方勇木 黃鶴光 周一農(nóng)

p38絲裂原活化蛋白激酶對急性壞死性胰腺炎大鼠低鈣血癥的影響

方勇木 黃鶴光 周一農(nóng)

目的探討p38絲裂原活化蛋白激酶(p38MAPK)信號轉(zhuǎn)導(dǎo)通路對急性壞死性胰腺炎(ANP)大鼠低鈣血癥和甲狀旁腺激素受體1(PTHR1)表達(dá)的影響。方法將雄性SD大鼠72只按完全隨機(jī)法分為ANP組、SB203580干預(yù)(SB)組和假手術(shù)(SO)組，每組分3 、6、12 h 3個(gè)時(shí)間點(diǎn)，每個(gè)時(shí)間點(diǎn)8只。以5%?；敲撗跄懰徕c逆行胰膽管注射建立ANP 模型，SB組在造模前30 min腹腔注射p38MAPK特異抑制劑SB203580 10 mg/kg體重。觀察各組血清鈣濃度，蛋白質(zhì)印跡法(Western blotting)分析骨組織磷酸化p38MAPK(P-p38 MAPK)和TNF-α變化，實(shí)時(shí)RT-PCR檢測骨組織PTHR1 mRNA表達(dá)。結(jié)果制模后6 h，SO組、ANP組和SB組血清鈣濃度分別為(2.50±0.08)mmol/L、(2.11±0.06)mmol/L和(2.35±0.10)mmol/L；骨組織P-p38 MAPK表達(dá)量分別為0.14±0.04、0.80±0.06和0.33±0.05；骨組織TNF-α表達(dá)量分別為0、0.91±0.04和0.44±0.03；骨組織PTHR1 mRNA表達(dá)量分別為1.00±0.12、0.23±0.04和0.44±0.06。SB組骨組織P-p38 MAPK及TNF-α表達(dá)較ANP組顯著降低(Plt;0.01)；骨組織PTHR1 mRNA表達(dá)量及血清鈣濃度較ANP組顯著增加(Plt;0.01)。結(jié)論p38MAPK信號轉(zhuǎn)導(dǎo)通路可介導(dǎo)ANP低鈣血癥的發(fā)生，抑制該通路可改善ANP低鈣血癥。

胰腺炎，急性壞死性； p38絲裂原活化蛋白激酶類； 低鈣血癥； 受體，甲狀旁腺激素； 腫瘤壞死因子

重癥急性胰腺炎(SAP)低鈣血癥的發(fā)病機(jī)制復(fù)雜，至今尚未完全闡明。我們前期研究證實(shí)，細(xì)胞因子與SAP低鈣血癥發(fā)生有關(guān)[1]。p38絲裂原活化蛋白激酶(p38MAPK)信號轉(zhuǎn)導(dǎo)通路在機(jī)體應(yīng)激和炎癥反應(yīng)調(diào)控過程起著極其重要的作用。本實(shí)驗(yàn)應(yīng)用p38MAPK特異的抑制劑SB203580干預(yù)急性壞死性胰腺炎(ANP)大鼠，探討p38MAPK信號轉(zhuǎn)導(dǎo)通路對ANP大鼠低鈣血癥和甲狀旁腺激素受體1(parathyrin receptor 1,PTHR 1)表達(dá)的影響。

材料和方法

一、實(shí)驗(yàn)動物分組

健康成年雄性SD大鼠72只，體重250～270 g，購自上海斯萊克實(shí)驗(yàn)動物有限責(zé)任公司(SCXK滬2007-0005，NO：0036178)。應(yīng)用完全隨機(jī)法分為ANP組、SB組和假手術(shù)(SO)組，每組又分造模后3、6、12 h 3個(gè)時(shí)間點(diǎn)，每個(gè)時(shí)間點(diǎn)8只大鼠。參照楊波等[2]方法，采用逆行膽胰管注射5%牛磺脫氧膽酸鈉1.0 ml/kg體重制備ANP模型。SB組在造模前30 min腹腔注射p38MAPK特異的抑制劑SB203580(美國InvivoGen公司)10 mg/kg體重[3]。SO組在開腹后，僅輕輕翻動十二指腸及胰腺后關(guān)腹。

各組大鼠在相應(yīng)時(shí)間點(diǎn)分別再次麻醉后開腹，下腔靜脈采血2 ml。迅速切取大鼠后肢股骨下端、脛骨上端，放入液氮速凍后置-80℃冰箱凍存。

二、檢測指標(biāo)和方法

1．血清鈣濃度測定：采用全自動生化分析儀檢測。

2．骨組織磷酸化p38MAPK(P-p38MAPK)和TNF-α表達(dá)檢測： 應(yīng)用蛋白提取試劑盒(Pierce公司)提取骨組織總蛋白，常規(guī)行Western blotting。抗P-p38MAPK一抗(Santa Cruz Biotechnology公司)1∶300，稀釋，抗TNF-α及β-actin一抗(博士德公司)1∶400稀釋，最后加入ECL試劑，壓片顯影。圖像采用Bandscan軟件分析，以目的條帶與β-actin的比值表示各組蛋白質(zhì)表達(dá)水平。

3．骨組織PTHR1mRNA檢測：采用實(shí)時(shí)定量RT-PCR法。用Trizol試劑盒(TaKaRa公司)提取骨組織總RNA，并用DNase I去除其中的DNA雜質(zhì)。PTHR1引物：上游5′-CCAGTGCTCAGCTCCGCATA-3′,下游5′- TCCTTGAGCAGCTTGTCACATTG -3′，片斷長度113 bp；β-actin(內(nèi)參)引物：上游5′- CCCATCTATGAGGGTTACGC -3′,下游5′- TTTAATGTCACGCACGATTTC -3′，片斷長度150 bp。進(jìn)行逆轉(zhuǎn)錄反應(yīng)后，使用SYBR Green I Real Time RT-PCR試劑盒，在Light Cycler Real Time PCR儀(Roche Diagnostics公司)上采用兩步法PCR 擴(kuò)增標(biāo)準(zhǔn)程序進(jìn)行基因擴(kuò)增。反應(yīng)結(jié)束進(jìn)行產(chǎn)物特異性驗(yàn)證(圖1)，根據(jù)2-△△Ct法[4]，以對照組△Ct平均值作為校正數(shù)，△△Ct=(Ct 目的基因-Ct 內(nèi)參基因)實(shí)驗(yàn)組-(Ct 目的基因-Ct 內(nèi)參基因)對照組,算出目的基因的相對表達(dá)量。

三、統(tǒng)計(jì)學(xué)方法

結(jié) 果

一、血清鈣濃度變化

ANP組各時(shí)間點(diǎn)血清鈣濃度較SO組均明顯降低(Plt;0.01)；SB組各時(shí)間點(diǎn)血清鈣濃度又較ANP組明顯回升(Plt;0.05或Plt;0.01，表1)。

二、骨組織P-p38MAPK和TNF-α的變化

SO組各時(shí)間點(diǎn)骨組織P-p38MAPK弱表達(dá)，TNF-α幾乎不表達(dá)；與SO組比較，ANP組各時(shí)間點(diǎn)P-p38MAPK和TNF-α表達(dá)量均顯著增高(Plt;0.01)； SB組各時(shí)間點(diǎn)P-p38MAPK和TNF-α表達(dá)量又較ANP組顯著降低(Plt;0.01，表1，圖1)。

三、骨組織PTHR1 mRNA表達(dá)的變化

ANP組各時(shí)間點(diǎn)PTHR1 mRNA表達(dá)量均低于SO組(Plt;0.01)；SB組各時(shí)間點(diǎn)PTHR1 mRNA表達(dá)量較ANP組均明顯上調(diào)(Plt;0.01，表1，圖2)。

表1 3、6、12 h各組骨組織中P-p38MAPK、TNF-α和PTHR1mRNA含量及血清鈣水平

注：與SO組比較，*Plt;0.01；與ANP組比較，△Plt;0.05，#Plt;0.01

圖1 制模后6 h骨組織中P-p38MAPK和TNF-α表達(dá)

A為PTHR1擴(kuò)增曲線，B為融解曲線的單峰圖(a為PTHR1，b為β-actin)

圖2實(shí)時(shí)RT-PCR產(chǎn)物特異性驗(yàn)證

討 論

p38MAPK信號轉(zhuǎn)導(dǎo)通路在機(jī)體應(yīng)激和炎癥反應(yīng)調(diào)控過程中具有極其重要作用，是調(diào)控炎癥細(xì)胞因子產(chǎn)生的重要通路之一[5]。應(yīng)激和各種刺激因素如各種炎癥細(xì)胞因子、生長因子、體液滲透壓改變、氧自由基、低氧血癥、休克和感染等均可激活胞質(zhì)的p38MAPK；p38MAPK激活后進(jìn)入胞核內(nèi)，調(diào)節(jié)效應(yīng)基因的表達(dá)，包括各種炎癥細(xì)胞因子基因，使介導(dǎo)炎癥細(xì)胞因子大量釋放。抑制p38MAPK活化阻斷該通路，可抑制炎癥細(xì)胞因子的產(chǎn)生。Chen等[6]研究表明，ANP時(shí)胰腺組織活化的p38MAPK和細(xì)胞因子表達(dá)量明顯升高，抑制ANP大鼠p38MAPK的活化，可減少炎癥細(xì)胞因子TNF-α、IL-1β釋放。Samuel等[7]和Denham等[8]均得出相同結(jié)論。本實(shí)驗(yàn)結(jié)果顯示，應(yīng)用p38MAPK特異抑制劑SB203580干預(yù)ANP大鼠，能抑制p38MAPK的活化，下調(diào)骨組織TNF-α的表達(dá)，進(jìn)一步證實(shí)了上述觀點(diǎn)。

我們前期研究證實(shí)：由于細(xì)胞因子的大量釋放，ANP大鼠抑制骨、腎PTHRmRNA表達(dá)，降低PTH及受體功能，產(chǎn)生低鈣血癥[1，9]；應(yīng)用甲強(qiáng)龍治療ANP大鼠，可使細(xì)胞因子水平顯著下降，PTHR1mRNA表達(dá)顯著上調(diào)，低鈣血癥得到明顯改善[10]。本實(shí)驗(yàn)應(yīng)用p38MAPK特異抑制劑SB203580預(yù)處理ANP大鼠后，骨組織TNF-α表達(dá)明顯下降，骨組織PTHR1mRNA和血清鈣明顯升高，表明是p38MAPK信號轉(zhuǎn)導(dǎo)通路介導(dǎo)了ANP低鈣血癥的發(fā)生。

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2008-07-14)

(本文編輯：屠振興)

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

福建省教育廳科研基金(JA03099)

350001 福州,福建醫(yī)科大學(xué)附屬協(xié)和醫(yī)院普外科

黃鶴光，Email: hhuang2@yahoo.com.cn