陳艷紅，孫瑩杰，張毅男，王麗晶，張鐵錚，陳克研

七氟烷后處理對失血性休克豬腸黏膜AQP8和I-FABP表達的影響

陳艷紅，孫瑩杰，張毅男，王麗晶，張鐵錚，陳克研

目的觀察七氟烷后處理對失血性休克巴馬小型豬腸黏膜水通道蛋白8(AQP8)和血清腸脂肪酸結(jié)合蛋白(I-FABP)表達的影響，探討七氟烷后處理對失血性休克引起的腸黏膜屏障損傷的保護作用及可能機制。方法18只巴馬小型豬隨機分為對照組(S組)、失血性休克組(HS組)和七氟烷后處理組(Post/Sev組)，每組6只。實驗動物術前禁食8h，給予丙泊酚3mg/kg實施麻醉。S組麻醉后經(jīng)股動脈和頸內(nèi)靜脈置管；HS組麻醉置管后建立失血性休克模型；Post/Sev組于麻醉置管建立失血性休克模型成功后給予2%七氟烷吸入30min。各實驗組均于麻醉前(T0)及失血性休克后30min(T1)、1h(T2)、1.5h(T3)、2h(T4)、3h(T5)、4h(T6)時間點頸內(nèi)靜脈取血，ELISA法檢測血清I-FABP的含量。失血性休克4h后放血處死實驗動物，取腸組織制作病理切片，HE染色觀察各組病理組織學變化；用干濕比法計算腸組織的含水量；ELISA法檢測腸黏膜AQP8的表達水平。結(jié)果與S組相比，HS組和Post/Sev組血清I-FABP含量、腸黏膜AQP8表達水平和腸組織含水量均顯著升高，差異有統(tǒng)計學意義(P<0.05)；與HS組比較，Post/Sev組上述指標明顯降低，差異有統(tǒng)計學意義(P<0.05)。光鏡觀察顯示，S組腸黏膜未見明顯變化；HS組腸黏膜損傷嚴重，可見腸黏膜出血、炎細胞浸潤、上皮細胞壞死；Post/Sev組腸黏膜損傷明顯減輕，僅見黏膜層腺體輕度擴張，上皮層和固有層中度分離，上皮下間隙輕度水腫，少許炎細胞浸潤。結(jié)論七氟烷后處理可減輕失血性休克引起的腸黏膜屏障損傷，其機制可能與降低I-FABP和AQP8表達、減輕腸黏膜水腫有關。

腸黏膜；休克，出血性；七氟烷；脂肪酸結(jié)合蛋白質(zhì)類；水通道蛋白質(zhì)8

失血性休克可以引起“休克腸”的發(fā)生，造成腸道損傷，是失血性休克和創(chuàng)傷后的嚴重并發(fā)癥。近年來有關失血性休克后腸道損傷及保護的研究逐漸受到關注[1-3]。既往研究表明，七氟烷不僅具有優(yōu)良的麻醉作用，而且具有一定的器官保護作用[4]。本研究通過建立失血性休克模型，觀察七氟烷后處理對巴馬小型豬腸黏膜水通道蛋白8(aquaporin 8，AQP8)和血清腸脂肪酸結(jié)合蛋白(intestinal fatty acid binding protein，I-FABP)表達的影響，從而探討七氟烷后處理對失血性休克引起的腸黏膜屏障損傷的保護作用及其可能機制。

1 材料與方法

1.1 動物分組 健康成年巴馬小型豬18只，由沈陽軍區(qū)總醫(yī)院醫(yī)學實驗動物科提供，雌雄不限，體重22～25kg，隨機分3組，每組6只。對照組(S組)：麻醉后股動脈置管、頸內(nèi)靜脈置管，觀察6h；失血性休克組(HS組)：麻醉置管后建立失血性休克模型，觀察4h；七氟烷后處理組(Post/Sev組)：麻醉置管后建立失血性休克模型，建模成功后吸入2%七氟烷30min，觀察4h。

1.2 失血性休克模型的建立 實驗動物術前禁食8h，禁水4h，于耳緣靜脈給予丙泊酚3mg/kg實施麻醉。麻醉成功后，將巴馬小型豬固定于手術臺上，進行氣管插管(ID 7.5mm)行機械通氣，給予2%七氟烷維持麻醉。手術部位備皮，碘伏消毒。右側(cè)股動脈穿刺插管，用于動脈采血和放血建立失血性休克模型；左側(cè)股動脈穿刺，用于監(jiān)測動脈血壓和平均動脈壓；右頸內(nèi)靜脈置入三腔中心靜脈導管，用于靜脈采血；右頸內(nèi)中心靜脈導管下方置入Swan-Ganz導管，用于測量心輸出量和溫度。置管等操作結(jié)束后，將導管縫合固定于皮膚上，停止吸入七氟烷，待豬自主呼吸恢復后，用氣體監(jiān)測儀監(jiān)測七氟烷濃度，至MAC為0時，在常溫(20～22℃)環(huán)境下，15min內(nèi)將40%的血容量(按30ml/kg計算)勻速放出，建立容量控制性失血性休克模型[5]。

1.3 標本采集及檢測 分別于T0(實驗前)、T1(失血性休克后30min)、T2(失血性休克后1h)、T3(失血性休克1.5h)、T4(失血性休克后2h)、T5(失血性休克后3h)、T6(失血性休克后4h)7個時間點經(jīng)頸內(nèi)靜脈采血3ml，立即在4℃下3000×g離心10min，分離出血清移入1.5ml離心管中，置于－80℃深低溫冰箱保存待測。采用ELISA法檢測血清腸I-FABP的含量，所用試劑盒由美國R&D公司提供。

失血性休克后4h放血處死各組動物，立即取空腸、回腸和結(jié)腸組織3cm置入組織凍存管中，置于－80℃冰箱保存，采用ELISA法檢測腸黏膜AQP8表達的變化，試劑盒由美國R&D公司提供。另取空腸組織3cm，置于4%甲醛溶液中，用于制作病理組織切片，HE染色觀察各組病理組織學變化。取空腸、回腸和結(jié)腸各5cm，去除腸道內(nèi)容物和腸系膜，冷鹽水沖洗干凈，用電子分析天平稱量濕重后，置于80℃恒溫干燥箱內(nèi)烤干24h至恒重(兩次稱重差別≤0.2mg)再稱干重，計算腸道組織含水量。腸道組織含水量(%)=(濕重－干重)/濕重×100%。

1.4 統(tǒng)計學處理 采用SPSS 19.0軟件進行統(tǒng)計分析，數(shù)據(jù)結(jié)果以表示，組間比較采用單因素方差分析，組內(nèi)比較采用重復測量設計的方差分析，進一步兩兩比較采用SNK-q檢驗，P<0.05為差異有統(tǒng)計學意義。

2 結(jié) 果

2.1 血清I-FABP含量的變化 與T0時點比較，各組血清I-FABP含量在T1時點開始升高，HS組和Pre/ Sev組T2-T6時點均顯著升高(P<0.05)，HS組在休克后1.5h(T3)達到峰值，然后呈下降趨勢，Post/Sev組在休克后2h(T4)達到峰值，然后呈下降趨勢。在T0時點3組血清I-FABP含量差異無統(tǒng)計學意義(P>0.05)，而在T1-T6時點HS組和Pre/Sev組血清中I-FABP含量均明顯高于S組，差異有統(tǒng)計學意義(P<0.05)，且HS組血清中I-FABP含量明顯高于Pre/ Sev組S組，差異有統(tǒng)計學意義(P<0.05，表1)。

2.2 各組腸黏膜AQP8表達水平比較 與S組比較，HS組和Post/Sev組失血性休克4h腸黏膜AQP8表達水平明顯升高，差異有統(tǒng)計學意義(P<0.05)，且Post/Sev組腸黏膜AQP8表達水平明顯低于HS組，差異有統(tǒng)計學意義(P<0.05，表2)。

2.3 各組腸組織含水量比較 與S組比較，HS組和Post/Sev組失血性休克4h空腸、回腸和結(jié)腸組織的含水量均明顯升高，差異有統(tǒng)計學意義(P<0.05)，且Post/Sev組明顯低于HS組，差異有統(tǒng)計學意義(P<0.05，表3)。

表1 各組動物血清I-FABP含量比較(ng/L，±s，n=6)Tab. 1 Comparison of the serum I-FABP contention in each group of pigs (ng/L,±s,n=6)

表1 各組動物血清I-FABP含量比較(ng/L，±s，n=6)Tab. 1 Comparison of the serum I-FABP contention in each group of pigs (ng/L,±s,n=6)

(1)P<0.05 compared with T0; (2)P<0.05 compared with S group; (3)P<0.05 compared with HS group

Time point S group HS group Post/Sev group T0 402.5±42.5 404.7±51.1 408.5±55.7 T1 403.0±39.3 551.1±54.8(1)(2) 478.7±51.4(1)(2)(3)T2 401.0±40.5 655.6±54.7(1)(2) 572.9±49.3(1)(2)(3)T3 407.8±50.5 868.2±87.1(1)(2) 642.0±48.6(1)(2)(3)T4 408.9±47.9 759.4±82.7(1)(2) 704.8±58.8(1)(2)(3)T5 401.3±44.6 705.1±55.2(1)(2) 606.1±66.1(1)(2)(3)T6 397.8±43.2 666.1±46.9(1)(2) 587.4±62.2(1)(2)(3)

表2 各組休克后4h時腸黏膜AQP8表達水平比較(ng/ mg，±s，n=6)Tab. 2 Comparison of AQP8 expression levels in intestinal mucosa 4h after hemorrhagic shock in each group of pigs (ng/mg,±s,n=6)

表2 各組休克后4h時腸黏膜AQP8表達水平比較(ng/ mg，±s，n=6)Tab. 2 Comparison of AQP8 expression levels in intestinal mucosa 4h after hemorrhagic shock in each group of pigs (ng/mg,±s,n=6)

(1)P<0.05 compared with S group; (2)P<0.05 compared with HS group

Location S group HS group Post/Sev group Jejunum 6.74±1.38 23.53±2.57(1) 9.36±2.05(1)(2)Ileum 4.08±1.02 12.88±1.88(1) 7.27±1.18(1)(2)Colon 5.68±1.23 18.37±2.30(1) 9.47±1.3(1)(2)

表3 各組休克后4h腸組織含水量比較(%，±s，n=6)Tab. 3 Comparison of the water content after hemorrhagic shock in each group of pigs (%,±s,n=6)

表3 各組休克后4h腸組織含水量比較(%，±s，n=6)Tab. 3 Comparison of the water content after hemorrhagic shock in each group of pigs (%,±s,n=6)

(1)P<0.05 compared with S group; (2)P<0.05 compared with HS group

Location S group HS group Post/Sev group Jejunum 75.4±5.3 86.0±8.9(1) 80.8±7.7(1)(2)Ileum 76.2±9.0 87.7±8.5(1) 79.3±8.5(1)(2)Colon 70.1±10.7 84.0±8.1(1) 75.5±8.2(1)(2)

2.4 巴馬小型豬腸黏膜形態(tài)學觀察 HE染色光鏡下觀察顯示，S組腸黏膜刷狀緣正常，無水腫充血，僅見少量炎細胞浸潤；HS組腸黏膜損傷嚴重，可見出血，黏膜層炎細胞浸潤，上皮細胞壞死；Post/Sev組腸黏膜損傷較HS組輕，僅見黏膜層腺體輕度擴張，上皮層和固有層中度分離，上皮下間隙輕度水腫，少許的炎細胞浸潤(圖1)。

3 討 論

近年研究發(fā)現(xiàn)，腸黏膜損傷后的腸道水腫是影響患者轉(zhuǎn)歸和住院時長的重要因素[6]。失血性休克后發(fā)生的腸道水腫不但與休克時微血管和腸黏膜通透性增加、自由基形成、中性粒細胞激活浸潤和細胞因子釋放有關，還可能與腸道內(nèi)的水轉(zhuǎn)運和含水量密切相關。AQP是一類結(jié)構(gòu)和功能類似的非選擇性的膜蛋白家族，AQP8在十二指腸、空腸、結(jié)腸、肝臟、胰腺中都有表達。研究發(fā)現(xiàn)，AQP在肺水腫和腦水腫的形成、腫瘤轉(zhuǎn)歸、尿崩癥的發(fā)展等方面發(fā)揮著重要作用[7-9]。Sakai等[10]對5-Fu引起的小鼠腹瀉進行研究發(fā)現(xiàn)，腹瀉小鼠的AQP8表達水平明顯下降。Nakano等[11]在研究腸道水代謝的過程中發(fā)現(xiàn)，小腸部分切除小鼠的AQP8表達水平增加。Zhao等[12]研究發(fā)現(xiàn)，在2,4,6-三硝基苯磺酸(TNBS)誘導的結(jié)腸炎大鼠模型中，AQP8和AQP3表達下調(diào)。以上研究均提示AQP8對于腸道水液調(diào)節(jié)具有重要作用。本研究結(jié)果顯示，休克后4h，HS組巴馬小型豬空腸、回腸、結(jié)腸AQP8的表達量增加，其腸組織含水量和對照組相比也呈增加趨勢，提示AQP8參與了失血性休克后腸組織水腫的形成。

圖1 3組腸黏膜病理改變(HE ×10)Fig. 1 Histopathological changes of intestinal mucosa in three groups of pigs (HE ×10) A. S group; B. HS group; C. Post/Sev group

I-FABP是一組存在于小腸的纖毛細胞內(nèi)、相對分子量為15kD的胞液蛋白。正常情況下外周血中檢測不到I-FABP，只有在腸黏膜發(fā)生損害時，I-FABP才會進入循環(huán)系統(tǒng)中，這一特點使I-FABP成為診斷腸道缺血、腸道屏障損傷的早期敏感指標[13]。近年來針對I-FABP和腸損傷的關系開展了大量研究。Niewold等[14]在大鼠腸道缺血再灌注損傷模型中發(fā)現(xiàn)，I-FABP在腸缺血損傷的早期即開始升高。Matsumoto等[15]在208例急性腸缺血患者中發(fā)現(xiàn)，對于腸缺血，I-FABP可能是最好的診斷指標。本研究結(jié)果顯示，HS組巴馬小型豬血清I-FABP含量明顯高于對照組，結(jié)合腸組織形態(tài)學觀察，提示I-FABP增高與失血性休克后腸道損傷有關。

七氟烷是臨床上常用的吸入麻醉藥，其對心、腦、肝、腎等器官的保護作用已經(jīng)得到基礎和臨床研究的證實[16]。失血性休克是一個不可預期的意外傷害和病理生理學過程，本研究觀察了七氟烷后處理對失血性休克巴馬小型豬腸屏障功能的影響，結(jié)果顯示，Post/Sev組巴馬小型豬血清I-FABP含量、腸黏膜AQP-8表達水平和腸組織含水量明顯低于HS組，可能與七氟烷抑制了腸黏膜細胞AQP8的表達，繼而對進入線粒體的水分起到調(diào)節(jié)作用，最終使線粒體基質(zhì)達到正常狀態(tài)，電子傳遞鏈激活，線粒體ATP合成增加，從而使腸道水腫和腸道損傷減輕有關。還有實驗發(fā)現(xiàn)七氟烷后處理可明顯增加循環(huán)血流量，升高平均動脈壓，穩(wěn)定血流動力學，減少組織灌注不足的發(fā)生率，降低乳酸釋放，由此推測其可以減輕失血性休克時乳酸堆積引發(fā)的滲透梯度改變，從而下調(diào)AQP8表達，減輕組織細胞水腫[17]。

綜上所述，七氟烷后處理可降低失血性休克巴馬小型豬血清I-FABP濃度，減輕休克導致的腸黏膜屏障損傷，其機制可能與七氟烷后處理降低腸黏膜AQP8表達水平、減輕腸組織水腫有關。

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Effects of post-conditioning with sevoflurane on the expressions of intestinal AQP8 and I-FABP in pigs with hemorrhagic shock

CHEN Yan-hong1,2, SUN Ying-jie1, ZHANG Yi-nan1*, WANG Li-jing1, ZHANG Tie-zheng1, CHEN Ke-yan11Department of Anesthesiology, General Hospital of Shenyang Command, Shenyang 110016, China
2Department of Anesthesiology, People's Hospital of Kangping, Kangping, Liaoning 110500, China
*< class="emphasis_italic">Corresponding author, E-mail: zynan13@126.com

, E-mail: zynan13@126.com
This work was supported by the“Twelfth Five-Year Plan” Medical Science Development Foundation of PLA (BWS12J008)

ObjectiveTo observe the effects of sevoflurane post-conditioning on the expression of Aquaporin 8 (AQP8) and intestinal fatty acid binding protein (I-FABP), in order to investigate the protective role of sevoflurane post-conditioning on intestinal injury and its underlying mechanism.MethodsEighteen bama miniature pigs were randomly divided into three groups (6 each) using a random number table: control group (S group), hemorrhagic shock group (HS group), and sevoflurane post-coditioning group (Post/ Sev group). Experimental animals were fasted for 8 hours before surgery, and propofol 3mg/kg was givenviathe ear vein. Endotracheal intubation was done when the animal fell asleep. Bloodletting from the femoral artery after anesthesia was done to reproduce hemorrhagic shock. In Post/Sev group, 2% sevoflurane was given by inhalation for 30min (post-conditioning) after successful reproduction of the model. Blood samples were collected prior to anesthesia (T0) and 30min (T1), 1h (T2), 1.5h (T3), 2h (T4), 3h (T5), 4h (T6) after hemorrhagic shock. The quantity of blood I-FABP and intestinal AQP8 levels were determined with ELISA. Water content in the intestinal tissue was determined by wet and dry weight method. Histopathological changes in the intestinal tissue were observed with HE staining.ResultsCompared with the control group, the serum I-FABP content, the expressions of intestinal AQP8, and water content inthe intestinal tissue were significantly increased in HS group and Post/Sev (P<0.05) group. Compared with HS group, the above indices in Post/Sev group were significantly lower (P<0.05). These results were confirmed by pathological examination.ConclusionPostconditioning with sevoflurane could improve, to some extent, pig's intestinal barrier function in hemorrhagic shock, and this effect is likely related with lowering of intestinal AQP8 and I-FABP expression and mucosal edema.

intestinal mucosa; shock, hemorrhagic; sevoflurane; fatty acid-binding proteins; aquaporin 8

R605.971

A

0577-7402(2015)11-0911-04

10.11855/j.issn.0577-7402.2015.11.11

2015-04-24；

2015-09-18)

(責任編輯：胡全兵)

全軍“十二五”醫(yī)學科研重點項目(BWS12J008)

陳艷紅，副主任醫(yī)師，碩士研究生。主要從事圍術期重要器官保護方面的研究

110016 沈陽 沈陽軍區(qū)總醫(yī)院麻醉科 (陳艷紅、孫瑩杰、張毅男、王麗晶、張鐵錚、陳克研)；110500 遼寧康平 康平縣人民醫(yī)院麻醉科(陳艷紅)

張毅男，E-mail：zynan13@126.com