瘦素在大鼠重症急性胰腺炎并发肺损伤中的作用
摘要
目的:通过制备重症急性胰腺炎(SAP)并发肺损伤的动物模型来了解其发病机制,探讨重症急性胰腺炎大鼠肺组织核因子-κB(NF-κB)活化和诱生型一氧化氮合酶(iN0S)、NO表达与重症急性胰腺炎并发肺损伤的关系,同时初步探讨瘦素的作用机制,为临床提供借鉴。
方法:36只Wistar大鼠随机分为正常对照组(A组)、重症急性胰腺炎组(B组)和外源性瘦素治疗组(C组)。腹腔麻醉成功后,采用胰管注射5%牛磺胆酸钠法建立重症急性胰腺炎动物模型。B组在胰管中注入5%牛磺胆酸钠形成SAP模型,A组注入生理盐水,C组以外源性瘦素于SAP模型大鼠关腹前行腹腔注射,在注入5%牛磺胆酸钠6h后分别取动脉血作血气分析,取静脉血作淀粉酶测定、检测左肺组织NF-κB活性、NO、iNOS含量,取右肺进行支气管肺泡灌洗,灌洗液做白细胞计数。同时对动物的肺脏和胰腺作组织学结构的观察。
结果:A组动物动脉血PaO2为(99.6±1.1)mmHg,B组动物下降为(76.1±7.6)mmHg,C组动物动脉血PaO2(92.9±11.0)mmHg。SAP时肺组织NF-κB活化及iNOS、NO、白细胞计数、血浆淀粉酶高表达,瘦素治疗组肺组织NF-κB活性、iNOS、NO含量、血浆淀粉酶、白细胞计数降低,肺组织病理学改变减轻。NF-κB活化与iNOS表达呈正相关,相关系数分别为0.79(P<0.01),iNOS表达与NO含量也呈正相关,相关系数0.66(P<0.01)。从肺、胰腺大体、组织学结构的观察,实验组达到重症急性胰腺炎并发肺损伤的诊断标准。瘦素治疗组较SAP组PaO2明显提高,肺泡灌洗液中白细胞计数明显下降。而治疗组和对照组之间的改变不明显。
结论:胰管注射5%牛磺胆酸钠是制备重症急性胰腺炎的有效方法; NF-κB活化、iNOS/NO过度表达介导重症急性胰腺炎并发肺损伤起重要作用;瘦素能显著改善重症急性胰腺炎并发的肺损伤。瘦素可能通过抑制NF-κB活化途径减少iNOS的活性表达,从而减少过量NO的生成,减轻肺损伤病理损害。
Objective:To explore the relationship between expression of iNOS、NO、NF-κB activation of lung tissue and lung injury in rats of severe acute pancreatitis(SAP) associated with lung injury and to explore the effect of leptin.
Methods:Thirty six Wistar rats were randomized into the three group:normal group(groupA);SAP group(groupB);leptin treatment group(groupC).The SAP model was induced by injection of 5%sodium taurocholate solution into biliopancreatic duct.All the animals were sacrificed in 6h.Activation of NF-κB was determined with immunohistoehemistry.Mean-while,iNOS、NO、plasma leve1 of amylase were determined.lavage was introduced in their lobes of right lung.The lavage fluid was counted leucocytes.In the end,lung and pancrea tissues histological were determined.
Results: In SAP group, the NF-κB activity、the NO levels and the iNOS expression increased markedly.In Leptin group, the activation of NF-κB was prevented and plasma level of amylase , and content of NO and iNOS remarkably decreased.Activation of NF-κB had positive correlation with expression of iNOS (r=O.79,P<0.01) and iNOS had positive correlation with NO (r=0.66,P<0.01).At sixth hour,the arterial blood gas、serum amylase、NO and were not obviously different between groupA and groupC.The leucocytes counts were higher in groupB than those in groupsA andC.By microscopic observation of the histological appearance and the cells of lung and pancrea resulted in the diagnostie standard of the serve acute pancreatitis with lung injury in groupB.
Conclusion: This is a successful method of making model of experimental serve acute pancreatitis associated with lung injury; NF-κB activation、iNOS expression and NO level were very important in inducing experimental acute pancreatitis associated with lung injury.Early blockage of NF-κB activation with leptin is effective for reducing severity of the lung injury.Leptin could reduce iNOS expression and NO level by inhibiting the NF-κB activation.
方法:36只Wistar大鼠随机分为正常对照组(A组)、重症急性胰腺炎组(B组)和外源性瘦素治疗组(C组)。腹腔麻醉成功后,采用胰管注射5%牛磺胆酸钠法建立重症急性胰腺炎动物模型。B组在胰管中注入5%牛磺胆酸钠形成SAP模型,A组注入生理盐水,C组以外源性瘦素于SAP模型大鼠关腹前行腹腔注射,在注入5%牛磺胆酸钠6h后分别取动脉血作血气分析,取静脉血作淀粉酶测定、检测左肺组织NF-κB活性、NO、iNOS含量,取右肺进行支气管肺泡灌洗,灌洗液做白细胞计数。同时对动物的肺脏和胰腺作组织学结构的观察。
结果:A组动物动脉血PaO2为(99.6±1.1)mmHg,B组动物下降为(76.1±7.6)mmHg,C组动物动脉血PaO2(92.9±11.0)mmHg。SAP时肺组织NF-κB活化及iNOS、NO、白细胞计数、血浆淀粉酶高表达,瘦素治疗组肺组织NF-κB活性、iNOS、NO含量、血浆淀粉酶、白细胞计数降低,肺组织病理学改变减轻。NF-κB活化与iNOS表达呈正相关,相关系数分别为0.79(P<0.01),iNOS表达与NO含量也呈正相关,相关系数0.66(P<0.01)。从肺、胰腺大体、组织学结构的观察,实验组达到重症急性胰腺炎并发肺损伤的诊断标准。瘦素治疗组较SAP组PaO2明显提高,肺泡灌洗液中白细胞计数明显下降。而治疗组和对照组之间的改变不明显。
结论:胰管注射5%牛磺胆酸钠是制备重症急性胰腺炎的有效方法; NF-κB活化、iNOS/NO过度表达介导重症急性胰腺炎并发肺损伤起重要作用;瘦素能显著改善重症急性胰腺炎并发的肺损伤。瘦素可能通过抑制NF-κB活化途径减少iNOS的活性表达,从而减少过量NO的生成,减轻肺损伤病理损害。
Objective:To explore the relationship between expression of iNOS、NO、NF-κB activation of lung tissue and lung injury in rats of severe acute pancreatitis(SAP) associated with lung injury and to explore the effect of leptin.
Methods:Thirty six Wistar rats were randomized into the three group:normal group(groupA);SAP group(groupB);leptin treatment group(groupC).The SAP model was induced by injection of 5%sodium taurocholate solution into biliopancreatic duct.All the animals were sacrificed in 6h.Activation of NF-κB was determined with immunohistoehemistry.Mean-while,iNOS、NO、plasma leve1 of amylase were determined.lavage was introduced in their lobes of right lung.The lavage fluid was counted leucocytes.In the end,lung and pancrea tissues histological were determined.
Results: In SAP group, the NF-κB activity、the NO levels and the iNOS expression increased markedly.In Leptin group, the activation of NF-κB was prevented and plasma level of amylase , and content of NO and iNOS remarkably decreased.Activation of NF-κB had positive correlation with expression of iNOS (r=O.79,P<0.01) and iNOS had positive correlation with NO (r=0.66,P<0.01).At sixth hour,the arterial blood gas、serum amylase、NO and were not obviously different between groupA and groupC.The leucocytes counts were higher in groupB than those in groupsA andC.By microscopic observation of the histological appearance and the cells of lung and pancrea resulted in the diagnostie standard of the serve acute pancreatitis with lung injury in groupB.
Conclusion: This is a successful method of making model of experimental serve acute pancreatitis associated with lung injury; NF-κB activation、iNOS expression and NO level were very important in inducing experimental acute pancreatitis associated with lung injury.Early blockage of NF-κB activation with leptin is effective for reducing severity of the lung injury.Leptin could reduce iNOS expression and NO level by inhibiting the NF-κB activation.
引文
[1].Interiano B,Stuard D,et al.Acute respiratory distress syndrome in Pancreatitis.Ann intern Med.1972,77:923.
[2].朱国英,李永渝核因子-KB作为急性胰腺炎治疗靶向的思考[J].国外医学外科学分册.2005,32(5):357-358.
[3].Shames BD,Barton HH,Reznikov LL.et a1.Ischemia alone is sufficient to induce TNF-alpha mRNA and peptied in the myocardium.Shock 2002,17:114-119.
[4].张喜平、李建秋、程琪辉.急性胰腺炎中NO和ET作用的两面性[J].中华研究杂志.2007,36(3):86.
[5].路琴、阴桢宏、诸欣平.瘦素对重症急性胰腺炎保护作用的实验研究[J].中华医学杂志.2006, 86(45):3222.
[6].Andrts Duarte-Rojof Ana Lezama-Barredaf Maria Teresa Ramirez-Iglesiasf,Is leptin related to systemic inflammatory response in acute pancreatitis? World,GastroenteroI 2006 July.
[7].A.k.Banergee,S.W. Gulloway,et al.Experimental models of acute Pancreatitis.Brit J Surg.1994,81:1096-1103.
[8] . C.R.B.Welbour and Y.Yong.Endotoxin , septic shock and acute lung injury:neutrophil , macrophages and inflammatory mediators in acute pancreatitis.Br J Surg.1992;79:998-1000.
[9].Takahashi K, Totsune K, Kikuchi K, Murakami O. Expression of endothelin-1 and adrenomedullin was not altered by leptin or resistin in bovine brain microvascular endothelial cells. Hypertens Res. 2006 Jun;29(6):443-448.
[10].Grewal HP,Moheyel Din A.Gaber L,et a1.Ameliomoratlon of the physiologic and biochemical changes of acute pancreatitis using an anti-TNF-a polyclonal an tibody.Am J Surg; 1994;167(1):214.
[11].Mattace Raso G,Esposito E,Iacono A,et al.Leptin induced nitric oxide synthase typeⅡi n c6 glioma cell. Role for nuclear factor-kappaB inhormone effect. Neurosci lett.2006,Mar.
[12].Tang CH,LUDY,Yang RS,Leptin-induced IL-6 producation is mediated by leptin eceptor,Insulinreceptorubstrate-1,phosphatidylinosito13-kinase,Akt,NF-kappaB,and p300 pathway in micoroglia, J Immunol.2007,Jul 15;179(2):1292-1302.
[13].Haraldsen P,Sun ZW,Bodesson A,et a1.Pancreatology,2003;3:14-25.
[14].Jaworek J,Bonior J.Nawrot K,et a1.Intracere broventricular administration of bactefial lipopoly saccharide prevents the development of acute experimental pancreatitis in the rat.Med Sci Monit,2002,8:136-143.
[15].Warzecha Z,Dembinski A,Ceranowicz P,et a1.Influence of leptin administration on the course of acute isehemic pancreatitis.J Physiol Pharmacol,2002,53:775-790.
[16].Correia MLG, Morgan DA , Sivitz WI , et al . Leptin acts in the centralnervous system to produce dose2 dependent changes in arterial pressure.Hypertension, 2001 ,37 :936-942.
[1].InterianoB,Stuard ID.Acute respiratory distress syndrome in Pancreatitis.Ann intern Med.1972,77:923.
[2].Guiee KS,Oldham KT,Johnson KJ,et al.Pancreatitis induced acute lung injury.An ARDS model.Ann Surg,1988,208:71-77.
[3].LiuXiaohong,Itsuro N,Hiroya Y,et al.Protective effect of nitric oxide on developm ent of AP in rats.Dig Dis Sci,1995,40(10):2162-2169.
[4].Yamanaka K,Saluja AK,Brown GE,et al.Protective effects of prostaglandin E2 on acute lung injury of cacruleirr induced acute pancreatitis in rats.Am J Physiol,1997,272:G23-G30.
[5].Nevalainen TJ,Gronroos JM,Kortesuo PT,et al.Pancreatic and Synovial type phospholipaseA2 in serum samples from patients with serve acute pancreatitis.Gut,1993,34(8):1133-1136.
[6].Hughes CB,Gaber IW,Mohey AB,el al.Inhibition of TNF improve Suruial in an experimental mode of acute pancreatitis J AM Surs1996,62(l):8-13.
[7].Scarpace PJ,Matheny M,Pollock BH, Tumer N: Leptin increases uncoupling protein expression and energy expenditure. Am J Physiol 1997;273:E226-E230.
[8].Takahashi K, Totsune K, Kikuchi K, Murakami O. Expression of endothelin-1 and adrenomedullin was not altered by leptin or resistin in bovine brain microvascular endothelial cells. Hypertens Res. 2006 Jun;29(6):443-448.
[9].Ckaya G, Ozata M, Bauraktar Z , et al.Is leptin associated with diabetic retinopathy Diabetes Care.2000 ,23 :371-376.
[10].Correia MLG, Morgan DA,Sivitz WI , et al . Leptin acts in the centralnervous system to produce dose-dependent changes in arterial pressure.Hypertension , 2001 ,37 :936-942.
[11].李燕书、闫军.外源性瘦素在急性胰腺炎时对一氧化氮、一氧化氮合酶表达的影响[J].中华肝胆外科学杂志.2007.
[12].张喜平、李建秋、程琪辉.急性胰腺炎中NO和ET作用的两面性[J].中华研究杂志.2007,36(3):86.
[13]. Q uehenberger P, Exner M , Sunder P lassmann R, et al. Leptin induces endo thelin-1 in endothelial cells in vitro. Circ Res,2002, 90 (6) : 711-718.
[14].曹莜佩、明文玉、程桦等.瘦素对培养肝细胞的糖原合成及糖原合成酶mRNA表达的影响[J].中华内分泌代谢杂志.2002.18(1):68-69.
[15].Kang SM,Kwon Hm,Kim D et al.Expression of leptin receptor in human atherosclerotic lesions:Potential role in intimal neovascularization.Yonsei Med.2000,41(1):68-75.
[16].Ciccone M,Vettor R,Pannacciulli N,et al[J].Int obes Ralat Metab Disord, 2001, 25(6):805-810.
[17].Bouloumie A,Marumo T,Lafontan M,et al.[J].FASEBJ,1999,13(10):1231-1238.
[18].Tang CH,LUDY,Yang RS,Leptin-induced IL-6 producation is mediated by leptin receptor,Insulinreceptorubstrate-1,phosphatidylinositol3-kinase,Akt,NF-kappaB, and p300 pathway in micoroglia, J Immunol.2007,Jul 15;179(2):1292-1302.
[19].Mattace Raso G,Esposito E,Iacono A,et al.Leptin induced nitric oxide synthase typeⅡin c6 glioma cell. Role for nuclear factor-kappaB in hormone effect. Neuroscilett.2006,Mar 27,396(2):121-126.
[20]. Bayon Y. Mechanisms of cell signaling in immune - mediated inflammation[J ] . Cytokins Cell Mol Ther. 1998 ,4(4) :275.
[21]. Jaworek J,Bonior J.Nawrot K,et a1.Intracerebroventricular administration of bactefial lipopolysaccharide prevents the development of acute exprimental pancreatitis in the rat.Med Sci Monit,2002,8:BR136-143.
[22]. Warzecha Z,Dembinski A,Ceranowicz P,et a1.Influence of leptinadministration on the course of acute ischemic pancreatitis.J Physiol Pharmacol, 2002,53(42):775-790.
[2].朱国英,李永渝核因子-KB作为急性胰腺炎治疗靶向的思考[J].国外医学外科学分册.2005,32(5):357-358.
[3].Shames BD,Barton HH,Reznikov LL.et a1.Ischemia alone is sufficient to induce TNF-alpha mRNA and peptied in the myocardium.Shock 2002,17:114-119.
[4].张喜平、李建秋、程琪辉.急性胰腺炎中NO和ET作用的两面性[J].中华研究杂志.2007,36(3):86.
[5].路琴、阴桢宏、诸欣平.瘦素对重症急性胰腺炎保护作用的实验研究[J].中华医学杂志.2006, 86(45):3222.
[6].Andrts Duarte-Rojof Ana Lezama-Barredaf Maria Teresa Ramirez-Iglesiasf,Is leptin related to systemic inflammatory response in acute pancreatitis? World,GastroenteroI 2006 July.
[7].A.k.Banergee,S.W. Gulloway,et al.Experimental models of acute Pancreatitis.Brit J Surg.1994,81:1096-1103.
[8] . C.R.B.Welbour and Y.Yong.Endotoxin , septic shock and acute lung injury:neutrophil , macrophages and inflammatory mediators in acute pancreatitis.Br J Surg.1992;79:998-1000.
[9].Takahashi K, Totsune K, Kikuchi K, Murakami O. Expression of endothelin-1 and adrenomedullin was not altered by leptin or resistin in bovine brain microvascular endothelial cells. Hypertens Res. 2006 Jun;29(6):443-448.
[10].Grewal HP,Moheyel Din A.Gaber L,et a1.Ameliomoratlon of the physiologic and biochemical changes of acute pancreatitis using an anti-TNF-a polyclonal an tibody.Am J Surg; 1994;167(1):214.
[11].Mattace Raso G,Esposito E,Iacono A,et al.Leptin induced nitric oxide synthase typeⅡi n c6 glioma cell. Role for nuclear factor-kappaB inhormone effect. Neurosci lett.2006,Mar.
[12].Tang CH,LUDY,Yang RS,Leptin-induced IL-6 producation is mediated by leptin eceptor,Insulinreceptorubstrate-1,phosphatidylinosito13-kinase,Akt,NF-kappaB,and p300 pathway in micoroglia, J Immunol.2007,Jul 15;179(2):1292-1302.
[13].Haraldsen P,Sun ZW,Bodesson A,et a1.Pancreatology,2003;3:14-25.
[14].Jaworek J,Bonior J.Nawrot K,et a1.Intracere broventricular administration of bactefial lipopoly saccharide prevents the development of acute experimental pancreatitis in the rat.Med Sci Monit,2002,8:136-143.
[15].Warzecha Z,Dembinski A,Ceranowicz P,et a1.Influence of leptin administration on the course of acute isehemic pancreatitis.J Physiol Pharmacol,2002,53:775-790.
[16].Correia MLG, Morgan DA , Sivitz WI , et al . Leptin acts in the centralnervous system to produce dose2 dependent changes in arterial pressure.Hypertension, 2001 ,37 :936-942.
[1].InterianoB,Stuard ID.Acute respiratory distress syndrome in Pancreatitis.Ann intern Med.1972,77:923.
[2].Guiee KS,Oldham KT,Johnson KJ,et al.Pancreatitis induced acute lung injury.An ARDS model.Ann Surg,1988,208:71-77.
[3].LiuXiaohong,Itsuro N,Hiroya Y,et al.Protective effect of nitric oxide on developm ent of AP in rats.Dig Dis Sci,1995,40(10):2162-2169.
[4].Yamanaka K,Saluja AK,Brown GE,et al.Protective effects of prostaglandin E2 on acute lung injury of cacruleirr induced acute pancreatitis in rats.Am J Physiol,1997,272:G23-G30.
[5].Nevalainen TJ,Gronroos JM,Kortesuo PT,et al.Pancreatic and Synovial type phospholipaseA2 in serum samples from patients with serve acute pancreatitis.Gut,1993,34(8):1133-1136.
[6].Hughes CB,Gaber IW,Mohey AB,el al.Inhibition of TNF improve Suruial in an experimental mode of acute pancreatitis J AM Surs1996,62(l):8-13.
[7].Scarpace PJ,Matheny M,Pollock BH, Tumer N: Leptin increases uncoupling protein expression and energy expenditure. Am J Physiol 1997;273:E226-E230.
[8].Takahashi K, Totsune K, Kikuchi K, Murakami O. Expression of endothelin-1 and adrenomedullin was not altered by leptin or resistin in bovine brain microvascular endothelial cells. Hypertens Res. 2006 Jun;29(6):443-448.
[9].Ckaya G, Ozata M, Bauraktar Z , et al.Is leptin associated with diabetic retinopathy Diabetes Care.2000 ,23 :371-376.
[10].Correia MLG, Morgan DA,Sivitz WI , et al . Leptin acts in the centralnervous system to produce dose-dependent changes in arterial pressure.Hypertension , 2001 ,37 :936-942.
[11].李燕书、闫军.外源性瘦素在急性胰腺炎时对一氧化氮、一氧化氮合酶表达的影响[J].中华肝胆外科学杂志.2007.
[12].张喜平、李建秋、程琪辉.急性胰腺炎中NO和ET作用的两面性[J].中华研究杂志.2007,36(3):86.
[13]. Q uehenberger P, Exner M , Sunder P lassmann R, et al. Leptin induces endo thelin-1 in endothelial cells in vitro. Circ Res,2002, 90 (6) : 711-718.
[14].曹莜佩、明文玉、程桦等.瘦素对培养肝细胞的糖原合成及糖原合成酶mRNA表达的影响[J].中华内分泌代谢杂志.2002.18(1):68-69.
[15].Kang SM,Kwon Hm,Kim D et al.Expression of leptin receptor in human atherosclerotic lesions:Potential role in intimal neovascularization.Yonsei Med.2000,41(1):68-75.
[16].Ciccone M,Vettor R,Pannacciulli N,et al[J].Int obes Ralat Metab Disord, 2001, 25(6):805-810.
[17].Bouloumie A,Marumo T,Lafontan M,et al.[J].FASEBJ,1999,13(10):1231-1238.
[18].Tang CH,LUDY,Yang RS,Leptin-induced IL-6 producation is mediated by leptin receptor,Insulinreceptorubstrate-1,phosphatidylinositol3-kinase,Akt,NF-kappaB, and p300 pathway in micoroglia, J Immunol.2007,Jul 15;179(2):1292-1302.
[19].Mattace Raso G,Esposito E,Iacono A,et al.Leptin induced nitric oxide synthase typeⅡin c6 glioma cell. Role for nuclear factor-kappaB in hormone effect. Neuroscilett.2006,Mar 27,396(2):121-126.
[20]. Bayon Y. Mechanisms of cell signaling in immune - mediated inflammation[J ] . Cytokins Cell Mol Ther. 1998 ,4(4) :275.
[21]. Jaworek J,Bonior J.Nawrot K,et a1.Intracerebroventricular administration of bactefial lipopolysaccharide prevents the development of acute exprimental pancreatitis in the rat.Med Sci Monit,2002,8:BR136-143.
[22]. Warzecha Z,Dembinski A,Ceranowicz P,et a1.Influence of leptinadministration on the course of acute ischemic pancreatitis.J Physiol Pharmacol, 2002,53(42):775-790.