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。

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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