缺血再灌注肝脏Kupffer细胞NF-kB激活及其意义
1重庆医科大学附一院普外科 重庆市 400016
2解放军第208医院 吉林省长春市 130062
徐明清，男，1968-11-25生，四川省乐山市人，汉族，1989年第三军医大学 毕业，2000年获第三军医大学博士学位，主治医师，讲师，主要从事肝脏疾病的研究，发表 论文6篇.
项目负责人 徐明清，400016，重庆市袁家岗医学院路1号重庆医科 大学附一院普外科.
Telephone: 13022303642
收稿日期 2001-01-08 接受日期 2001-01-11
Significance of Kupffer cell NF-kB activation during hepatic ischemia /reperfusion in rats
Ming-Qing Xu1, Lan Xue2 and Jian-Ping Gong1
1Department of General Surgery,First Affiliated Hospital,Chong qing University of Medical science, Chongqing 400016,China.
2The 208th hospital of PLA, Changchun 130062, Jilin Province, China.
Correspondence to: Xu Ming-Qing, Department of General Surgery, First Affiliated Hospital, Chongqing University of Medical science, Chongqing 4 00016, China.
Received 2001-01-08 Accepted 2001-01-11
Abstract
AIM To explore the effect of Kupffer cells (KCs) NF-kB act ivation on liver injury during hepatic ischemia/reperfusion(HIR) in rats.
METHODS Wistar rats were divided randomly into HIR group in whi ch hepatic reperfusion was given after 90 minutes of ischemia by interruption of the arterial and portal venous blood supply to the left lobes and middle lobes of the liver; HIR IL-10/saline treatment group in which recombinant murine I L-10(10mg/kg) or normal saline were injected via the dorsum vein of penis b efore ischemia and reperfusion; and sham control group in which midline laparotomy was performed without vascular occlusion and treatment. NF-kB activities of KCs were determined with EMSA. Expression levels of TNFαmRNA,IL-6mRNA and IL -1βmRNA of KCs were measured with RT-PCR. Serum levels of AL T and AST were measured.
RESULTS KCs NF-kB were activated 0-12h after reperfusion and activities of NF-kB were maximal 3h after reperfusion in HIR group. Expression levels of TNFαmRNA,IL-6 mRNA and IL-1βmRNA of KCs were incr eased markedly from 0 to 12h and peaked 3h after reperfusion in HIR group, and t he mRNA expression levels of KCs were significantly higher in HIR group than in sham control group. Serum levels of ALT and AST were increased significantly af ter reperfusion in HIR group rats compared with sham control group; KCs NF-kB activities were significantly lower in HIR IL-10 treatment group than in HIR g roup from 0 to 12h after reperfusion. Expression levels of TNFαmRNA, IL-6 mRNA and IL-1βmRNA of KCs were lowered markedly in HIR IL-10 trea tment group as compared with HIR group from 0 to 12h after reperfusion. Serum le vels of ALT and AST were decreased significantly after reperfusion in HIR IL-10 treatment group as compared with HIR group.
CONCLUSIONS NF-kB activation of KCs after HIR induces hepatic injury by promoting expressions of TNFαmRNA, IL-6mRNA and IL-1β mRNA of KCs. IL-10 protects against hepatic injury by suppressing excessiv e NF-kB activation of KCs and the subsequent mRNA expressions of proinflammator y mediators of KCs during HIR.
Subject headings Liver/pathology；Reperfusion injury/me tabolism；Reperfusion injury/physiopathology；Kupffer cells/metabolism；NF- κB/metabolism
Xu MQ, Xue L， Gong JP.Significance of Kupffer cell NF-kB activation d uring hepatic ischemia/reperfusion in rats.
Shijie Huaren Xiaohua Zazhi,2001;9( 11):1250-1253
摘 要目的 探讨Kupffer细胞(KCs)NF-kB激活在缺血再灌注肝损伤中的 作用.
方法 Wistar大鼠随机分为肝缺血再灌注(HIR)组、HIR IL-10治疗组 、HIR生理盐水治疗组及对照组.HIR组大鼠肝左叶及中肝叶缺血90min后再灌注，治疗组分别 于肝缺血前及再灌注前从阴茎背静脉注入重组鼠IL-10(10mg/kg)或生理盐水，对照组 仅显露肝门部血管但不进行缺血再灌注与治疗.分别采用EMSA法及RT-PCR法检测再灌注后0h 、1h、3h、6h及12h KCs NF-kB活性和KCs TNF-αmRNA、IL-6mRNA、IL-1βmRNA表达， 检测HIR 后0h与12h血清ALT及AST水平.
结果 HIR后0h～12h KCs NF-kB明显激活并于HIR后3h达到高峰；HIR后0 h～12h KCs TNFαmRNA、IL-6mRNA及IL-1βmRNA表达显著高于对照组，并于HIR后3h达到 高峰；HIR后0h与12h血ALT及AST活性明显高于对照组.IL-10治疗能显著降低HIR后KCs NF- kB活性及减少KCs源性 TNFαmRNA、IL-1βmRNA与IL-6mRNA表达，从而缓解HIR后肝损伤.
结论 HIR后KCs NF-kB高水平激活并促进KCs源性肝细胞损害递质的表达 从而促进肝损害.
主题词 肝/病理学；再灌注损伤/代谢；再灌注损伤/病理生 理学；枯否氏细胞/代谢；NF-κB/代谢 英文：
徐明清，薛兰，龚建平.缺血再灌注肝脏Kupffer细胞NF-kB激活及其意义.世界华 人消化杂志,2001;9(11):1250-1253
0 引言肝内核转录因子NF-kB的激活对肝脏缺血再灌 注(HIR)损伤起着十分重要的作用，NF-kB激活引发一系列炎症因子(TNF-α、IL-1及I L-6)及细胞因子源性中性粒细胞趣化物(CINC)的表达，从而介导肝细胞损伤[1，2 ]，但肝内何种细胞NF-kB激活导致HIR损伤尚不清楚.既往研究发现Kupffer细胞(KCs) 是肝内上述炎症递质的主要来源细胞[3]，而NF-kB激活则是KCs源性炎症因子产生 的关键信号传导[4]，但HIR损伤是否亦与KCs NF-kB激活有关、通过调控KCs NF- kB激活能否缓解HIR损伤，目前并未得到直接证明.
1 材料和方法
1.1 动物模型制作 健康♂Wistar大鼠，体重220g～2 50g，随机分为HIR组、HIR IL-10或生理盐水治疗组及对照组.HIR模型制作方法如下：大鼠 乙醚麻醉后开腹，阻断进入肝左叶与肝中叶的动静脉血流90min后恢复入肝血流，治疗组在 阻断肝血流前及再灌注前从阴茎背静脉注入重组鼠IL-10(10mg/kg)或无菌生理盐水， 对照组仅显露肝门血管但不阻断入肝血流.观察时相为HIR后0h、1h、3h、6h及12h，每时相 点检测6只大鼠.
1.2 KCs分离与培养 采用Wanner[5]介 绍的Seglen的原位灌注胶原酶消化法分离、培养并收集KCs待测.
1.3 EMSA检测KCs核NF-kB激活 按Chang CK等[6 ]介绍的方法提取KCs核蛋白，参照Jefferson等[7]介绍的方法进行EMSA测定.NF -kB寡核苷酸探针(序列为：5’-GCC TCC AAT GTT CGC GAA CTT TCG-3’)购于Santa Cruz生物公司，32P标记.结果判定：测定每条电泳滞后带的吸光度A值，所得结 果表示核转录因子NF-kB相对活性.
1.4 RT-PCR法检测KCs TNFα IL-6及IL-1βmRNA表达 KCs总RNA提取.二步法RT-PCR扩增.引物序列参照文献[8]设计，由上海生工合成.扩增 产物大小为TNFα 346bp、IL-1β378bp、IL-6 298bp、β2-Microglobulin 231bp， 其中β2-Microglobulin为参照基因.引物退火温度为60℃，扩增30个循环.扩增产物在2% 琼脂糖凝胶上电泳.以目的基因mRNA扫描密度值/参照基因mRNA扫描密度值的百分数(%) 作为目的基因mRNA的相对表达量.
1.5 血ALT及AST水平检测 运用美国贝克曼公司的CX7全 自动生化分析仪检测血清ALT、AST水平，试剂盒购自美国贝克曼公司.统计学处理 所有实验结果数据资料以x±s表达，差 异显著性采用t检验.
2 结果
2.1 HIR后KCs NF-kB活性变化 HIR后0h～12h KCs NF-kB均保 持激活状态，KCs NF-kB活性明显高于对照组，并于HIR后3h 达到峰值.采用IL-10治疗能 显著下调HIR后KCs NF-kB活性(见表1，图1)，而生理盐水则无此作用.
图1 HIR后KCs NF-kB激活变化(EMSA) S道为对照组KCs NF-kB激活 , 1、3、5、7道，2、4、6、8道分别为HIR组、HIR+IL-10组HIR后0h、3h、6h、12h KCs N F-kB激活.
图2 HIR后KCs TNFα、IL-6、 IL-1βmRNA表达(RT-PCR)M：mark er；S泳道为对照组KCs TNFα、IL-6、 IL-1β mRNA表达,1、3泳道及2、4泳道分别为HIR 组、HIR+IL-10组HIR后0h、3h KCs TNFα、IL-6、 IL-1βmRNA表达.
2.2 HIR后KCs TNFαmRNA、IL-6mRNA及IL-1βmRNA表达 HIR后0～12h KCs TNFαmRNA、IL-6mRNA及IL-1βmRNA表达明显高于对照组，并于HIR 后3h达到最高值.IL-10治疗能明显降低HIR后KCs TNFα、IL-6及IL-1βmRNA表达(见表2 ～表4，图2)，而生理盐水无此作用.
2.3 血ALT、AST水平变化 如表5所示： HIR后0h、12h 血ALT、AST明显增高，而IL-10能显著降低HIR后血ALT、AST水平，说明IL-10通过下调KCs NF-kB活性而缓解HIR损伤.
表1 HIR后KCs NF-kB相对活性(A，x±s，n=6)
aP<0.001, vs对照组；bP<0.001, vs HIR组.
表2 HIR后KCs TNFαmRNA 表达变化(%，x±s，n=6)
aP<0.001, vs对照组； bP<0.05, cP<0.01, vs HIR组.
表3 HIR后KCs IL-6mRNA表达变化(%，x±s，n=6)
aP<0.001, vs对照组；bP<0.05, cP<0.01, vs HIR组.
表4 HIR后KCs IL-1βmRNA表达 (%，x±s，n=6)
aP<0.001, vs对照组； bP<0.05, cP<0.01, vs HIR组.
表5 HIR后 血ALT及AST水平(IU/L，x±s，n=6)
aP<0.001, vs对照组；bP<0.05, cP<0.001 , vsHIR组.
3 讨论HIR损伤仍然是肝部分切除及肝移植的 严重制约之一，他可导致肝脏功能衰竭，可使约4%的移植肝因缺血再灌注损伤而失去功能， 甚至可导致远隔器官衰竭及死亡.因此，进一步探讨HIR损伤机制对肝移植及肝部分切除术后 肝功能的保护具有特别重要的意义.NF-kB是近年发现的转录因子家族中的新成员，是细胞内最重要的核转录因子，他在LPS等 细胞外刺激介导的细胞信息的转录调控中起核心作用，参与多种基因的表达和调控，是细胞激活的标志.在静息细胞中，NF-kB二聚体通过非共价键的形式与其抑制蛋白(IkB)结合而 分隔在细胞质内，包括内质网应激在内的许多因素可激活NF-kB，激活后的NF-kB进入细胞 核，与DNA模块上的特异蛋白结合，诱导特异mRNA的产生，最后转录、产生和释放各种细胞 因子.最近研究发现NF-kB激活是HIR损伤的重要信号传导.Yoshidome et al[1]报 道 大鼠部分肝缺血90min再灌注后30min内肝内NF-kB核转移增高，HIR后4h NF-kB激活达到峰 值，肝内炎症递质如TNFα和巨噬细胞炎症蛋白-2(MIP-2)的产生亦随之增高，IL-10能 通过抑制肝内NF-kB激活及肝内炎症递质的产生而缓解HIR损伤.HIR过程中肝内NF-kB是通 过细胞内活性氧递质与Ca2+的增高从而导致IkB酪氨酸化而激活的[9].目前 肝内何种细胞NF-kB激活介导HIR损伤并不清楚，鉴于KCs是HIR损伤的重要介导细胞[1 -3,5,10,11]，以及NF-kB激活是内毒素血症和酒精性肝损害等模型中KCs活化并释放TN Fα、IL-6及IL-1β等炎症因子的重要信号传导[4,12]，可以推测KCs NF-kB激 活亦可能是HIR损伤的重要信号传导，通过调控KCs NF-kB活性应能缓解HIR损伤.为证实以上推论，我们检测了部分肝缺血90min再灌注后肝KCs NF-kB激活调控对HIR损伤的 影响，实验发现HIR后0～12h KCs NF-kB均保持激活状态，KCs NF-kB活性于HIR后3h达到 高峰，HIR后12h前KCs NF-kB活性均明显高于对照组，伴随着KCs NF-kB的激活，KCs源性T NFαmRNA、IL-6mRNA 及IL-1βmRNA表达均显著增高，同时血ALT及AST水平增高，提示有H IR损伤的存在；IL-10治疗能明显下调HIR后KCs NF-kB活性、减少KCs 源性促炎症因子(T NFα、IL-6、IL-1β)mRNA表达并降低血ALT及AST水平，提示IL-10通过调控KCs NF-kB激活而缓解HIR损伤.实验结果表明HIR后KCs NF-kB高水平激活导致KCs 源性TNFα、IL-6 及IL-1β等肝损害炎症因子mRNA表达增高，从而促进肝缺血再灌注损伤，这就说明KCs NF -kB激活是HIR损伤的重要信号传导.IL-10能缓解大鼠肝移植过程中的肝损伤和LPS导致的实验性肝损伤[13]，虽然 其机制尚不清楚，但可能与IL-10抑制KCs合成TNFα及TNFα源性细胞因子有关.IL-10是强 有力的抗炎因子，他能抑制Th1 细胞、单核/巨噬细胞及中性粒细胞源性促炎症因子的合成 ，还能抑制巨噬细胞NO和活性氧中间产物的产生，从细胞信号传导途径方面他能抑制NF-kB的激活[1].我们在实验中证实IL-10抑制KCs源性TNFα、IL-1及IL-6的产生从而 缓解HIR损伤是通过抑制KCs NF-kB激活而实现的，这就提示KCs NF-kB激活确是HIR损伤 的重要机制之一，通过调节KCs NF-kB活性可缓解HIR损伤，从而为临床肝移植及肝部分切 除过程中肝损伤的防治提供可能的理论依据.
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13 Louis H,Le Moine O,Peny MO.Hepatoprotective role of IL-10 in galactosamine/LPS mouse live injury. Gastroenterology，1997;112:935-942, 百拇医药(徐明清1 薛 兰2 龚建平1)
2解放军第208医院 吉林省长春市 130062
徐明清，男，1968-11-25生，四川省乐山市人，汉族，1989年第三军医大学 毕业，2000年获第三军医大学博士学位，主治医师，讲师，主要从事肝脏疾病的研究，发表 论文6篇.
项目负责人 徐明清，400016，重庆市袁家岗医学院路1号重庆医科 大学附一院普外科.
Telephone: 13022303642
收稿日期 2001-01-08 接受日期 2001-01-11
Significance of Kupffer cell NF-kB activation during hepatic ischemia /reperfusion in rats
Ming-Qing Xu1, Lan Xue2 and Jian-Ping Gong1
1Department of General Surgery,First Affiliated Hospital,Chong qing University of Medical science, Chongqing 400016,China.
2The 208th hospital of PLA, Changchun 130062, Jilin Province, China.
Correspondence to: Xu Ming-Qing, Department of General Surgery, First Affiliated Hospital, Chongqing University of Medical science, Chongqing 4 00016, China.
Received 2001-01-08 Accepted 2001-01-11
Abstract
AIM To explore the effect of Kupffer cells (KCs) NF-kB act ivation on liver injury during hepatic ischemia/reperfusion(HIR) in rats.
METHODS Wistar rats were divided randomly into HIR group in whi ch hepatic reperfusion was given after 90 minutes of ischemia by interruption of the arterial and portal venous blood supply to the left lobes and middle lobes of the liver; HIR IL-10/saline treatment group in which recombinant murine I L-10(10mg/kg) or normal saline were injected via the dorsum vein of penis b efore ischemia and reperfusion; and sham control group in which midline laparotomy was performed without vascular occlusion and treatment. NF-kB activities of KCs were determined with EMSA. Expression levels of TNFαmRNA,IL-6mRNA and IL -1βmRNA of KCs were measured with RT-PCR. Serum levels of AL T and AST were measured.
RESULTS KCs NF-kB were activated 0-12h after reperfusion and activities of NF-kB were maximal 3h after reperfusion in HIR group. Expression levels of TNFαmRNA,IL-6 mRNA and IL-1βmRNA of KCs were incr eased markedly from 0 to 12h and peaked 3h after reperfusion in HIR group, and t he mRNA expression levels of KCs were significantly higher in HIR group than in sham control group. Serum levels of ALT and AST were increased significantly af ter reperfusion in HIR group rats compared with sham control group; KCs NF-kB activities were significantly lower in HIR IL-10 treatment group than in HIR g roup from 0 to 12h after reperfusion. Expression levels of TNFαmRNA, IL-6 mRNA and IL-1βmRNA of KCs were lowered markedly in HIR IL-10 trea tment group as compared with HIR group from 0 to 12h after reperfusion. Serum le vels of ALT and AST were decreased significantly after reperfusion in HIR IL-10 treatment group as compared with HIR group.
CONCLUSIONS NF-kB activation of KCs after HIR induces hepatic injury by promoting expressions of TNFαmRNA, IL-6mRNA and IL-1β mRNA of KCs. IL-10 protects against hepatic injury by suppressing excessiv e NF-kB activation of KCs and the subsequent mRNA expressions of proinflammator y mediators of KCs during HIR.
Subject headings Liver/pathology；Reperfusion injury/me tabolism；Reperfusion injury/physiopathology；Kupffer cells/metabolism；NF- κB/metabolism
Xu MQ, Xue L， Gong JP.Significance of Kupffer cell NF-kB activation d uring hepatic ischemia/reperfusion in rats.
Shijie Huaren Xiaohua Zazhi,2001;9( 11):1250-1253
摘 要目的 探讨Kupffer细胞(KCs)NF-kB激活在缺血再灌注肝损伤中的 作用.
方法 Wistar大鼠随机分为肝缺血再灌注(HIR)组、HIR IL-10治疗组 、HIR生理盐水治疗组及对照组.HIR组大鼠肝左叶及中肝叶缺血90min后再灌注，治疗组分别 于肝缺血前及再灌注前从阴茎背静脉注入重组鼠IL-10(10mg/kg)或生理盐水，对照组 仅显露肝门部血管但不进行缺血再灌注与治疗.分别采用EMSA法及RT-PCR法检测再灌注后0h 、1h、3h、6h及12h KCs NF-kB活性和KCs TNF-αmRNA、IL-6mRNA、IL-1βmRNA表达， 检测HIR 后0h与12h血清ALT及AST水平.
结果 HIR后0h～12h KCs NF-kB明显激活并于HIR后3h达到高峰；HIR后0 h～12h KCs TNFαmRNA、IL-6mRNA及IL-1βmRNA表达显著高于对照组，并于HIR后3h达到 高峰；HIR后0h与12h血ALT及AST活性明显高于对照组.IL-10治疗能显著降低HIR后KCs NF- kB活性及减少KCs源性 TNFαmRNA、IL-1βmRNA与IL-6mRNA表达，从而缓解HIR后肝损伤.
结论 HIR后KCs NF-kB高水平激活并促进KCs源性肝细胞损害递质的表达 从而促进肝损害.
主题词 肝/病理学；再灌注损伤/代谢；再灌注损伤/病理生 理学；枯否氏细胞/代谢；NF-κB/代谢 英文：
徐明清，薛兰，龚建平.缺血再灌注肝脏Kupffer细胞NF-kB激活及其意义.世界华 人消化杂志,2001;9(11):1250-1253
0 引言肝内核转录因子NF-kB的激活对肝脏缺血再灌 注(HIR)损伤起着十分重要的作用，NF-kB激活引发一系列炎症因子(TNF-α、IL-1及I L-6)及细胞因子源性中性粒细胞趣化物(CINC)的表达，从而介导肝细胞损伤[1，2 ]，但肝内何种细胞NF-kB激活导致HIR损伤尚不清楚.既往研究发现Kupffer细胞(KCs) 是肝内上述炎症递质的主要来源细胞[3]，而NF-kB激活则是KCs源性炎症因子产生 的关键信号传导[4]，但HIR损伤是否亦与KCs NF-kB激活有关、通过调控KCs NF- kB激活能否缓解HIR损伤，目前并未得到直接证明.
1 材料和方法
1.1 动物模型制作 健康♂Wistar大鼠，体重220g～2 50g，随机分为HIR组、HIR IL-10或生理盐水治疗组及对照组.HIR模型制作方法如下：大鼠 乙醚麻醉后开腹，阻断进入肝左叶与肝中叶的动静脉血流90min后恢复入肝血流，治疗组在 阻断肝血流前及再灌注前从阴茎背静脉注入重组鼠IL-10(10mg/kg)或无菌生理盐水， 对照组仅显露肝门血管但不阻断入肝血流.观察时相为HIR后0h、1h、3h、6h及12h，每时相 点检测6只大鼠.
1.2 KCs分离与培养 采用Wanner[5]介 绍的Seglen的原位灌注胶原酶消化法分离、培养并收集KCs待测.
1.3 EMSA检测KCs核NF-kB激活 按Chang CK等[6 ]介绍的方法提取KCs核蛋白，参照Jefferson等[7]介绍的方法进行EMSA测定.NF -kB寡核苷酸探针(序列为：5’-GCC TCC AAT GTT CGC GAA CTT TCG-3’)购于Santa Cruz生物公司，32P标记.结果判定：测定每条电泳滞后带的吸光度A值，所得结 果表示核转录因子NF-kB相对活性.
1.4 RT-PCR法检测KCs TNFα IL-6及IL-1βmRNA表达 KCs总RNA提取.二步法RT-PCR扩增.引物序列参照文献[8]设计，由上海生工合成.扩增 产物大小为TNFα 346bp、IL-1β378bp、IL-6 298bp、β2-Microglobulin 231bp， 其中β2-Microglobulin为参照基因.引物退火温度为60℃，扩增30个循环.扩增产物在2% 琼脂糖凝胶上电泳.以目的基因mRNA扫描密度值/参照基因mRNA扫描密度值的百分数(%) 作为目的基因mRNA的相对表达量.
1.5 血ALT及AST水平检测 运用美国贝克曼公司的CX7全 自动生化分析仪检测血清ALT、AST水平，试剂盒购自美国贝克曼公司.统计学处理 所有实验结果数据资料以x±s表达，差 异显著性采用t检验.
2 结果
2.1 HIR后KCs NF-kB活性变化 HIR后0h～12h KCs NF-kB均保 持激活状态，KCs NF-kB活性明显高于对照组，并于HIR后3h 达到峰值.采用IL-10治疗能 显著下调HIR后KCs NF-kB活性(见表1，图1)，而生理盐水则无此作用.
图1 HIR后KCs NF-kB激活变化(EMSA) S道为对照组KCs NF-kB激活 , 1、3、5、7道，2、4、6、8道分别为HIR组、HIR+IL-10组HIR后0h、3h、6h、12h KCs N F-kB激活.
图2 HIR后KCs TNFα、IL-6、 IL-1βmRNA表达(RT-PCR)M：mark er；S泳道为对照组KCs TNFα、IL-6、 IL-1β mRNA表达,1、3泳道及2、4泳道分别为HIR 组、HIR+IL-10组HIR后0h、3h KCs TNFα、IL-6、 IL-1βmRNA表达.
2.2 HIR后KCs TNFαmRNA、IL-6mRNA及IL-1βmRNA表达 HIR后0～12h KCs TNFαmRNA、IL-6mRNA及IL-1βmRNA表达明显高于对照组，并于HIR 后3h达到最高值.IL-10治疗能明显降低HIR后KCs TNFα、IL-6及IL-1βmRNA表达(见表2 ～表4，图2)，而生理盐水无此作用.
2.3 血ALT、AST水平变化 如表5所示： HIR后0h、12h 血ALT、AST明显增高，而IL-10能显著降低HIR后血ALT、AST水平，说明IL-10通过下调KCs NF-kB活性而缓解HIR损伤.
表1 HIR后KCs NF-kB相对活性(A，x±s，n=6)
| 组别 | 0h | 1h | 3h | 6h | 12h |
| 对照组 | 0.04±0.03 | 0.05±0.04 | 0.02±0.01 | 0.03±0.02 | 0.04±0. 02 |
| HIR组 | 4.38±0.82a | 6.81±1.94a | 11.36±2.19a | 8.42±1.65a | 5.76±1.88a |
| HIR+IL-10组 | 1.54±0.66b | 2.68±0.87b | 4.36±1.21b | 3.11±1.09b | 1.35±0.42b |
aP<0.001, vs对照组；bP<0.001, vs HIR组.
表2 HIR后KCs TNFαmRNA 表达变化(%，x±s，n=6)
| 组别 | 0h | 1h | 3h | 6h | 12h |
| 对照组 | 13.2±5.3 | 10.5±4.1 | 9.2±3.4 | 12.6±6.3 | 8.6±3.2 |
| HIR组 | 32.5±7.5a | 44.8±10.6a | 63.7±12.8a | 47.6±10.2a | 39.2±8.3a |
| HIR+IL-10组 | 20.3±6.2b | 29.5±9.6b | 41.8±9.2c | 32.4±8.3b | 25.8±5.7c |
aP<0.001, vs对照组； bP<0.05, cP<0.01, vs HIR组.
表3 HIR后KCs IL-6mRNA表达变化(%，x±s，n=6)
| 组别 | 0h | 1h | 3h | 6h | 12h |
| 对照组 | 12.4±5.9 | 11.7±5.2 | 9.8±3.1 | 13.2±6.8 | 12.8±5.8 |
| HIR组 | 30.8±7.2a | 45.6±10.5a | 60.4±11.6a | 51.2±8.6a | 36.7±6.7a |
| HIR+IL-10组 | 19.2±6.3b | 28.8±8.9b | 42.9±10.2b | 35.7±7.6c | 25.2±5.3c |
aP<0.001, vs对照组；bP<0.05, cP<0.01, vs HIR组.
表4 HIR后KCs IL-1βmRNA表达 (%，x±s，n=6)
| 组别 | 0h | 1h | 3h | 6h | 12h |
| 对照组 | 10.5±6.2 | 12.5±5.8 | 11.8±6.7 | 14.1±7.1 | 13.6±5.2 |
| HIR组 | 29.4±8.1a | 42.1±10.5a | 64.6±11.6a | 52.5±10.8a | 36.4±7.6a |
| HIR+IL-10组 | 18.8±6.5b | 27.8±7.3b | 42.7±8.4c | 33.4±8.1c | 24.7±6.4b |
aP<0.001, vs对照组； bP<0.05, cP<0.01, vs HIR组.
表5 HIR后 血ALT及AST水平(IU/L，x±s，n=6)
| 组别 | ALT | AST | ||
| 0h | 12h | 0h | 12h | |
| 对照组 | 45.25±13.58 | 34.82 ±11.47 | 53.51±16.13 | 61.72±13.95 |
| HIR组 | 136.73±46.82a | 435.18±53.34a | 241.62±44.25a | 557.84±65.73a |
| HIR+IL-10组 | 78.21±27.46b | 182.53±41.26c | 93.48±32.65c | 203.37±46.29c |
aP<0.001, vs对照组；bP<0.05, cP<0.001 , vsHIR组.
3 讨论HIR损伤仍然是肝部分切除及肝移植的 严重制约之一，他可导致肝脏功能衰竭，可使约4%的移植肝因缺血再灌注损伤而失去功能， 甚至可导致远隔器官衰竭及死亡.因此，进一步探讨HIR损伤机制对肝移植及肝部分切除术后 肝功能的保护具有特别重要的意义.NF-kB是近年发现的转录因子家族中的新成员，是细胞内最重要的核转录因子，他在LPS等 细胞外刺激介导的细胞信息的转录调控中起核心作用，参与多种基因的表达和调控，是细胞激活的标志.在静息细胞中，NF-kB二聚体通过非共价键的形式与其抑制蛋白(IkB)结合而 分隔在细胞质内，包括内质网应激在内的许多因素可激活NF-kB，激活后的NF-kB进入细胞 核，与DNA模块上的特异蛋白结合，诱导特异mRNA的产生，最后转录、产生和释放各种细胞 因子.最近研究发现NF-kB激活是HIR损伤的重要信号传导.Yoshidome et al[1]报 道 大鼠部分肝缺血90min再灌注后30min内肝内NF-kB核转移增高，HIR后4h NF-kB激活达到峰 值，肝内炎症递质如TNFα和巨噬细胞炎症蛋白-2(MIP-2)的产生亦随之增高，IL-10能 通过抑制肝内NF-kB激活及肝内炎症递质的产生而缓解HIR损伤.HIR过程中肝内NF-kB是通 过细胞内活性氧递质与Ca2+的增高从而导致IkB酪氨酸化而激活的[9].目前 肝内何种细胞NF-kB激活介导HIR损伤并不清楚，鉴于KCs是HIR损伤的重要介导细胞[1 -3,5,10,11]，以及NF-kB激活是内毒素血症和酒精性肝损害等模型中KCs活化并释放TN Fα、IL-6及IL-1β等炎症因子的重要信号传导[4,12]，可以推测KCs NF-kB激 活亦可能是HIR损伤的重要信号传导，通过调控KCs NF-kB活性应能缓解HIR损伤.为证实以上推论，我们检测了部分肝缺血90min再灌注后肝KCs NF-kB激活调控对HIR损伤的 影响，实验发现HIR后0～12h KCs NF-kB均保持激活状态，KCs NF-kB活性于HIR后3h达到 高峰，HIR后12h前KCs NF-kB活性均明显高于对照组，伴随着KCs NF-kB的激活，KCs源性T NFαmRNA、IL-6mRNA 及IL-1βmRNA表达均显著增高，同时血ALT及AST水平增高，提示有H IR损伤的存在；IL-10治疗能明显下调HIR后KCs NF-kB活性、减少KCs 源性促炎症因子(T NFα、IL-6、IL-1β)mRNA表达并降低血ALT及AST水平，提示IL-10通过调控KCs NF-kB激活而缓解HIR损伤.实验结果表明HIR后KCs NF-kB高水平激活导致KCs 源性TNFα、IL-6 及IL-1β等肝损害炎症因子mRNA表达增高，从而促进肝缺血再灌注损伤，这就说明KCs NF -kB激活是HIR损伤的重要信号传导.IL-10能缓解大鼠肝移植过程中的肝损伤和LPS导致的实验性肝损伤[13]，虽然 其机制尚不清楚，但可能与IL-10抑制KCs合成TNFα及TNFα源性细胞因子有关.IL-10是强 有力的抗炎因子，他能抑制Th1 细胞、单核/巨噬细胞及中性粒细胞源性促炎症因子的合成 ，还能抑制巨噬细胞NO和活性氧中间产物的产生，从细胞信号传导途径方面他能抑制NF-kB的激活[1].我们在实验中证实IL-10抑制KCs源性TNFα、IL-1及IL-6的产生从而 缓解HIR损伤是通过抑制KCs NF-kB激活而实现的，这就提示KCs NF-kB激活确是HIR损伤 的重要机制之一，通过调节KCs NF-kB活性可缓解HIR损伤，从而为临床肝移植及肝部分切 除过程中肝损伤的防治提供可能的理论依据.
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