低分子肝素對大鼠急性壞死性胰腺炎并發肺損傷時TLR4及VEGF表達的影響

張啟杰 唐丙喜 李新立

·論著·

低分子肝素對大鼠急性壞死性胰腺炎并發肺損傷時TLR4及VEGF表達的影響

張啟杰 唐丙喜 李新立

目的觀察低分子肝素對急性壞死性胰腺炎(ANP)大鼠并發的肺損傷的影響，探討其作用機制。方法90只Wistar大鼠隨機分為對照組、ANP組、低分子肝素干預(LH)組。采用逆行胰膽管注射4%牛磺膽酸鈉的方法制備ANP模型，LH組于造模后皮下注射低分子肝素10 U/100 g體質量。術后6、12、24 h分批處死大鼠，取胰腺及肺組織常規行病理學檢查并評分，采用免疫組化法檢測肺組織Toll樣受體4(TLR4)、VEGF表達，采用ELASA法測定血及肺組織IL-6、IL-10、TNF-α水平。結果對照組大鼠肺臟及胰腺組織結構正常。ANP組見胰腺片狀出血及壞死，大量炎癥細胞浸潤；肺臟見肺泡壁破裂，間質充血、水腫、大量中性粒細胞浸潤。LH組胰腺及肺組織病理改變均較ANP組顯著減輕。ANP 12 h組胰腺及肺組織病理評分分別為(6.34±1.09)、(7.01±1.16)分，LH組分別為(5.48±0.86)、(6.24±0.86)分，LH組均較ANP組顯著下降(P值均<0.05)，且肺臟與胰腺組織損傷呈正相關(r=0.812，P<0.01)。ANP 12 h組肺組織的TLR4、VEGF、IL-6、TNF-α、IL-10表達量分別為0.68±0.10、0.50±0.11和(2617.2±485.3)、(1603.1±519.7)、(608.3±137.5)pg/g，LH組分別為0.61±0.09、0.41±0.06和(2398.5±503.7)、(1302.4±389.8)、(753.2±100.0)pg/g，LH組的TLR4、VEGF、IL-6、TNF-α表達量均較ANP組顯著下調，而IL-10表達量顯著上調，差異均有統計學意義(P值均<0.05)。ANP 12 h組大鼠血清IL-6、TNF-α、IL-10水平分別為(184.3±45.7)、(289.7±60.4)、(143.2±30.4)μg/L，LH組分別為(143.8±31.8)、(256.4±40.7)、(189.3±50.9)μg/L，LH組血IL-6、TNF-α水平均較ANP組顯著下降，而IL-10表達量顯著升高，差異均有統計學意義(P值均<0.05)。肺組織TLR4、VEGF表達量與肺組織損傷程度呈正相關(r值分別為0.524、0.503，P值均<0.05)。結論低分子肝素可改善ANP并發的肺損傷，其機制可能與抑制TLR4、VEGF、IL-6、TNF-α表達，上調IL-10表達有關。

胰腺炎，急性壞死性； 肝素，低分子量； 肺損傷； Toll樣受體4； 血管內皮生長因子

重癥急性胰腺炎(SAP)病情發展迅速、預后兇險，是臨床常見危重急癥之一，病死率高達20%。急性肺損傷是SAP最常見的并發癥，相當一部分患者可進展為急性呼吸窘迫綜合征(acute respiratory distress syndrome，ARDS)，成為SAP患者早期病死的重要原因[1]。目前關于低分子肝素的抗凝作用已比較明確，但其對炎癥反應的影響尚不十分清楚。有報道低分子肝素對SAP患者的臨床療效明顯[2-3]。本研究觀察低分子肝素對急性壞死性胰腺炎(ANP)大鼠并發的肺損傷的保護作用，探討其機制。

材料與方法

一、實驗動物與分組

清潔級Wistar大鼠90只，雌雄各半，體質量220～270 g，由山東大學實驗動物中心提供。按數字表法隨機分為假手術組、ANP組、低分子肝素治療組(LH組)，每組30只。采用膽胰管逆行注入4%牛磺膽酸鈉(Sigma公司)0.1 ml/100 g體質量的方法制備ANP模型。LH組于造模后皮下注射低分子肝素(葛蘭素史克有限公司)10 U/100 g體質量。對照組開腹輕翻胰腺組織后關腹。ANP組和對照組術后皮下注射等容積生理鹽水。術后6、12、24 h分批處死大鼠，取血分離血清，取脾門前胰尾部組織和左肺上葉置10%甲醛固定。

二、胰腺及肺組織病理學檢查

取固定的胰腺及肺組織，常規石蠟包埋、切片、HE染色。由病理科醫師盲法讀片，并分別采用改良的Schimidt法[4]及Osma標準[5]對胰腺及肺組織進行病理評分。

三、胰腺及肺組織Toll樣受體4(TLR4)、VEGF蛋白表達檢測

采用免疫組化法檢測胰腺及肺組織TLR4、VEGF蛋白表達，抗TLR4、VEGF一抗購自北京博奧森生物技術有限公司，按照說明書操作，最后用zmage-proplus 5.0軟件測量灰度值。

四、血清及肺組織IL-6、IL-10、TNF-α水平檢測

血清IL-6、IL-10、TNF-α水平采用ELISA方法檢測，檢測試劑盒均購自武漢博士德生物工程有限公司，按說明書操作。新鮮肺組織制備勻漿后同樣用ELISA方法檢測。

五、統計學處理

結 果

一、各組大鼠胰腺及肺組織病理學改變

對照組胰腺及肺組織結構正常，無明顯病理變化。ANP組光鏡下可見胰腺間質和胰腺小葉炎癥細胞浸潤，片狀及彌漫性出血、壞死；肺臟可見肺泡壁破裂，間質充血、水腫、增寬，肺泡和肺間質中有大量的中性粒細胞浸潤。LH組光鏡下見胰腺細胞變性，腺小葉結構破壞程度較ANP組減輕，炎癥細胞浸潤減少；肺組織輕度水腫，間質充血、水腫較ANP組明顯減輕，肺泡和肺間質中性粒細胞浸潤減少(圖1)。ANP組胰腺及肺組織的病理評分均隨時間的延長而增加，LH組的評分均較同時間點ANP組降低，差異有統計學意義(P值均<0.05，表1)。胰腺組織與肺組織的病理學評分呈正相關(r=0.812,P<0.01)。

二、各組大鼠肺組織TLR4、VEGF表達變化

對照組肺組織TLR4、VEGF低表達；ANP組大鼠肺組織TLR4、VEGF高表達，均在12 h達峰值；LH組的表達水平較同時間點ANP組均顯著降低，但仍顯著高于對照組(P值均<0.05，圖2、表2)。肺組織TLR4與VEGF表達水平呈正相關(r=0.586,P<0.05)；TLR4、VEGF表達與肺組織病理評分均呈正相關(r值分別為0.524、 0.503,P值均<0.05)。

三、各組大鼠血清及肺組織IL-6、IL-10、TNF-α水平

對照組各時間點的血清及肺組織IL-6、IL-10、TNF-α水平均無顯著變化。ANP組及LH組大鼠血清及肺組織IL-6、TNF-α水平隨時間延長逐漸升高；而IL-10水平隨時間延長逐漸降低。ANP組、LH組大鼠血清及肺組織IL-6、IL-10、TNF-α表達水平均顯著高于對照組(P值均<0.01)；LH組的血清及肺組織IL-6、TNF-α水平較ANP組顯著下降，而IL-10水平較ANP組顯著升高(P值均<0.05,表3)。

圖1 ANP組、LH組12 h點的胰腺(1A、1B)及肺組織(1C、1D)病理改變(HE ×400)

表1 對照組、ANP組、LH組大鼠胰腺及肺組織的病理學評分(分

注：與對照組比較，aP<0.01；與ANP組比較，bP<0.05

圖2 ANP組、LH組12 h點肺組織TLR4(2A、2B)及VEGF(2C、2D)表達(免疫組化 ×400)

表2 對照組、ANP組、LH組大鼠肺組織TLR4、VEGF表達

注：與對照組比較，aP<0.01；與ANP組比較，bP<0.05

表3 對照組、ANP組、LH組大鼠血清及肺組織IL-6、IL-10、TNF-α水平變化

注：與對照組比較，aP<0.01；與ANP組比較，bP<0.05

討 論

SAP的發病機制是一個多因素多環節的過程，涉及一系列事件，且常伴有胰外臟器損傷，尤以急性肺損傷最為常見，有人稱之為AP相關性肺損傷(acute pancreatitis associated lung injury, APALI)[6]，它是SAP早期最常見、最嚴重的并發癥。

1997年Medzhitov和Poltorak相繼發現人類Toll樣蛋白和Toll樣受體(Toll-like receptors，TLRs)，后被證實為哺乳動物胞外抗原信息傳遞到胞內的關鍵跨膜蛋白[7-9]，其中TLR4最早被發現，也是被研究最多[10]。實驗證明TLR4 -/-老鼠可明顯減輕胰腺炎的嚴重程度并可減輕肺臟損傷[11]。Li等[12]研究發現，TLR4主要在胰腺微動脈、微靜脈、腺泡毛細血管網、竇狀毛細血管網表達，腸道中主要在微血管內皮、黏膜固有層內白細胞中表達，因此推測TLR4與SAP時胰腺微循環障礙、腸道菌群易位、機體二次感染有關。它可高效誘導細胞因子、趨化因子、黏附因子、生長因子、急性反應蛋白等表達[13]。

VEGF是一種通過二硫鍵相連的分泌型同源二聚體糖蛋白，是目前最強的促血管生長因子。現研究發現其可增加毛細血管通透性，導致組織水腫，微循環障礙，并參與全身炎癥反應。它作為一種炎癥調節因子，對細胞因子、黏附因子、趨化因子均具有重要作用，在動脈粥樣硬化、類風濕性關節炎、急性肺損傷等疾病發生中也具有重要作用[14-19],在水腫型胰腺炎向壞死型胰腺炎發展變化中同樣具有重要作用[20]。Li等[21]認為，VEGF與細胞因子相互作用可引發全身炎癥反應綜合征，損傷胰腺及胰外臟器功能，抑制其表達可改善胰腺炎的嚴重程度。

IL-6、TNF-α、IL-10在SAP的發生、發展中具有重要作用[22]。Inagaki等[23]認為，當IL-6>122 μg/L時預測SAP及器官衰竭的敏感度和特異度分別為81.8%和77.7%。在SAP早期即可檢測到胰腺組織TNF-α及其mRNA升高，隨后在肺、肝中TNF-α大量產生，最終導致胰腺及胰腺外組織的損傷[24]。IL-10作為一種重要的抗炎細胞因子，可抑制Th1細胞的增殖，減少巨噬細胞MHC-2分子的表達，是維持細胞因子網絡平衡的重要負調節機制[25]。

本研究結果表明，ANP大鼠的胰腺與肺臟組織損傷呈正相關。ANP大鼠肺組織TLR4表達水平在6 h時即顯著升高，12 h達峰值，之后開始下降，與Xiping等[26]報道的TLR4在肝臟、腎臟、小腸的表達規律一致。此外，ANP大鼠肺組織VEGF水平也明顯升高，24 h表達水平略有下降，但無統計學意義，推測可能與肺組織嚴重損傷，肺泡壁破裂有關。低分子肝素干預后胰腺及肺組織的損傷減輕，肺組織TLR4及VEGF的表達降低，血清及肺組織IL-6、TNF-α水平下降，IL-10水平升高，提示低分子肝素改善SAP并發的肺損傷作用可能與其抑制肺組織TLR4、VEGF、IL-6、TNF-α的表達，上調IL-10的表達有關。

[1] Moore FA，Moore EE.Eolvintg concepts in the pathogenesis of post injury multiple organ failure[J]. Surg Clin North Am, 1995,75(2):257-277.

[2] 袁楚明，陳世勇，李奕璉，等.小劑量低分子肝素對急性胰腺炎的影響[J]. 中華胰腺病雜志, 2009,9(4):253-255.

[3] 李保存，陳元清.低分子肝素治療重癥急性胰腺炎20例臨床分析[J]. 臨床內科雜志, 2011,28(1):68-69.

[4] Schimidt J, Lewandrowsi K，Warshaw AL，et al.Morphometric characteristics and homogeneity of a new model of acute pancreatitis in the rat[J].Int J Pancreatol，1992,12(1)：41-51.

[5] Osman MO，Kristensen JU，Jacobsen NO, et al. A monoclonal anti-inteleukin 8 antibody (WS-4) inhibits cytokine response and acute necrotising pancreatic in rabbits[J].Gut，1998,43(2)：232-239.

[6] Hartwig W,Werner J,Jimenez RE,et al.Trypsin and activation of circulating trypsinogen contribute to pancreatitis-associated lung injury[J]. Am J Physiol, 1999, 277(5Pt1):G1008-G1016.

[7] Gordon S. Pattern recognition receptors: doubling up for the innate immune response[J]. Cell, 2002,111(7): 927-930.

[8] Randhawa AK, Hawn TR. Toll-like receptors: their roles in bacterial recognition and respiratory infections[J]. Expert Rev Anti Infect Ther, 2008, 6(4):479-495.

[9] Jaekal J, Abraham E, Azam T, et al. Individual LPS responsiveness depends on the variation of toll-like receptor (TLR)expression level[J]. J Microbiol Biotechnol, 2007,17(11):1862-1867.

[10] Zhang X, Liu D, Wu D, et al. Effect of salvia miltio-rrhizae on the expressions of TLR4 protein in the liver of rats with SAP or OJ[J]. Inflammation, 2009, 32(3):151-162.

[11] Sharif R, Dawra R, Wasiluk K, et al. Impact of toll-like receptor 4 on the severity of acute pancreatitis and pancreatitis-associated lung injury in mice[J]. Gut, 2009, 58(6):813-819.

[12] Li Y, Zhou ZG, Zhang J, et al.Chen YD，et al.Microcirculatory detection of Toll-like receptor 4 in rat pancreas and intestine[J]. Clin Hemorheol Microcirc, 2006, 34(1-2):213-219.

[13] Liu RL, Liu ML, Ma LL, et al. Effect of ulinastatin on nuclear factor-κB expression in acute necrotic pancreatitis in rats[J]. Shijie Huaren Xiaohua Zazhi, 2005,13:2700-2703.

[14] Lucerna M, Zernecke A,de Nooijer R, et al.Vascular endothelial growth factor-A induces plaque expansion in ApoE knock-out mice by promoting de novo leukocyte recruitment[J]. Blood, 2007, 109(1):122-129.

[15] Ohtani K, Egashira K, Hiasa K, et al. Blockade of vascular endothelial growth factor suppresses experimental restenosis after intraluminal injury by inhibiting recruitment of monocyte lincage cells[J]. Crrculation, 2004, 110(16):2444-2452.

[16] Kuryliszyn-Moskal A, Klimiuk PA, Sicrakowski S, et al. A study on vascular endothelial growth factor and endothelim-1 in patients with extra-articular involvement of rheumatoid arthritiss[J]. Clin Rheumatol, 2006, 25(3):314-319.

[17] Nagashima M, Asano G, Yoshino S. Imbalance in production between vascular endothelial growth factor and endostatin in patients with rhcumatoid arthritis[J]. J Rheumatol, 2000, 27(10):2339-2342.

[18] Nolan A, Weiden MD, Thurston G, et al. Vascular endorhclial growth factor blockade reduces plasma cytokines in a murine model of polymicrobial sepsis[J]. Inflammation, 2004, 28(5):171-178.

[19] van der Flier M, van Leeuween HJ, van Kessel KP, et al. Plasma vascular endothelial growth factor in severe scpsis[J]. Shock, 2005, 23(1):35-38.

[20] 戴璟瑜,鄭通標,許燕平，等. 水腫型胰腺炎向壞死型胰腺炎進展中VEGF的表達及意義[J]，中國現代普通外科進展，2013,16(1):11-14.

[21] Li S, Chen X, Wu T, et al. Role of heparin on serum VEGF levels and local VEGF contents in reducing the severity of experimental severe acute pancreatitis in rats[J]. Scand J Gastroenterol, 2012, 47(2): 237-244.

[22] 唐丙喜, 鄧芝云, 孔祥才,等.大黃甘草湯對急性壞死性胰腺炎大鼠并發的肺損傷的影響[J]，中華胰腺病雜志，2010,10(3)：180-183.

[23] Inagaki T, Hoshino M, Hayakawa T, et al. Interleukin-6 is a useful marker for early prediction of the severity actu pancreatitis[J]. Pancreas, 1997,14(1):1-8.

[24] Sathyanarayan G, Garg PK, Prasad H,et al. Elevated level of interleukin-6 predicts organ failure and severe disease in patients with acute pancreatitis[J]. J Gastroenterol hepatol, 2007, 22(4):550-554.

[25] Pooran N, Indaram A, Singh P, et al. Cytokines (IL-6 , IL-8 , TNF): early and reliable predictors of severe acute pancreatitis[J]. J Clin Gastroenterol, 2003, 37:263-266.

[26] Xiping Z, Jie Z, Qin X,et al.Influence of baicalin and octreotide on NF-κB and p-selectin expression in liver and kidney of rats with severe acute pancreatitis[J]. Inflammation, 2009, 32(1):1-11.

(本文編輯：呂芳萍)

Effect of low molecular weight heparin on the expressions of TLR4 and VEGF expression in severe acute pancreatitis combined with lung injury

ZhangQijie,TangBingxi,LiXinli.

DepartmentofGastroenterology,ZiboCentralHospital,Zibo255036,China

ZhangQijie,Email:qijiezhang@medmail.com.cn

Objective To investigate the effect of low molecular weight heparin on lung Injury complicated by severe acute pancreatitis and explore its mechanism. Methods Ninety Wistar rats were randomly divided into 3 groups, namely sham operation (SO) group, acute necrotizing pancreatitis (ANP) group, low molecular weight heparin treatment (LH) group. 4% sodium taurocholate was injected into the pancreatic duct to induce ANP model. Subcutaneous low molecular weight heparin (10 U/100 g body weight) was injected in the LH group, the equivalent amount of normal saline was injected in the SO and ANP group. After 6, 12, 24 h, rats were sacrificed respectively, pancreas and lung tissues were harvested to observe the pathological changes and the pathological changes were scored; and the changes of TLR4 and VEGF protein expression in lung tissue was determined by immunohistochemical method. Serum and lung levels of IL-6, IL-10, TNF-α were determined by ELASA method. Results The pancreas and lungs tissues were normal in SO group, diffuse hemorrhage, necrosis and a large number of inflammatory cells infiltration was observed in pancreas tissue in ANP group. Lung alveolar wall rupture, interstitial hyperemia, edema, a large number of infiltrating neutrophils could be seen in lung tissue in ANP group. The pancreas and lungs tissues injuries were significantly alleviated. The pancreas and lungs pathological scores of ANP group at 12 h were 6.34±1.09, 7.01±1.16, and those were 5.48±0.86, 6.24±0.86 in LH group, the values in LH group were significantly lower than those in ANP group (P<0.05), and there was a positive association between lung and pancreas scores (r=0.812,P<0.01). The expressions of TLR4, VEGF, IL-6, TNF-α, IL-10 in lung tissue of ANP group at 12 h were 0.68±0.10, 0.50±0.11 and (2617.2±485.3), (1603.1±519.7), (608.3±137.5)pg/g, which were 0.61±0.09,0.41±0.06 and (2398.5±503.7), (1302.4±389.8), (753.2±100.0)pg/g in LH group, and the expressions of TLR4, VEGF, IL-6, TNF-α in LH group were significantly lower than those in ANP group, but the expression of IL-10 was significantly up-regulated, and the difference between the two groups was statistically significant (P<0.05). The serum levels of IL-6, TNF-α, IL-10 in ANP group at 12 h were (184.3±45.7), (289.7±60.4), (143.2±30.4)μg/L, which were (143.8±31.8), (256.4±40.7), (189.3±50.9)μg/L in LH group, and the levels of IL-6, TNF-α in LH group were significantly lower than those in ANP group, but the expression of IL 10 was significantly increased, and the difference was statistically significant (P<0.05). The expression of TLR4, VEGF in lung tissue was positively associated with the degree of lung injuries (r=0.524, 0.503,P<0.05). Conclusions Low molecular weight heparin may improve lung injury complicated by ANP. The mechanism may involve inhibiting the expression of TLR4 and VEGF protein, IL-6, TNF-α, and up-regulation of the expression of IL-10.

Pancreatitis, acute necrogizing; Heparin, low-molecular-weight; Lung injury； Toll-like receptor 4; Vascular endothelial growth factor

10.3760/cma.j.issn.1674-1935.2015.04.010

255036 淄博，淄博市中心醫院消化科(張啟杰、唐丙喜)，檢驗科(李新立)

張啟杰，Email: qijiezhang@medmail.com.cn

2014-04-23)