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100023 北京市2345信箱	世界华人消化杂志 2001年11月15日;9(11):1242-1245
Email: wcjd@public.bta.net.cn	世界华人消化杂志 ISSN 1009-3079 CN 14-1260/R
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⊙研究原著⊙

实验性大鼠肝纤维化进程中TNF-α、IL-6与IL-10的作用

李丹张莉娟陈治新黄月红王小众

福建医科大学附属协和医院消化内科福建省福州市 350001
李丹，女，1976-11-22生，福建省福清市人，汉族，1999年福建医科大学临床医疗系毕业.同年考取福建医科大学硕士研究生，主要从事肝脏疾病研究.
福建省教委基金资助项目. No. JA 01070
项目负责人 王小众, 350001, 福州市新权路29号福建医科大学附属协和医院消化内科. drwangxz@public6.fz.fj.cn
Telephone: 0591-3357896 Ext 8482
收稿日期 2001-07-05 接受日期 2001-07-11

Effects of TNFα, IL-6 and IL-10 on the development of experimental r at liver fibrosis

Dan Li, Li-Juan Zhang, Zhi-Xin Chen, Yue-Hong Huang and Xiao-Zhong Wang
Department of Gastroenterology, Union Hospital, Fu Jian Medical Univ ersity, Fu zhou 350001, Fu jian Province, China
Supported by the Fund of Education Commision, Fu jian Province, China No . JA 01070
Correspondence to: Dr. Xiao-Zhong Wang, Department of Gastroent erology, Union Hospital, Fu Jian Medical University, Fu zhou 350001, Fu jian Pro vince, China
Received 2001-07-05 Accepted 2001-07-11

Abstract
AIM To investigate the role of tumor necrosis factorα, inte rleukin-6 and interleukin-10 on experimental rat liver fibrosis.

METHODS One hundred clean SD rats were divided randomly into 3 groups: control group (intraperitoneal injection of saline 2mL·kg^-1,2· wk^-1), model group(intraperitoneal injection of 500mL·L^-1CCl₄ 2 mL·kg^-1,2·wk^-1), IL-10 group (in addition to the same dosage o f CCl₄, intraperitoneal injection of IL-10 4μg·kg^-1 was made from week 3). In week 5, 7 and 9, the rats of the three groups were sel ected randomly to collect plasma and resect liver. The serum levels of TNF-α, IL-6 and IL-10 were measured by enzyme linked immunosorbent assay (ELISA), li ver tissues were made into paraffin section with sliver staining.

RESULTS The CCl₄-induced experimental rat hepatic fibrosis model was successfully established and liver fibrosis was developed with the inj ection of CCl₄. The TNF-α, IL-6 and IL-10 levels in serum of group C were 15.2±3.8ng·L^-1，63.6±33.0ng·L^-1 and 132.9±22.1ng·L^-1 respectively; group M were 18.9±5.3ng·L^-1, 89.1±25.4ng·L^-1 an d 57.6±18.9ng·L^-1 respectively; group T were 14.0±1.9ng·L^-1, 7 4.7±21.2ng·L^-1 and 88.2±20.8ng·L^-1. The TNF-α levels in group M at week 5,7, 9 were 16.5±2.7ng·L^-1, 17.1±0.9ng·L^-1, 22.6±6 .8 ng·L^-1 and 13.4±1.0ng·L^-1，13.6±1.2ng·L^-1，14.5±2. 9g·L^-1 in group T respectively; the IL-6 levels in group M at three sta ge were 63.8±12.2ng·L^-1, 71.5±16.1ng·L^-1, 112.3±20.2ng·L ^-1 and 63.1±13.1ng·L^-1, 70.1±11.8ng·L^-1, 85.8±11.6ng·L ^-1 in group Trespectively; the IL-10 levels in group Min the three stage w ere 82.1±5.2ng·L^-1, 65.7±8.4ng·L^-1 and 50.0±8.5ng·L^-1 . The levels of TNF-α and IL-6 in group M were significantly higher than tha t in group C and group T（P<0.05） and tended to increase in the progress of hepatic fibrosis, however, the levels of TNF-α and IL-6 in group T after tre ated by IL-10 were significantly lower than that in group M（P<0.05）. The level of IL-10 in group M was significantly lower than that in group C（P<0 .05）.

CONCLUSIONS TNF-α, IL-6 and IL-10 play important roles in t he progress of hepatic fibrosis, IL-10 has fibrogenesis-inhibiting effect on the CCl₄-induced rat hepatic fibrosis.

Subject headings Liver cirrhosis, experimental/physiopath ology; tumor necrosis factor; Interleukin-6; interleukin-10

Li D, Zhang LJ, Chen ZX, Huang YH，Wang XZ.Effects of TNFα, IL-6 and IL-10 on the development of experimental rat liver fibrosis.
Shijie Huaren Xiao hua Zazhi,2001;9(11):1242-1245

摘要
目的探讨TNF-α，IL-6及IL-10在实验性大鼠肝纤维化进程中的作用.

方法清洁级♂SD大鼠100只，随机分为3组.正常对照组（C组）24只，腹腔内注射生理盐水2mL·kg^-1 ，2次·wk^-1；模型组（M组）40只及治疗组（ T组）36只，腹腔内注射500mL·kg^-1CCl₄,2mL·kg^-1，2次·wk^-1 ；E组从3wk起，注射CCl₄前腹腔内注射IL-10 4ug·kg^-1.于5wk, 7wk, 9wk分别随机选取C组动物7只，M, E组动物各10只颈动脉插管取血，留取肝脏，ELISA法测定血清中T NF-α, IL-6及IL-10水平,肝脏常规石蜡切片，银染.

结果肝脏病理组织学检查证实成功构建CCl₄诱导的实验性大鼠肝纤维模型，且随注射CCl₄次数增多，肝纤维化程度逐渐加重，同时期动物T组较M组肝纤维化程度减轻.C组血清TNF-α、IL-6及IL-10水平分别为(15.2±3.8)ng·L^-1，(63.6± 33.0) ng·L^-1和(132.9±22.1) ng·L^-1；M组水平分别为(18.9±5.3)ng· L^-1, (89.1±25.4)ng·L^-1和(57.6±18.9)ng·L^-1；T组水平分别为(14.0±1.9)ng·L^-1,( 74.7±21.2)ng·L^-1及(88.2±20.8)ng·L^-¹.wk5, 7,9，M组TNF-α动态变化水平分别为(16.5±2.7)ng·L^-1,(17.1±0.9)ng·L^-1,(22.6±6.8)ng·L^-1；T组水平分别为(13.4±1.0)ng·L^-1， (13.6±1.2)ng·L^-1，(14.5±2.9)ng·L^-1；M组IL-6动态变化水平分别为 (63.8±12.2)ng·L^-1, (71.5±16.1)ng·L^-1, (112.3±20.2)ng·L^-¹, T组水平分别为(63.1±13.1)ng·L^-1, (70.1±11.8)ng·L^-1, (85.8 ±11.6)ng·L^-1；M组IL-10动态变化水平分别为(82.1±5.2)ng·L^-1, ( 65.7±8.4)ng·L^-1，(50.0±8.5)ng·L^-1.M组TNF-α及 IL-6水平显著高于C组和T组（P<0.05），且在肝纤维化进程中呈动态上升趋势，经IL-10干预后，二者水平下降与M组有明显差异（P<0.05）.M组IL-10水平低于N组（P<0.05），在肝纤维化进程中呈动态下降趋势.

结论 TNF-α，IL-6升高及IL-10降低在肝纤维化进程中起重要促进作用，予以外源性IL-10对CCl₄诱导的肝纤维化有明显拮抗作用.

主题词 肝硬化，实验性/病理生理学；肿瘤坏死因子；白介素6 ；白介素10

李丹，张莉娟，陈治新，黄月红，王小众.实验性大鼠肝纤维化进程中TNF -α、IL-6与IL-10的作用.世界华人消化杂志,2001;9(11):1242-1245

0 引言
肝纤维化（liver fibrosis, LF）是各种原因引起的慢性肝损伤所共有的病理改变，其特征为大量细胞外基质（ECM）在Disse间隙沉积，被视为肝硬变的早期阶段^［^1-3^].近年来，随着细胞生物学和免疫学的进展，越来越多的证据表明，各种细胞因子通过复杂的调节机制在肝纤维化的进程中扮演重要角色^［^4-6^].我们建立CCl₄诱导的大鼠肝纤维化模型并行IL-10干预实验，检测各组大鼠血清中TNF-α，IL-6及IL-10水平，旨在探讨细胞因子TNF-α、IL-6及IL-10在肝纤维化进程中的作用.

1 材料和方法
1.1 材料清洁级♂SD大鼠100只, 体质量140g-180g ，购自上海实验动物中心，随机分为3组.正常对照组（C组）24只，模型组（M组）40只,治疗组（T组）36只，按常规条件饲养温度（22±2）℃，湿度（55±5）%，每天人工照明12h ，自由取食取水，饲料由上海BK公司提供，水为饮用自来水.
1.2 方法正常对照组（C组）腹腔内注射生理盐水2mL ·kg^-1 ，2次·wk^-1；模型组（M组）及治疗组（T组）腹腔内注射500mL·L ^-1CCl₄ （溶于蓖麻油中）2mL·kg^-1，2次·wk^-1；T组从wk3起，注射CCl₄前20min腹腔内注射IL-10（溶于生理盐水中）4μg·kg^-1，2次·wk ^-1.每周一、四注射，注射前称质量.至wk5 M组动物死亡3只，T组动物死亡2只，至wk7 M组动物共死亡8只，T组动物共死亡4只，至wk9 M组动物共死亡10只，T组动物共死亡6只， C组大鼠共死亡3只.于wk5，wk7，wk9分别随机选取C组动物7只，M、T组动物各10只,10mg·k g-1氯氨酮腹腔内麻醉，颈动脉插管取血，留取肝脏，40g·L^-1甲醛固定，常规石蜡切片，银染.ELISA法测定血清中TNF-α, IL-6及IL-10水平，大鼠TNF-α，IL -6，IL-10药盒购自美国Endogen公司，操作按说明书进行. 统计学处理数据均采用x±s表示，统计采用t检验方法，数据分析软件为SPSS软件.

2 结果
2.1 血浆TNF-α, IL-6和IL-10水平模型组大鼠血浆TNF-α和IL-6水平明显高于正常组（P<0.05），IL-10水平明显低于正常组（P<0.05），经IL-10治疗，实验组血浆TNF-α和IL-6水平较治疗组明显下降（P<0.05，表1), 且随着CCl₄注射次数增多，肝纤维化程度加重，血浆TNF-α和IL-6水平呈上升趋势，IL -10水平呈下降趋势（表2）.

表1 肝纤维化大鼠血浆TNF-α，IL-6 及IL-10水平比较 (x± s, ng·L^-1)

分组	n	TNF-α	IL-6	IL-10
C组	21	15.2±3.8	63.6 ±33.0	132.9±22.1
M组	30	18.9±5.3	89.1±25.4	57.6±18.9
T组	30	14.0±1.9	74.7±21.2	88.2±20.8

表2 大鼠血浆TNF-α，IL-6及IL-10动态变化 (x±s, ng·L^-1)

分组	n	TNF-α			IL-6			IL-10
分组	n	5wk	7wk	9wk	5wk	7wk	9wk	5wk	7wk	9wk
C组	7	15.3±3.5	15.0±3.8	15.4±3.8	62.8±10.2	61.8±11.3	63.5±10.9	131.9±18.2	129.6±8.5	126.8±12.2
M组	10	16.5±2.7	17.1±0.9	22.6±6.8	63.8±12.2	71.5±16.1	112.3±20.2	82.1±5.2	65.7±8.4	50.0±8.5
T组	10	13.4±1.0	13.6±1.2	14.5±2.5	63.1±13.1	70.1±11.8	85.8±11.6	101.1±8.9	93.2±7.7	80.7±7.4

2.2 肝脏病理组织形态学 随着CCl₄注射次数增多，肝纤维化程度逐渐加重, wk5以肝细胞脂肪变性，气球样变为主，并有少量纤维组织沉积，wk7除肝细胞脂肪变性，气球样变仍明显外，胶原纤维沿肝板延伸呈纤维隔状，但无完整分界形成，wk9肝纤维化继续加重，小叶间隔形成完整纤维隔，部分假小叶形成(图 1.2).经IL-10干预后，wk5肝组织病理改变不明显，wk7肝细胞坏死及脂肪变性程度减轻，w k9可见到小叶间隔胶原纤维减少，未见假小叶形成(图3).

图1 正常大鼠肝脏银染×100
图2 模型组wk9肝脏改变银染×100
图3 治疗组wk9肝脏改变银染×100

3 讨论
近年来，肝纤维化的形成机制研究取得了令人鼓舞的进展，越来越多的实验及临床证据表明，细胞因子在肝细胞再生的调控，肝星形细胞的激活，细胞外基质的合成与降解方面均有重要作用^［⁷^］，因此，细胞因子与肝纤维化的关系成为目前研究热点之一.TNF-α是在肝纤维化研究中最早受到关注的细胞因子之一，各种原因引起肝脏病变时，肝实质细胞和库普弗细胞在内毒素的介导下产生包括 TNF-α在内的多种细胞因子^［^8,9^].研究证实实验性动物模型^［¹⁰^］及肝炎后肝硬化患者^［^11-13^］血清TNF-α随纤维化程度增加呈上升趋势，而对每周注射刀豆蛋白A致肝纤维化的BALB/C小鼠以抗TNF-α抗体干预后，能阻止肝纤维化的发展 ^［14］.TNF-α通过多种途径参与肝纤维化的调控：作为重要的刺激原使肝星形细胞活化 ^[¹⁵^］，导致细胞外基质合成增加并分泌多种细胞因子^［^16-18^］；激活转录因子c-jun/AP-1，胶原酶表达下降^［¹⁹^］而基质金属蛋白酶表达升高^［²⁰^] ；刺激产生单核细胞趋化因子^［²¹^］和中性粒细胞趋化因子^［¹⁵^］；在转录抑制剂的协同作用下介导肝细胞的坏死^［²²^］和凋亡^［^23-25^]. 近来，Lang et al^[²⁶^］发现TNF-α对已活化的肝星形细胞可产生凋亡诱导，此作用能被TNF -α的主要效应物NF-kB所阻断.NF-kB对肝脏再生及炎症均有调节作用^［²⁷^],他能通过其基因产物协同作用抑制TNF-α诱发的Caspase-8激活来阻止凋亡^［²⁸^].肝成纤维细胞和活化的肝星形细胞是肝内IL-6的主要来源，TNF-α、LPS、IL-1β是上述细胞合成分泌的有效刺激物^［²⁹^］，NF-kB被抑制后IL-6的表达下降.国外研究表明，不同病因肝硬化患者IL-6含量均高于正常对照组^［^30，31^］，国内也报道同正常对照组相比，病毒性肝炎患者IL-6活性和可溶性白介素受体（sIL-6R)水平均明显升高，并指出，血清IL-6和sIL-6R升高同肝坏死密切相关^［³²^].Cresssman et al ^[³³^］发现IL-6基因敲除小鼠行部分肝切除后能导致肝坏死和肝衰竭，若术前单次注射IL-6，术后肝组织再生恢复近正常水平，提示IL-6是损伤后促进细胞增殖的重要物质. 目前认为IL-6主要通过直接刺激肝细胞上调TIMP-1表达^［³⁴^］及作为急性相反应蛋白的强力诱导剂调控蛋白酶、I 型前胶原的转录表达等参与肝纤维化的进程^［^35-38^] .本实验结果表明，模型组大鼠 TNF-α，IL-6水平高于正常对照组，且随肝纤维化程度加重而升高，予外源性IL-10干预后，肝纤维化程度减轻，二者水平也下降至同模型组有明显区别，提示TNF-α，IL-6升高在CCl₄诱导的大鼠肝纤维化中起重要作用.IL-10作为继IFN-γ之后又一抗纤维化细胞因子在新近研究中倍受瞩目^［^39,40^］，其主要由TH2细胞，巨噬细胞，星状细胞，肝细胞等产生.Thompson et al^［41］和Louis et al^[⁴²^］在1998年进行CCl₄诱导IL-10基因敲除小鼠肝纤维化实验中发现，IL-10-/-小鼠较野生型小鼠肝内炎症细胞侵润明显，肝纤维化程度要严重的多，故一致认为IL-10缺乏促进肝纤维化的发展.有证据表明，IL-10通过多种途径阻止肝纤维化的进展：抑制巨噬细胞合成分泌TNF-α^［⁴³^］及下调TNF-α下游效应物NF -kB表达^［⁴⁴^］；有效抑制促纤维化因子TGF-β1的产生^［^40,45^］；通过下调 I 型胶原的合成及促进胶原蛋白酶基因表达重构细胞外基质^［^46,47^］；广泛抑制多种细胞特别是单核细胞和巨噬细胞合成化学致炎因子^［^48,49^];急性期减轻炎症反应影响肝细胞坏死，修复期限制肝细胞再生而阻止肝细胞过度增殖反应.本研究中，肝纤维化组IL-10水平显著低于正常组，且随肝纤维化进展递减，予外源性IL-10干预后，病理证实肝纤维化程度明显减轻，且伴随TNF-α，IL-6水平下降，故有理由认为，外源性IL -10对CCl₄诱导的肝纤维化有明显拮抗作用，通过以NF-kB为中心环节的TNF信号环路调节TNF-α，IL-6生物学活性可能是IL-10发挥抗纤维化作用途径之一.近来已有IL-10应用于慢性丙型肝炎的报道^［⁵⁰^］，但例数尚少，因此，IL-10应用于慢性肝炎和肝纤维化的可靠性和临床疗效仍有待于进一步验证.

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