促肝细胞生长素对肺纤维化大鼠的干预作用及其途径

Interventional effect and possible mechanism of hepatocyte growth-promoting factor in pulmonary fibrosis rats

目的:观察早期和晚期给予促肝细胞生长素对大鼠肺纤维化模型的干预作用,并分析其可能作用途径。方法:实验于2005-05/09在哈尔滨医科大学附属第二医学院实验中心完成。选择雄性Wistar大鼠85只,按随机数字表法分为5组,正常对照组、博来霉素模型组、地塞米松治疗组及促肝细胞生长素早期治疗组每组各20只,促肝细胞生长素晚期治疗组5只。除正常对照组外,其他大鼠均经气管内灌注博来霉素A5(5mg/kg)生理盐水溶液0.2～0.3mL,诱导肺纤维化。地塞米松治疗组和促肝细胞生长素早期治疗组于造模后第2天开始腹腔注射地塞米松3mg/kg和促肝细胞生长素1mg/kg,1次/d。正常对照组和博来霉素模型组注射等体积生理盐水。各组分别于气管内灌注后的第3,7,14,28天经麻醉腹主动脉放血法处死5只大鼠。促肝细胞生长素晚期治疗组于造模后第14天开始腹腔注射促肝细胞生长素1mg/kg,1次/d,于造模后第28天经麻醉腹主动脉放血法处死5只大鼠。分离肺组织,用免疫组化方法检测转化生长因子β1和基质金属蛋白酶9、基质金属蛋白酶抑制剂1蛋白在大鼠肺组织的表达水平。通过苏木精-伊红染色、Masson胶原染色及测定肺组织羟脯氨酸浓度来评价治疗效果,肺泡炎及肺纤维化程度分为4级(1分:无肺泡炎或无纤维化;4分:重度肺泡炎或纤维化,受累面积>50%)。结果:85只大鼠全部进入结果分析,无脱失。①促肝细胞生长素早期治疗组造模后第7,28天肺组织羟脯氨酸含量均显著低于博来霉素模型组(P<0.05～0.01)。②促肝细胞生长素早期治疗组造模后第28天肺纤维化评分显著低于博来霉素模型组(P<0.05)。③促肝细胞生长素早期治疗组造模后第7,28天肺组织转化生长因子β1蛋白表达水平显著低于博来霉素模型组(P<0.05)。④促肝细胞生长素早期治疗组造模后第3,7,14天基质金属蛋白酶抑制剂1蛋白表达水平均显著低于博来霉素模型组(P<0.05);造模后第14,28天基质金属蛋白酶9蛋白表达水平均显著高于博来霉素模型组(P<0.05)。⑤促肝细胞生长素早期治疗组和晚期治疗组各项指标比较差异无显著性意义(P>0.05)。结论:早期和晚期应用促肝细胞生长素能减轻博来霉素诱导的大鼠肺纤维化,这种作用可能通过抑制转化生长因子β1和调节基质金属蛋白酶9、基质金属蛋白酶抑制剂1蛋白的表达而实现。

AIM： To observe the interventional effect of hepatocyte growth-promoting factor on pulmonary fibrosis in rats in early period and late period and analyze its possible mechanism. METHODS： The experiment was conducted at experimental center of Second Affiliated Hospital, Harbin Medical University from May to September 2005. A total of 85 male Wistar rats were selected and assigned randomly into 5 groups： normal control group, bleomycin model group, dexamethasone treatment group and hepatocyte growth-promoting factor early treatment group with 20 rats in each group, hepatocyte growth-promoting factor late treatment group with 5 rats. Except the normal control group, other rats received 0.2-0.3 mL bleomycin A5 （5 mg/kg） saline solution by perfusion via weasand to induce pulmonary fibrosis. The rats were treated with 3 mg/kg dexamethasone and 1 mg/kg hepatocyte growth- promoting factor via abdominal cavity at day 2 after establishing models in the dexamethasone treatment group and hepatocyte growth-promoting factor early treatment group, once a day. The rats in the normal control group and bleomycin model group were injected with the same volume of saline. Five rats in each group were sacrificed at days 3, 7, 14 and 28 after intratracheal instillation with anaesthesia abdominal aorta depletion method. 1 mg/kg hepatocyte growth-promoting factor was injected via intraperitoneal injection into rats of the hepatocyte growth-promoting factor late treatment group at day 14 after establishing models, once a day. Five rats were killed at day 28 after establishing models with anaesthesia abdominal aorta depletion method. Lung tissue was isolated. Levels of transforming growth factor β1 and matrix metalloproteinase 9, matrix metallo-proteinase inhibitor 1 protein in lung tissue of rats were determined with immunohistochemical method. Therapeutic efficacy was evaluated by hematoxylin-esoin （HE） staining, Masson staining and determination of concentration of hydroxyproline of lung tissue. Alveolitis and pulmonary fibrosis were divided into 4 grades （1 point represented no alveolitis or pulmonary fibrosis; 4 points represented severe alveolitis or pulmonary fibrosis, more than a half of area were involved）. RESULTS： A total of 85 rats were involved in the result analysis, without dropout.①The content of hydroxyproline in the hepatocyte growth- promoting factor early treatment group was significantly lower than that in the bleomycin model group at days 7 and 28 after establishing models （P 〈 0.05-0.01）. ②Score of pulmonary fibrosis in the hepatocyte growth-promoting factor early treatment group was significantly lower than that in the bleomycin model group at day 28 （P 〈 0.05）. ③The level of transforming growth factor β1 in the hepatocyte growth-promotlng factor early treatment group was significantly lower than that in the bleomycin model group at days 7 and 28 （P 〈 0.05）.④Level of matrix metalloproteinase inhibitor 1 protein in the hepatocyte growth-promoting factor early treatment group was significantly lower than that in the bleomycin model group at days 3, 7 and 14 （P 〈 0.05）. Level of matrix metalloproteinase 9 at days 14 and 28 was significantly higher than that in the bleomycin model group （P 〈 0.05）.⑤There was insignificant difference in each index between the hepatocyte growth-promoting factor early treatment group and hepatocyte growth-promoting factor late treatment group （P〉 0.05）. CONCLUSION： The hepatocyte growth-promoting factor used in early and late stages can alleviate bleomycin-induced pulmonary fibrosis in rats, This effect can come true by inhibiting the expression of transforming growth factor β1, regulating the expression of matrix metalloproteinase 9 and matrix metallo-proteinase inhibitor 1.