曲克蘆丁對大鼠肝臟缺血再灌注損傷的保護作用(1)

1、曲克蘆丁對大鼠肝臟缺血再灌注損傷的保護作用(1) 作者：齊貴勝， 裴素霞， 任家勝【關鍵詞】 曲克蘆?。?肝臟；,缺血再灌注摘要：目的探討曲克蘆丁對大鼠肝臟缺血再灌注損傷的影響及其機理。方法將Wistar大鼠40只隨機分為假手術對照組、缺血再灌注組(I/R 組)和I/R 加曲克蘆丁預處理組。I/R 加曲克蘆丁預處理組動物術前腹腔內(nèi)注射曲克蘆丁20 mg/kg, 1次/d, 連續(xù)3 d。 然后制作大鼠肝缺血再灌注損傷模型，檢測大鼠血清中谷丙轉(zhuǎn)氨酶(ALT)和谷草轉(zhuǎn)氨酶(AST)的含量及肝組織中脂質(zhì)過氧化反應產(chǎn)物丙二醛(MDA)、超氧化物歧化酶(SOD)的含量, 并通過光鏡觀察肝組織結(jié)構(gòu)的形態(tài)學改

2、變。結(jié)果用曲克蘆丁處理組大鼠血清中ALT 和AST及肝組織中MDA含量明顯低于缺血再灌注組(P 0.01) , 而肝組織中SOD含量則高于缺血再灌注組(P 0.01) , 與假手術組差異較小(P 0.05), 肝細胞形態(tài)學異常改變較I/R 組也明顯減輕。結(jié)論曲克蘆丁對肝臟I/R損傷具有保護作用，其機理與抑制氧自由基的生成,增強氧自由基的清除, 減輕肝細胞脂質(zhì)過氧化作用有關。關鍵詞：曲克蘆??； 肝臟； 缺血再灌注Preventive Effect of Troxerutin Injection on the Damage of IschemiaReperfusion in Rat LiverAb

3、stract：ObjectiveTo study the preventive effect on damage of ischemia-reperfusion in rat liver by applying Troxerutin Injection.Methods30 rats were put randomly into sham operated control(SOC)group ,ischemia reperfusion ( I/ R) group and ischemia reperfusion + Troxerutin ( I/ R + Troxerutin)group.The

4、 animal model of hepatic ischemia-reperfusion was made by temporarily blocking the hepatic portal blood vessels and releasing 30 minutes later. The malondialdehyde (MDA) ,superoxide dismutase (SOD) in liver tissue and ALT, AST levels in the blood were determined after the liver had been reperfused f

5、or 120 minutes ,the hepatic tissue was examined under light microscope. ResultsMDA, ALT, AST decreased and SOD increased significantly in I/ R + Troxerutin group when compared those in the I/ R group （P 0.01) . The histologic changes of the liver tissue under light microscope in the I/ R + Troxeruti

6、n group was more easer than in the I/ R group.ConclusionTroxerutin has the ability to scavenge oxygen free radical (OFR) and to protect from the damage of ischemia-reperfusion in rat liver.Key words：Troxerutin; Liver; Ischemia-reperfusion; Oxygen free radical 在復雜的肝膽手術、嚴重肝創(chuàng)傷的處理及肝移植時均不可避免地會導致肝缺血再灌注損傷(

7、Ischemia/Reperfusion，I/R) ,甚至引發(fā)肝功能衰竭,而藥物預適應是減輕I/ R 損傷的有效方法之一。蘆丁( rutin) 又名蕓香苷, 為一來源很廣的黃酮類化合物。蘆丁及其衍生物具有廣泛的藥理活性, 具有降低毛細血管通透性、抗炎、抗病毒、鎮(zhèn)痛、抗氧化及抑制醛糖還原酶等多方面作用1，2。 臨床上主要用于防治腦出血、高血壓、視網(wǎng)膜出血、紫癜和急性出血性腎炎等。文獻上對蘆丁在心腦血管疾病方面的作用機制研究和探討較多,但其是否對肝臟的缺血再灌注損傷具有保護作用目前尚未見文獻報道。本實驗復制大鼠肝缺血再灌注損傷模型，并應用蘆丁的可溶性羥乙基衍生物曲克蘆丁(Troxerutin)對動

8、物進行術前預處理。測定各組大鼠血清中谷丙轉(zhuǎn)氨酶(ALT)和谷草轉(zhuǎn)氨酶(AST)的含量及肝組織中脂質(zhì)過氧化反應產(chǎn)物丙二醛(MDA) 和超氧化物歧化酶(SOD)的含量,光鏡檢查肝細胞結(jié)構(gòu)的形態(tài)學改變，觀察曲克蘆丁對大鼠肝臟I/R 損傷的保護作用并探討其作用機理。1 材料與方法1.1 材料實驗動物與分組：成年健康Wistar大鼠30 只, 由山東大學醫(yī)學院動物中心提供, 體重（25030）g, 雌雄不限, 隨機分為3 組, 每組10 只大鼠。假手術對照組:（sham operated control , SOC） 常規(guī)手術和術后處理; 肝缺血再灌注組:（ischemia- reperfusion ,

9、 I/ R，模型組）, 夾閉肝固有動脈和門靜脈30 min 后, 再灌注120 min; 曲克蘆丁預處理組:（I/R +Troxerutin）術前腹腔內(nèi)注射曲克蘆丁20 mg/kg, 1次/d, 連續(xù)3 d, 然后制作肝缺血再灌注損傷模型。1.2 藥品與試劑曲克蘆丁注射液由山東華魯制藥有限公司生產(chǎn)（批號：A0507121；規(guī)格：60 mg/支）。試劑：血清谷丙轉(zhuǎn)氨酶(ALT)、谷草轉(zhuǎn)氨酶(AST)、丙二醛(MDA )、超氧化物歧化酶(SOD) 測定試劑盒, 均購于南京建成生物工程研究所。1.3 方法1.3.1 模型制作各組動物均手術前12 h禁食,不禁飲水。用1% 戊巴比妥鈉麻醉后固定于手術臺

10、上, 常規(guī)脫毛、消毒、鋪巾后, 上腹正中切口,打開腹腔，分離出肝蒂, 游離開肝固有動脈、門靜脈和肝總管， 用小血管夾同時夾閉肝固有動脈和門靜脈,造成肝臟缺血。30 min后取下血管夾恢復肝臟血供, 再灌注120 min后采集標本進行指標測定。1.3.2 血清谷丙轉(zhuǎn)氨酶(ALT)、谷草轉(zhuǎn)氨酶(AST) 的活性測定分別以標準試劑盒（按照試劑盒說明書）測血清谷丙轉(zhuǎn)氨酶(ALT)、谷草轉(zhuǎn)氨酶(AST) 的活性。1.3.3 肝組織丙二醛(MDA )、超氧化物歧化酶(SOD)含量測定頸椎脫臼法處死大鼠后, 置于冰環(huán)境中, 迅速切取肝組織, 分析天平稱取組織濕重, 用蒸餾水混后制成勻漿, 按MDA 和SOD

11、 試劑盒操作程序進行測定MDA、SOD 的含量。1.3.4 肝組織病理形態(tài)學檢查將大鼠以頸椎脫臼法處死后, 快速取其肝組織，置于4%甲醛溶液中。將肝組織固定、脫水后，做石蠟切片，HE（蘇木素伊紅）染色，用光學顯微鏡觀察。1.4 主要觀察指標各組動物血清谷丙轉(zhuǎn)氨酶(ALT)、谷草轉(zhuǎn)氨酶(AST) 的活性及肝組織丙二醛(MDA )、超氧化物歧化酶(SOD)含量測定結(jié)果；各組大鼠肝組織病理形態(tài)學變化。1.5 統(tǒng)計學分析實驗數(shù)據(jù)以 s表示，各組間差別的顯著性用t檢驗判定。 1 operating system. The desktop configuration consisted of a P II

12、I processor, standard 24-bit video-graphics card on it and a monitor with 85 Hz. horizontal frequencies and resolution of 800x600 pixels. The first image of the program is in a circular order of 30 round shapes, each having a different color (see Figure 1A). These colored circles are numbered on the

13、 screen and are separated by equal distance in a circular fashion. These 30 different color hues were created using HLS color naming space with a programming algorithm derived from RGB system of Microsoft so as to have constant saturation and brightness but differing hue components. The hue componen

14、t presents the changed parameters of each color. This avoids the possibility of color blinds to discriminate colors by saturation and/or brightness. Application of the NCACVT All subjects were well informed by the tester about the test procedure. There is an introductory mini-test at the beginning o

15、f the principal testing program. This introductory phase there were 7 different color caps were showed, which are recognizable for both in normal and the color blinds. The subjects compared the sample test color with the circle colors by moving the mouse anticlockwise for matching until subject reac

16、hes the corresponding color cap and clicks on the mouse (Figure 1A). After this mini-test illustration the principal test was started. The colored circles are numbered on the screen and are separated by equal distance in a circular fashion. The principal colors representing red, green and blue (RGB)

17、 are numbered as 1, 11, and 21 respectively and they are shown with bold arrows. Numbers 6, 16 and 26 are the colors created by mixing two of the principal colors of the RGB system and they are shown with light arrows. In other words, these are the cyan-magenta-yellow (CMYK) systems principal colors

18、. There are 4 more intermediate hues in between these marked ones (Figure 1B). This presentation allows an even distribution of colors having close wavelengths in different quadrants. Consecutive presentations of the comparing test colors are also random and successive colors have distant wavelengths. Thirty colors are presented one after another and the subject moves the comparing test color anticlockwise until subject reaches the corresponding color cap and clicks on the mouse (Figure 1C). If the s