17β-雌二醇对大鼠肝纤维化影响作用的实验研究

Study the effects of 17β-estrodiol on CCl_4-induced liver fibrosis in rats

目的探讨17β-雌二醇（E2）对大鼠肝纤维化及肝窦毛细血管化的影响作用及其分子生物学机制.方法健康Wistar大鼠80只,体重（200±17）g,分成三组.CCl4模型组（30只）以60%CCl4橄榄油剂行腹部皮下注射制备肝纤维化模型,每次2 ml/kg体重,每周2次.E2预防组（30只）CCl4组以同样方法制备肝纤维化模型,同时皮下注射E2,剂量为200μg/kg体重,每周1次.对照组（20只）给予生理盐水.每周处死各实验组大鼠4～6只,记录实验结果.自肝脏右叶取材,作常规H-E染色和Masson胶原染色、原位杂交和透射电镜检查,检测大鼠肝组织中NF-kB P65 mRNA基因表达情况.由图像分析仪半定量分析结果.结果CCl4模型组大鼠血清ALT、AST、球蛋白水平及大鼠肝纤维化程度明显高于E2预防组（P＜0.05）,白蛋白水平在造模第12周末,明显低于E2预防组（P＜0.05）.原位杂交结果显示,于CCl4造模第6周及第12周,大鼠肝组织中NF-kB P65 mRNA表达阳性染色细胞数分别为21.70%±5.93%和46.19%±6.73%;而在E2预防组则分别为15.36%±4.41%和31.51±6.02%,明显低于CCl4模型组（P＜0.05）,E2可明显下调NF-kB P65 mRNA基因表达.与E2预防组比较,CCl4模型组大鼠肝细胞损害程度明显,可见大量细胞外基质沉积及胶原纤维.结论E2能下调大鼠肝组织NF-kB P65 mRNA基因表达,阻断HSC活化增殖信号传导通路,减少细胞外基质分泌与沉积.通过抗脂质过氧化作用,减少肝细胞损伤,改善肝功能,抑制肝窦毛细血管化,具有抗肝纤维化作用.

Objective To investigate the effects of 17β-estrodiol （E2） on capillarization of CCl4-i-lduced rat liver fibrosis. Methods Eighty Wistar rats （40M,40F）, weight （200 ± 17） g were divided into thres group. For CCl4 group （ n = 30）, 60% CCl4 in peanut oil was injected subcutaneously at a dose of 2 ml/kg twice weekly, and the first dosage was doubled. The E2 group（ n = 30）, in addition to CCl4, rats were injected subcutaneausly with E2 200μg/kg once weekly. Twenty rats were served as control 4-6 rats were fasted overnight and sacrified every 3 weeks. The serum obtained was analysed for liver function tested. Liver tissues from each rat were stained with hematoxylin-eosin （H-E） and masson＇ s trichrome. Expression of NF-kB P65 mRNA were detected by immunohistochemistry, hybfization in situ and TEM were performed. Results The results revealed that CCl4 produced a marked increase in the activities of serum ALT, AST, hepatic fibrosis stage in both male and female rats. After administration of 17β-estradiol, the enzyme levels and the stage of hepatic fibrosis were significantly decreased than that of CCl4 only groups （ P 〈 0.05, P 〈 0.01）. Analysis of NF-kB P65 mRNA expression by hybridization in situ showed that positive staining cells was （21.70 ± 5.93） % and （46.19 ± 6.73） % at the 6 th and 12 th week in CCl4 group respectively. They were higher significantly compared with that in E2 group （ 15.36 ± 4.41 ） %, （31.51± 6.02） % （ P 〈 0.05, P 〈 0.01 ）. Transmission electron microscopy showed that the hepatocellular damage of CCl4 group rats was more serious than E2 group, with much deposition of extracellular matrix and collagen fibers. Conclusion E2 could downregulate the expression of NF-kB P65 mRNA in rat liver tissues and block the signal transconduct that led to liver fibrosis. E2 could improve liver function and reduce the damages of hepatocytes induced by CCl4, it may play an important role as an endogenous fibrosuppressant in fibrosis, accounting for sex-assoeiated differences in the progression from hepatic fibrosis to cirrhosis.