tidal volume group and control group (P>0.05).
3 Comparing with control group, the levels of MPO activity in plasma and BALF were highly significant in high tidal volume group and XueBiJing pretreatment group(P＜0.01); Comparing with high tidal volume group, the levels of MPO activity in plasma and BALF were decreased significant in XueBiJing pretreatment group(P<0.01).
4 Comparing with control group, the expression of HMGB1 mRNA and protein was highly significant in high tidal volume group and XueBiJing pretreatment group (P＜0.01); Comparing with high tidal volume group, the expression of HMGB1 mRNA and protein was decreased significant in XueBiJing pretreatment group(P<0.01).
Conclusion:
1 The over expression of HMGB1 mRNA and protein can be induced by high tidal volume ventilation, which can lead to increased inflammatory response in lung tissue to expand. It is one of the most important factors in VILI.
2 There is small effect on the expression of HMGB1 in lung tissue in the L-VT group and no obvious lung injury.
3 XueBiJing injection may play a role of prevention and treatment in the ventilator induced lung injury by inhibiting the expression of HMGB1.
引文
[1]张志寰,蔡绍曦,赵海金.呼吸机相关性肺损伤的炎症机制研究进展[J].实用医学杂志,2007,23(3)：297-298.
[2]Slutsky AS. Lung injury caused by mechanical ventilation[J]. Chest,1999,116:9s-15s.
[3]Abraham E, Arcaroli J, Carmody A et al. Cutting edge:HMGB1 as a mediator of acute lung inflammation. J Immunol,2000,165(6):2950.
[4]Wang H, Zhu S, Zhou R, Li W, Sama AE. Therapeutic potential of HMGB1-targeting agents in sepsis.Expert Rev Mol Med.2008 Nov 4;10:e32.
[5]Feng Y, Yang Q, Xu J, Qian G, Liu Y. Effects of HMGB1 on PMN apoptosis during LPS-induced acute lung injury.Exp Mol Pathol.2008Dec;85(3):214-22. Epub 2008 Oct 8.
[6]姚咏明,柴家科,林洪远.现代脓毒症理论与实践.北京科学出版社,20051 1112-1152.
[7]姚咏明,盛志勇. 高迁移率族蛋白1在脓毒症发病中的作用与意义.解放军医学杂志,2002,27(9)：753.
[8]李银平,乔佑杰,武子霞。血必净注射液对脓毒症大鼠高迁移率族蛋白B1的影响中国危重病急救医学,2007,19(4)：9-12.
[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]Tremblay LN,Slutsky AS. Ventilator-induced injury:from barotraumas to biotrauma[J].Proc Assoc Am Physicians,1998,110:482-488.
[12]slustsky AS.Imai Y.Ventilator-induced lung injury,cytokines,PEEP,and mortality: implication for practice and for clinical trials [J].Intensive Care Med,2003,29(8):1218-1219.
[13]Bradley P P,Priebat D A,Christensen R D,et al.Measurement of cutaneous inflammation:estimation of neutrophil content with an enzyme marker[J].J Invest Dermatol,1982,78:206-209.
[14]cell death,caspase activation, and HMGB1 realease of porcine choroid plexus epithelial cell during strepeococcus suis infection.in Vitro[J].brain.res.2006.1100(1):1.
[15]olireira FM,Abreu.IC.Rumjanek FD.et.al.cloning the geneand DNA binding properties of high mobility group B1 proteins from the human blood.flukes schis to osoroa mansoni and schisto soma.Dapunuum Gene.2006.377:33.
[16]Wang H, Bloom O, Zhang M, et al. HMGB-1 as a late mediator of endotoxin lethality in mice [J]. Science,1999,285(5425):248-251.
[17]Lindersson EK, Hojrup P, Gai WP, et al. alpha-Synuclein filaments bind the transcriptional regulator HMGB-1[J].Neuroreport,2004,15(18):2735.
[18]Hicking KG.Permissive hypercapnia[J]. Respir Care Clin N Am,2002,8(2):155-169.
[19]Fink MP. Bench-to-bedside review:High-mobility group box land critical illness. Crit Care,2007,11:229.
[20]Kim JY,Park JS,Strassheim D,et al.HMGB1 contributes to the development of acute lung injury after hemorrhage.Am J Physiol Lung Cell Mol Physiol,2005,288:L958-L965.
[21]Pugin J,Jolliet P.Ventilator-induced lung:an inflammatory disease? [J].Clin Pulm Med,1998,5:290-299.
1.张会云;曹书华;王今达等,活血化瘀法对多脏器功能障碍综合征内毒素血症作用的临床研究。中华医院感染学杂志,2005,15(12)：1342-1343
2. Jiang Jianxia, Diao Youfang, Tian Kunlun, et al. Effect of hemorrhagic shock on endotoxin-inducing TNF production and intra-tissue lipopolysaccharide-binding proteinm RNA experession and their relationship. Shock,1997,7:206-210.
3.孙元莹,郭茂松等.血必净注射液对急性肺损伤肿瘤坏死因子的影响.时珍国医国药,2006.17(4)：251-252.
4.曹书华;高红梅;王永强;常文秀等.神农33号对多器官功能障碍综合征大鼠细胞因子的影响.中国急诊医学杂志。2003.2：102-103.
5.王旭东;李明;张超;杜波;韩大贺。血必净治疗ARDS疗效及其对炎症相关细胞因子的影响。徐州医学院学报,2007.11：67-68.
6. Abraham E, Arcaroli J, Carmody A et al. Cutting edge:HMGB1 as a mediator of acute lung inflammation. J Immunol,2000,165(6):2950
7. Wang H, Zhu S, Zhou R, Li W, Sama AE. Therapeutic potential of HMGB1-targeting agents in sepsis. Expert Rev Mol Med.2008 Nov 4;10:e32.
8. Feng Y, Yang Q, Xu J, Qian G, Liu Y. Effects of HMGB1 on PMN apoptosis during LPS-induced acute lung injury. Exp Mol Pathol.2008Dec;85(3):214-22. Epub 2008 Oct
9.姚咏明,柴家科,林洪远1现代脓毒症理论与实践1北京：科学出版社,20051 1112-1152
10.姚咏明,盛志勇1 高迁移率族蛋白21在脓毒症发病中的作用与意义1解放军医学杂志,2002,27(9)：753
11.李银平,乔佑杰,武子霞。血必净注射液对脓毒症大鼠高迁移率族蛋白B1的影响中国危重病急救医学,2007,19(4)：9-12.
12.李磊,王兴鹏.炎症反应的晚期介质HMGB1[J].国外医学生理、病理科学与临床分册,2004,24(16)：568
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[2]Slutsky AS. Lung injury caused by mechanical ventilation[J]. Chest,1999,116:9s-15s.
[3]Abraham E, Arcaroli J, Carmody A et al. Cutting edge:HMGB1 as a mediator of acute lung inflammation. J Immunol,2000,165(6):2950.
[4]Wang H, Zhu S, Zhou R, Li W, Sama AE. Therapeutic potential of HMGB1-targeting agents in sepsis.Expert Rev Mol Med.2008 Nov 4;10:e32.
[5]Feng Y, Yang Q, Xu J, Qian G, Liu Y. Effects of HMGB1 on PMN apoptosis during LPS-induced acute lung injury.Exp Mol Pathol.2008Dec;85(3):214-22. Epub 2008 Oct 8.
[6]姚咏明,柴家科,林洪远.现代脓毒症理论与实践.北京科学出版社,20051 1112-1152.
[7]姚咏明,盛志勇. 高迁移率族蛋白1在脓毒症发病中的作用与意义.解放军医学杂志,2002,27(9)：753.
[8]李银平,乔佑杰,武子霞。血必净注射液对脓毒症大鼠高迁移率族蛋白B1的影响中国危重病急救医学,2007,19(4)：9-12.
[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]Tremblay LN,Slutsky AS. Ventilator-induced injury:from barotraumas to biotrauma[J].Proc Assoc Am Physicians,1998,110:482-488.
[12]slustsky AS.Imai Y.Ventilator-induced lung injury,cytokines,PEEP,and mortality: implication for practice and for clinical trials [J].Intensive Care Med,2003,29(8):1218-1219.
[13]Bradley P P,Priebat D A,Christensen R D,et al.Measurement of cutaneous inflammation:estimation of neutrophil content with an enzyme marker[J].J Invest Dermatol,1982,78:206-209.
[14]cell death,caspase activation, and HMGB1 realease of porcine choroid plexus epithelial cell during strepeococcus suis infection.in Vitro[J].brain.res.2006.1100(1):1.
[15]olireira FM,Abreu.IC.Rumjanek FD.et.al.cloning the geneand DNA binding properties of high mobility group B1 proteins from the human blood.flukes schis to osoroa mansoni and schisto soma.Dapunuum Gene.2006.377:33.
[16]Wang H, Bloom O, Zhang M, et al. HMGB-1 as a late mediator of endotoxin lethality in mice [J]. Science,1999,285(5425):248-251.
[17]Lindersson EK, Hojrup P, Gai WP, et al. alpha-Synuclein filaments bind the transcriptional regulator HMGB-1[J].Neuroreport,2004,15(18):2735.
[18]Hicking KG.Permissive hypercapnia[J]. Respir Care Clin N Am,2002,8(2):155-169.
[19]Fink MP. Bench-to-bedside review:High-mobility group box land critical illness. Crit Care,2007,11:229.
[20]Kim JY,Park JS,Strassheim D,et al.HMGB1 contributes to the development of acute lung injury after hemorrhage.Am J Physiol Lung Cell Mol Physiol,2005,288:L958-L965.
[21]Pugin J,Jolliet P.Ventilator-induced lung:an inflammatory disease? [J].Clin Pulm Med,1998,5:290-299.
1.张会云;曹书华;王今达等,活血化瘀法对多脏器功能障碍综合征内毒素血症作用的临床研究。中华医院感染学杂志,2005,15(12)：1342-1343
2. Jiang Jianxia, Diao Youfang, Tian Kunlun, et al. Effect of hemorrhagic shock on endotoxin-inducing TNF production and intra-tissue lipopolysaccharide-binding proteinm RNA experession and their relationship. Shock,1997,7:206-210.
3.孙元莹,郭茂松等.血必净注射液对急性肺损伤肿瘤坏死因子的影响.时珍国医国药,2006.17(4)：251-252.
4.曹书华;高红梅;王永强;常文秀等.神农33号对多器官功能障碍综合征大鼠细胞因子的影响.中国急诊医学杂志。2003.2：102-103.
5.王旭东;李明;张超;杜波;韩大贺。血必净治疗ARDS疗效及其对炎症相关细胞因子的影响。徐州医学院学报,2007.11：67-68.
6. Abraham E, Arcaroli J, Carmody A et al. Cutting edge:HMGB1 as a mediator of acute lung inflammation. J Immunol,2000,165(6):2950
7. Wang H, Zhu S, Zhou R, Li W, Sama AE. Therapeutic potential of HMGB1-targeting agents in sepsis. Expert Rev Mol Med.2008 Nov 4;10:e32.
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