基础研究 Open Access
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
世界华人消化杂志. 2003-08-15; 11(8): 1168-1171
在线出版日期: 2003-08-15. doi: 10.11569/wcjd.v11.i8.1168
卡托普利对肝纤维化模型鼠MMP-2, 3 TIMP-2, 3表达的影响
李乾, 张桂英, 李新华, 徐美华
李乾, 张桂英, 李新华, 徐美华, 中南大学湘雅医院消化内科 湖南省长沙市 410008
基金项目: 湖南省卫生厅课题, No. Y 02-008
通讯作者: 张桂英, 410008, 湖南省长沙市湘雅路141号, 中南大学湘雅医消化内科. liqian0816@hotmail.com
电话: 0731-4327249
收稿日期: 2002-11-19
修回日期: 2002-11-30
接受日期: 2002-12-07
在线出版日期: 2003-08-15

目的

探讨卡托普利抗大鼠肝纤维化的作用及对MMP-2, MMP-3, TIMP-2, TIMP-3的表达影响.

方法

Wistar大鼠40只随机分为正常对照组, 实验对照组A、B, 卡托普利预防组、治疗组, 采用混合损害因素构建肝纤维化模型. 行HE和VG染色, 判断炎症和肝纤维化程度. 免疫组化检测MMP-2, MMP-3, TIMP-2, TIMP-3的表达.

结果

实验对照组A平均肝纤维化积分值为2.17±0.75、卡托普利预防组为1.33±0.52; 实验对照组B为2.86±0.69、卡托普利治疗组为1.67±0.82, 二者比较均P<0.05. 实验对照组A、卡托普利预防组的MMP-2阳性反应面积比分别为8.20±0.24%, 4.43±0.25%, 实验对照组B、治疗组分别为5.67±0.32%, 3.21±0.16%, 二者比较均P<0.01; 实验对照组A、卡托普利预防组MMP-3阳性反应面积比分别为1.54±0.36%, 4.25±0.37%, 实验对照组B、治疗组分别为3.69±0.27%, 10.75±1.69%, 二者比较均P<0.01; 实验对照组A、卡托普利预防组TIMP-2阳性反应面积比分别为3.61±0.46%, 2.16±0.17%, 实验对照组B、治疗组分别为6.68±0.52%, 7.87±0.59%, 二者比较均P<0.01; 实验对照组A、卡托普利预防组TIMP-3阳性反应面积比分别为4.13±0.29%, 3.06±0.28%, 实验对照组B、治疗组分别为8.54±0.45%, 5.35±0.34%, 二者比较均P<0.01.

结论

卡托普利可抑制MMP-2, TIMP-3蛋白表达, 增强MMP-3蛋白表达, 可能是其抗纤维化作用的机制之一.

关键词: N/A
引文著录: 李乾, 张桂英, 李新华, 徐美华. 卡托普利对肝纤维化模型鼠MMP-2, 3 TIMP-2, 3表达的影响. 世界华人消化杂志 2003; 11(8): 1168-1171
Effects of captopril on expression of MMP-2, 3 and TIMP-2, 3 in rat hepatic fibrosis
Qian Li, Gui-Ying Zhang, Xin-Hua Li, Mei-Hua Xu
Qian Li, Gui-Ying Zhang, Xin-Hua Li, Mei-Hua Xu, Department of Digestion, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
Correspondence to: Dr. Gui-Ying Zhang, Department of Digestion, Xiangya Hospital, Central South University, 141 Xiangya Road, Changsha 410008, Hunan Province, China. liqian0816@hotmail.com
Received: November 19, 2002
Revised: November 30, 2002
Accepted: December 7, 2002
Published online: August 15, 2003

AIM

To investigate the effects of captopril on the expression of MMP-2, 3 and TIMP-2, 3 in rat hepatic fibrosis.

METHODS

Forty healthy Wistar rats were randomly divided into five groups: normal control group, experiment group A, experiment group B, captopril-prevention group, captopril-treatment group. Hepatic fibrosis models were induced in the latter 4 groups by a combination of several factors. Liver sections were stained by hematoxylin-eosin and Van Gieson to evaluate the degree of inflammation and hepatic fibrosis. Expression of MMP-2, 3 and TIMP-2, 3 in liver were assayed by immunohistochemistry.

RESULTS

There was significant difference between captopril-prevention group(1.33±0.52)and experimental group A (2.17±0.75) on histologic assessment of hepatic fibrosis, and between captopril-treated group (1.67±0.82)and experimental group B (2.86±0.69) (P<0.05). The ratios of positive expression area of MMP-2 in captopril-prevention group and captopril-treatment group (4.43±0.25% and 3.21±0.16%, respectively) was smaller than in experimental group A and B (8.20±0.24% and 5.67±0.32%, respectively), (P<0.01). Expression of MMP-3 in experimental group A and experimental group B (1.54±0.36% and 3.69±0.27%, respectively) was weaker than in captopril-prevention group and captopril-treated group (4.25±0.37% and 10.75±1.69%, respectively) (P<0.01). Expression of TIMP-2 in captopril-prevention group (2.16±0.17%) was weaker than in experimental group A (3.61±0.46%)(P<0.01); but TIMP-2 in captopril-treatment group (7.87±0.59%) was stronger than in experimental group B (6.68±0.52%) (P<0.01). Expression of TIMP-3 in captopril-prevention group and captopril-treatment group (3.06±0.28% and 5.35±0.34%, respectively) was weaker than in experimental group A and experimental group B(4.13±0.29% and 8.54±0.45%, respectively) (P<0.01).

CONCLUSION

Captopril could suppress expression of MMP-2, TIMP-3, and enhance the expression of MMP-3, which might be related with its anti-hepatic fibrosis activity.

Key Words: N/A
0 引言

肝纤维化是一种或几种病因反复作用于肝脏, 组织发生修复反应导致细胞外基质(ECM)合成、降解与沉积平衡混乱而引起的病理过程, 其主要特征是ECM在肝脏的过度沉积. 卡托普利是一种血管紧张素转化酶抑制剂(ACEI), 能够抑制血管紧张素I向血管紧张素II的转化, 早期主要用于抗高血压治疗, 在临床使用过程中逐渐发现其能够逆转心脏、肾脏、肝脏的纤维化[1-5]. 但ACEI通过哪些环节起作用？是否与ECM合成与降解过程中起主要作用的基质金属蛋白酶(MMPs)和基质金属蛋白酶组织抑制物(TIMPs)有关？目前国内外未见报道. 我们通过ACEI处理大鼠肝纤维化模型, 检测肝脏组织中与肝纤维化密切相关的MMP-2, MMP-3, TIMP-2和TIMP-3的表达变化, 探讨ACEI抗大鼠肝纤维化的可能作用机制.

1 材料和方法
1.1 材料

同一品系清洁级健康Wistar大白鼠40只, 雌雄各半, 质量(180±45) g及普通饲料均由中南大学湘雅医学院实验动物学部提供; 卡托普利片, 江苏常州制药厂; 无水酒精, CCL4购自湖南师范大学生化试剂厂; 高脂饲料由850 g/kg普通饲料加150 g/kg的猪油组成.

1.2 方法

大鼠实验前自由喂养1 wk, 按雌雄随机分为5组: (1)正常对照组 (C) 6只: 正常普通饮食饮水; (2)实验对照组A(Ea)9只: 均予高脂饮食, 以无水乙醇每天给大鼠10 g/Kg加入饮水中代替饮水; 实验第1天予400 mL/L CCl4 菜籽油皮下注射4 mL/kg体重, 以后皮下注射3 mL/kg, 1次/wk; 生理盐水 2 mL灌胃1次/d, 共6 wk; (3)实验对照组B (Eb)9只: 造模条件同实验对照组A, 共12 wk; (4)卡托普利预防组(P)8只: 造模条件同实验对照组, 造模开始即每天给予卡托普利按100 mg/kg (溶于2 mL稀释液中)灌胃, 1次/d, 共6 wk; (5)卡托普利治疗组(T)8只: 造模条件同实验对照组, 造模12 wk, 从第7周起给予卡托普利, 给药剂量, 方法均同卡托普利预防组至造模结束. 动物均在20 °C左右环境温度, 明暗各12 h的清洁动物实验室内饲养. 冰冻切片分别予以HE和van Gieson (VG)染色. 按肝纤维结缔组织增生程度将肝纤维化分为0-4级[6] : 0级: 无肝纤维化; 1级: 纤维结缔组织增生仅局限于汇管区或有汇管区扩大, 有向小叶发展倾向; 2级: 纤维组织增生进入肝小叶2/3及有1级同样的改变; 3级: 纤维组织进入肝小叶中央静脉周围; 4级: 纤维组织在全小叶呈多处弥漫性增生, 有假小叶形成, 并有3级同样改变. MMP-2, MMP-3, TIMP-2, TIMP-3的免疫组化测定按试剂盒说明操作. 结果用彩色病理图像分析仪分析: 阳性产物主要位于胞质, 阳性判断标准为该位置出现棕黄色颗粒并测阳性反应面积比, 每张切片选取四周及中央5个区域, 均取该区域中阳性反应最多的视野20倍物镜下测定阳性反应面积比(阳性面积/肝组织面积×100%), 取平均值[7].

统计学处理 计量资料以mean±SD表示, 运用统计软件SPSS 10.0进行方差齐性检验, 并在此基础上进行方差分析, 以P<0.05为差异具有显著性.

2 结果
2.1 大鼠肝纤维化组织学分级

正常对照组大鼠VG胶原染色仅见少许纤细纤维分布于血管壁; 实验对照组大鼠VG胶原染色见汇管区大量胶原纤维和网状纤维沉积, 相互连接形成纤维间隔, 小部分重新分割肝小叶形成假小叶; 卡托普利预防组、治疗组大鼠VG胶原染色仅于汇管区可见轻度增生的胶原纤维和网状纤维及纤维间隔吸收后的残留. 按纤维化分级标准, 卡托普利预防组1.33±0.52、治疗组1.67±0.82平均纤维化积分分别与实验对照组A 2.17±0.75, B 2.86±0.69比较降低 P<0.05 (图1, 图2).

图1
图1 Eb组大鼠肝组织VG染色×100.
图2
图2 T组大鼠肝组织VG染色×100.
2.2 大鼠肝组织MMP-2, TIMP-2, MMP-3, TIMP-3的表达

正常对照组MMP-2, TIMP-2, MMP-3, TIMP-3的阳性表达极少; 实验对照组A、对照组B、卡托普利预防组、治疗组的MMP-2, MMP-3阳性表达均增加, 主要分布于血管内皮细胞、窦周内皮细胞及肝星状细胞的胞质中; 其中实验对照组B的MMP-2阳性表达低于对照组A, 卡托普利预防组、治疗组的MMP-2阳性表达均低于对应的实验对照组A、对照组B; 实验对照组B的MMP-3阳性表达高于对照组A, 卡托普利预防组、治疗组MMP-3阳性表达分别高于实验对照组A、对照组B. 实验对照组A、对照组B、卡托普利预防组、治疗组TIMP-2、TIMP-3阳性表达均增加, 主要分布于窦周内皮细胞及肝星状细胞胞质中; 其中实验对照组B的TIMP-2阳性表达高于对照组A, 卡托普利预防组TIMP-2阳性表达低于对照组A, 而卡托普利治疗组阳性表达高于对照组B; 实验对照组B的TIMP-3阳性表达高于A, 卡托普利预防组、治疗组TIMP-3阳性表达分别低于对应实验对照组A、对照组B(表1, 图3, 图4).

表1 大鼠肝组织MMP-2, MMP-3, TIMP-2, TIMP-3的阳性表达面积比(％ mean±SD).
分组MMP-2TIMP-2MMP-3TIMP-3
C0.17±0.050.13±0.050.21±0.080.18±0.06
Ea8.20±0.243.61±0.461.54±0.364.13±0.29
P4.43±0.25b2.16±0.17b4.25±0.37b3.06±0.28b
Eb5.67±0.32b6.68±0.52b3.69±0.27b8.54±0.45b
T3.21±0.16d7.87±0.59d10.75±1.69d5.35±0.34d
bP<0.01 vs Ea;
dP<0.01 vs Eb.
图3
图3 Eb组大鼠肝组织MMP-3表达×400.
图4
图4 T组大鼠肝组织MMP-3表达×400.
3 讨论

AngII在心、肾纤维化形成过程中的作用已得到证实[8,9], 如环孢素引起的肾小管间质纤维化可被ACEI所减轻[10]; 同样, ACEI可减轻梗阻性肾病大鼠的IV型胶原的表达和间质的沉积[11-13] . 有研究证实心, 肾间质纤维母细胞和HSC均表达AT1受体[14-16]. 早期在体研究也表明血管紧张素II可以通过AT1受体增加心肌成纤维细胞的I, III型胶原, 前胶原α1和纤维连接素的mRNA表达水平[10]. 近期国内外有研究表明ACEI类及血管紧张素II受体拮抗剂亦具有抗肝纤维化作用, 但均是在肝纤维化形成前即予以药物处理[17,18]. Ohishi et al [3]用lisinopril预处理CCL4诱导的肝纤维化实验, 能减缓肝纤维化的发生, 并发现血管紧张素II能上调前胶原α1和TGF-β1的mRNA表达水平. 从临床应用角度看, 治疗肝纤维化有效的药物应该能阻止已形成的肝纤维化的进一步发展、甚至逆转肝纤维化. 本实验中无论是预防组抑或治疗组在肝细胞坏死, 脂肪变性, 炎性细胞浸润、胶原纤维增生等病理组织学方面, 及按纤维化分级标准所得到的平均纤维化积分方面均较相应实验对照组明显改善, 在治疗组甚至可见到纤维间隔吸收后的残留痕迹, 结果显示卡托普利不仅可以预防肝纤维的发生, 而且可以逆转肝纤维化. 提示血管紧张素II可能是肝纤维化发生、发展的重要递质, 使用卡托普利能够阻止肝纤维化的发生、发展, 甚至逆转肝纤维化.

Benyon et al发现离体HSC受损早期短暂表达MMP-3[16,18], MMP-13和PA, 呈现基质降解相. 在大鼠和人类慢性肝病, 均存在MMP-1下调和MMP-2, MMP-9上调, 导致基底膜胶原降解增加, 间质胶原降解减少[19-23]. Ninomiya et al用干扰素治疗慢性丙型肝炎后肝纤维化患者[24,25], 发现有纤维化逆转的患者血清MMP-1/TIMP-1比值增加, 而非干扰素治疗组血清MMP-1/TIMP-1比值下降; Rooprai et al [26]在肿瘤研究中也发现卡托普利能降低MMP-2蛋白和基因的表达水平. 我们发现MMP-2在肝纤维化早期表达明显, 而MMP-3的表达随着肝纤维化的进展逐渐增加, 同时卡托普利预防组、治疗组的MMP-2表达明显低于相应实验对照组, MMP-3的表达高于相应实验对照组. 在肝纤维化过程中MMP-2与MMP-3表达的不一致, 提示不同的MMPs对纤维化的形成作用不同. 卡托普利能够抑制降解基底膜胶原的明胶酶MMP-2的表达, 而增加降解间质胶原的MMP-3的表达, 从而阻止了构架肝小叶的正常胶原的破坏, 促进了异常沉积的胶原降解.

我们发现随纤维化进展, TIMP-2, TIMP-3表达均增强, 卡托普利可明显抑制TIMP-3表达, 而对TIMP-2表达无抑制作用. 近年来研究发现激活pro-MMP-2不仅需要MT1-MMP, 还需要低浓度的TIMP-2的碳端结合pro-MMP-2的血红素蛋白区, 使pro-MMP-2接近活化的MT1-MMP, 而高浓度TIMP-2则抑制pro-MMP-2的活化[27-30]. 这提示TIMP-2对MMP-2的调节可能具有双向作用.

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