p38丝裂原活化蛋白激酶对重症急性胰腺炎大鼠低钙血症的影响
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摘要
目的:探讨p38丝裂原活化蛋白激酶(p38MAPK)信号转导通路对重症急性胰腺炎(SAP)大鼠低钙血症和甲状旁腺激素受体1(PTHR1)表达的影响。
方法:将雄性SD大鼠72只随机分为:SAP组;治疗组(SB组);假手术组(SO组),每组分3h,6h,12h三个时间点,每个时间点8只。以5%牛磺脱氧胆酸钠逆行胰胆管注射建立SAP模型,SB组在造模前30分钟腹腔注射p38MAPK特异抑制剂SB203580。观察各组血清钙浓度,蛋白免疫印迹技术(Western blot)分析骨组织磷酸化p38MAPK(P-p38MAPK)和肿瘤坏死因子-α(TNF-α)变化,实时逆转录聚合酶链反应(Real-time RT-PCR)检测骨组织PTHR1mRNA表达。
结果:SAP组与SO组比较骨组织P-p38MAPK和TNF-α的表达量明显增高,骨组织PTHR1mRNA表达量和血清钙浓度显著下降。SB组与SAP组比较骨组织P-p38MAPK [3h:(0.20±0.05)vs.( 0.40±0.06),t=-7.044,P=0.000;6h:(0.33±0.05)vs.(0.80±0.06),t=-18.099,P=0.000;12h:(0.27±0.05)vs.(0.51±0.03),t=-12.385,P=0.000]和TNF-α[3h:(0.31±0.03)vs.(0.70±0.03),t=-23.870,P=0.000;6h:(0.44±0.03)vs.(0.91±0.04),t=-27.755,P=0.000;12h:(0.37±0.04)vs.(0.80±0.03),t=-23.652,P=0.000]表达显著下调,骨组织PTHR1mRNA表达量[3h:(0.53±0.03)vs.( 0.28±0.03),t=17.858,P=0.000;6h:(0.44±0.06)vs.( 0.23±0.04),t=8.804,P=0.000;12h:(0.40±0.11)vs.( 0.20±0.03),t=5.060,P=0.001]和血清钙浓度[3h:(2.39±0.06)mmol/L vs.( 2.31±0.06) mmol/L,t=2.900,P=0.012;6h:(2.35±0.10) mmol/L vs.( 2.11±0.06) mmol/L,t=5.507,P=0.000;12h:(2.26±0.12) mmol/L vs.( 2.01±0.04) mmol/L,t=5.881,P=0.000]显著升高。
结论:p38MAPK信号转导通路可介导SAP低钙血症的发生,抑制该通路可改善SAP低钙血症。
Objective To investigate the role of p38 mitogen-activated protein kinase(MAPK) signal transduction pathway in hypocalcaemia and parathyroid hormone receptor 1 (PTHR1) of rats with severe acute pancreatitis (SAP). Methods Seventy-two male health adult Sprague-dawley rats were completely randomized into three groups:SAP group,SAP treated with SB203580 group (SB group),sham operation group(SO group).SAP model was induced by bili-pancreatic duct retrograde infusion with 5% sodium taurocholate solution.In the SB group,rats were treated with the specific inhibior (SB203580) of p38MAPK 30 minutes before the induction of SAP model. Rats in each group were killed at 3h,6h,12h after operation to determine the Serum calcium, the change of phosphorylated p38MAPK and tumor necrosis factor (TNF)-alpha (by western blot) and the expression of PTHR1mRNA(by quantitative real-time RT-PCR),8 rats in each time point.
Results Compared with sham group,the expression of bone phosphorylated p38MAPK and TNF-alpha were increased significantly,the mRNA level of bone PTHR1 down-regulated significantly and the concentration of serum calcium decreased significantly in SAP group.Compared with SAP group,the expression of bone phosphorylated p38MAPK [3h:(0.20±0.05)vs.( 0.40±0.06),t=-7.044,P=0.000;6h:(0.33±0.05)vs.(0.80±0.06),t=-18.099,P=0.000;12h:(0.27±0.05)vs.(0.51±0.03),t=-12.385,P=0.000] and TNF-alpha [3h:(0.31±0.03)vs.(0.70±0.03),t=-23.870,P=0.000;6h:(0.44±0.03)vs.(0.91±0.04),t=-27.755,P=0.000;12h:(0.37±0.04)vs.(0.80±0.03),t=-23.652,P=0.000] were decreased significantly, the mRNA level of bone PTHR1 [3h:(0.53±0.03)vs.( 0.28±0.03),t=17.858,P=0.000;6h:(0.44±0.06)vs.( 0.23±0.04),t=8.804,P=0.000;12h:(0.40±0.11)vs.( 0.20±0.03),t=5.060,P=0.001] up-regulated significantly and the concentration of serum calcium [3h:(2.39±0.06) mmol/L vs.( 2.31 ±0.06) mmol/L,t=2.900,P=0.012;6h:(2.35±0.10) mmol/L vs.( 2.11±0.06) mmol/L,t=5.507,P=0.000;12h:(2.26±0.12) mmol/L vs.( 2.01±0.04) mmol/L,t = 5.881,P = 0.000] increased significantly in SB group.
Conclusion P38MAPK signal transduction pathway may mediates the development of hypocalcaemia,the level of serum calcium could be increased by blocking this pathway in rats with SAP.
方法:将雄性SD大鼠72只随机分为:SAP组;治疗组(SB组);假手术组(SO组),每组分3h,6h,12h三个时间点,每个时间点8只。以5%牛磺脱氧胆酸钠逆行胰胆管注射建立SAP模型,SB组在造模前30分钟腹腔注射p38MAPK特异抑制剂SB203580。观察各组血清钙浓度,蛋白免疫印迹技术(Western blot)分析骨组织磷酸化p38MAPK(P-p38MAPK)和肿瘤坏死因子-α(TNF-α)变化,实时逆转录聚合酶链反应(Real-time RT-PCR)检测骨组织PTHR1mRNA表达。
结果:SAP组与SO组比较骨组织P-p38MAPK和TNF-α的表达量明显增高,骨组织PTHR1mRNA表达量和血清钙浓度显著下降。SB组与SAP组比较骨组织P-p38MAPK [3h:(0.20±0.05)vs.( 0.40±0.06),t=-7.044,P=0.000;6h:(0.33±0.05)vs.(0.80±0.06),t=-18.099,P=0.000;12h:(0.27±0.05)vs.(0.51±0.03),t=-12.385,P=0.000]和TNF-α[3h:(0.31±0.03)vs.(0.70±0.03),t=-23.870,P=0.000;6h:(0.44±0.03)vs.(0.91±0.04),t=-27.755,P=0.000;12h:(0.37±0.04)vs.(0.80±0.03),t=-23.652,P=0.000]表达显著下调,骨组织PTHR1mRNA表达量[3h:(0.53±0.03)vs.( 0.28±0.03),t=17.858,P=0.000;6h:(0.44±0.06)vs.( 0.23±0.04),t=8.804,P=0.000;12h:(0.40±0.11)vs.( 0.20±0.03),t=5.060,P=0.001]和血清钙浓度[3h:(2.39±0.06)mmol/L vs.( 2.31±0.06) mmol/L,t=2.900,P=0.012;6h:(2.35±0.10) mmol/L vs.( 2.11±0.06) mmol/L,t=5.507,P=0.000;12h:(2.26±0.12) mmol/L vs.( 2.01±0.04) mmol/L,t=5.881,P=0.000]显著升高。
结论:p38MAPK信号转导通路可介导SAP低钙血症的发生,抑制该通路可改善SAP低钙血症。
Objective To investigate the role of p38 mitogen-activated protein kinase(MAPK) signal transduction pathway in hypocalcaemia and parathyroid hormone receptor 1 (PTHR1) of rats with severe acute pancreatitis (SAP). Methods Seventy-two male health adult Sprague-dawley rats were completely randomized into three groups:SAP group,SAP treated with SB203580 group (SB group),sham operation group(SO group).SAP model was induced by bili-pancreatic duct retrograde infusion with 5% sodium taurocholate solution.In the SB group,rats were treated with the specific inhibior (SB203580) of p38MAPK 30 minutes before the induction of SAP model. Rats in each group were killed at 3h,6h,12h after operation to determine the Serum calcium, the change of phosphorylated p38MAPK and tumor necrosis factor (TNF)-alpha (by western blot) and the expression of PTHR1mRNA(by quantitative real-time RT-PCR),8 rats in each time point.
Results Compared with sham group,the expression of bone phosphorylated p38MAPK and TNF-alpha were increased significantly,the mRNA level of bone PTHR1 down-regulated significantly and the concentration of serum calcium decreased significantly in SAP group.Compared with SAP group,the expression of bone phosphorylated p38MAPK [3h:(0.20±0.05)vs.( 0.40±0.06),t=-7.044,P=0.000;6h:(0.33±0.05)vs.(0.80±0.06),t=-18.099,P=0.000;12h:(0.27±0.05)vs.(0.51±0.03),t=-12.385,P=0.000] and TNF-alpha [3h:(0.31±0.03)vs.(0.70±0.03),t=-23.870,P=0.000;6h:(0.44±0.03)vs.(0.91±0.04),t=-27.755,P=0.000;12h:(0.37±0.04)vs.(0.80±0.03),t=-23.652,P=0.000] were decreased significantly, the mRNA level of bone PTHR1 [3h:(0.53±0.03)vs.( 0.28±0.03),t=17.858,P=0.000;6h:(0.44±0.06)vs.( 0.23±0.04),t=8.804,P=0.000;12h:(0.40±0.11)vs.( 0.20±0.03),t=5.060,P=0.001] up-regulated significantly and the concentration of serum calcium [3h:(2.39±0.06) mmol/L vs.( 2.31 ±0.06) mmol/L,t=2.900,P=0.012;6h:(2.35±0.10) mmol/L vs.( 2.11±0.06) mmol/L,t=5.507,P=0.000;12h:(2.26±0.12) mmol/L vs.( 2.01±0.04) mmol/L,t = 5.881,P = 0.000] increased significantly in SB group.
Conclusion P38MAPK signal transduction pathway may mediates the development of hypocalcaemia,the level of serum calcium could be increased by blocking this pathway in rats with SAP.
引文
1.陆逢春,黄鹤光.肿瘤坏死因子-α对重症急性胰腺炎低钙血症的影响及作用机制.中华普通外科杂志,2006,2l: 827-8.
2. BoneRC.Sir Isaac Newton, sepsis, SIRS, and CARS.Crit Care Med. 1996, 24:1125-8.
3. Ren C, Liu X, Guo Z,etal. Systemic inflammatory response syndrome in critical patients - an analysis of 1,292 cases. Zhonghua Nei Ke Za Zhi. 1999 ,38:40-3.
4.黄鹤光,冷希圣.重症急性胰腺炎大鼠骨组织甲状旁腺素受体mRNA的表达.中华实验外科杂志,2001,18:303-4.
5.周一农,黄鹤光.甲强龙对重症急性胰腺炎大鼠低钙血症和甲状旁腺激素受体表达的影响.中华实验外科杂志,2006,23:567-9.
6. Obata T, Brown GE, Yaffe MB. MAP kinase pathways activated by stress:the p38MAPK pathway. Crit Care Med, 2000 ,28:67-77.
7. Nick JA,Avdi NJ,Young SK,et a1.An intracellular signaling pathway linhing lipopolysaccaride stimulation to cellular responses of the human neitrophil:the p38 MAP kinase cascade and its functional significance.Chest,1999,116 :54S-55S.
8. Dong X, Liu Y, Du M, et al. p38 mitogen-activated protein kinase inhibition attenuates pulmonary inflammatory response in a rat cardiopulmonary bypasss model. Eur J Cardiothorac Surg,2006, 30:77-84.
9. Nick JA, Avdi NJ, Young SK, et al. Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils. J Clin Invest 1999,103:851–8.
10. Lee JC,Laydon JT,McDonnell PC,et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis.Nature,1994, 372:739-746.
11.杨波,黄鹤光,陈大良,等.逆行性胰胆管注射法制作重症急性胰腺炎大鼠模型.福建医科大学学报,2002,36:71-72.
12. Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods.2001,25:402-8.
13.中华医学会外科学分会胰腺外科学组.重症急性胰腺炎诊治指南.中华外科杂志,2007,45:727-9.
14. Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 2001,81:807–69.
15. Chen P,Zhang Y,Qiao M,et al. Activated protein C, an anticoagulant polypeptide, ameliorates severe acute pancreatitis via regulation of mitogen-activated protein kinases. J Gastroenterol.2007,42:887-896.
16. Samuel I, Zaheer A, Fisher RA. In vitro evidence for role of ERK, p38, and JNK in exocrine pancreatic cytokine production. J Gastrointest Surg,2006,10:1376-1383.
17. Denham W,Yang J,Wang H,etal. Inhibition of p38 mitogen activate kinase attenuates the severity of pancreatitis-induced adult respiratory distress syndrome. Crit Care Med, 2000,28: 2567-2572.
1 Ono K, Han J. The p38 signal transduction pathway: activation and function. Cell Signal 2000,12:1-13.
2 Foltz IN, Lee JC, Young PR, et al. Hemopoietic growth factors with the exception of interleukin-4 activate the p38 mitogen-activated protein kinase pathway. J Biol Chem, 1997;272: 296–301.
3 Lee JC,Kumar S.Griswold DE,et al.Inhibition of p38 MAP kinase as a therapeutic strategy.Immunopharmacology,2000,47:185-201.
4 Obata T,Brown GE,Yaffe MB.MAP kinase pathways activated by stress:the p38MAPK pathway.Crit Care Med,2000,28:N67-77.
5 Nick JA,Avdi NJ,Young SK,et a1.An intracellular signaling pathway linhing lipopolysaccaride stimulation to cellular responses of the human neitrophil:the p38 MAP kinase cascade and its functional significance.Chest,1999, 116:54S-55S.
6 Dong X,Liu Y,Du M,et al.p38 mitogen-activated protein kinase inhibition attenuates pulmonary inflammatory response in a rat cardiopulmonary bypasss model.Eur J Cardiothorac Surg,2006,30:77-84.
7 Nick JA, Avdi NJ, Young SK, et al. Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils.J Clin Invest 1999,103:851-858.
8 Bone RC.Sir Isaac Newton, sepsis, SIRS, and CARS.Crit Care Med. 1996,24:1125-28.
9 Ren C, Liu X, Guo Z,etal. Systemic inflammatory response syndrome in critical patients - an analysis of 1,292 cases. Zhonghua Nei Ke Za Zhi.1999,38:40-43.
10 Ren HB, Li ZS,Xu GM,et al.Dynamic changes of mitogen-activated protein kinase signal transduction in rats with severe acute pancreatitis. Chin J Dig Dis, 2004,5:123-5.
11 Denham W, Yang J, Wang H, etal. Inhibition of p38 mitogen activate kinase attenuates the severity of pancreatitis-induced adult respiratory distress syndrome. Crit Care Med, 2000,28:2567-72.
12 Samuel I, Zaheer A, Fisher RA. In vitro evidence for role of ERK, p38, and JNK in exocrine pancreatic cytokine production. J Gastrointest Surg,2006,10:1376-83.
13 Blinman TA,Gukovsky I, Mouria M, et al . Activation of pancreatic acinar cells on isolation from tissue:cytokine upregulation via p38 MAP kinase.Am J Physiol Cell Physiol,2000,279:C1993-2003.
14 Van Laethem JL,Eskinazi R,Louis H,et a1.Multisystemic production of interleukin 10 limits the severity of acute pancreatitis in mice.Gut,1998,43:408-13.
15 Friess H,L u Z,Riesle E.et al . Enhanced expression of TGFbetas and their receptors in human acute pancreatitis. Surgery,1998,227:95- 104.
16 Koprak S, Staruch MJ, Dumont FJ. A specific inhibitor of the p38 mitogen activated protein kinase affects differentially the production of various cytokines by activated human T cells: dependence on CD28 signaling and preferential inhibition of IL-10 production. Cell Immunol. 1999 ,192:87-95.
17 Pahl A, Zhang M, Kuss H, et al. Regulation of IL-13 synthesis in human lymphocytes: implications for asthma therapy.Br J Pharmacol,2002,135:1915-26.
18 David M, Ford D, Bertoglio J, et al. Induction of the IL-13 receptor alpha2-chain by IL-4 and IL-13 in human keratinocytes: involvement of STAT6, ERK and p38 MAPK pathways. Oncogene, 2001,20:6660-68.
19 Fleischer F, Dabew R, G?ke B, Wagner AC. Stress kinase inhibition modulates acute experimental pancreatitis.World J Gastroenterol,2001,7:259-65.
20 Lee JC, Laydon JT, McDonnell PC, et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature,1994, 372:739-46.
2. BoneRC.Sir Isaac Newton, sepsis, SIRS, and CARS.Crit Care Med. 1996, 24:1125-8.
3. Ren C, Liu X, Guo Z,etal. Systemic inflammatory response syndrome in critical patients - an analysis of 1,292 cases. Zhonghua Nei Ke Za Zhi. 1999 ,38:40-3.
4.黄鹤光,冷希圣.重症急性胰腺炎大鼠骨组织甲状旁腺素受体mRNA的表达.中华实验外科杂志,2001,18:303-4.
5.周一农,黄鹤光.甲强龙对重症急性胰腺炎大鼠低钙血症和甲状旁腺激素受体表达的影响.中华实验外科杂志,2006,23:567-9.
6. Obata T, Brown GE, Yaffe MB. MAP kinase pathways activated by stress:the p38MAPK pathway. Crit Care Med, 2000 ,28:67-77.
7. Nick JA,Avdi NJ,Young SK,et a1.An intracellular signaling pathway linhing lipopolysaccaride stimulation to cellular responses of the human neitrophil:the p38 MAP kinase cascade and its functional significance.Chest,1999,116 :54S-55S.
8. Dong X, Liu Y, Du M, et al. p38 mitogen-activated protein kinase inhibition attenuates pulmonary inflammatory response in a rat cardiopulmonary bypasss model. Eur J Cardiothorac Surg,2006, 30:77-84.
9. Nick JA, Avdi NJ, Young SK, et al. Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils. J Clin Invest 1999,103:851–8.
10. Lee JC,Laydon JT,McDonnell PC,et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis.Nature,1994, 372:739-746.
11.杨波,黄鹤光,陈大良,等.逆行性胰胆管注射法制作重症急性胰腺炎大鼠模型.福建医科大学学报,2002,36:71-72.
12. Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods.2001,25:402-8.
13.中华医学会外科学分会胰腺外科学组.重症急性胰腺炎诊治指南.中华外科杂志,2007,45:727-9.
14. Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 2001,81:807–69.
15. Chen P,Zhang Y,Qiao M,et al. Activated protein C, an anticoagulant polypeptide, ameliorates severe acute pancreatitis via regulation of mitogen-activated protein kinases. J Gastroenterol.2007,42:887-896.
16. Samuel I, Zaheer A, Fisher RA. In vitro evidence for role of ERK, p38, and JNK in exocrine pancreatic cytokine production. J Gastrointest Surg,2006,10:1376-1383.
17. Denham W,Yang J,Wang H,etal. Inhibition of p38 mitogen activate kinase attenuates the severity of pancreatitis-induced adult respiratory distress syndrome. Crit Care Med, 2000,28: 2567-2572.
1 Ono K, Han J. The p38 signal transduction pathway: activation and function. Cell Signal 2000,12:1-13.
2 Foltz IN, Lee JC, Young PR, et al. Hemopoietic growth factors with the exception of interleukin-4 activate the p38 mitogen-activated protein kinase pathway. J Biol Chem, 1997;272: 296–301.
3 Lee JC,Kumar S.Griswold DE,et al.Inhibition of p38 MAP kinase as a therapeutic strategy.Immunopharmacology,2000,47:185-201.
4 Obata T,Brown GE,Yaffe MB.MAP kinase pathways activated by stress:the p38MAPK pathway.Crit Care Med,2000,28:N67-77.
5 Nick JA,Avdi NJ,Young SK,et a1.An intracellular signaling pathway linhing lipopolysaccaride stimulation to cellular responses of the human neitrophil:the p38 MAP kinase cascade and its functional significance.Chest,1999, 116:54S-55S.
6 Dong X,Liu Y,Du M,et al.p38 mitogen-activated protein kinase inhibition attenuates pulmonary inflammatory response in a rat cardiopulmonary bypasss model.Eur J Cardiothorac Surg,2006,30:77-84.
7 Nick JA, Avdi NJ, Young SK, et al. Selective activation and functional significance of p38alpha mitogen-activated protein kinase in lipopolysaccharide-stimulated neutrophils.J Clin Invest 1999,103:851-858.
8 Bone RC.Sir Isaac Newton, sepsis, SIRS, and CARS.Crit Care Med. 1996,24:1125-28.
9 Ren C, Liu X, Guo Z,etal. Systemic inflammatory response syndrome in critical patients - an analysis of 1,292 cases. Zhonghua Nei Ke Za Zhi.1999,38:40-43.
10 Ren HB, Li ZS,Xu GM,et al.Dynamic changes of mitogen-activated protein kinase signal transduction in rats with severe acute pancreatitis. Chin J Dig Dis, 2004,5:123-5.
11 Denham W, Yang J, Wang H, etal. Inhibition of p38 mitogen activate kinase attenuates the severity of pancreatitis-induced adult respiratory distress syndrome. Crit Care Med, 2000,28:2567-72.
12 Samuel I, Zaheer A, Fisher RA. In vitro evidence for role of ERK, p38, and JNK in exocrine pancreatic cytokine production. J Gastrointest Surg,2006,10:1376-83.
13 Blinman TA,Gukovsky I, Mouria M, et al . Activation of pancreatic acinar cells on isolation from tissue:cytokine upregulation via p38 MAP kinase.Am J Physiol Cell Physiol,2000,279:C1993-2003.
14 Van Laethem JL,Eskinazi R,Louis H,et a1.Multisystemic production of interleukin 10 limits the severity of acute pancreatitis in mice.Gut,1998,43:408-13.
15 Friess H,L u Z,Riesle E.et al . Enhanced expression of TGFbetas and their receptors in human acute pancreatitis. Surgery,1998,227:95- 104.
16 Koprak S, Staruch MJ, Dumont FJ. A specific inhibitor of the p38 mitogen activated protein kinase affects differentially the production of various cytokines by activated human T cells: dependence on CD28 signaling and preferential inhibition of IL-10 production. Cell Immunol. 1999 ,192:87-95.
17 Pahl A, Zhang M, Kuss H, et al. Regulation of IL-13 synthesis in human lymphocytes: implications for asthma therapy.Br J Pharmacol,2002,135:1915-26.
18 David M, Ford D, Bertoglio J, et al. Induction of the IL-13 receptor alpha2-chain by IL-4 and IL-13 in human keratinocytes: involvement of STAT6, ERK and p38 MAPK pathways. Oncogene, 2001,20:6660-68.
19 Fleischer F, Dabew R, G?ke B, Wagner AC. Stress kinase inhibition modulates acute experimental pancreatitis.World J Gastroenterol,2001,7:259-65.
20 Lee JC, Laydon JT, McDonnell PC, et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature,1994, 372:739-46.