何文華　夏亮　謝川　祝蔭　劉丕　朱勇　曾皓　朱萱　呂農(nóng)華

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夾竹桃麻素治療大鼠急性壞死性胰腺炎的療效

何文華夏亮謝川祝蔭劉丕朱勇曾皓朱萱呂農(nóng)華

目的觀察NADPH氧化酶(NOX)抑制劑夾竹桃麻素治療急性壞死性胰腺炎(ANP)大鼠的療效，探討其作用機(jī)制。方法60只SD大鼠按數(shù)字表法隨機(jī)分為對照組、ANP組、夾竹桃麻素組。采用胰膽管逆行注射5%牛磺膽酸鈉方法制備ANP模型。夾竹桃麻素組在制模同時(shí)腹腔注射夾竹桃麻素10 mg·kg-1·d-1。對照組僅開腹及關(guān)腹。術(shù)后12、24 h分批處死大鼠。觀察24 h內(nèi)各組大鼠存活率及呼吸、腎臟衰竭情況。取動(dòng)脈血檢測PaO2、淀粉酶和肌酐，采用ELISA法檢測血TNF-α、IL-6水平，蛋白質(zhì)印跡法檢測胰腺組織NOX亞基NOX2、p22phox、p67phox及p-NF-κB p65表達(dá)。 結(jié)果ANP組大鼠24 h的急性呼吸衰竭發(fā)生率為71.4%(5/7)，急性腎衰竭發(fā)生率為100%(7/7)；夾竹桃麻素組發(fā)生率分別為22.2%(2/9)和0。兩組差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。24 h時(shí)夾竹桃麻素組血PaO2顯著高于ANP組[(68.4±6.8)mmHg比(56.5±6.1)mmHg，1 mmHg=0.133 kPa]，血淀粉酶、肌酐、TNF-α、IL-6水平顯著低于ANP組[(2 907±849)U/L比(6 421±690)U/L，(122.3±19.4)μmol/L比(213.0±39.2)μmol/L,(120.0±11.9)ng/L比(302.5±41.6)ng/L，(174.4±19.3)ng/L比(378.6±13.6)ng/L]，胰腺病理評分顯著低于ANP組[(3.16±0.91)分比(7.95±1.23)分]，胰腺組織的p22phox、p67phox和p-NF-κB p65表達(dá)顯著低于ANP組[0.79(0.75,0.84)比1.36(1.18,1.51)，0.82(0.80,0.87)比1.70(1.47,1.80)，0.82(0.80,0.84)比1.50(1.31.1.61)] ，NOX2表達(dá)完全被抑制[0比0.93(0.87,1.06)] ，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。結(jié)論夾竹桃麻素對ANP大鼠有治療作用，其機(jī)制可能是通過抑制NOX介導(dǎo)的NF-κB活化和TNF-α、IL-6釋放實(shí)現(xiàn)的。

胰腺炎，急性壞死性；NADP氧化酶；夾竹桃麻素；治療結(jié)果

Fund Program：Natural Science Youth Foundation of Jiangxi Province (20142BAB215010); Science and Technology Research Project of Education Department of Jiangxi Province(GJJ14019)

急性胰腺炎(AP)是世界范圍內(nèi)的常見疾病，發(fā)病率高，總體病死率達(dá)5%～10%[1-2]。最新的指南將AP的嚴(yán)重程度分為輕度AP(MAP)、中度AP(MSAP)和重度AP(SAP)；根據(jù)病程分為早期(1周內(nèi))和后期(1周至數(shù)月)[2-3]。臨床發(fā)現(xiàn)早期全身炎癥反應(yīng)綜合征(SIRS)與SAP的發(fā)生密切相關(guān)[4-5]，早期阻斷 SIRS可望阻止病情進(jìn)展。煙酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶(NADPH oxidase,NOX)在胰腺炎的發(fā)病中起重要作用，它通過產(chǎn)生活性氧自由基(ROS)不僅直接引起氧化應(yīng)激，參與胰蛋白酶的激活和胰腺自我消化，還介導(dǎo)促炎信號通路的激活[6-10]。但大多數(shù)研究是在細(xì)胞水平上進(jìn)行的，體內(nèi)研究較少。本研究以NOX抑制劑夾竹桃麻素干預(yù)急性壞死性胰腺炎(ANP)大鼠，觀察其療效，并探討其機(jī)制。

材料與方法

一、實(shí)驗(yàn)材料與主要試劑

清潔級健康雄性SD大鼠60只，4～6周齡，體重160～200 g，由南昌大學(xué)實(shí)驗(yàn)動(dòng)物中心提供，在標(biāo)準(zhǔn)環(huán)境下飼養(yǎng)，自由進(jìn)食、進(jìn)水，實(shí)驗(yàn)過程中對動(dòng)物的處理嚴(yán)格遵循《實(shí)驗(yàn)動(dòng)物管理?xiàng)l例》。 ?；悄懰徕c、夾竹桃麻素購自美國Sigma-Aldrich公司，抗NOX2、p22phox抗體購自英國Abcam公司，抗p67phox、p-NF-κB p65 (Ser536)抗體購自美國Cell Signaling Technology公司，大鼠細(xì)胞因子試劑盒(TNF-α、IL-6)為美國R&D 公司產(chǎn)品，免疫組化檢測試劑盒(PV-6002)及DAB 試劑盒為北京中杉金橋生物技術(shù)有限公司產(chǎn)品。

二、方法

1．動(dòng)物模型制備及分組：60只SD大鼠入室適應(yīng)1周后按數(shù)字表法隨機(jī)分為對照組、ANP組、夾竹桃麻素組，每組20只。采用逆行胰膽管內(nèi)加壓注射5%?；悄懰徕c0.1 ml/100 g體重方法制備ANP模型[11]；夾竹桃麻素組在制模同時(shí)腹腔注射夾竹桃麻素10 mg·kg-1·d-1；對照組僅行開、關(guān)腹手術(shù)。術(shù)后12、24 h分批處死大鼠。

2．觀察大鼠存活及臟器衰竭情況：觀察24 h內(nèi)3組大鼠存活情況。采用改良Mashall標(biāo)準(zhǔn)判定3組大鼠呼吸衰竭和腎衰竭發(fā)生率。

3．血生物化學(xué)指標(biāo)檢測：腹部動(dòng)脈取血8～10 ml，應(yīng)用血?dú)夥治鰞x檢測動(dòng)脈血氧分壓(PaO2)、全自動(dòng)血生物化學(xué)分析儀檢測血淀粉酶和肌酐水平，采用ELISA法檢測血清TNF-α、IL-6水平。

4．胰腺組織病理學(xué)檢查：取部分胰腺組織，置10%甲醛中固定過夜，常規(guī)石蠟包埋、切片，HE染色，光鏡下觀察胰腺組織病理學(xué)改變，參照Schmidt等[12]標(biāo)準(zhǔn)進(jìn)行胰腺病理損傷程度評分。

5．胰腺組織NOX2、p22phox、p67phox和p-NF-κB p65蛋白表達(dá)檢測：取液氮保存的胰腺組織，研磨成粉末后用蛋白提取液提取蛋白，常規(guī)行蛋白質(zhì)印跡法檢測NOX2、p22phox、p67phox和p-NF-κB p65蛋白表達(dá)，以GAPDH為內(nèi)參?？筃OX2、p67phox、p22phox、p-NF-κB p65抗體工作濃度均為1∶1 000，HRP標(biāo)記的羊抗兔或羊抗鼠二抗工作濃度1∶5 000。最后用SuperSignal West Femto敏感曝光試劑盒曝光底片，采用Gel-Proanalyzer 4軟件分析蛋白條帶灰度值，以目的條帶與內(nèi)參條帶的灰度值比表示蛋白的相對表達(dá)量。

三、統(tǒng)計(jì)學(xué)處理

結(jié)　　果

一、大鼠存活情況、臟器衰竭發(fā)生率及血PaO2、淀粉酶、肌酐水平

對照組大鼠全部存活， ANP組大鼠24 h內(nèi)死亡3只，夾竹桃麻素組大鼠24 h內(nèi)死亡1只，各組間差異無統(tǒng)計(jì)學(xué)意義(P=0.13)。ANP組24 h的急性呼吸衰竭發(fā)生率為71.4%(5/7)，急性腎衰竭發(fā)生率為100%；夾竹桃麻素組發(fā)生率分別為22.2%(2/9)和0，均顯著低于ANP組(P值分別為0.004、0.000)。

ANP組PaO2顯著低于對照組，夾竹桃麻素組顯著高于ANP組；ANP組血淀粉酶、肌酐水平均顯著高于對照組，夾竹桃麻素組又顯著低于ANP組。差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.001，表1)。

二、大鼠胰腺組織病理改變

24 h時(shí)對照組大鼠胰腺組織結(jié)構(gòu)清晰，細(xì)胞形態(tài)正常(圖1A)；ANP組胰腺的腺小葉排列紊亂，片狀壞死，壞死區(qū)腺泡結(jié)構(gòu)消失，有炎癥細(xì)胞浸潤(圖1B)；夾竹桃麻素組胰腺組織水腫，無明顯壞死，腺泡細(xì)胞顆粒樣或空泡變性，間質(zhì)有炎癥細(xì)胞浸潤(圖1C)。對照組、ANP組、夾竹桃麻素組胰腺組織病理評分分別為(0.18±0.23)、(7.95±1.23)和(3.16±0.91)分，組間差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.01)。

三、大鼠血清TNF-α、IL-6水平

ANP組血清TNF-α、IL-6水平顯著高于對照組，夾竹桃麻素組顯著低于ANP組，但仍顯著高于對照組，差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.001，表2)。

四、胰腺組織p22phox、p67phox、NOX2和p-NF-κB p65蛋白表達(dá)

對照組胰腺組織NOX2未能檢出，p22phox、p67phox和p-NF-κB p65低表達(dá)；ANP組胰腺組織NOX2、p22phox、p67phox和p-NF-κB p65表達(dá)均顯著高于對照組；夾竹桃麻素組NOX2未能檢出，p22phox、p67phox和p-NF-κB p65表達(dá)顯著低于ANP組。3組間差異均有統(tǒng)計(jì)學(xué)意義(P值均<0.001，表3、圖2)。

表1　各組大鼠動(dòng)脈血氧分壓、血清淀粉酶、血清肌酐的變化±s)

注：與對照組比較，aP<0.001；與ANP組比較，bP<0.001；1 mmHg=0.133 kPa

圖1　對照組(1A)、ANP組(1B)、夾竹桃麻素組(1C)胰腺病理改變(HE　×200)

組別只數(shù)12hTNF-αIL-624hTNF-αIL-6對照組2070.8±8.2117.5±10.474.2±7.7125.2±9.6ANP組20321.8±25.4a332.7±27.7a302.5±41.6a378.6±13.6a夾竹桃麻素組20109.2±12.8b149.9±12.0b120.0±11.9b174.4±19.3bF值613.70388.20218.16662.34P值<0.001<0.001<0.001<0.001

注：與對照組比較，aP<0.001；與ANP組比較，bP<0.001

表3　各組胰腺組織NOX亞基和p-NF-κB p65表達(dá)[M(P25,P75)]

注：與對照組比較，aP<0.001；與ANP組比較，bP<0.001

圖2　對照組(1)、夾竹桃麻素組(2)、ANP組(3)胰腺組織p22phox、p67phox 、NOX2和p-NF-κB p65蛋白表達(dá)

討　　論

最近的研究發(fā)現(xiàn)胰腺腺泡細(xì)胞內(nèi)早期觸發(fā)且持續(xù)激活的炎癥信號通路是AP發(fā)病的關(guān)鍵[13]。胰腺腺泡內(nèi)胰蛋白酶原的激活雖然引起細(xì)胞損傷，但并不引起AP的炎癥反應(yīng)[14]。而胰腺腺泡細(xì)胞早期NF-κB被活化，它與胰蛋白酶原的激活是相互獨(dú)立的事件[14-15]。 NF-κB的激活可引起局部損傷和全身炎癥反應(yīng)，導(dǎo)致SAP的發(fā)生[16]。

近年研究發(fā)現(xiàn)，NOX在AP發(fā)病中起重要作用。吞噬細(xì)胞型NOX由細(xì)胞膜上的催化亞基NOX2 (gp91phox)、調(diào)節(jié)亞基p22phox和胞質(zhì)的調(diào)節(jié)亞基p47phox、p40phox、p67phox、Rac組成，目前發(fā)現(xiàn)有6種NOX2的同源蛋白(NOX1、NOX3、NOX4、NOX5、DUOX1和DUOX2)，NOX是專職產(chǎn)生ROS的多蛋白復(fù)合體，它們產(chǎn)生的ROS介導(dǎo)炎癥信號通路的激活[6,8-9]，參與多種生理活動(dòng)，也與疾病的發(fā)生密切相關(guān)[17-19]。有研究在胰腺腺泡細(xì)胞(AR42J)檢測到NOX1的表達(dá)，給予雨蛙素可激活NOX產(chǎn)生ROS，引起NF-κB活化和IL-6表達(dá)，應(yīng)用NOX抑制劑DPI可抑制NF-κB活化和IL-6表達(dá)[8]。另有研究發(fā)現(xiàn)，胰腺腺泡細(xì)胞NOX產(chǎn)生的ROS介導(dǎo)JAK2/STAT3和MAPKs信號通路的激活，DPI同樣可抑制這些信號通路的活化[9]。

本研究結(jié)果顯示，NOX抑制劑夾竹桃麻素干預(yù)ANP大鼠后，大鼠血淀粉酶、TNF-α、IL-6水平下降，胰腺組織損傷減輕，急性呼吸衰竭和急性腎衰竭的發(fā)生率降低。此外胰腺組織NOX2的表達(dá)完全被抑制，p22phox、p67phox、p-NF-κB p65表達(dá)也顯著低于ANP大鼠，表明夾竹桃麻素對大鼠ANP有治療作用，其作用機(jī)制可能是通過抑制NOX2的表達(dá)，進(jìn)而抑制NF-κB的活化，減少炎癥細(xì)胞因子TNF-α、IL-6的表達(dá)，阻斷SIRS的發(fā)生，從而阻止器官功能損傷。

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(本文編輯：呂芳萍)

Therapeutic efficacy of apocynin on acute necrotizing pancreatitis rats

HeWenhua,XiaLiang,XieChuan,ZhuYin,LiuPi,ZhuYong,ZengHao,ZhuXuan,LyuNonghua.

DepartmentofGastroenterology,FirstAffiliatedHospitalofNanchangUniversity,Nanchang330006,China

Correspondingauthor:LyuNonghua,Email:lunonghua@163.com

ObjectiveTo explore the effect and mechanism of NADPH oxidase (NOX) inhibitor apocynin in treating acute necrotizing pancreatitis (ANP) rat. MethodsSixty SD rats were randomly divided into three groups: control group, ANP group and apocynin treated group. ANP rat model was established by retrograde injection of 5% sodium taurocholate into pancreatic duct. ANP rats in apocynin group were treated by intraperitoneal injection of 10 mg·kg-1·d-1apocynin. Rats in control group underwent sham surgery of opening and closing abdominal cavity. Rats were killed at 12 h and 24 h, respectively. Survival, respiration and renal failure were observed within 24 h. PaO2, amylase and creatinine in arterial blood specimen were detected. Serum TNF-α and IL-6 level was detected by enzyme-linked immunosorbent assay (ELISA). NOX2, p22phox, p67phoxand p-NF-κB p65 expression in pancreatic tissue were detected by Western blot. ResultsIn ANP group, the incidence of acute respiratory failure and acute renal failure was 71.42%(5/7) and 100% (7/7)at 24 h, respectively. The incidence of acute respiratory failure and acute renal failure was 22.2%(2/9) and 0(0/9) in apocynin treated group, and the difference was statistically significant (bothP<0.01). In Apocynin treated group, PaO2level was higher than that in ANP group at 24 h [(68.4±6.8)vs(56.5±6.1)mmHg, 1 mmHg=0.133 kPa]. Serum amylase, creatinine, TNF-α and IL-6 level was obviously lower than those in ANP group at 24 h [(2 907±849)vs(6 421±690)U/L,(122.3±19.4)vs(213.0±39.2)μmol/L,(120.0±11.9)vs(302.5±41.6)ng/L,(174.4±19.3)vs(378.6±13.6)ng/L. Pancreatic pathological score was significantly lower than that in ANP group [(3.16±0.91)vs(7.95±1.23); p22phox, p67phox, and p-NF-κB p65 expression in pancreatic tissue was also significantly lower than those in ANP model group[0.79(0.75, 0.84)vs1.36(1.18,1.51)，0.82(0.80, 0.87)vs1.7(1.47,1.8), 0.82(0.80, 0.84)vs1.50(1.31, 1.61)]; NOX2 expression was completely inhibited by apocynin[0vs0.93(0.87,1.06)], which were statistically different (allP<0.001). ConclusionsApocynin could exert a therapeutic effect in ANP rats, and the potential mechanism may be associated with NOX mediated activation of NF-κB and TNF-α, IL-6 release.

Pancreatitis, acute necrotizing;NADP oxidase;Apocynin;Treatment outcome

10.3760/cma.j.issn.1674-1935.2016.04.009

330006南昌，南昌大學(xué)第一附屬醫(yī)院消化內(nèi)科

呂農(nóng)華，Email: lunonghua@163.com

江西省青年自然基金(20142BAB215010)；江西省教育廳科學(xué)技術(shù)研究項(xiàng)目(GJJ14019)

2015-11-26)