Melatonin对大鼠肝再灌注损伤的保护作用

摘要

目的: 探讨褪黑素对大鼠肝再灌注损伤的影响作用及其机制.

方法: 150只健康♂ Wistar大鼠(质量190-210 g, 6-7周龄), 随机分为褪黑素处理组(Mel)、酒精溶媒对照组(Alc)和生理盐水对照组(NS). 建立肝缺血再灌注损伤模型, 缺血均为60 min, 之后每组分别按再灌注后30 min、1、6、12、24 h采集标本. M组(20 mg/kg)于缺血前30 min腹腔注射melatonin; A组采取与Mel组相同浓度的酒精液, N组则注射同比例的生理盐水. 测定血清丙氨酸氨基转移酶(ALT), 肝组织中超氧化物岐化酶(SOD)及肝组织过氧化的终产物-丙二醛(MDA), 对肝组织进行HE染色及ICAM-1免疫组化染色.

结果: Mel组在再灌注后各时点的ALT均显著低于Alc及NS对照组(P<0.05), 且Alc组与NS组相比无显著性差异. Mel组在再灌注后6、12、24 h时点的MDA显著低于Alc及NS对照组(P<0.05), 且各时点内Alc组与NS组相比无显著性差异. Mel组在再灌注后12、24 h时点的SOD显著高于Alc及NS对照组(P<0.05), 且各时点内Alc组与NS组相比无显著性差异. Mel组在再灌注后各时点ICAM-1染色的阳性细胞率均显著低于Alc组和NS组(P<0.05), 且每时点内的Alc组与NS组相比无显著性差异.

结论: 外源性Mel可以抑制再灌注后血清丙氨酸氨基转移酶(ALT), 增加肝组织超氧化物歧化酶(SOD)、的活性, 减少肝组织MDA的浓度, 抑制肝组织ICAM-1蛋白的表达, 对缺血再灌注肝损伤有明确的保护作用.

关键词: N/A

Protective effect of melatonin on liver ischemia reperfusion injury in rats

Jian-Yi Li, Wen-Hai Zhang, Yong Zhou, Jun Yang, Yi-Min Qin

Jian-Yi Li, Fourth Department of General Surgery, The Second Affiliated Hospital of China Medical University, Shenyang 110003, Liaoning Province, China

Wen-Hai Zhang, Yong Zhou, Department of General Surgery, The Second Affiliated Hospital of China Medical University, Shenyang 110022, Liaoning Province, China

Jun Yang, Department of Oncology, The First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning Province, China

Yi-Min Qin, Department of General Surgery, The Tenth Municipal People Hospital of Shenyang, Shenyang 110044, Liaoning Province, China

Supported by: the National Science Foundation of Liaoning Province, No. 619025.

Correspondence to: Dr. Wen-Hai Zhang, Department of General Surgery, Second Affiliated Hospital, China Medical University, 39 Huaxiang Road,Shenyang 110022, Liaoning Province, China. surgeonzwh@163.net

Received: November 18, 2003
Revised: December 1, 2003
Accepted: February 1, 2004
Published online: April 15, 2004

Abstract

AIM: To investigate the effect of melatonin (Mel) on liver ischemia reperfusion (I/R) injury in rats.

METHODS: 150 male Wistar rats (190-210 g, 6-7weeks age) were divided into three groups at random: Mel exposure group, alcohol solvent control group and saline control group. The left branches of portal vein, hepatic artery, hepatic duct were blocked up for 60 min and then opened to establish liver I/R I models in rats. In each group, samples were collected in 0.5, 1, 6, 12, and 24 h after reperfusion respectively. 20 mg/kg of Mel was injected peritoneally in rats 30 min before experimentation in Mel exposure group. The duplicate concentration of alcohol and the same volume of saline were injected in control group as a substitution. Serum alanine aminotransferase (ALT) by auto biochemical analyzer, and superoxide dismutase (SOD) and terminal productions of lipid peroxidationin (MDA) in liver tissue were measured. Pathological changes in liver and immunohistochemical straining of ICAM-1 were determined with optical microscope.

RESULTS: The level of ALT measured in various time after reperfusion in Mel group was totally significantly lower than that in alcohol and saline control groups (P < 0.05). The level of MDA measured in 6 h, 12 h, and 24 h after reperfusion in Mel group was significantly lower than that in alcohol and saline control groups (P < 0.05). The level of SOD measured in 12, 24 h after reperfusion in Mel group was significantly higher than that in alcohol and saline control groups (P < 0.05). The expression level of ICAM-1 (%) measured in various time after reperfusion in Mel group was significantly lower than that in alcohol and saline control groups (P < 0.05).

CONCLUSION: Exotic Mel inhibits the activities of ALT, increases activities of superoxide dismutase (SOD), and decreases the cumulation of MDA in liver reperfusion tissue and expression of ICAM-1 in liver reperfusion tissue. Therefore, it can improve the hepatic function after reperfusion and plays a definitely protective role in liver I/R.

Key Words: N/A

0 引言

肝移植目前已成为治疗终末期肝病的有效方法, 但是仍然存在着术后原发无功能肝、排斥反应等亟待解决的并发症. 肝的再灌注损伤能够导致移植术后原发无功能肝(primary non-function liver)的发生, 是导致肝移植失败的主要原因[1]. 再灌注损伤的发病机制至今尚未被完全阐明[2], 与如下因素有关: 一氧化氮与内皮素[3-4]、细胞因子(包括IL, TNF, IFN等)[5]、Kupffer细胞的激活[6-7]、黏附分子的表达与中性粒细胞的浸润[8]、线粒体能量变化及通透性的改变[9]、核转录因子的表达、热休克蛋白的表达[10]、凋亡相关蛋白的表达及凋亡的发生等[11]. 褪黑素(melatonin, Mel)由于其在生殖系统、脂肪代谢和毛发替换、调节睡眠、免疫系统、心血管系统及抗氧化损伤及抗衰老方面的作用日益受到人们的重视而成为研究的热点[12]. 在肝脏方面的研究则主要限于中毒及免疫性肝损伤[13], 我们以大鼠为研究对象, 建立肝再灌注损伤模型, 按时点采集标本, 对血清丙氨酸氨基转移酶(ALT), 肝组织脂质过氧化物的终产物-丙二醛(MDA)及肝组织超氧化物岐化酶(SOD)进行测定; 对肝组织进行HE染色及ICAM-1免疫组化染色, 光镜下观察肝组织细胞形态学改变, 探讨Mel对大鼠肝再灌注损伤的影响作用及其机制, 为Mel在临床肝脏外科的应用提供依据.

1 材料和方法

1.1 材料

健康♂Wistar大鼠(质量190-210 g, 6-7周龄)150只, 随机分为3组, 即褪黑素处理组(Mel)、酒精溶媒对照组(Alc)、生理盐水对照组(NS). 分别建立肝再灌注损伤模型, 缺血时间均为60 min, 之后每组分别按再灌注后30 min、1、6、12、24 h采集标本. 在缺血手术前将以上3组大鼠分别饲养在12 h光照, 12 h黑暗的条件下(07: 30-19: 30为光明, 其余为黑暗)至少1 wk, 自由摄食饮水. 动物模型制备 参照Kobayashi法, 采取以动脉夹夹闭肝门静脉左支、肝动脉左支、左肝管60 min之后放开, 建立大鼠肝左叶、中叶再灌注损伤模型[14]. 褪黑素(Melatonin) 1 g购自美国Sigma公司, 经400 mL/L酒精生理盐水溶解后低温保存, 每次用时稀释10倍, 按20 mg/kg计算给药量后, Mel组于缺血前30 min腹腔注射; Alc组采取与Mel组相同浓度的酒精液; NS组则注射同比例的生理盐水. 麻醉采取用硫喷妥钠(40 mg/kg)缺血手术前45 min(即腹腔给药前15 min)大鼠股二头肌肌肉注射. 40 g/L多聚甲醛0.1 moL/L磷酸缓冲液(PH =7.3); SOD、MDA试剂盒均购自南京建成生物工程研究所; 血生化相关试剂购自德国宝灵曼公司; ICAM-1试剂盒购自英国Serotech公司. OLYMPUS-BX60光学显微镜及照像系统; HITACHI-7600A全自动生化分析仪.

1.2 方法

经膈肌心脏穿刺取血; 取左叶边缘肝组织200-210 g行SOD及MDA测定; 取中叶中央部肝组织2 cm×1 cm×0.3 cm行ICAM-1免疫组化染色. 利用自动生化分析仪测定ALT, TBA法测定MDA, NBT还原法测定SOD, SP法对肝细胞进行ICAM-1染色. 免疫组化结果在400倍光镜下观察, 胞质内出现株黄色细颗粒者为阳性细胞(包括肝血窦内皮细胞和肝细胞). 计数每个高倍视野中100个细胞中ICAM-1阳性细胞个数, 每张切片计数5个视野, 求出均值用mean±SD表示, 得出阳性细胞百分率. 所有数据均用专业软件SPSS 11.0进行统计, 采用方差分析及SNK检验[11].

2 结果

2.1 血清ALT

Mel组在再灌注后各时点的ALT均显著低于Alc组及NS组(P<0.05), 且Alc组与NS组相比无显著性差异(表1).

表1 缺血再灌注大鼠血清ALT(mean±SD, kat/L).

分组	30 min	1 h	6 h	12 h	24 h
Melatonin	3.4±1.2a	3.1±1.0a	3.1±1.0a	3.2±1.1a	4.5±1.8a
Alcohol	6.7±2.3	6.2±2.7	6.0±2.5	5.6±2.1	5.5±2.3
N.S.	6.6±2.2	6.4±2.4	6.0±1.7	5.4±1.9	5.2±1.2

^aP<0.05 vs Alcohol, NS组.

2.2 肝组织MDA和SOD

Mel组在再灌注后6、12、24 h时点的MDA显著低于Alc及NS对照组(P<0.05), 且各时点内Alc组与NS组相比无显著性差异; Mel组在再灌注后12、24 h时点的SOD显著高于Alc及NS对照组(P<0.05), 且各时点内Alc组与NS组相比无显著性差异(表2).

表2 缺血再灌注大鼠肝MDA(mean±SD ).

分组	30 min	1 h	6 h	12 h	24 h
MDA(nmol/g)Melatonin	436±139	496±67	556±99a	693±158a	1001±318a
Alcohol	396±85	548±112	742±102	1 001±186	1815±458
N.S.	445±93	559±117	724±144	974±115	1844±668
SOD (kNU/g)Melatonin	125±40	153±55	164±61	244±63c	234±51c
Alcohol	103±6	121±23	130±31	181±53	151±48
N.S.	99±24	132±24	135±20	167±33	142±26

^aP<0.05 vs Alcohol, NS组;

^cP<0.05 vs Alcohol, NS组.

2.3 肝组织ICAM-1染色

Mel组再灌注后各时点的阳性细胞率均显著低于Alc组和NS组(P<0.05), 且每时点内的Alcohol组与N.S. 组相比无显著性差异. 阳性细胞多为肝血窦内皮细胞, 或为靠近中央静脉的肝细胞(图1-6). 两组对照组的阳性细胞率的变化有明显的规律性, 早在30 min及1 h时与Mel组相比就有较高的表达, 且在再灌注后6-24 h呈平台值(表3).

图1 Melatonin组再灌注1 h×400.

图2 Alcoho组再灌注1 h×400.

表3 缺血再灌注肝组织ICAM-1染色 (mean±SD,%).

分组	30 min	1 h	6 h	12 h	24 h
Melatonin	4.1±2.4a	7.8±2.4a	12.6±4.2a	16.3±3.0a	14.8±4.0a
Alcohol	15.9±3.2	22.7±3.7	40.1±4.4	39.2±6.0	39.0±5.2
N.S.	17.9±2.8	23.0±4.5	42.8±4.8	42.0±5.9	40.4±5.5

^aP<0.05, vs Alcohol, NS组.

图3 NS组再灌注1 h×400.

图4 Melatonin组 再灌注6 h×400.

图5 Alcohol组再灌注6 h×400.

图6 NS组再灌注6 h×400.

2.4 肝组织HE染色

在再灌注12 h (图7-9)及其之后的24 h (图10-12)时点的Mel处理组的肝细胞变性、坏死的程度与范围, 肝索结构的破环程度与范围均明显的轻于Alc组和NS组. 并且对各组别内的各时点作了比较, 发现随着再灌注的时间的延长, 两个对照组肝细胞变性坏死的程度及肝索破环的范围也逐渐扩大, 到再灌注24 h时达到高峰; 而这一变化在Mel组则表现不明显, 从再灌注12及24 h的病理切片来看, 区别不明显. 提示外源性Mel可以减轻I/R后肝细胞的变性和坏死的程度及范围.

图7 Melatonin组再灌注12 h×400.

图8 Alcohol组再灌注12 h×400.

图9 NS组再灌注12 h×400.

图10 Melatonin组再灌注24 h ×400.

图11 Alcohol组再灌注24 h ×400.

图12 NS组再灌注24 h ×400.

3 讨论

一般认为, I/R可以造成ROS的大量释放[15-16] ; ROS可以攻击生物膜, 造成后者通透性的改变[17] ; 生物膜通透性的改变诱发Ca²⁺在细胞内及线粒体等浓度升高(Overload)[18]; 细胞内Ca²⁺ Overload又加速ROS的产生, 同时可激活多种磷脂酶, 促进膜磷脂分解, 造成膜磷脂降解产物花生四烯酸、溶血磷脂等增多; 花生四烯酸的代谢产物 (如白三烯)能吸引大量中性粒细胞黏附于血管内皮并进入组织[19]; 中性粒细胞与血管内皮细胞黏附后进一步激活, 自身合成释放如TNF、IL、黏附分子[20-21]等多种具有趋化作用的炎性递质[22-23], 使白细胞浸润进一步加重, 同时对血液流变学、微血管口径及血管通透性的影响最终造成了再灌注后的无复流现象[24-25]. 另一方面, 早在1991年Ianas et al首次报道了Mel的具有抗氧化作用[26], 之后2a Tan的研究说明与GSH及甘露醇相比, Mel能更有效地清除稯H[27]. 1996年, Marshall的研究发现Mel清除ROO. 的能力是VE的2倍[28], 因此Mel是目前为止发现的最有效和最理想的自由基清除剂之一.

血清中ALT, 是最敏感的肝功检测指标之一[29]. 表1显示各组的ALT在再灌注后30 min已普遍升高, 而且持续到再灌注后24 h. 从每个时点内组别情况来看, Mel组的ALT值均显著低于其他两组对照组(^aP<0.05), 肝功能的损害明显减轻. 实验分别测定肝组织MDA, SOD作为评价ROS损伤及内源性抗ROS的指标[30-31]. 表2第一组数据显示的是再灌注后肝组织MDA各组各时点的变化情况, 总体来看, 随着时间的延长, 各组MDA在肝组织内逐渐堆积, 呈逐渐增高的趋势, 从再灌注6 h开始, 与对照组相比Mel组MDA增加的速度逐渐放慢, 在6、12、24 h时点MDA值均显著性的低于对照组(^aP< 0.05), 说明外源性Mel可以有效的降低再灌注后肝组织中MDA的浓度. 2001年Sinha et al通过闭塞大鼠的大脑中动脉来模拟脑I/R过程中, 发现Mel组的MDA的水平显著低于溶媒对照组(P<0.05)[32]; 之后1 a Inci et al在以Mel为处理因素用于肺I/R的模型研究中同样发现, Mel组肺组织内的MDA、MPO的活性以及支气管肺泡的亚硝酸水平都明显的低于对照组(P<0.05)[33]. 表2的第二组数据显示由于再灌注损伤释放了大量ROS, 反应性的造成SOD随着再灌注时间的延长而保护性的增高, 这种增高在再灌注后12 h达到了高峰. 与对照组相比, Mel组这种保护性的增高更为明显, 且延续的时间更长, 在12 h及24 h时这种明显的增高差异具有显著性(^cP<0.05). 说明外源性Mel可以提高再灌注后肝组织SOD的活性. 与此同时以Mel为处理因素, 以MDA、SOD、GSH. px、GSSG、LPO等为测定的相关指标的再灌注损伤研究分别在胃[34]、心脏[35]、小肠[36-37]等脏器中得到了相类似的结果. 近年来有关Mel结构研究提示吲哚环5位上的甲氧基和侧链上的N-乙酰基是Mel作为自由基清除剂的必需基团[38]. Mel主要通过提供电子来清除自由基, 失去电子后Mel本身变成了毒性很低的吲哚阳离子自由基, 而他又能清除超氧阴离子自由基(O₂^-), 最后变成AMFK并经肾脏排出[39]. 另外, Mel还可以通过增强诸如SOD、GSH. px等抗自由基酶系的活性来发挥其清除自由基的作用[40]. 更有报道证明, 外源性应用Mel可以在通过提升SOD mRNA 的表达, 来发挥其对再灌注损伤的保护作用[41]. 最近有关Mel外周受体的研究显示, Mel可与除中枢外的多系统多脏器中的受体相结合, 来发挥多种生理作用[42]. 通过统计肝细胞ICAM-1染色的阳性细胞率, 其结果显示Mel组再灌注后各时点的阳性细胞率均显著低于对照组(^aP<0.05), 且每时点内的Alc组与NS组相比无显著性差异. 验证了I/R诱发肝细胞ICAM-1的强表达, 而外源性Mel可以有效的抑制ICAM-1的表达. 2000年Cuzzocrea et al 通过钳夹肠系膜上动脉及腹腔干动脉建立大鼠的内脏器官缺血再灌注模型, 不仅通过测定MDA、LPO等指标证实了Mel可以通过清除ROS及抗氧化的途径来发挥对再灌注损伤的保护作用, 并且通过对回肠组织的免疫组织化学染色发现, 与对照组相比, Mel组的ICAM-1及P-selectin的蛋白表达明显减少(P<0.05)[43]; 1年以后他们在用Mel治疗二硝基苯磺酸所引起的结肠炎时再次证实了Mel可以通过抑制ICAM-1的表达, 有效地抑制中性粒细胞的浸润而发挥治疗作用[44].

备注

编辑: N/A

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