蒙花苷体内外抗肺纤维化作用及其机制研究

Study on the anti-pulmonary fibrosis effect of linarin in vivo and in vitro and its mechanism

目的 考察蒙花苷的体内外抗纤维化作用,并初步探讨其作用机制。方法 将C57BL/6J小鼠随机分为正常组(羧甲基纤维素钠)、模型组(羧甲基纤维素钠)、阳性对照组(吡非尼酮,200 mg/kg)和蒙花苷低、高剂量组(12.5、25 mg/kg),每组8只。除正常组外,其余各组小鼠均制备肺纤维化模型。造模结束后,灌胃给药,每天1次,连续14 d。观察小鼠一般情况,测定其肺指数,检测其血清中肿瘤坏死因子α(TNF-α)、转化生长因子β1(TGF-β1)水平以及肺组织中白细胞介素6(IL-6)水平;采用苏木素-伊红(HE)染色及Masson染色观察其肺组织病理学变化,并参照Ashcroft评分标准进行肺纤维化评分;检测其肺组织中α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(CollagenⅠ)、磷酸化胞外信号调节激酶(p-ERK1/2)、TGF-β1的表达水平。使用TGF-β1刺激人胚肺成纤维细胞HFL1建立体外肺纤维化模型,实验设置正常组、模型组和蒙花苷低、中、高浓度组(3.7、7.4、14.8 mg/L),培养48 h后,检测细胞中α-SMA、CollagenⅠ、p-ERK1/2蛋白表达水平。结果 在体内实验中,与正常组比较,模型组小鼠肺指数和TNF-α、TGF-β1、IL-6水平均显著升高(P<0.01);肺泡中出现大量炎症浸染及细胞纤维化等病变,且有大量胶原蛋白沉积,HE染色和Masson染色评分均显著升高(P<0.01);肺组织中α-SMA、CollagenⅠ、p-ERK1/2及TGF-β1蛋白表达均显著上调(P<0.01)。与模型组比较,蒙花苷高剂量组小鼠上述指标均显著改善(P<0.05或P<0.01),蒙花苷低剂量组大部分指标(除肺指数外)均显著改善(P<0.05或P<0.01)。在体外实验中,与空白组比较,模型组细胞出现密度变大、明显增殖等变化,细胞中α-SMA、CollagenⅠ和p-ERK1/2蛋白表达均显著上调(P<0.05或P<0.01)。与模型组比较,蒙花苷各浓度组细胞密度变小,形态逐渐恢复正常;蒙花苷高浓度组细胞中上述蛋白及蒙花苷中浓度组细胞中p-ERK1/2蛋白表达均显著下调(P<0.05或P<0.01)。结论 蒙花苷可能通过调控ERK及炎症相关通路,减轻炎症反应,从而发挥抗肺纤维化作用。

OBJECTIVE To investigate the anti-pulmonary fibrosis effect of linarin in vivo and in vitro, and investigate its mechanism preliminarily. METHODS C57BL/6J mice were randomly divided into normal group(carboxymethylcellulose sodium), model group(carboxymethylcellulose sodium), positive control group(pirfenidone, 200 mg/kg), linarin low-dose and high-dose groups(12.5, 25 mg/kg), with 8 mice in each group. Except for normal group, pulmonary fibrosis model was induced in other groups. After modeling, they were given relevant medicine intragastrically, once a day, for consecutive 14 d. The general situation of mice was observed, and their lung indexes were measured;the levels of tumor necrosis factor-α(TNF-α) and transforming growth factor-β1(TGF-β1) in serum and interleukin-6(IL-6) in lung tissue were determined. Hematoxylin-eosin(HE) staining and Masson staining were used to observe the histopathological morphology of lung. The pulmonary fibrosis was scored according to Ashcroft score standard. The expressions of α-smooth muscle actin(α-SMA) and(type Ⅰ collagen, Collagen Ⅰ), phosphorylated extracellular signal-regulated kinase(p-ERK1/2) and TGF-β1 in lung tissues were detected. HFL1 cells were stimulated by TGF-β1 to form pulmonary fibrosis model in vitro, which were divided into normal group, model group and linarin low-, medium-and high-concentration groups(3.7, 7.4, 14.8 mg/L). After being cultured for 48 h, the protein expressions of α-SMA, Collagen Ⅰ and p-ERK1/2 in HFL1 cells were detected. RESULTS In vivo, compared with normal group, the lung index of model group and the levels of TNF-α, TGF-β1 and IL-6 were significantly increased(P<0.01). There were a large number of inflammatory infiltration and cellular fibrosis lesions in the alveoli, and a large number of collagen depositions. The scores of HE staining and Masson staining were significantly increased(P<0.01). The protein expressions of α-SMA, Collagen Ⅰ, p-ERK1/2 and TGF-β1 in lung tissue were up-regulated significantly(P<0.01). Compared with model group, above indexes of mice were improved significantly in linarin high-dose group(P<0.05 or P<0.01), and most of indexes(except for lung index) were improved significantly in linarin low-dose group(P<0.05 or P<0.01). In vitro, compared with blank group, the density of cells in the model group increased, and obvious proliferation and other changes occurred;protein expressions of α-SMA, Collagen Ⅰ and p-ERK1/2 were significantly up-regulated(P<0.05 or P<0.01). Compared with model group, the cell density of each concentration group was decreased and the morphology gradually returned to normal;the expressions of above proteins in linarin high-concentration group and the protein expression of p-ERK1/2 in linarin medium-concentration group were down-regulated significantly(P<0.05 or P<0.01). CONCLUSIONS Linarin may regulate ERK and inflammatory pathways to reduce the inflammatory response, thereby exerting anti-pulmonary fibrosis effect.