α-硫辛酸對大鼠急性胰腺炎的保護作用及其抗氧化機制

王艷紅 馮志杰 魏亞寧

·論著·

α-硫辛酸對大鼠急性胰腺炎的保護作用及其抗氧化機制

王艷紅 馮志杰 魏亞寧

目的探討抗氧化劑α-硫辛酸對急性胰腺炎(AP)的治療作用以及可能的機制。方法3.5%牛磺膽酸鈉逆行胰膽管注射制備AP 大鼠模型，數(shù)字表法隨機分為假手術(shù)組、AP組、生理鹽水組和α-硫辛酸組，每組30只。α-硫辛酸組于造模后腹腔內(nèi)注射α-硫辛酸1 mg/kg體重，生理鹽水組注射等量生理鹽水。分別于術(shù)后1、3、6、9、12 h處死大鼠，檢測血清淀粉酶、TNF-α、ICAM-1水平，觀察胰腺病理改變，測定胰腺組織超氧化物歧化酶(SOD)活力、丙二醛(MDA)含量。結(jié)果AP組胰腺水腫、粘連、壞死，腹腔內(nèi)可見血性腹水。術(shù)后6 h AP組血清淀粉酶、TNF-α、ICAM-1水平以及胰腺組織MDA含量分別為(2211.0±547.4)U/L、(174.8±7.9)ng/ml、(49.3±8.0)ng/ml和(32.2±5.9)U/mg prot，較假手術(shù)組的(160±23)U/L、(6.5±1.1)mg/ml、(13.9±3.4)mg/ml、(16.2±3.2)U/mg prot明顯升高(Plt;0.05);胰腺組織SOD活力為(38.5±9.5)U/mg prot，顯著低于假手術(shù)組(56.7±6.6)U/mg prot(Plt;0.05)。α-硫辛酸6 h組的血清淀粉酶、TNF-α、ICAM-1水平以及胰腺組織MDA含量分別為(1478±642)U/L、(164.8±6.2)ng/ml、(37.5±3.9)ng/ml和(20.2±8.4)U/mg prot，較AP組顯著降低(Plt;0.05)；胰腺組織SOD活力為(66.0±8.6)U/mg prot，較AP組顯著升高(Plt;0.05)。結(jié)論AP發(fā)病與氧化應激有關(guān)，抗氧化劑α-硫辛酸對AP 大鼠具有較好的治療作用，其機制可能與抑制TNF-α、ICAM-1活性有關(guān)。

胰腺炎； 抗氧化劑； 硫辛酸； 超氧化物歧化酶； 丙二醛

急性胰腺炎(acute pancreatitis，AP)易并發(fā)多臟器功能衰竭，危及患者的生命。在AP發(fā)病機制中，氧化應激可能發(fā)揮著重要作用，并與AP時胰腺外器官損傷有密切的聯(lián)系[1-2]。α-硫辛酸(α-lipoic acid)作為一種抗氧化劑，具有較強的抗氧化活性。本實驗應用α-硫辛酸干預實驗性AP大鼠，觀察其對AP的抗氧化治療作用。

材料和方法

一、實驗分組及模型制備

健康雄性Wister大鼠120只，由河北醫(yī)科大學實驗動物中心提供(合格證編號：607046)，清潔級，體重230～280 g。按數(shù)字表法隨機分為假手術(shù)組、AP組、生理鹽水對照(NS)組和α-硫辛酸治療(α-硫辛酸)組，每組30只。以胰膽管逆行注入3.5%?；悄懰徕c0.1 ml/100 g體重方法制備AP模型。α-硫辛酸組于造模后腹腔內(nèi)注射α-硫辛酸1 mg/kg體重；NS組造模后腹腔內(nèi)注射等量生理鹽水；假手術(shù)組僅翻動胰腺后關(guān)腹。各組于術(shù)后1、3、6、9、12 h分批處死大鼠，取血及胰腺組織。

二、觀察指標及檢測方法

1．血淀粉酶、TNF-α及細胞間黏附分子-1(ICAM-1)測定：血淀粉酶采用全自動生化分析儀檢測。血TNF-α及ICAM-1檢測采用ELISA方法，測試盒購自上海森雄生物有限公司。

2．胰腺組織學檢查：取部分胰腺組織，常規(guī)病理檢查。

3．胰腺組織超氧化物歧化酶(SOD)活力及丙二醛(MDA)含量測定：取新鮮胰腺組織制成組織勻漿，用化學比色法測定SOD及MDA，測試盒購自南京建成生物工程研究所。

三、統(tǒng)計學分析

結(jié) 果

一、大鼠胰腺病理學變化

假手術(shù)組胰腺及周圍組織大體結(jié)構(gòu)正常。AP組和NS組腹腔內(nèi)可見血性腹水，胰腺局部出現(xiàn)水腫，與周圍組織粘連，表面呈灰褐色并可見壞死灶及皂化斑。α-硫辛酸組較AP組病變明顯減輕。光鏡下假手術(shù)組胰腺及周圍組織結(jié)構(gòu)正常；AP組和NS組胰腺及周圍組織水腫、出血和壞死；α-硫辛酸組較AP組損害相對減輕(圖1)。

二、血清淀粉酶、TNF-α、ICAM-1水平的變化

AP組和NS組血清淀粉酶、TNF-α、ICAM-1水平均較假手術(shù)組顯著升高(Plt;0.01)；α-硫辛酸組則顯著低于AP組(Plt;0.01)，但仍高于假手術(shù)組(Plt;0.01，表1)。

三、胰腺組織SOD活力和MDA含量變化

AP組和NS組于術(shù)后3 h起SOD活力明顯下降(Plt;0.05或Plt;0.01)；α-硫辛酸組術(shù)后3 h后SOD活力較AP組明顯增加(Plt;0.05或Plt;0.01)。AP組及NS組胰腺組織MDA含量均較同時點假手術(shù)組顯著升高(Plt;0.01)；α-硫辛酸組各時點MDA含量均顯著低于AP組(Plt;0.05或Plt;0.01，表1)。

討 論

在AP的發(fā)展過程中，氧自由基(oxygen free radicals，OFRs)及其衍生物通過脂肪酸過氧化作用造成類脂膜破壞及溶酶體膜破壞對胰腺損害起著重要作用。OFRs可以激活補體，促進白細胞黏附、活化和遷移[3]，損傷內(nèi)皮細胞的完整性，增加毛細血管的通透性，造成循環(huán)血量的丟失，引起微循環(huán)障礙，加重胰腺損傷[4-5]。在AP的炎癥應答過程中，致炎因子和氧化應激發(fā)揮協(xié)同作用，導致炎癥的級聯(lián)擴增[6]。研究發(fā)現(xiàn)，在大鼠AP中胰腺損傷伴隨著組織中MDA水平增加以及過氧化氫酶、SOD、谷胱甘肽過氧化物酶活力和谷胱甘肽(GSH)水平下降[7]。本實驗結(jié)果顯示，AP組大鼠胰腺組織SOD活力顯著降低，而MDA顯著升高，表明氧化應激參與AP的發(fā)病[8]，測定血清氧化應激指標可以反映AP的嚴重程度[9]。

A：假手術(shù)組(HE ×200)；B：AP組(HE ×200)；C：NS組(HE ×400)；D：α-硫辛酸組(HE ×400)

時間點組別只數(shù)血清淀粉酶(U/L)TNF?α水平(ng/ml)ICAM?1水平(ng/ml)SOD活力(U/mgprot)MDA含量(U/mgprot)1h假手術(shù)組6127±306．3±1．410．8±2．859．5±6．915．2±3．2AP組61149±274b52．4±7．8b30．7±6．9b50．0±7．226．7±6．5bNS組61173±319b59．3±5．9b31．3±5．3b48．4±7．427．2±6．3bα?硫辛酸組6 832±298ac44．4±6．4bc23．3±3．7b51．4±8．719．7±3．6ac3h假手術(shù)組6135±366．9±1．310．1±1．958．9±7．814．4±4．2AP組61525±370122．2±13．3b41．1±7．4b42．9±6．7a27．5±5．7bNS組61651±373a124．1±14．0b41．8±8．1b41．0±6．5b31．3±7．1bα?硫辛酸組61143±342ac102．8±4．8bc32．2±6．0bc52．6±10．0c19．9±8．0ac6h假手術(shù)組6160±23a6．5±1．113．9±3．456．7±6．616．2±3．2AP組62211±547a174．8±7．9b49．3±8．0b38．5±9．5b32．2±5．9bNS組62335±417a179．6±6．8b49．0±9．4b34．7±6．7b33．9±6．3bα?硫辛酸組61478±642ac164．8±6．2bc37．5±3．9bc66．0±8．6d20．2±8．4ad9h假手術(shù)組6163±37 5．6±0．914．0±3．456．7±9．416．1±8．2AP組62484±365b164．4±9．0b62．3±8．9b35．9±6．4b36．7±7．9bNS組62633±392b169．7±7．6b64．2±10．7b33．9±7．2b39．1±9．5bα?硫辛酸組62015±488bc155．7±6．0bc49．0±8．1bc66．5±7．6d25．1±9．6ad12h假手術(shù)組6160±32 6．1±1．114．5±3．76．1±1．117．2±10．2AP組62995±354b112．7±9．3b70．5±9．2b112．9±9．3b38．1±10．4bNS組63126±413b111．8±8．0b73．7±9．0b111．8±8．0b43．1±11．2bα?硫辛酸組62545±413bc97．2±4．6bc58．0±8．9bc97．2±4．6bc30．2±7．1ad

注：與假手術(shù)組比較，aPlt;0.05，bPlt;0.01；與AP組比較，cPlt;0.05，dPlt;0.01

TNF-α是導致AP時胰腺及胰外器官組織損傷的主要細胞因子。TNF-α水平與疾病的嚴重程度、病死率和預后呈正相關(guān)[10]。本實驗研究顯示，AP組血清TNF-α水平明顯升高，在AP發(fā)生6 h后達高峰。TNF-α是AP早期增加的細胞因子，能促進炎癥部位白細胞聚集和活化，活化的白細胞又可產(chǎn)生大量OFRs，兩者形成一個惡性循環(huán)[1]。ICAM-1是一種主要表達于內(nèi)皮細胞表面的蛋白質(zhì)，能介導白細胞黏附和穿越內(nèi)皮細胞，促使白細胞通過血管內(nèi)皮屏障遷移至炎性區(qū)域[11]。Frossard等[12]研究發(fā)現(xiàn)，AP時存在器官灌注急劇減少及ICAM-1表達增加。本實驗結(jié)果顯示，AP組血清ICAM-1含量呈時間依賴性升高，它的上調(diào)是導致AP發(fā)生與發(fā)展的重要因素之一。

硫辛酸屬于維生素B類化合物，在丙酮酸脫氫酶、α-酮戊二酸脫氫酶、氨基己酸脫羧酶等多酶復合體中作為輔酶發(fā)揮作用。硫辛酸具有強效的抗氧化作用，可以直接清除OFRs，阻斷脂質(zhì)過氧化，誘導GSH的合成，恢復細胞氧化還原穩(wěn)態(tài)，減少細胞氧化損傷[13]。Park等[14]報道，給予雨蛙肽誘導的AP大鼠腹腔注射α-硫辛酸能夠明顯減輕血清脂肪酶和淀粉酶水平。本研究顯示，給予實驗性AP大鼠α-硫辛酸后血清淀粉酶活性明顯降低，胰腺病理損傷改善，胰腺組織中MDA含量減少，SOD活力增加，表明α-硫辛酸通過抗氧化作用對AP大鼠起到較好的治療效果。另外，α-硫辛酸能夠降低AP大鼠血清TNF-α和ICAM-1水平，由此推測α-硫辛酸對AP的治療作用可能也與抑制細胞因子TNF-α、ICAM-1活性有關(guān)。

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2009-03-09)

(本文編輯：屠振興)

Protectiveeffectandantioxidativemechanismofα-lipoicacidinratswithacutepancreatitis

WANGYan-hong,FENGZhi-jie,WEIYa-ning.

DepartmentofGastroenterology,SecondAffilitedHospital,HeBeiMedicalUniversity,Shijiazhuang050000,China

FENGZhi-jie,Email:zhijiefeng2005@126.com

ObjectiveTo investigate the protective effects of α-lipoic acid in rats with acute pancreatitis (AP) and its potential mechanism.MethodsWistar rats were randomly divided into four groups according to random number table: sham operation (SO) group, AP group, normal saline (NS) group and α-lipoic acid group with 30 rats in each group. AP model was induced by retrograde injection of 3.5% sodium taurocholate into the pancreatobiliary duct. Rats in α-lipoic acid group immediately

α-lipoic acid intra-peritoneal injection at the dose of 1 mg/kg. Rats in NS group received same amount of normal saline. The rats were sacrificed at 1, 3, 6, 9 and 12 h after AP induction. The serum levels of amylase, TNF-α and ICAM-1 were measured. Pancreatic histological changes were observed. The activities of pancreatic SOD and MDA were measured.ResultsIn rats of AP group, optical microscopy showed pancreatic edema, adhesion and necrosis. The serum amylase, TNF-α, ICAM-1 and MDA levels in pancreatic tissue 6h after operation were (2211±547)U/L, (174.8±7.9)ng/ml, (49.3±8.0)ng/ml and (32.2±5.9)U/mg prot, respectively, in AP group; which were significantly increased when compared with those of SO group (Plt;0.05). Pancreatic SOD activity was (38.5±9.5)U/mg prot, which was significantly lower than (56.7±6.7)U/mg prot of SO group

(Plt;0.05). The serum amylase, TNF-α, ICAM-1 and MDA levels in pancreatic tissue 6 h after operation in α-lipoic acid group were (1478±642)U/L, (164.8±6.2)ng/ml, (37.5±3.9)ng/ml and (20.2±8.4)U/mg prot, respectively; which were significantly decreased when compared with those of AP group (Plt;0.05). Pancreatic SOD activity was (66.0±8.6)U/mg prot, which were significantly higher than (38.5±9.5)U/mg prot of AP group (Plt;0.05).ConclusionsThe pathogenesis of AP was associated with oxidative stress, and α-lipoic acid as an antioxidant played a role in the treatment of AP, the possible mechanisms included inhibited production of TNF-α and ICAM-1.

Pancreatitis; Antioxidants; Thioctic acid; Superoxide dismutase; Malondialdehyde

10.3760/cma.j.issn.1674-1935.2009.06.014

050000 石家莊，河北醫(yī)科大學第二醫(yī)院消化內(nèi)科(王艷紅，現(xiàn)在河北省邢臺市人民醫(yī)院內(nèi)鏡室工作)

馮志杰，Email: zhijiefeng2005@163.com