雌二醇对肝纤维化大鼠I, III型胶原及TGF β1表达的影响

摘要

目的

观察雌二醇对肝纤维化大鼠肝脏胶原沉积和转化生长因子β₁ (TGF β₁) 表达的影响, 研究雌激素对肝纤维化形成的抑制作用, 并探讨其可能的机制.

方法

设立模型组、治疗对照组、雌二醇组和正常对照组, 以四氯化碳复合因素诱导大鼠肝纤维化动物模型, 雌二醇组在四氯化碳应用的同时皮下注射苯甲酸雌二醇1 mg/kg, 2 次/wk, 共8 wk. 大鼠肝脏HE染色与Masson染色, 分级观察肝组织的炎性坏死与胶原纤维沉积变化, 并观察对大鼠肝纤维化形成过程中肝脏表达I, III型胶原蛋白及对TGF β₁的影响.

结果

与正常对照组比较, 四氯化碳模型大鼠出现典型的肝纤维化表现, 肝脏胶原纤维间隔广泛形成, 肝小叶与肝窦内胶原增生沉积明显, I, III型胶原(0.58±0.26 vs 6.34±2.24, 1.07±0.49 vs 5.28±1.28, P值均<0.001)及TGF β₁基因表达明显增多; 雌二醇应用可以明显减轻肝脏内胶原纤维增生沉积(P<0.05), 抑制肝脏I、III型胶原蛋白(2.47±0.76 vs 6.34±2.24, 3.02±1.20 vs 5.28±1.28, P值均<0.05)及TGF β₁的合成表达.

结论

雌二醇可抑制肝纤维化大鼠肝脏I, III型胶原蛋白及TGF β₁的合成表达, 发挥对肝纤维化的抑制作用.

关键词: N/A

Effects of estradiol on type I, III collagens and TGF β₁ in hepatic fibrosis in rats

Jun-Wang Xu, Jun Gong, Xin-Li Feng, Xin-Ming Chang, Jin-Yan Luo, Lei Dong, Ai Jia, Gui-Ping Xu

Jun-Wang Xu, Xin-Li Feng, Xin-Ming Chang, Ai Jia, Gui-Ping Xu, Department of Gastroenterology, First Hospital of Xi'an Jiaotong University, Xi'an 710061, Shaanxi Province, China

Jun Gong, Jin-Yan Luo, Lei Dong, Department of Gastroenterology, Second Hospital of Xi'an Jiaotong University, Xi'an 710031, Shaanxi Province, China

Supported by: the Doctorate Foundation of Xi'an Jiaotong University, No. 2001-13.

Correspondence to: Dr. Jun-Wang Xu, Department of Gastroenterology, First Hospital of Xi'an Jiaotong University, Xi'an 710061, Shaanxi Province, China. xujw@pub.xaonline.com

Received: March 8, 2003
Revised: March 20, 2003
Accepted: March 26, 2003
Published online: August 15, 2003

Abstract

AIM

To study the effects of estradiol on the production of collagen I, III and transforming growth factor β₁ (TGF β₁) in experimental fibrosis in rats induced by carbon tetrachloride (CCL4), and to investigate the suppressive effects of estrogen on liver fibrosis.

METHODS

Rats were randomly allocated into a normal control group, a model control group, a therapy control group and an estradiol group. Liver fibrosis was induced by CCL₄ administration. The estradiol group, apart from the administration of CCL₄, was treated subcutaneously with estradiol (benzoic estradiol) 1 mg/kg twice weekly. At the end of week 8, all the rats were sacrificed. Liver inflammation and collagen deposition were observed with HE and Masson's collagen stains, analyzed with scoring and staging systems. Type I, III collagens and TGF β₁ were observed with immunohistochemical method.

RESULTS

CCL₄ group had the typical liver fibrosis compared with normal control group. The fibrous septa were formed in CCL₄ group rats, and collagens were accumulated and deposited in the sinusoids and liver lobules. The expression of type I , III collagens (0.58±0.26 vs 6.34±2.24, 1.07±0.49 vs 5.28±1.28, P<0.001) and TGF β₁ was significantly increased. Estradiol significantly attenuated collagen accumulation (P<0.05) in the fibrotic livers, and decreased type I , III collagens (2.47±0.76 vs 6.34±2.24, 3.02±1.20 vs 5.28±1.28, P<0.05) and TGF β₁ expression in the liver.

CONCLUSION

Estradiol treatment reduces the synthesis of hepatic type I , III collagens and TGF β₁ in the fibrotic liver induced by CCL₄ administration, and attenuates hepatic fibrosis.

Key Words: N/A

0 引言

肝纤维化是许多肝脏疾病共同的病理过程, 是肝损伤过程中机体抗损伤修复的一种表现, 以肝脏胶原等细胞外基质的沉积为其主要特征. 肝硬化在男性的发病比女性更普遍, 男性和女性的发病率之比为2.3:1-2.6:1[1,2]. 虽然肝脏并非性激素作用的基本耙器官, 但肝脏中存在有雌激素受体, 能对雌激素产生反应, 从而调节肝脏的功能[3]. 因此, 性激素在肝纤维化至肝硬化的发展中可能发挥一定的作用. 在肝纤维化的动物模型和人类肝脏疾病的研究发现, TGF β₁在肝纤维化的过程中具有重要作用[4,5]. 有研究发现, 抗雌激素药物他莫昔芬可使女性患者肺纤维化的危险性明显升高, 而他莫昔芬的致肺纤维化是由TGF β₁介导的[6]. 因此设想, 肝纤维化时雌激素可能在调节TGF β₁等一些细胞因子的表达中发挥了作用. 本实验通过四氯化碳复合因素诱导大鼠肝纤维化动物模型, 结合I, III型胶原蛋白及TGF β₁的免疫组化, 探讨雌二醇的抗肝脏纤维化作用.

1 材料和方法

1.1 材料

♂Sprague-Dawley 大鼠40只, 清洁级, 质量220±21 g, 10周龄左右, 由陕西省实验动物中心提供. 苯甲酸雌二醇由上海第九制药厂生产, 批号00080103; 秋水仙碱片为云南昆明植物药厂出品; 四氯化碳和胆固醇分析纯由西安化学试剂厂生产; 市售猪油、玉米粉; 精制花生油(山东鲁华集团出品); I, III型胶原抗体购自武汉博士德生物工程有限公司, 工作浓度1:50; TGF β₁的免疫组化试剂盒购自Dako公司, 工作浓度1:1000; ST试剂盒购自福建迈新生物技术开发公司.

1.2 方法

试验动物饲养于控温控湿12 h光照的环境中, 不限饮食. 每10只1组, 随机分为４组. CCL₄组皮下注射400 mL/L CCL₄花生油溶液, 剂量为2 mL/kg, 2次/wk, 首剂加倍. 雌二醇(E)组, 注射CCL₄的同时皮下注射苯甲酸雌二醇1 mg/kg, 2 次/wk. 秋水仙碱(Col)组注射CCL₄的同时, 每日0.11 mg/kg秋水仙碱罐胃. 以上3组动物均给予含5 g/kg胆固醇和200 g/kg猪油的高脂饮食. 对照组给予普通饮食并皮下注射等容量花生油溶剂, 2次/wk. 8 wk后, 全部实验动物禁食1晚, 经肌肉注射苯巴比妥钠(40 mg/kg)麻醉后行颈椎错位处死, 迅速取出肝脏, 被切除的大鼠肝脏取右叶, 经40 g/L甲醛固定, 石蜡包埋和HE、Masson染色, 由半定量的方法[7]对肝纤维化程度进行评估. 胶原增生程度半定量标准: 0分, 正常肝脏或无明显胶原纤维增生; 1分, 胶原纤维增生, 中央静脉和汇管区有少量纤维索放散延伸, 但无间隔形成; 2分, 胶原纤维明显增生, 中央静脉和汇管区结缔组织变厚, 由此向四周延出纤维索, 形成不完全间隔; 3分, 胶原纤维大量增生, 有个别菲薄的完全间隔形成(假小叶), 或较厚的不完全间隔, 即将形成假小叶; 4分, 完全间隔较厚, 假小叶大量形成. I, III型胶原及TGF β₁免疫组化采用PAP法DAB显色. I、III型胶原表达程度, 应用CMIAS彩色医学图像分析仪, 对各组切片随机选取10个视野测量阳性染色面积占肝脏视野面积比进行定量分析, 以他们的染色面积在标本中所占百分比表示_[8].

统计学处理 将数据表示为mean±SD, 进行非配对的秩和检验、t检验或F检验. 数据输入计算机采用SPSS 10.0统计软件分析.

2 结果

2.1 雌二醇对大鼠实验性肝纤维化的影响

HE染色显示对照组的肝脏具有正常的肝小叶结构; CCL₄组表现出严重的损伤性病理改变, 肝小叶失去正常结构, 肝板排列紊乱, 肝细胞普遍变性, 大多数为脂肪变性, 少数水样变, 炎症及坏死明显, 汇管区纤维组织增生, 并可见厚薄不一的纤维分隔包绕肝小叶, 部分有肝细胞再生及假小叶形成; 雌二醇组肝脏损伤性病理改变明显减轻, 纤维有部分分割肝小叶, 但未见完整的假小叶形成, 肝组织有部分再生. Masson胶原染色(表1), 肝脏胶原纤维增生程度半定量测量, 表明雌二醇可明显抑制大鼠实验性肝纤维化的形成.

表1 雌二醇对大鼠实验性肝纤维化的作用.

分组	n	肝纤维化分级(级)
		-	+	++	+++	++++
Control	10	10	0	0	0	0
CCL4	9	0	2	2	3	1
CCL4+Ea	8	0	4	2	1	0
CCL4+Cola	9	0	3	4	2	0

^aP<0.05, vs CCL₄组.

2.2 I, III型胶原免疫组化染色分析

用免疫组织化学PAP法对肝脏组织切片I, III型胶原进行观察, 正常对照组肝脏I型胶原蛋白主要存在于中央静脉、门脉区三联管管周及管间纤维组织, III型胶原除上述区域外还存在于肝窦四周. CCL₄组中I, III型胶原阳性细胞染色均明显增多, 其分部除汇管区、中央静脉及肝窦四周增多外, 在纤维隔内呈弥漫分布. 雌二醇组及秋水仙碱组I型胶原蛋白主要位于中央小叶和门静脉周围纤维带, 而III型胶原蛋白以门脉区及肝细胞坏死区较多. 各组I, III型胶原蛋白面密度(胶原面积/肝组织视野面积100%)测量值(表2).

表2 雌二醇对大鼠肝纤维化肝脏I, III型胶原蛋白的影响(mean±SD).

组别	n	I 型胶原	III型胶原
Control	10	0.58±0.26	1.07±0.49
CCL4	9	6.34±2.24	5.28±1.28
CCL4+E a	8	2.47±0.76	3.02±1.20
CCL4+Col a	9	2.63±0.91	3.31±1.43

^aP<0.05, vs CC_L4组.

2.3 TGF β₁的免疫组化染色分析

CCL₄组的TGF β₁阳性细胞主要位于中央小叶和门静脉周围纤维带及肝纤维间隔中, 主要见于肝星状细胞、类间质细胞和炎症细胞质及其周围. 而雌二醇及秋水仙碱组中阳性染色程度较CCL₄组明显为轻 (图1、图2).

图1 CCL₄组TGF β₁的免疫组化染色PAP法×100.

图2 雌二醇组TGF β₁的免疫组化染色PAP法×100.

3 讨论

肝纤维化通常始于肝细胞的破坏, 继而炎症细胞和血小板聚集, 刺激枯否细胞释放细胞因子和生长因子(如TGF β₁等), 这些因子通过活化肝星状细胞, 肝星状细胞活化后增生并转化为成纤维母细胞样的细胞, 合成并大量沉积细胞外基质(extracellular matrix, ECM), 从而将炎症和肝硬化的形成过程联系起来[9-13]. ECM包括胶原、非胶原糖蛋白及蛋白多糖, 其中胶原是最重要的ECM. 肝脏胶原有I, III, IV, V, VI型等, 以I, III型为主, 约占肝脏胶原总量的60％以上[14-17]. 我们以CCL₄等复合因素诱导大鼠肝纤维化模型, 可见正常对照组肝脏仅有少量I型胶原存在于中央静脉、门脉区三联管管周及管间纤维组织, III型胶原除上述区域外还少量存在于肝窦四周, 而CCL₄组中I, III型胶原阳性细胞染色均明显增多, 其分布除汇管区、中央静脉及肝窦四周增多外, 在纤维隔内亦呈弥漫分布, 包绕肝小叶. 雌二醇组的肝纤维化程度明显低于CCL₄组, 且I, III型胶原阳性细胞免疫组化染色也弱于CCL₄组, I型胶原蛋白主要位于中央小叶和门静脉周围纤维带, 而III型胶原蛋白以门脉区及肝细胞坏死区为主. 表明雌二醇能抑制I, III型胶原的合成而发挥抗肝纤维化作用.

肝损害时肝星状细胞被认为是ECM的主要来源, 而肝星状细胞的活化是肝细胞、内皮细胞、血小板、枯否细胞、炎症细胞、细胞因子和生长因子间相互作用的结果[18-22]. 细胞因子在肝纤维化的每个阶段都发挥着重要作用[23-26]. 在肝纤维化的动物模型和人类肝脏疾病的研究发现, TGF β₁在肝纤维化的过程中具有重要作用[27-31], 他是一种多功能生长因子, 能使单个肝星状细胞胶原合成增加60-80％, 被认为是肝纤维化形成的关键因子. Bentzen et al [6]在研究放疗女性肺纤维化时发现, 给予他莫昔芬(tamoxifen, 一种抗雌激素的药物)可使患者肺纤维化的危险性明显升高, 而他莫昔芬的致肺纤维化是由TGF β₁介导的. 因此设想, 肝纤维化时雌激素可能在调节TGF β₁等一些细胞因子的表达中发挥了作用. 本实验显示, 雌二醇组肝纤维组织中TGF β₁的表达量明显弱于CCL₄组, 表明雌二醇可能通过下调TGF β₁的表达, 从而间接地发挥对肝纤维化的抑制作用.

肝纤维化时肝星状细胞由静止状态转变为活化细胞, 增加I, III型胶原等ECM的表达, 激活的肝星状细胞还可通过自分泌途径扩大激活细胞群, TGF β₁是肝星状细胞自分泌扩增的重要细胞因子, 既增加胶原的合成, 又进一步刺激TGF β₁的产生, 使更多静止的肝星状细胞被激活[32-35]. 本研究雌二醇能抑制TGF β₁的产生, 阻断自分泌放大过程, 从而减少I, III型胶原等ECM的合成, 这一作用可能是肝纤维化及肝硬化的发病率存在性别差异的原因之一.

备注

$[Footnotes]

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