前列腺素E1对梗阻性黄疸大鼠肝脏缺血再灌注损伤时白介素-1β表达的影响

摘要

目的: 探讨前列腺素(PG)E1对梗阻性黄疸肝脏缺血再灌注损伤的保护作用以及对IL-1β表达的影响.

方法: 将♂Wistar大鼠随机分为PGE1处理组(PG组, n = 18)和生理盐水对照组(NS组, n = 18). 参照Yoshidome法结扎并切断胆总管建立梗阻性黄疸模型, 1 wk后Pringle法阻断肝门15 min, 再灌注后建立胆道再通. 于缺血前15 min至再灌注60 min, PG组经门静脉持续泵入PGE1 0.5 µg/(kg•min), NS组给予等量生理盐水. 于再灌注1、6和24 h 3个时点取材, 检测血清ALT, AST, 总胆红素(total bilirubin, TBIL), 直接胆红素(direct bilirubin, DBIL)水平, 测定肝组织GSH, MDA含量. ELISA法测定肝组织IL-1β表达, 并观察肝脏病理组织学改变.

结果: 再灌注各时点PG组血清ALT水平(1 h: 1939±1427 nkat/L vs 5596±2975 nkat/L; 6 h: 3409±1708 nkat/L vs 9279±4404 nkat/L; 24 h: 1434±274 nkat/L vs 2264±630 nkat/L)和AST(1 h: 21746±12083 nkat/L vs 37552±12382 nkat/L; 6 h: 55039±35471 nkat/L vs 98811±11126 nkat/L; 24 h: 9394±1662 nkat/L vs 27664±15856 nkat/L)、肝组织MDA含量(1 h: 0.89±0.18 μmol/g vs 1.21±0.24 μmol/g; 6 h: 1.08±0.23 μmol/g vs 1.45±0.13 μmol/g; 24 h: 1.03±0.08 μmol/g vs 1.45±0.26 μmol/g)以及肝组织IL-1β表达水平(1 h: 304.1±67.9 ng/L vs 362.8±137.1 ng/L; 6 h: 376.8±74.6 ng/L vs 618.8±217.8 ng/L; 24 h: 273.0±69.0 ng/L vs 373.0±71.7 ng/L)均显著低于NS组(P<0.05), 而肝组织GSH含量显著高于NS组(1 h: 945.1±121.2 mg/g vs 720.0±80.1 mg/g; 6 h: 753.5±118.6 mg/g vs 553.6±140.0 mg/g; 24 h: 768.0±135.9 mg/g vs 596.3±36.4 mg/g, P<0.05), 但两者胆红素水平无明显差异(P>0.05). PG组肝脏病理组织学损伤程度也较NS组明显减轻.

结论: PGE1下调肝组织IL-1β表达, 对梗阻性黄疸肝脏缺血再灌注损伤具有保护作用.

关键词: 梗阻性黄疸; 肝脏; 缺血再灌注损伤; 白介素-1β; 前列腺素E1

Effect of prostaglandin E1 on expression of interleukin-1β in hepatic ischemic reperfusion injury in rats with obstructive jaundice

Feng Xu, Chao-Liu Dai, Chuang Zhao, Chang-Jun Jia, Yong-Qing Xu, Song-Lin Peng, Xi Gu, Yang Zhao

Feng Xu, the First Department of Biliary Surgery, Shengjing Hospital, China Medical University, Shenyang 110004, Liaoning Province, China

Chao-Liu Dai, Chuang Zhao, Chang-Jun Jia, Yong-Qing Xu, Song-Lin Peng, Xi Gu, Yang Zhao, Department of Hepatobiliary Surgery, Shengjing Hospital, China Medical University, Shenyang 110004, Liaoning Province, China

Correspondence to: Professor Chao-Liu Dai, Department of Hepatobiliary Surgery, Shengjing Hospital, China Medical University, 36 Sanhao Street, Heping District, Shenyang 110004, Liaoning Province, China. daicl-sy@tom.com

Received: April 1, 2007
Revised: September 28, 2007
Accepted: September 28, 2007
Published online: November 18, 2007

Abstract

AIM: To investigate the protective effect of prostaglandin E1 against hepatic ischemic reperfusion injury and effect on expression of interleukin-1β in rats with obstructive jaundice.

METHODS: Male Wistar rats were divided randomly into two groups: PG group received prostaglandin E1 (n = 18) and NS group received normal saline served as controls (n = 18). All rats underwent ligation and division of common bile duct to establish obstructive jaundice model according to Yoshidome's maneuver. After 1 wk, total liver ischemia for 15 min according to Pringle's maneuver and internal biliary drainage during hepatic reperfusion were performed. The hepatoprotective effect of prostaglandin E1 in PG group was studied throughout treatment with PGE1 by continuously pumping via portal vein from 15 min before hepatic ischemia to 60 min after reperfusion, while rats of NS group were only treated with normal saline. All rats were sacrificed at 1, 6, and 24 h after reperfusion. IL-1β expression was determined by ELISA, and glutathione (GSH) and malondialdehyde (MDA) in liver tissue homogenate, activities of alanine transaminase (ALT) and aspartate transaminase (AST) in serum, in addition to serum level of bilirubin (total and conjugated) were evaluated. Liver tissue sections were examined to observe histopathological changes.

RESULTS: Analysis of data showed that treatment with prostaglandin E1 significantly attenuated hepatic IL-1β expression (1 h: 304.1 ± 67.9 ng/L vs 362.8 ± 137.1 ng/L; 6 h: 376.8 ± 74.6 ng/L vs 618.8 ± 217.8 ng/L; 24 h: 273.0 ± 69.0 ng/L vs 373.0 ± 71.7 ng/L), and lowered serum activities of ALT (1 h: 1939 ± 1427 nkat/L vs 5596 ± 2975 nkat/L; 6 h: 3409 ± 1708 nkat/L vs 9279 ± 4404 nkat/L; 24 h: 1434 ± 274 nkat/L vs 2264 ± 630 nkat/L), AST (1 h: 21 746 ± 12 083 nkat/L vs 37  552 ± 12 382 nkat/L; 6 h: 55 039 ± 35  471 nkat/L vs 98 811 ± 11 126 nkat/L; 24 h: 9394 ± 1662 nkat/L vs 27 664 ± 15 856 nkat/L) and MDA (1 h: 0.89 ± 0.18 μmol/g vs 1.21 ± 0.24 μmol/g; 6 h: 1.08 ± 0.23 μmol/g vs 1.45 ± 0.13 μmol/g; 24 h: 1.03 ± 0.08 μmol/g vs 1.45 ± 0.26 μmol/g) levels in tissue homogenate while elevating GSH (1 h: 945.1 ± 121.2 mg/g vs 720.0 ± 80.1 mg/g; 6 h: 753.5 ± 118.6 mg/g vs 553.6 ± 140.0 mg/g; 24 h: 768.0 ± 135.9 mg/g vs 596.3 ± 36.4 mg/g, P < 0.05) levels, while there was no difference in serum bilirubin levels between PG group and NS group (P > 0.05). Additionally, histological evaluation revealed improvement of liver damage in PG group compared with NS group.

CONCLUSION: The data indicate that prostaglandin E1 administered via the portal vein reduces expression of IL-1β in hepatic tissue and protects against hepatic ischemia/reperfusion injury in rats with obstructive jaundice.

Key Words: Obstructive jaundice; Liver; Ischemic reperfusion injury; Interleukin-1β; Prostaglandin E1

0 引言

胆道恶性肿瘤引起的梗阻性黄疸正逐年增加. 为了彻底根除病灶和延长生存期, 越来越多的患者在解除胆道梗阻的同时需联合切除部分肝脏. 切肝时常用肝门阻断法控制出血, 当解除阻断后不可避免地会发生肝脏缺血再灌注损伤(hepatic ischemia reperfusion injury, HIRI). HIRI常导致患者术后肝功能衰竭甚至死亡, 但目前对此仍缺少有效的防护方法. HIRI时肝脏非实质细胞产生的细胞因子如IL-1β介导肝细胞损伤, 甚至导致肝功能衰竭. 近来研究发现, 前列腺素E1(prostaglandin E1, PGE1)能有效地治疗肝移植后原发性无功能, 说明对肝脏具有保护作用[1-4]. 因此我们通过梗阻性黄疸大鼠肝脏缺血再灌注损伤模型探讨PGE1抑制肝组织IL-1β表达, 达到对梗阻性黄疸HIRI的保护作用, 为临床应用提供实验依据.

1 材料和方法

1.1 材料

Wistar♂大鼠, 体质量270±20 g, 中国医科大学盛京医院实验动物中心提供. IL-1β ELISA试剂盒购自上海森雄科技实业有限公司, 谷胱甘肽(GSH)及丙二醛(MDA)试剂盒均购自南京建成生物工程研究所, PGE1购自北京赛生药业有限公司.

1.2 方法

将36只♂Wistar大鼠随机分为PGE1处理组(PG组, n = 18)和生理盐水对照组(NS组, n = 18). 参照Yoshidome et al[5]和Wang et al[6]报道的方法建立梗阻性黄疸模型, 稍加改动. 各组大鼠用100 g/L水合氯醛(按3 μL/g)ip麻醉, 取正中切口入腹, 5/0丝线结扎并切断胆总管. 术后大鼠均单笼饲养, 常规进食水. 造模1 wk后再次麻醉, 取正中及右肋缘下联合切口入腹, 游离扩张的胆总管, 离断肝周韧带. PG组PGE1按0.5 µg/(kg•min)(100 µg PGE1溶于20 mL生理盐水)经门静脉持续泵入15 min, NS组生理盐水按相同速度给予. 随后用无创伤动脉夹Pringle法完全阻断肝门15 min, 移除动脉夹恢复肝脏血供. 肝脏再灌注后再持续泵入PGE1或生理盐水60 min, 并用硬膜外导管连通胆总管与十二指肠, 建立胆道再通. 分别于肝脏再灌注后1, 6, 24 h 3个时点(各6只)取材, 自腹主动脉采血3 mL, 静置20 min, 3000 r/min离心10 min. 将血清转移至塑料弹头中, 置于-20℃冰箱中保存, 待测血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、总胆红素(TBIL)以及直接胆红素(DBIL)水平(用Hitachi 7600全自动生化分析仪检测上述血清指标)方法如下: 快速夹取肝组织, 用锡箔纸包裹后置于液氮中, 再转至-80℃冰箱中保存, 待做肝组织GSH和MDA含量测定和白介素-1β(IL-1β)ELISA检测. 处死大鼠, 取1 cm×1 cm×1 cm大小肝组织置于40 g/L甲醛中固定, 待做病理组织学检查.

1.2.1 肝组织IL-1β、GSH、MDA含量测定: 称取-80℃冰箱保存的肝组织, <100 mg, 加生理盐水使其浓度为1 kg/L, 冰水中匀浆, 4℃ 16 000 r/min离心10 min, 取上清液. 按试剂盒说明书操作, 测定GSH和MDA含量. 建立IL-1β标准曲线, 待测品孔每孔加入待测样品100 μL, 将反应板充分混匀后置37℃水浴120 min, 用洗涤液将反应板充分洗涤4-6次, 向滤纸上印干. 每孔中加入一抗体50 μL, 置37℃水浴60 min, 洗板: 每孔加酶标抗体100 μL, 将反应板置37℃水浴60 min. 洗板; 每孔加底物100 μL, 置37℃暗处反应5-10 min. 每孔加1滴终止液, 混匀, 于492 nm处用全自动酶标仪测定吸光度A值和IL-1β含量.

1.2.2 肝组织病理组织学检查: 肝组织块常规石蜡包埋, 切片, HE染色, 光镜下观察肝病理组织学变化.

统计学处理 所有数据采用统计软件SPSS11.5进行t检验分析, P<0.05为有统计学意义.

2 结果

2.1 血清生化酶和胆红素水平

PG组再灌注各时点血清ALT和AST水平均显著低于NS组(P<0.05). 肝组织损伤虽以再灌注6 h最明显, 但随再灌注时间延长呈逐渐恢复趋势. PG组血清TBIL和DBIL水平虽较NS组有下降, 但无统计学差异(P>0.05, 表1).

表1 大鼠肝IR血清ALT、AST、TBIL和DBIL水平(mean±SD, n = 6).

指标	分组	再灌注1 h	再灌注6 h	再灌注24 h
ALT(nkat/L)	NS组	5596±2975	9279±4404	2264±630
	PG组	1939±1427a	3409±1708a	1434±274a
AST(nkat/L)	NS组	37 552±12 382	98 811±11 126	27 664±15 856
	PG组	21 746±12 083a	55 039±35 471a	9394±1662a
TBIL(μmol/L)	NS组	123.3±24.8	65.2±10.2	83.6±30.1
	PG组	101.1±31.0	58.8±26.9	81.9±9.1
DBIL(μmol/L)	NS组	100.4±20.0	51.9±8.7	70.2±24.4
	PG组	83.2±24.8	46.4±23.7	70.0±8.6

^aP<0.05 vs NS组.

2.2 肝组织GSH和MDA含量

PG组再灌注6 h肝组织GSH水平虽达低值, 但再灌注各时点GSH水平均显著高于NS组(P<0.05). PG组再灌注6 h肝组织MDA水平虽达峰值但随再灌注时间延长, MDA水平有逐渐下降趋势, 且再灌注各时点MDA水平均显著低于NS组水平(P<0.05). 而NS组再灌注24 h肝组织MDA则仍维持于较高水平(表2).

表2 大鼠肝IR肝组织GSH、MDA和IL-1β含量(mean±SD, n = 6).

指标	分组	再灌注1 h	再灌注6 h	再灌注24 h
GSH(mg/g)	NS组	720.0±80.1	553.6±140.0	596.3±36.4
	PG组	945.1±121.2a	753.5±118.6a	768.0±135.9a
MDA(μmol/g)	NS组	1.21±0.24	1.45±0.13	1.45±0.26
	PG组	0.89±0.18a	1.08±0.23a	1.03±0.08a
IL-1β(ng/L)	NS组	362.8±137.1	618.8±217.8	373.0±71.7
	PG组	304.1±67.9	376.8±74.6a	273.0±69.0a

^aP<0.05 vs NS组.

2.3 肝组织IL-1β含量

PG组再灌注各时点肝组织IL-1β水平均低于NS组, 且再灌注6, 24 h两组肝组织IL-1β水平均有统计学差异(P<0.05). 再灌注24 h PG组肝组织IL-1β水平已明显低于再灌注1 h水平, 而NS组却仍高于再灌注1 h水平(表2).

2.4 肝组织病理形态学

PG组再灌注各时点肝组织损伤均较NS组轻, 表现为肝细胞肿胀较轻、肝细胞索排列较规则、肝窦结构较宽且较清晰、肝细胞坏死较少. 而NS组肝组织结构不清, 肝细胞索排列紊乱, 肝窦明显变窄, 肝细胞坏死明显, 尤其再灌注6 h中央静脉周围出现大量肝细胞坏死(图1).

图1 大鼠肝IR 6 h肝组织改变(HE×400). A: NS组; B: PG组.

3 讨论

如何减轻梗阻性黄疸患者肝脏缺血再灌注损伤是肝脏外科面临的一个重要临床问题. 虽然目前对梗阻性黄疸肝脏缺血再灌注损伤的作用机制进行了初步研究[5,7-12], 但至今仍无有效的防护方法. 研究发现, PGE1具有扩张血管、增加肝脏血流量作用, 能改善再灌注时微循环障碍所致的无复流现象. 治疗原发性肝移植物无功能[1-4]. 但PGE1经外周静脉给药后约70%在肺部被迅速代谢, 此外动脉和肠道也可利用一部分[4]. 这势必需要加大剂量才能使PGE1对肝脏产生满意的作用, 由此也容易出现低血压等全身不良反应. 如利用肝脏独特的供血系统, 采用经门静脉途径给药, PGE1可直接经肝脏循环对肝细胞产生作用, 则可减少药物用量. 但至今为止, PGE1经门静脉途径给药在梗阻性黄疸HIRI中作用尚无研究报道. 本研究显示PGE1治疗组再灌注后各时点血清ALT和AST水平均显著下降, 且肝脏病理组织学改变也明显减轻, 提示应用PGE1可减轻缺血再灌注对瘀疸肝脏的损伤, 具有保护作用. HIRI时肝细胞内还原性GSH显著下降, 且其损伤程度与缺血前细胞内GSH含量成反比, GSH水平可作为组织损伤严重程度的敏感指标之一[13-14]. 缺血再灌注时产生的氧自由基是造成肝脏损伤的一个重要因素. GSH作为体内重要的抗氧化剂, 可对抗氧自由基的损害, 减轻对肝脏组织的损伤[15-16]. MDA则是自由基连锁反应-脂质过氧化的最终产物, 测定其含量可反映机体内脂质过氧化的程度, 间接反映细胞膜损伤的程度, 两者的变化可反映肝脏的抗氧化能力[17-18]. 我们发现, PG组, 再灌注后GSH水平虽有下降但明显高于NS组, 且MDA水平虽有增加但不明显, 提示应用PGE1可通过提高肝组织GSH水平, 降低MDA水平, 增强肝脏的抗氧化能力, 有效地清除氧自由基, 从而减轻HIRI. 这与Bulbuller et al[15]实验结果类似.

瘀疸肝脏缺血再灌注后肝细胞肿胀进一步加重, 压迫微血管, 肝脏白细胞浸润增加, 并通过ICAM-1表达增加黏附于内皮细胞, 进而嵌顿、阻塞肝窦; 肝窦内大量白细胞黏附、附壁的同时, 释放大量IL-1β等炎性介质, 从而进一步加重肝脏损伤[5,7-12,19]. 我们应用PGE1后, 再灌注各时点肝组织白细胞浸润减少, IL-1β表达水平显著下降. 这可能与以下因素有关: (1)PGE1具有直接舒张血管平滑肌作用, 增加肝脏血流量, 并阻止缩血管物质的释放, 降低血管平滑肌对血管收缩物质的敏感性, 抑制白细胞黏附, 从而减少IL-1β等细胞因子介导的损伤, 稳定溶酶体膜及抑制溶酶释放, 保护细胞膜[20-24]. (2)PGE1具有阻止血小板聚集的作用, 抑制血小板聚集, 减少血小板释放TXA2, 防止微血栓形成, 减少白细胞黏附[25-27]. (3)PGE1直接提高Kupffer细胞内cAMP含量, 抑制IL-1β等炎性介质的合成和释放[20]. (4)PGE1可通过激活腺苷酸环化酶, 提高肝细胞内cAMP含量, 明显抑制肝窦ICAM-1等黏附分子表达, 抑制白细胞与肝窦内皮细胞的黏附和渗出, 增加红细胞的变形能力, 改善肝窦微循环障碍[28-30]. 总之, PGE1可减少梗阻性黄疸HIRI时肝组织IL-1β表达、减轻肝脏损伤, 但其具体的分子机制仍有待进一步研究.

评论

背景资料

如何防护梗阻性黄疸肝脏缺血再灌注损伤是肝脏外科面临的一个重要临床问题, 但至今仍缺少有效的防护手段.

研发前沿

肝脏缺血再灌注损伤的防护方法有多种, 其中应用药物防护是目前的研究热点, 也更符合临床实际.

创新盘点

本文以梗阻性黄疸肝脏缺血再灌注损伤的肝脏微循环障碍为切入点, 应用前列腺素E1对微循环的作用进行研究, 具有一定的创新性.

应用要点

本文提示前列腺素E1对梗阻性黄疸肝脏缺血再灌注损伤具有保护作用, 因此在临床上可以适度地推广应用.

名词解释

梗阻性黄疸: 指外科黄疸. 由于肝外胆道梗阻, 导致胆汁返流, 血中胆红素水平升高, 并出现黄疸表现的一类疾病.

同行评价

本文实验结果真实可靠, 数据处理严谨, 具有一定的创新性和应用前景.

备注

编辑:程剑侠 电编:李军亮

参考文献

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