PAF受體拮抗劑對內毒素血癥幼年大鼠胃黏膜PGE2含量及COX-2表達的影響

劉春英，李奇玉，王琳，蔣靜，王麗杰，孫梅

PAF受體拮抗劑對內毒素血癥幼年大鼠胃黏膜PGE2含量及COX-2表達的影響

劉春英，李奇玉，王琳，蔣靜，王麗杰，孫梅

目的探討前列腺素E2(PGE2)、環(huán)氧化酶-2(COX-2)、血小板活化因子(PAF)受體拮抗劑在內毒素(LPS)腹腔注射誘導的幼年大鼠急性胃黏膜損傷中的作用。方法18日齡Wistar大鼠192只，隨機分為對照組、LPS組、PAF受體拮抗劑預防組和治療組。采用LPS(O55:B5脂多糖)5mg/kg腹腔注射制備幼年大鼠LPS血癥模型，預防組和治療組分別于LPS腹腔注射前后0.5h應用5mg/kg PAF受體拮抗劑BN52021(Ginkgolide B)，對照組腹腔注射等量生理鹽水。于LPS注射后1.5、3、6、24、48、72h處死動物，每組每時間點8只，肉眼及光學顯微鏡下觀察胃黏膜損傷情況，采用放射免疫法測定胃黏膜PGE2含量，免疫組織化學SP法測定胃黏膜COX-2蛋白的表達，半定量RT-PCR法測定胃黏膜COX-2 mRNA的表達。結果LPS組腹腔注射LPS后1.5h黏膜上皮細胞水腫，3h組織充血、水腫，6h胃黏膜損傷最重，黏膜內有出血，細胞核碎裂、固縮，凋亡小體出現；24h上皮脫落、中性粒細胞浸潤，48h黏膜層變薄、腺體減少，72h未見明顯異常。預防組和治療組改變輕微。與對照組比較，LPS組胃黏膜PGE2含量在3h時明顯降低(P<0.05)，6h時達最低(P<0.01)，預防組胃黏膜PGE2含量在3、6h明顯增高(P<0.05)，治療組PGE2含量在6h時明顯增高(P<0.05)。與LPS組比較，預防組、治療組胃黏膜PGE2含量在6h時均明顯增高(P<0.01)。對照組胃黏膜組織未見明顯COX-2蛋白及mRNA表達；與對照組比較，LPS組腹腔注射LPS后6h胃黏膜組織胞質即有COX-2蛋白表達，24、48、72h時明顯增高(P<0.01)，其mRNA水平亦上調；預防組和治療組6h COX-2蛋白、mRNA水平明顯增高(P<0.01)；預防組和治療組6h COX-2蛋白、24h COX-2 mRNA與LPS組比較亦明顯增高(P<0.01)。結論PAF受體拮抗劑可上調COX-2 mRNA及蛋白表達，使PGE2含量增加，對胃黏膜有保護作用。

胃黏膜；脂多糖類；地諾前列酮；環(huán)氧化酶2；血小板活化因子；PAF受體拮抗劑

重癥感染是急性胃黏膜損傷的常見原因之一[1]。急性胃黏膜損傷是由于胃黏膜保護機制削弱，損傷因素相對增強所致[2]。近年研究發(fā)現前列腺素E2(PGE2)、環(huán)氧化酶-2(COX-2)在胃黏膜損傷修復方面發(fā)揮重要作用。本研究通過給幼年大鼠腹腔注射脂多糖(lipopolysaccharide，LPS)建立急性胃黏膜損傷模型，觀察應用血小板活化因子(platelet activating factor，PAF)受體拮抗劑后胃黏膜PGE2含量和COX-2表達的變化，旨在探討其在胃黏膜損傷修復中的作用。

1 材料與方法

1.1 動物分組及模型建立 192只健康18日齡Wistar大鼠，體重32.37±6.32g，與母鼠共同飼養(yǎng)(由中國醫(yī)科大學第二臨床學院實驗動物中心提供)。LPS(O55:B5脂多糖)、PAF受體拮抗劑BN52021(Ginkgolide B)為美國Sigma公司產品。PGE2放射免疫試劑盒由北京東亞免疫研究所提供，液體閃爍計數器為美國Beckman公司產品。COX-2免疫組化試劑盒由北京中杉生物工程有限公司提供，總RNA提取系統試劑由河南華美生物工程公司提供。COX-2、內參照β-actin引物由上海生工生物工程技術公司合成，TaKaRa試劑盒由大連寶生物工程有限公司提供。將大鼠隨機分為4組：對照組、LPS組、PAF受體拮抗劑預防組(預防組)和治療組，每組于1.5、3、6、24、48、72h各個時間點設動物8只。對照組腹腔注射生理鹽水1ml/kg；LPS組腹腔注射LPS(O55:B5脂多糖)5mg/kg，配比濃度5g/L，用生理鹽水溶解。預防組于LPS腹腔注射前0.5h、治療組于LPS腹腔注射后0.5h給予PAF受體拮抗劑BN52021(Ginkgolide B)5mg/kg，配比濃度5g/L。用藥后各組均放回鼠籠，繼續(xù)哺乳。分別于LPS腹腔注射后相應時間點處死動物各8只。

1.2 方法

1.2.1 標本采集及保存 大鼠處死后迅速開腹取胃，沿胃大彎剪開，肉眼觀察胃黏膜大體損傷情況，在腺胃區(qū)胃大彎處取0.5cm×0.5cm胃組織置入40g/L甲醛溶液中固定，其余部分用生理鹽水沖洗3次，置入去RNA酶試管中，深低溫冰箱－70℃保存。

1.2.2 胃黏膜PGE2含量測定 采用放射免疫法，取低溫保存腺胃區(qū)黏膜20mg，加生理鹽水及乙醇制成10%勻漿，2000r/min低溫離心8min，取上清液，－20℃以下保存。測量時在上清液中加入重蒸餾乙酸乙酯，混勻，離心，吸取上清液，按試劑盒說明加入3H標記物等試劑，離心后將上清液倒入預先加有閃爍液的瓶中，在液體閃爍計數器上測放定射性。根據放射性得出PGE2含量。

1.2.3 胃黏膜COX-2蛋白表達水平測定 取胃組織于40g/L甲醛溶液中固定，包埋，石蠟切片。應用免疫組化SP法(過氧化物酶標記的鏈霉卵白素法)測定COX-2表達水平。抗體工作濃度：兔抗鼠-COX-2 IgG抗體稀釋比例1:150。顯微鏡觀察，細胞質中有棕褐色顆粒者為COX-2陽性表達，以PBS代替一抗作為陰性對照。各時間點選取染色清晰的切片5～6張，于光鏡下(×40)隨機選取2～3個視野，應用Olympus-BX41圖像采集系統，Meta Morph/Dp10/ BX41軟件分析系統測定平均光密度值。

1.2.4 RT-PCR檢測胃黏膜COX-2 mRNA的表達

Trizol裂解待測胃黏膜組織，用酚-氯仿抽提總RNA，反轉錄合成cDNA后行PCR擴增，反應總體積25μl，PCR擴增引物由本實驗室參照美國國立圖書館PubMed基因庫中電腦軟件Primer 5.0自行設計。COX-2：上游5'-ACGGACTTGCTCACTTTG-3'，下游5'-AGGAGAACAGATGGGATT-3'，產物大小377bp。β-actin：上游5'-CACCCTGTGCTGCTCACC GAGGCC-3'，下游5'-CCACACAGATGACTTGCGC TCAGG-3'，產物大小690bp。COX-2反應條件為：94℃預變性3min；94℃變性40s、51.5℃退火1min、72℃延伸1min，擴增35個循環(huán)；最后72℃ 7min終止反應。β-actin反應條件：94℃變性3min、55℃退火30s、72℃延伸30s，擴增35個循環(huán)；最后60℃延伸10min終止反應。PCR擴增后，取PCR反應產物10μl加入2g/L瓊脂糖凝膠中電泳，應用計算機凝膠成像分析系統進行半定量分析。以β-actin為內對照，COX-2的mRNA的相對表達量用COX-2的PCR電泳結果掃描值與β-actin的PCR電泳結果掃描值的比值來確定。

1.3 統計學處理 采用SPSS 10.0軟件進行分析，數據以表示，多組間比較采用方差分析，進一步兩兩比較采用SNK-q檢驗。P<0.05為差異有統計學意義。

2 結 果

2.1 病理改變 大體觀察可見LPS組1.5h胃黏膜輕度水腫，3h可見充血、水腫，6h黏膜表面可見大片糜爛、出血、條索狀壞死，與胃縱軸平行，損傷主要見于腺胃區(qū)，竇部少見，前胃部無損傷；24h黏膜糜爛、出血，壞死灶陳舊、縮小，48h黏膜輕度萎縮，72h胃黏膜基本恢復正常。光鏡下LPS組1.5h黏膜上皮細胞水腫，3h組織充血、水腫，6h胃黏膜損傷最重，黏膜表面上皮廣泛脫落，黏膜內有出血，炎性細胞浸潤，核碎裂、固縮，凋亡小體出現，腺體受損，24h上皮脫落、中性粒細胞浸潤，48h黏膜層變薄、腺體減少，72h未見明顯異常。預防組和治療組6h大體觀僅見胃黏膜充血水腫，光鏡下可見黏膜上皮細胞腫脹、充血；兩組其他時間點病理改變均較輕。對照組胃黏膜表面上皮隱窩細胞形態(tài)學正常，腺體結構完整(圖1)。

2.2 胃黏膜PGE2含量 與對照組比較，LPS組腹腔注射LPS后1.5h胃黏膜PGE2含量輕度下降，3h明顯降低(P<0.05)，6h最低(P<0.01)，24h開始上升，48h升至與對照組相同的水平；預防組胃黏膜PGE2含量在3、6h明顯增高(P<0.05)；治療組PGE2含量在6h明顯增高(P<0.05)。與LPS組比較，預防組、治療組胃黏膜PGE2含量在6h明顯增高(P<0.01)。預防組與治療組各時間點比較差異無統計學意義(P>0.05，表1)。

圖1 6h時點胃黏膜的病理改變(HE ×400)Fig.1 Pathologic changes of gastric mucosa at 6h time point (HE ×400)

表1 各組各時間點胃黏膜PGE2含量(μg/L，±s，n=8)Tab.1 PGE2contents of gastric mucosa in different groups at each time point (μg/L,±s,n=8)

表1 各組各時間點胃黏膜PGE2含量(μg/L，±s，n=8)Tab.1 PGE2contents of gastric mucosa in different groups at each time point (μg/L,±s,n=8)

(1)P<0.05, (2)P<0.01 compared with control group; (3)P<0.05, (4)P<0.01 compared with LPS group

Group 1.5h 3h 6h 24h 48h 72h Control 225.3±21.1 239.2±12.5 245.1±8.9 263.6±13.1 246.1±9.7 269.0±23.6 LPS 216.9±66.1 195.7±22.1(1) 134.5±9.3(2) 236.7±62.2 250.2±14.0 253.4±7.6 Prevention 235.0±20.0 276.0±64.0(1)(4) 304.4±15.0(1)(4) 278.3±24.6(3) 262.7±32.4 247.0±11.2 Treatment 213.0±34.5 252.2±21.0(3) 315.9±43.7(1)(4) 295.9±43.7(4) 276.4±69.5 267.4±87.4

2.3 胃黏膜COX-2蛋白及mRNA的表達 對照組胃黏膜組織基本未見COX-2蛋白及mRNA表達。LPS組腹腔注射LPS后6h胃黏膜組織胞質COX-2蛋白開始表達，24、48、72h時與對照組比較明顯增高(P<0.01)，其mRNA水平亦上調；預防組和治療組6h COX-2蛋白表達與對照組比較即明顯增高(P<0.01)，mRNA水平明顯上調(P<0.05)，且預防組和治療組6h COX-2蛋白、24h COX-2 mRNA與LPS組比亦明顯增高(P<0.01，表2、圖2，表3、圖3)。

3 討 論

前列腺素(PG)是一種脂質，主要在精囊、腎髓質、肺和胃腸道中合成。胃腸道黏膜可合成PG，包括PGE、PGF、PGI，其中以PGE2含量最多。PG不在細胞內貯存，而在分泌瞬間合成，所以PG的產生和釋放是組織損害的結果[3-4]。早在1978年Robert等[5]首先觀察到PG可抑制胃酸分泌，且非抗酸劑量的PG可完全防止乙醇、強酸、強堿等物質引起的急性胃黏膜損傷。國外大量研究證實，十二指腸潰瘍患者胃竇部及十二指腸球部黏膜PGE2含量下降，且在潰瘍活動期下降明顯[6]。本研究結果顯示，幼年大鼠腹腔注射LPS后6h胃黏膜損傷最重，PGE2濃度最低；此后，PGE2濃度逐漸上升，胃黏膜損傷逐漸愈合。因而，LPS血癥時急性胃黏膜損傷可能與PGE2缺乏有關，PG的不斷產生和釋放可促進胃黏膜細胞愈合，發(fā)揮保護作用。這種不依賴于抗酸的作用被稱之為“細胞保護”[5]。文獻報道PG可能通過刺激胃黏液的生成和分泌，促進HCO–3分泌，增加表面磷脂生成，加強胃黏膜屏障的疏水性，保護胃黏膜微循環(huán)結構的完整，維持胃黏膜血液供應，保護增殖區(qū)細胞，促進黏膜上皮的更新及修復，以及抑制胃運動過強等機制來實現對胃黏膜的保護作用[7-8]。本實驗中腹腔注射LPS之前及之后給予PAF受體拮抗劑，均增加了胃黏膜PGE2的含量，減輕了幼年大鼠的急性胃黏膜損傷。

表2 各組各時間點胃黏膜COX-2 蛋白的表達(±s，n=8)Tab.2 Expressions of COX-2 protein in different groups at each time point (±s,n=8)

表2 各組各時間點胃黏膜COX-2 蛋白的表達(±s，n=8)Tab.2 Expressions of COX-2 protein in different groups at each time point (±s,n=8)

(1)P<0.01 compared with control group; (2)P<0.01 compared with LPS group

Group 1.5h 3h 6h 24h 48h 72h Control 19.1±2.1 20.2±1.5 20.0±1.9 19.8±1.1 20.3±1.7 19.9. ±2.6 LPS 18.1±1.9 19.5±2.1 23.5±2.3 29.7±2.2(1) 30.2±1.5(1) 31.4±2.7(1)Prevention 19.0±2.0 18.0±1.9 29.0±2.7(1)(2) 30.0±3.1(1) 31.7±3.4(1) 30.1±1.2(1)Treatment 20.1±1.7 21.2±1.6 28.9±3.1(1)(2) 29.7±3.2(1) 30.1±2.3(1) 31.7±3.4(1)

圖2 免疫組化SP法檢測胃黏膜6h時點COX-2蛋白的表達(DAB ×400)Fig.2 Expression of COX-2 protein in gastric mucosal at 6h time point detected by immunohistochemistry SP method (DAB ×400)

表3 各組各時間點胃黏膜COX-2 mRNA的表達(±s，n=8)Tab.3 Expressions of COX-2 mRNA in different groups at each time point (±s,n=8)

表3 各組各時間點胃黏膜COX-2 mRNA的表達(±s，n=8)Tab.3 Expressions of COX-2 mRNA in different groups at each time point (±s,n=8)

(1)P<0.01 compared with control group; (2)P<0.05 compared with LPS group

Group 1.5h 3h 6h 24h 48h 72h Control 0.18±0.03 0.17±0.02 0.18±0.02 0.18±0.02 0.17±0.02 0.18 ±0.03 LPS 0.16±0.04 0.19±0.05 0.85±0.15(1) 0.96±0.13(1) 1.22±0.17(1) 1.23±0.16(1)Prevention 0.18±0.03 0.19±0.06 0.99±0.16(1) 1.20±0.12(1)(2) 1.22±0.12(1) 1.25±0.17(1)Treatment 0.18±0.02 0.16±0.03 0.96±0.13(1) 1.22±0.13(1)(2) 1.20±0.13(1) 1.23±0.13(1)

圖3 胃黏膜COX-2 mRNA表達Fig.3 Expressions of COX-2 mRNA in gastric mucosa

環(huán)氧化酶(cyclooxygenase，COX)是PG合成過程中重要的限速酶，它將花生四烯酸(arachidonic acid，AA)代謝成各種前列腺素產物，維持機體的各種生理病理過程[9-10]。目前已知COX具有兩種亞型，即COX-1、COX-2。COX-1為結構型，在大多數正常細胞中穩(wěn)定表達。COX-2為誘導型，在正常生理狀態(tài)下幾乎不表達或表達甚少，僅在細胞受到生長因子、細胞因子、內皮素觸發(fā)活動及一氧化氮等刺激后迅速從頭合成，參與多種病理生理過程。COX-2在維持胃腸道完整性等方面的作用較COX-1更大[11-12]。研究發(fā)現，應用COX-2選擇性抑制劑可顯著減少PGE2生成，降低潰瘍邊緣肉芽組織內微血管的密度，影響肉芽組織的成熟，延遲潰瘍愈合，并呈明顯的劑量反應關系[13-14]。故目前認為COX-2產物在潰瘍愈合過程中起著積極作用。本實驗結果顯示，幼年大鼠LPS血癥急性胃黏膜損傷發(fā)生時，胃黏膜組織COX-2 mRNA的表達明顯高于對照組，免疫組織化學染色證實了存在COX-2蛋白表達。應用PAF受體拮抗劑可使COX-2蛋白表達增加、mRNA水平上調提前。當細胞受到各種刺激后，可通過C蛋白偶聯機制、生長因子受體以及一系列信號傳導通路作用于COX-2的5'端轉錄起始點上游區(qū)的轉錄調控序列，促進COX-2的轉錄，誘導COX-2的表達[15-16]。COX-2在急性胃黏膜損傷中表達明顯增強，從而發(fā)揮對胃黏膜的保護作用并促進胃黏膜損傷的愈合。

PAF是迄今發(fā)現的內源性潰瘍形成介質中最強的一種，作為炎性細胞因子放大網絡的中心放大器，可促進對其他炎性細胞因子的作用[17-18]。PAF受體拮抗劑能顯著改變LPS或PAF引起的血流動力學改變及組織器官損傷，抑制花生四烯酸類介質釋放和氧自由基的產生[19-20]。本實驗采用預先或在腹腔注射LPS后給予PAF受體拮抗劑的方式減輕了胃黏膜損傷，證明PAF受體拮抗劑可增加COX-2的表達和PGE2濃度。

綜上，本實驗結果提示，幼年大鼠LPS血癥時胃黏膜COX-2 mRNA表達上調，COX-2表達增強，PGE2含量增加，對胃黏膜起保護作用并促進胃黏膜損傷的愈合。PAF受體拮抗劑可增加COX-2的表達、提高PGE2的濃度，在減輕胃黏膜損傷的同時可能也促進了胃黏膜損傷的愈合。

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Effect of PAF receptor antagonist on PGE2content and COX-2 expression in gastric mucosa of young rats with endotoxemia

LIU Chun-ying1, LI Qi-yu1, WANG Lin1, JIANG Jing1, WANG Li-jie2, SUN Mei2*1Department of Pediatrics, 202 Hospital of PLA, Shenyang 110812, China
2Department of Pediatrics, Second Clinical College of China Medical University, Shenyang 110004, China

ObjectiveTo investigate the protective effect of prostaglandin E2(PGE2), cyclooxygenase-2 (COX-2) and platelet activating factor (PAF) receptor antagonist on endotoxin-induced acute gastric mucosal injury in young rats.MethodsEighteen-day old Wistar rats were randomly divided into 4 groups: normal control group, model group (LPS group), PAF antagonist prevention group, and PAF antagonist treatment group. The model of endotoxemia in young rats was reproduced by intraperitoneal injection of endotoxin (5mg/kg of O55:B5 lipopolysaccharide). The rats in PAF prevention and treatment group

PAF antagonist BN52021 (Ginkgolide B, 5mg/kg) 0.5h before or after modeling. The rats in control group were given intraperitoneal injection of same amount of normal saline (1ml/kg). The animals were sacrificed 1.5, 3, 6, 24, 48 and 72h after intraperitoneal injection of endotoxin (8 in each group). The pathologic changes in gastric mucosa were observed after HE staining. The content of PGE2was measured by radioimmunoassay, the expression of COX-2 protein was determined by immunohistochemistry SP method, and the expression of COX-2 mRNA was assessed with RT-PCR method.ResultsPathological changes in gastric mucosa were found to be edema of epithelial cells at 1.5h, and hyperemia and edema 3h after intraperitoneal injection of endotoxin in LPSgroup. The changes were most marked at 6h, including bleeding, karyorrhexis, pyknosis and apoptosis of epithelial cells of gastric mucosa. Exfoliation of the epithelium and neutrophil infiltration were observed at 24h, thinning of mucosa and a decrease in glands were observed at 48h, but no further changes were observed at 72h. However, all the above changes were significantly alleviated in prevention and treatment groups. The PGE2content of gastric mucosa was lowered at 3h (P<0.05), and it was lowest at 6h (P<0.01) after endotoxin injection in LPS group, and significant difference was found between LPS group and control group. The PGE2content of gastric mucosa was obviously increased at 3h and 6h in prevention group (P<0.05), and at 6h in treatment group (P<0.05). The differences at 6h were significant (P<0.01) among prevention group, treatment group and LPS group. No expression of COX-2 protein or mRNA was seen in gastric mucosal tissue of control group. In contrast with control group, cytoplasm COX-2 protein of gastric mucosal tissue was seen to express at 6h after endotoxin injection in LPS group, and it was obviously enhanced at 24, 48 and 72h (P<0.01), and the COX-2 mRNA level was also elevated. The expressions of COX-2 protein and mRNA were increased obviously at 6h in PAF antagonist prevention group and treatment group (P<0.01). The expressions of COX-2 protein at 6h and COX-2 mRNA at 24h were obviously elevated in prevention group and treatment group compared with those of LPS group (P<0.01). Conclusion PAF receptor antagonist may up-regulate the expression level of COX-2 protein and mRNA, increase PGE2content, alleviate acute gastric mucosal injury, and promote the healing of gastric mucosal injury.

gastric mucosa; lipopolysaccharides; dinoprostone; cyclooxygenase 2; platelet activating factor; PAF receptor antagonist

R573

A

0577-7402(2015)01-0040-06

10.11855/j.issn.0577-7402.2015.01.09

2014-04-28；

2014-11-20)

(責任編輯：張小利)

劉春英，醫(yī)學博士，副主任醫(yī)師。主要從事兒童消化系統疾病的基礎與臨床研究

110812 沈陽 解放軍202醫(yī)院兒科(劉春英、李奇玉、王琳、蔣靜)；110004 沈陽 中國醫(yī)科大學第二臨床學院兒科(王麗杰、孫梅)

]孫梅，E-mail：sunm@cmu2h.com

*Corresponding author, E-mail: sunm@ cmu2h.com