p38丝裂原活化蛋白激酶对急性坏死性胰腺炎大鼠低钙血症的影响

方勇木 黄鹤光 周一农

p38丝裂原活化蛋白激酶对急性坏死性胰腺炎大鼠低钙血症的影响

方勇木 黄鹤光 周一农

目的探讨p38丝裂原活化蛋白激酶(p38MAPK)信号转导通路对急性坏死性胰腺炎(ANP)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只按完全随机法分为ANP组、SB203580干预(SB)组和假手术(SO)组，每组分3 、6、12 h 3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ANP 模型，SB组在造模前30 min腹腔注射p38MAPK特异抑制剂SB203580 10 mg/kg体重。观察各组血清钙浓度，蛋白质印迹法(Western blotting)分析骨组织磷酸化p38MAPK(P-p38 MAPK)和TNF-α变化，实时RT-PCR检测骨组织PTHR1 mRNA表达。结果制模后6 h，SO组、ANP组和SB组血清钙浓度分别为(2.50±0.08)mmol/L、(2.11±0.06)mmol/L和(2.35±0.10)mmol/L；骨组织P-p38 MAPK表达量分别为0.14±0.04、0.80±0.06和0.33±0.05；骨组织TNF-α表达量分别为0、0.91±0.04和0.44±0.03；骨组织PTHR1 mRNA表达量分别为1.00±0.12、0.23±0.04和0.44±0.06。SB组骨组织P-p38 MAPK及TNF-α表达较ANP组显著降低(Plt;0.01)；骨组织PTHR1 mRNA表达量及血清钙浓度较ANP组显著增加(Plt;0.01)。结论p38MAPK信号转导通路可介导ANP低钙血症的发生，抑制该通路可改善ANP低钙血症。

胰腺炎，急性坏死性； p38丝裂原活化蛋白激酶类； 低钙血症； 受体，甲状旁腺激素； 肿瘤坏死因子

重症急性胰腺炎(SAP)低钙血症的发病机制复杂，至今尚未完全阐明。我们前期研究证实，细胞因子与SAP低钙血症发生有关[1]。p38丝裂原活化蛋白激酶(p38MAPK)信号转导通路在机体应激和炎症反应调控过程起着极其重要的作用。本实验应用p38MAPK特异的抑制剂SB203580干预急性坏死性胰腺炎(ANP)大鼠，探讨p38MAPK信号转导通路对ANP大鼠低钙血症和甲状旁腺激素受体1(parathyrin receptor 1,PTHR 1)表达的影响。

材料和方法

一、实验动物分组

健康成年雄性SD大鼠72只，体重250～270 g，购自上海斯莱克实验动物有限责任公司(SCXK沪2007-0005，NO：0036178)。应用完全随机法分为ANP组、SB组和假手术(SO)组，每组又分造模后3、6、12 h 3个时间点，每个时间点8只大鼠。参照杨波等[2]方法，采用逆行胆胰管注射5%牛磺脱氧胆酸钠1.0 ml/kg体重制备ANP模型。SB组在造模前30 min腹腔注射p38MAPK特异的抑制剂SB203580(美国InvivoGen公司)10 mg/kg体重[3]。SO组在开腹后，仅轻轻翻动十二指肠及胰腺后关腹。

各组大鼠在相应时间点分别再次麻醉后开腹，下腔静脉采血2 ml。迅速切取大鼠后肢股骨下端、胫骨上端，放入液氮速冻后置-80℃冰箱冻存。

二、检测指标和方法

1．血清钙浓度测定：采用全自动生化分析仪检测。

2．骨组织磷酸化p38MAPK(P-p38MAPK)和TNF-α表达检测： 应用蛋白提取试剂盒(Pierce公司)提取骨组织总蛋白，常规行Western blotting。抗P-p38MAPK一抗(Santa Cruz Biotechnology公司)1∶300，稀释，抗TNF-α及β-actin一抗(博士德公司)1∶400稀释，最后加入ECL试剂，压片显影。图像采用Bandscan软件分析，以目的条带与β-actin的比值表示各组蛋白质表达水平。

3．骨组织PTHR1mRNA检测：采用实时定量RT-PCR法。用Trizol试剂盒(TaKaRa公司)提取骨组织总RNA，并用DNase I去除其中的DNA杂质。PTHR1引物：上游5′-CCAGTGCTCAGCTCCGCATA-3′,下游5′- TCCTTGAGCAGCTTGTCACATTG -3′，片断长度113 bp；β-actin(内参)引物：上游5′- CCCATCTATGAGGGTTACGC -3′,下游5′- TTTAATGTCACGCACGATTTC -3′，片断长度150 bp。进行逆转录反应后，使用SYBR Green I Real Time RT-PCR试剂盒，在Light Cycler Real Time PCR仪(Roche Diagnostics公司)上采用两步法PCR 扩增标准程序进行基因扩增。反应结束进行产物特异性验证(图1)，根据2-△△Ct法[4]，以对照组△Ct平均值作为校正数，△△Ct=(Ct 目的基因-Ct 内参基因)实验组-(Ct 目的基因-Ct 内参基因)对照组,算出目的基因的相对表达量。

三、统计学方法

结 果

一、血清钙浓度变化

ANP组各时间点血清钙浓度较SO组均明显降低(Plt;0.01)；SB组各时间点血清钙浓度又较ANP组明显回升(Plt;0.05或Plt;0.01，表1)。

二、骨组织P-p38MAPK和TNF-α的变化

SO组各时间点骨组织P-p38MAPK弱表达，TNF-α几乎不表达；与SO组比较，ANP组各时间点P-p38MAPK和TNF-α表达量均显著增高(Plt;0.01)； SB组各时间点P-p38MAPK和TNF-α表达量又较ANP组显著降低(Plt;0.01，表1，图1)。

三、骨组织PTHR1 mRNA表达的变化

ANP组各时间点PTHR1 mRNA表达量均低于SO组(Plt;0.01)；SB组各时间点PTHR1 mRNA表达量较ANP组均明显上调(Plt;0.01，表1，图2)。

表1 3、6、12 h各组骨组织中P-p38MAPK、TNF-α和PTHR1mRNA含量及血清钙水平

注：与SO组比较，*Plt;0.01；与ANP组比较，△Plt;0.05，#Plt;0.01

图1 制模后6 h骨组织中P-p38MAPK和TNF-α表达

A为PTHR1扩增曲线，B为融解曲线的单峰图(a为PTHR1，b为β-actin)

图2实时RT-PCR产物特异性验证

讨 论

p38MAPK信号转导通路在机体应激和炎症反应调控过程中具有极其重要作用，是调控炎症细胞因子产生的重要通路之一[5]。应激和各种刺激因素如各种炎症细胞因子、生长因子、体液渗透压改变、氧自由基、低氧血症、休克和感染等均可激活胞质的p38MAPK；p38MAPK激活后进入胞核内，调节效应基因的表达，包括各种炎症细胞因子基因，使介导炎症细胞因子大量释放。抑制p38MAPK活化阻断该通路，可抑制炎症细胞因子的产生。Chen等[6]研究表明，ANP时胰腺组织活化的p38MAPK和细胞因子表达量明显升高，抑制ANP大鼠p38MAPK的活化，可减少炎症细胞因子TNF-α、IL-1β释放。Samuel等[7]和Denham等[8]均得出相同结论。本实验结果显示，应用p38MAPK特异抑制剂SB203580干预ANP大鼠，能抑制p38MAPK的活化，下调骨组织TNF-α的表达，进一步证实了上述观点。

我们前期研究证实：由于细胞因子的大量释放，ANP大鼠抑制骨、肾PTHRmRNA表达，降低PTH及受体功能，产生低钙血症[1，9]；应用甲强龙治疗ANP大鼠，可使细胞因子水平显著下降，PTHR1mRNA表达显著上调，低钙血症得到明显改善[10]。本实验应用p38MAPK特异抑制剂SB203580预处理ANP大鼠后，骨组织TNF-α表达明显下降，骨组织PTHR1mRNA和血清钙明显升高，表明是p38MAPK信号转导通路介导了ANP低钙血症的发生。

[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,2006,21:827-828.

[2] 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.

[3] Lee JC,Laydon JT,McDonnell PC,et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis.Nature,1994,372:739-746.

[4] Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods,2001,25:402-408.

[5] Kyriakis JM,Avruch J.Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation.Physiol Rev,2001,81:807-869.

[6] 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.

[7] 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.

[8] Denham W,Yang J,Wang H,et al.Inhibition of p38 mitogen activate kinase attenuates the severity of pancreatitis-induced adult respiratory distress syndrome.Crit Care Med,2000,28:2567-2572.

[9] 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,2001,18:303-304.

[10] 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,2006,23:567-569.

2008-07-14)

(本文编辑：屠振兴)

Effectofp38mitogen-activatedproteinkinaseonhypocalcaemiaofratswithacutenecrotizingpancreatitis

FANGYong-mu,HUANGHe-guang,ZHOUYi-nong.

DepartmentofGeneralSurgery,UnionHospital,FujianMedicalUniversity,Fuzhou350001,China

HUANGHe-guang,Email:hhuang2@yahoo.com.cn

ObjectiveTo investigate the role of p38 mitogen-activated protein kinase (MAPK) signal transduction pathway in hypocalcaemia and parathyroid hormone receptor 1 (PTHR1) of rats with acute necrotizing pancreatitis (ANP).MethodsSeventy-two male health adult Sprague-Dawley rats were randomized into three groups: ANP group, ANP treated with SB203580 group (SB group), sham operation group (SO group). Every group was sub-divided into 3, 6, 12 h group with 8 rats in each one. ANP model was induced by retrograde infusion with 5% sodium taurocholate solution into the biliopancreatic duct. In the SB group, rats were treated with the specific p38MAPK inhibitor: SB203580 30 minutes before the induction of ANP model. The serum level of calcium was determined, the change of phosphorylated p38MAPK and TNF-alpha were measured by western blot and the expression of PTHR1mRNA was determined by quantitative real time RT-PCR.Results6 h after ANP model induction, the serum levels of calcium in ANP, SB and SO group were (2.50±0.08)mmol/L, (2.11±0.06)mmol/L and (2.35±0.10)mmol/L,respectively;the expression levels of phosphorylated p38MAPK in bone tissue were 0.14±0.04, 0.80±0.06 and 0.33±0.05, respectively; the expression levels of p38MAPK TNF-alpha were 0, 0.91±0.04 and 0.44±0.03, respectively; the expression levels of PTHR1 mRNA were 1.00±0.12, 0.23±0.04 and 0.44±0.06, respectively. The expression levels of p38MAPK and TNF-α in SB group were significantly lower than those in the ANP group (Plt;0.01); while the expression levels of PTHR1 mRNA and calcium were significantly higher than those in the ANP group (Plt;0.01).ConclusionsP38MAPK signal transduction pathway may mediate the development of hypocalcaemia in the course of ANP, and hypocalcaemia could be improved by blocking this pathway.

Pancreatitis, acute necrotizing; p38 Mitogen-activated protein kinases; Hypocalcaemia; Receptors, parathyroid hormone; Tumor necrosis factor-alpha

10.3760/cma.j.issn.1674-1935.2009.02.010

福建省教育厅科研基金(JA03099)

350001 福州,福建医科大学附属协和医院普外科

黄鹤光，Email: hhuang2@yahoo.com.cn