P38MAPK在急性胰腺炎肺损伤发病机制中的作用及清胰汤影响的实验研究
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摘要
目的: 探讨P38MAPK在大鼠重症急性胰腺炎急性肺损伤发病机制中的作用,观察肺组织中炎症因子水平、血中内毒素、淀粉酶含量等相关指标的变化及中药清胰汤的影响,进一步明确清胰汤的作用机理,为临床上提高疗效提供理论指导。
方法: 健康SD大鼠随机分为假手术组、模型组、抑制剂组、清胰汤组。除假手术组外,其余三组大鼠经胰胆管逆行注入去氧胆酸钠复制大鼠重症急性胰腺炎急性肺损伤模型。抑制剂组为术前1h腹腔注射SB203580,1mg/Kg。清胰汤组于术前1h及术后清醒时灌胃,1ml/kg。术后24h采取标本做病理检查和各项指标检测。
肺组织ICAM-1、P38MAPK的检测方法采用Western Blot法。肺组织TNF-а检测采用放免法。肺组织NO含量检测采用硝酸还原酶法。
结果: 与假手术组相比较模型组肺组织P38MAPK、ICAM-1活性明显增强,TNF-а、NO水平明显升高(P<0.05)。血中淀粉酶含量、内毒素水平以及肺组织湿/干比值亦明显增高。与模型组相比清胰汤组和抑制剂组肺组织P38MAPK及ICAM-1活性,肺组织TNF-а、NO含量则显著下降(P<0.05);血中淀粉酶、内毒素水平也显著降低(p<0.05)。
肺组织病理观察,模型组表现为明显的肺损伤,而清胰汤组及抑制剂组则显著改善,假手术组肺组织损伤最轻。
结论: 重症急性胰腺炎肺损伤时,P38MAPK活化,将炎症信号传递给核转录因子NF-kappaB,使之活化,增加了TNF-а、NO等多种炎症因子的转录,以及ICAM-1的大量表达,使炎症因子大量释放入血。同时在胰腺炎发生发展过程中血中内毒素,淀粉酶水平明显升高。抑制剂SB203580能特异性抑制肺组织中P38MAPK的活化,明显减少炎症因子的产生,进而减轻肺组织的炎症损伤。中药清胰汤组肺组织P38MAPK的活性较模型组明显降低,接近于抑制剂组,略高于假手术组。其血中炎症因子水平以及肺组织中炎症介质(介导炎症细胞局部反应的ICAM-1等)也与抑制剂组相仿。从而说明,中药在治疗重症急性胰腺炎肺损伤时可以通过下调P38MAPK活性等环节影响多种炎症介质的产生,进而起到保护肺组织的积极作用。中西药物在急性胰腺炎肺损伤发病的不同环节中,发挥着十分重要的作用,且各有所长,因此,在重症急性胰腺炎肺损伤的治疗过程中应该遵循:内外结合、中西并用、辨证施治、标本兼治。
Objective: To investigate the potential role of P38MAPK activation in acute lung injury induced by severe acute pancreatitis and also to observe inflammatory cytokines and endotoxin in pulmonary tissue and blood. Chinese medicine QingYiTang Decoction was used and the results may give us some new ideas for curing acute lung injury.
Methods: The rats were divided into four groups:Sham operated controls (n=16); SAP model group (n=16); QingYiTang Decoction treated group (n=16); Inhibitor treated group (n=16). 1.5% sodium deoxycholate was injected inversely into the common biliopancreatic duct of rats to improve the model of SAP. All the rats were killed at 24 hours after operation. Pathological examination and some indexes of ALI were measured.
Pulmonary P38MAPK and ICAM-1 activity were determined by Western Blot. And we also measured the level of NO、TNF-а、Endotoxin、Amylase, et al.
Results: The levels of P38MAPK and ICAM-1 in pulmonary of model group were significantly higher than those of sham operated control group (p<0.05). Some other indexes of ALI showed obvious pathogenic changes in model group and so did the pathogenic examination of lung
tissue. QingYiTang Decoction could down-regulate the expression of the P38MAPK and ICAM-1 activity. The decline of NO、TNF-а in QingYiTang Decoction group was more significant (p<0.05).
Conclusions: During acute lung injury induced by severe acute pancreatitis, many factors have very important effects, such as P38MAPK activity which stimulates the activity of NF-kB, and start up the volume release of NO and TNF-а, and increase the activity of ICAM-1 obviously. QYT decoction could exert its protecting effects through eliminating P38MAPK activity, alleviating the level of NO、TNF-а, eliminating ICAM-1 activity and decreasing the higher level of endotoxin and amylase,then alleviating acute pulmonary injury. In a word, traditional and western medicine make very important effects in acute lung injury induced by severe acute pancreatitis. If applying traditional and western medicine reasonably, we will find more useful methods to prevent and cure acute lung injury induced by severe acute pancreatitis in clinic.
方法: 健康SD大鼠随机分为假手术组、模型组、抑制剂组、清胰汤组。除假手术组外,其余三组大鼠经胰胆管逆行注入去氧胆酸钠复制大鼠重症急性胰腺炎急性肺损伤模型。抑制剂组为术前1h腹腔注射SB203580,1mg/Kg。清胰汤组于术前1h及术后清醒时灌胃,1ml/kg。术后24h采取标本做病理检查和各项指标检测。
肺组织ICAM-1、P38MAPK的检测方法采用Western Blot法。肺组织TNF-а检测采用放免法。肺组织NO含量检测采用硝酸还原酶法。
结果: 与假手术组相比较模型组肺组织P38MAPK、ICAM-1活性明显增强,TNF-а、NO水平明显升高(P<0.05)。血中淀粉酶含量、内毒素水平以及肺组织湿/干比值亦明显增高。与模型组相比清胰汤组和抑制剂组肺组织P38MAPK及ICAM-1活性,肺组织TNF-а、NO含量则显著下降(P<0.05);血中淀粉酶、内毒素水平也显著降低(p<0.05)。
肺组织病理观察,模型组表现为明显的肺损伤,而清胰汤组及抑制剂组则显著改善,假手术组肺组织损伤最轻。
结论: 重症急性胰腺炎肺损伤时,P38MAPK活化,将炎症信号传递给核转录因子NF-kappaB,使之活化,增加了TNF-а、NO等多种炎症因子的转录,以及ICAM-1的大量表达,使炎症因子大量释放入血。同时在胰腺炎发生发展过程中血中内毒素,淀粉酶水平明显升高。抑制剂SB203580能特异性抑制肺组织中P38MAPK的活化,明显减少炎症因子的产生,进而减轻肺组织的炎症损伤。中药清胰汤组肺组织P38MAPK的活性较模型组明显降低,接近于抑制剂组,略高于假手术组。其血中炎症因子水平以及肺组织中炎症介质(介导炎症细胞局部反应的ICAM-1等)也与抑制剂组相仿。从而说明,中药在治疗重症急性胰腺炎肺损伤时可以通过下调P38MAPK活性等环节影响多种炎症介质的产生,进而起到保护肺组织的积极作用。中西药物在急性胰腺炎肺损伤发病的不同环节中,发挥着十分重要的作用,且各有所长,因此,在重症急性胰腺炎肺损伤的治疗过程中应该遵循:内外结合、中西并用、辨证施治、标本兼治。
Objective: To investigate the potential role of P38MAPK activation in acute lung injury induced by severe acute pancreatitis and also to observe inflammatory cytokines and endotoxin in pulmonary tissue and blood. Chinese medicine QingYiTang Decoction was used and the results may give us some new ideas for curing acute lung injury.
Methods: The rats were divided into four groups:Sham operated controls (n=16); SAP model group (n=16); QingYiTang Decoction treated group (n=16); Inhibitor treated group (n=16). 1.5% sodium deoxycholate was injected inversely into the common biliopancreatic duct of rats to improve the model of SAP. All the rats were killed at 24 hours after operation. Pathological examination and some indexes of ALI were measured.
Pulmonary P38MAPK and ICAM-1 activity were determined by Western Blot. And we also measured the level of NO、TNF-а、Endotoxin、Amylase, et al.
Results: The levels of P38MAPK and ICAM-1 in pulmonary of model group were significantly higher than those of sham operated control group (p<0.05). Some other indexes of ALI showed obvious pathogenic changes in model group and so did the pathogenic examination of lung
tissue. QingYiTang Decoction could down-regulate the expression of the P38MAPK and ICAM-1 activity. The decline of NO、TNF-а in QingYiTang Decoction group was more significant (p<0.05).
Conclusions: During acute lung injury induced by severe acute pancreatitis, many factors have very important effects, such as P38MAPK activity which stimulates the activity of NF-kB, and start up the volume release of NO and TNF-а, and increase the activity of ICAM-1 obviously. QYT decoction could exert its protecting effects through eliminating P38MAPK activity, alleviating the level of NO、TNF-а, eliminating ICAM-1 activity and decreasing the higher level of endotoxin and amylase,then alleviating acute pulmonary injury. In a word, traditional and western medicine make very important effects in acute lung injury induced by severe acute pancreatitis. If applying traditional and western medicine reasonably, we will find more useful methods to prevent and cure acute lung injury induced by severe acute pancreatitis in clinic.
引文
方步武,邱奇,周振理,秦明放,孔棣,崔乃强,吴咸中。急腹症内毒素血症期的全身炎症反应。中国中西医结合外科杂志,2001,10,7(5):295-297。
Fein AM,Calalang-Colucci MG.Acute lung injury and acute respiratory disress syndrome in sepsis and septic shock.Crit Care Clin,2000,16(2):289-317.
Doersohuk CM,Mizgerd JP,Kubo H,Karton D.Adhension molecules and cellular biomechanical changes in acute lung injury.Chest,1999,166:37-43.
姚咏明,盛志勇。脓毒症信号转导机制的现代认识。中国危重病急救医学,2003,1,15(1):3-6.
黄翠萍。p38MAPK与内毒素性肺损伤。国外医学生理病理科学与临床分册,2001,10,21(5):373-375。
Nick JA,Avdi NJ,Young SK,McDonsld PP,Billstrom MA,Henson PM,Johnson GL,Worthen GS.An intracellular signaling pathway linking lipopolysaccharide stimulation to cellular responses of the human neutrophil: the p38 MAP kinase cascade and its functional significance.Chest. 1999 Jul;116(1 Suppl):54S-55S. No abstract available.
Jiang Y,Liu AN,Zhang L,Zhao KS.Activation of p38 mitogen-activated protein kinase induced by lipopolysaccharide and it’s the in TNF-аgene expression.J Med Coll PLA,1999,14(2):138-143.
Guan Z,Baier LD,Morrison AR.P38 mitogen-activated protein kinase down-regulates nitric oxide and up regulate sprostaglandin E2 biosynthesis stimulated by interleukin-1beta.J Biol Chem,1997,272(12):8083-8089.
Guan Z,Buckman SY,Miller BW,Springer.Interleukin-1 betainduced cyclooxygenase-2 expression requires activation of both c-jun NH2-terminal kinase and p38MAPK signal pathways in rat renal messangial cells.J Biol Chem,1998,273(44):28670-28676.
Read MA,Whitley MZ,Gupta S,Botti C.Tumor necrosis factor alpha-induced E-selectin expression is activated bythe nuclear factor-Kappa B and c-Jun N-terminal kinase/p38 mitogen-activated protein kinase pathways.J Biol Chem,
1997,272(5):2753-2761.
张刚。实验性急性胰腺炎大鼠肺组织肿瘤坏死因子mRNA的表达及其意义。华西医科大学学报,1999,30:379-380。
闫文生,阙文宏,黄巧冰,姜勇,赵克森。脂多糖诱导小鼠肠组织ICAM-1表达的变化及p38MAPK在其中的作用。中国病理生理杂志,2002,18(9):1029-1033。
毛华,陈宏。p38蛋白激酶活性测定方法.国外医学临床生物化学与检验学分册,2000,21(4):211-212。
陈海龙。急性胰腺炎的中西医结合研究与治疗。中华中西医临床杂志,2003,12,3(12):1409-1412。
李泉,邹最。炎症信号转道于危急疾患。国外医学麻醉学与复苏分册,2002,23(2),66-71。
罗富荣。中性粒细胞与急性肺损伤。 国外医学麻醉学与复苏分册,2002,23(2),71-73。
黄翠萍,张珍祥,徐永健。P38蛋白激酶对大鼠肺泡巨噬细胞活化机制的调控。中国病理生理杂志,2003,19(5):661-663。
Tamura DY, Moore EE, Johnson JL, Zallen G, Aiboshi J, Silliman CC.p38 mitogen-activated protein kinase inhibition attenuates intercellular adhesion molecule-1 up-regulation on human pulmonary microvascular endothelial cells.Surgery 1998 Aug;124(2):403-7; discussion 408.
王蛟。粘附分子与急性胰腺炎。国外医学消化系疾病分册,2001,21(2),104-107。
Carlton B,Sijmonsma TP, Verseyden C, Jansen EH, De Koning EJ, Rabelink TJ, Castro Cabezas M.Postprandial recruitment of neutrophils may contribute to endothelial dysfunction.Res Exp Med(Berl),1995,195(3):125-144.
郭禹标,陈永雄,谢灿茂,杨惠玲,尹培达。巨噬细胞移动抑制因子在油酸致急性肺损伤动物模型肺组织中的表达。中国病理生理杂志,2001,17(1):40-42。
Jun Yang,Colleen Murphy,Woody Denham,Galina Botchkina,Phd,Kevin J,Tracey, James Norman.Evidence of a central role for P38 map kinase induction of tumor
necrosis factorаin pancreatitis-associated pulmonary injury.Surgery, 1999, 126:216-22.
You-Li Zu,Jiafan Qi,Annette Gilchirist,Gustavo A,Fernandez,Dolores Vazquez- Abad, Donald L,Kreutzer,Chi-Kuang Huang,Ramadan I.p38 Mitogen-Activated Protein Kinase Activation Is Required for Human Neutrophil Function Triggered by TNF-а or FMLP Stimulation.The Journal of Immunology, 1998, 160: 1982-1989.
Christopher D,Beaty,Terry L.Franklin,Yoshimsas Ueharn and Christopher B.Wilson.Lipopolysaccharide-induced cytokine production un human monocytes: role of tyrosine phosphorylation in transmembrane signal transduction. Eur.J. Immunol,1994,24:1278-1284.
Douglas Y.Tamura,Ernest E.Moore,Jeffrey L.Johnson.p38 Mitogen-activated protein kinase inhibition attenuates intercellular adhesion molecule-1 up-regualation on human pulmonary microvascular endothelial cells.Surgery, 1998, 124:403-8.
Brown,K.,S.Park,T.Kanno,G.Franzoso,and U.Siebenlist.Mutual regulation of the transcriptional activator NF-kB and its inhibitor,Ikb-а.Proc.Natl.Acad. Sci. USA,1993 90:2532.
Jerry A.Nick,Natalie J.Avdi,Scott K.Young,Lisa A.Lehman,Patrick P,McDonald S,Courtney Frasch,Marcella A,Billstrom,Peter M Henson,Gary L,Johnson G,Scott Worthen.Selectiv activation and functional significance of p38 mitigen-antivated protein kinase in lipopolusaccharide-stimulated nertrophils.J. Clin.Invest, 1999, 103:851-858.
Jamrl El Benna,Jiahuai Han,Jeen-Woo Park,Elmar Schmid,Richard J,Ulevitch,Bernard M,Babior.Activation of p38 in stimulated human neutrophils: Phospohrylation of the oxidase componentp47phoxby p38 and ERK but not by JNK.Archives of Biochemistry and Biophysics, 1996, Oct. 15, 334(2):395-400.
姜勇,刘爱华,张琳,赵克森。脂多糖激活p38在诱导肿瘤坏死因子а基因表达中的作用。中华医学杂志,1999,5,79(5):360-364。
Chen XL, Xia ZF, Ben DF, Wang GQ, Wei D..Role of p38 mitogen-activated
protein kinase in lung injury after burn trauma. Shock. 2003 May;19(5):475-9.
Arbabi S, Garcia I, Bauer GJ, Maier RV.Alcohol (ethanol) inhibits IL-8 and TNF: role of the p38 pathway.J Immunol,99 Jun 15;162(12):7441-5.
Zu YL, Qi J, Gilchrist A, Fernandez GA, Vazquez-Abad D, Kreutzer DL, Huang CK, Sha'afi RI.p38 mitogen-activated protein kinase activation is required for human neutrophil function triggered by TNF-alpha or FMLP stimulation.J Immunol,1998 Feb 15;160(4):1982-9.
李海龙,陈海龙,张波。清胰汤对急性胰腺炎时急性肺损伤防治作用的实验研究。 中华中西医临床杂志,2003,12,3(12):1480-1483。
Fein AM,Calalang-Colucci MG.Acute lung injury and acute respiratory disress syndrome in sepsis and septic shock.Crit Care Clin,2000,16(2):289-317.
Doersohuk CM,Mizgerd JP,Kubo H,Karton D.Adhension molecules and cellular biomechanical changes in acute lung injury.Chest,1999,166:37-43.
姚咏明,盛志勇。脓毒症信号转导机制的现代认识。中国危重病急救医学,2003,1,15(1):3-6.
黄翠萍。p38MAPK与内毒素性肺损伤。国外医学生理病理科学与临床分册,2001,10,21(5):373-375。
Nick JA,Avdi NJ,Young SK,McDonsld PP,Billstrom MA,Henson PM,Johnson GL,Worthen GS.An intracellular signaling pathway linking lipopolysaccharide stimulation to cellular responses of the human neutrophil: the p38 MAP kinase cascade and its functional significance.Chest. 1999 Jul;116(1 Suppl):54S-55S. No abstract available.
Jiang Y,Liu AN,Zhang L,Zhao KS.Activation of p38 mitogen-activated protein kinase induced by lipopolysaccharide and it’s the in TNF-аgene expression.J Med Coll PLA,1999,14(2):138-143.
Guan Z,Baier LD,Morrison AR.P38 mitogen-activated protein kinase down-regulates nitric oxide and up regulate sprostaglandin E2 biosynthesis stimulated by interleukin-1beta.J Biol Chem,1997,272(12):8083-8089.
Guan Z,Buckman SY,Miller BW,Springer.Interleukin-1 betainduced cyclooxygenase-2 expression requires activation of both c-jun NH2-terminal kinase and p38MAPK signal pathways in rat renal messangial cells.J Biol Chem,1998,273(44):28670-28676.
Read MA,Whitley MZ,Gupta S,Botti C.Tumor necrosis factor alpha-induced E-selectin expression is activated bythe nuclear factor-Kappa B and c-Jun N-terminal kinase/p38 mitogen-activated protein kinase pathways.J Biol Chem,
1997,272(5):2753-2761.
张刚。实验性急性胰腺炎大鼠肺组织肿瘤坏死因子mRNA的表达及其意义。华西医科大学学报,1999,30:379-380。
闫文生,阙文宏,黄巧冰,姜勇,赵克森。脂多糖诱导小鼠肠组织ICAM-1表达的变化及p38MAPK在其中的作用。中国病理生理杂志,2002,18(9):1029-1033。
毛华,陈宏。p38蛋白激酶活性测定方法.国外医学临床生物化学与检验学分册,2000,21(4):211-212。
陈海龙。急性胰腺炎的中西医结合研究与治疗。中华中西医临床杂志,2003,12,3(12):1409-1412。
李泉,邹最。炎症信号转道于危急疾患。国外医学麻醉学与复苏分册,2002,23(2),66-71。
罗富荣。中性粒细胞与急性肺损伤。 国外医学麻醉学与复苏分册,2002,23(2),71-73。
黄翠萍,张珍祥,徐永健。P38蛋白激酶对大鼠肺泡巨噬细胞活化机制的调控。中国病理生理杂志,2003,19(5):661-663。
Tamura DY, Moore EE, Johnson JL, Zallen G, Aiboshi J, Silliman CC.p38 mitogen-activated protein kinase inhibition attenuates intercellular adhesion molecule-1 up-regulation on human pulmonary microvascular endothelial cells.Surgery 1998 Aug;124(2):403-7; discussion 408.
王蛟。粘附分子与急性胰腺炎。国外医学消化系疾病分册,2001,21(2),104-107。
Carlton B,Sijmonsma TP, Verseyden C, Jansen EH, De Koning EJ, Rabelink TJ, Castro Cabezas M.Postprandial recruitment of neutrophils may contribute to endothelial dysfunction.Res Exp Med(Berl),1995,195(3):125-144.
郭禹标,陈永雄,谢灿茂,杨惠玲,尹培达。巨噬细胞移动抑制因子在油酸致急性肺损伤动物模型肺组织中的表达。中国病理生理杂志,2001,17(1):40-42。
Jun Yang,Colleen Murphy,Woody Denham,Galina Botchkina,Phd,Kevin J,Tracey, James Norman.Evidence of a central role for P38 map kinase induction of tumor
necrosis factorаin pancreatitis-associated pulmonary injury.Surgery, 1999, 126:216-22.
You-Li Zu,Jiafan Qi,Annette Gilchirist,Gustavo A,Fernandez,Dolores Vazquez- Abad, Donald L,Kreutzer,Chi-Kuang Huang,Ramadan I.p38 Mitogen-Activated Protein Kinase Activation Is Required for Human Neutrophil Function Triggered by TNF-а or FMLP Stimulation.The Journal of Immunology, 1998, 160: 1982-1989.
Christopher D,Beaty,Terry L.Franklin,Yoshimsas Ueharn and Christopher B.Wilson.Lipopolysaccharide-induced cytokine production un human monocytes: role of tyrosine phosphorylation in transmembrane signal transduction. Eur.J. Immunol,1994,24:1278-1284.
Douglas Y.Tamura,Ernest E.Moore,Jeffrey L.Johnson.p38 Mitogen-activated protein kinase inhibition attenuates intercellular adhesion molecule-1 up-regualation on human pulmonary microvascular endothelial cells.Surgery, 1998, 124:403-8.
Brown,K.,S.Park,T.Kanno,G.Franzoso,and U.Siebenlist.Mutual regulation of the transcriptional activator NF-kB and its inhibitor,Ikb-а.Proc.Natl.Acad. Sci. USA,1993 90:2532.
Jerry A.Nick,Natalie J.Avdi,Scott K.Young,Lisa A.Lehman,Patrick P,McDonald S,Courtney Frasch,Marcella A,Billstrom,Peter M Henson,Gary L,Johnson G,Scott Worthen.Selectiv activation and functional significance of p38 mitigen-antivated protein kinase in lipopolusaccharide-stimulated nertrophils.J. Clin.Invest, 1999, 103:851-858.
Jamrl El Benna,Jiahuai Han,Jeen-Woo Park,Elmar Schmid,Richard J,Ulevitch,Bernard M,Babior.Activation of p38 in stimulated human neutrophils: Phospohrylation of the oxidase componentp47phoxby p38 and ERK but not by JNK.Archives of Biochemistry and Biophysics, 1996, Oct. 15, 334(2):395-400.
姜勇,刘爱华,张琳,赵克森。脂多糖激活p38在诱导肿瘤坏死因子а基因表达中的作用。中华医学杂志,1999,5,79(5):360-364。
Chen XL, Xia ZF, Ben DF, Wang GQ, Wei D..Role of p38 mitogen-activated
protein kinase in lung injury after burn trauma. Shock. 2003 May;19(5):475-9.
Arbabi S, Garcia I, Bauer GJ, Maier RV.Alcohol (ethanol) inhibits IL-8 and TNF: role of the p38 pathway.J Immunol,99 Jun 15;162(12):7441-5.
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