环氧合酶-2在CCl_4诱导大鼠肝纤维化形成中的作用

Effect of cyclooxygenase-2 on liver fibrogenesis induced by CCl_4 in rats

目的探讨环氧合酶-2(cyclooxygenase-2,COX-2)在四氯化碳(CCl4)诱导大鼠肝纤维化形成过程中的作用。方法用50%CCl4经腹腔注射,每周2次共8周诱导雄性S-D大鼠肝纤维化模型。将S-D大鼠随机分3组:正常对照组给橄榄油腹腔注射;罗非昔布组在CCl4诱导肝纤维化模型的同时给予选择性COX-2抑制剂罗非昔布(按10 mg.d-1.kg-1体重溶于10 mL生理盐水中灌胃);模型对照组在CCl4诱导肝纤维化模型的同时给予同等体积的生理盐水灌胃。第8周末处死大鼠,病理组织学观察肝纤维化严重程度并评分,并通过Masson三色染色法将胶原染成蓝色,用图像分析仪自动分析胶原面积。用Western blot检测肝组织COX-2表达和Ⅳ型胶原。计数肝窦内α-SMA阳性的细胞数来观察肝星状细胞的活化。结果与肝纤维化模型组相比,罗非昔布干预组肝组织纤维化严重程度明显减轻(平均积分分别为:4.00±0.00vs2.89±0.60,P<0.05)、胶原面积也减少(分别为:30.7±8.9vs23.5±6.5,P<0.05)、肝窦内平均α-SMA阳性细胞数明显减少(分别为:12.5±1.3vs7.4±1.4,P<0.01);Western blot分析提示:与肝纤维化模型组相比,罗非昔布能降低Ⅳ型胶原表达、对COX-2表达无明显影响。结论COX-2在肝纤维化的形成过程中发挥重要作用,选择性COX-2抑制剂罗非昔布具有减轻或阻止肝纤维生成的作用。

Purpose To investigate the effect of cyclooxygenase-2（COX-2） on liver fibrogenesis induced by CCl4. Methods Male S-D rats were used. Hepatic fibrosis was induced by 50% CCl4 twice a week for 8 weeks. Control rats received olive oil （ip） only; CCl4 induced rats were divided into 2 groups： one concurrently received placebo （saline solution by gavage）, the other received rofecoxib （10 mg ·kg^-1·d^-1 by gavage） for 8 weeks respectively. After 8 weeks, histopathological examination was performed using a histological scoring system, liver fibrogenesis was also assessed by digital image analysis of Masson＇s trichrome stained sections. The collagen type Ⅳ and COX-2 proteins levels in liver were determined by Western blot. Numbers of activated hepatic stellate cells were quantified after alpha-smooth muscle actin （alpha-SMA） staining. Results Livers from CCl4-treated rats exhibited substantial damage, including fibrosis and extensive expression of smooth muscle alpha-actin （alpha-SMA）. Compared to controls, rats administered rofecoxib revealed significant decrease in the degrees of fibrosis （fibrosis score respectively, 2.89 ± 0.60 vs 4. 00 ± 0. 00, P〈0. 05） and the fibrotic area was significantly higher in the control group than rofecoxib group （30.7 ± 8.9 vs 23.5± 6.5, P〈0.05）. Compared to placebo, administration of rofecoxib resulted in a marked decrease in the number of alphaSMA-positive cells （respectively, 12.5 ± 1. 3 vs 7.4 ± 1. 4, P〈0.01）. Western blot analysis confirmed that type Ⅳ collagen and COX-2 significantly increased in CCl4 induced cirrhotic rats compared with normal rats, but rofecoxib reduced type Ⅳ collagen levels compared with the model control rats which treated by CCl4 plus saline, and COX-2 unchanged. Conclusions COX-2 play an important role in liver fibrogenesis, and chronic administration of rofecoxib may inhibit liver fibrogenesis in CCl4 induced cirrhotic rats.