摘要
目的观察博莱霉素（BLM）致肺纤维化动物模型中NOTCH信号通路对成纤维细胞（FB）表型转化的影响，探索中药银杏叶提取物（GBE）治疗肺纤维化的作用机制。方法20只昆明小鼠按随机数字表法分为对照组、模型组、1分泌酶抑制剂DAPT组、GBE组，每组5只。采用气管切开注入BLM5mg／kg复制小鼠肺纤维化模型，对照组给予生理盐水1．25mUkg。GBE组于制模当日开始灌胃GBE0．6g·kg^-1·d^-1，DAPT组于制模后次日灌胃DAPT5mg-kg^-1·d^-1，隔日1次。28d处死小鼠，取左肺组织行苏木素-伊红（HE）染色，观察肺泡炎和肺纤维化程度；用免疫组化染色及蛋白质免疫印迹试验（WesternBlot）检测NOTCH1及α-平滑肌肌动蛋白（α-SMA）的蛋白表达。结果模型组肺纤维化程度最重，肺泡间隔增宽，肺泡连续性受到破坏，肺泡直径和面积大小不均匀，终末小气道周边炎性细胞浸润明显，血管周边也发现明显的炎性细胞浸润现象，同时伴有纤维化形成，以支气管周边区域较为明显，外周局部区域也有局灶性的纤维化表现，GBE组及DAPT组肺纤维化程度较模型组明显减轻。免疫组化显示，模型组NOTCH1及α-SMA的蛋白含量较对照组明显增高[NOTCH1（A值，×10^3）：129．21±1．28比59．60±8．25，α-SMA（A值，×10^3）：108．23±21．73比55．42±9．23，均P〈0．05]；GBE组和DAPT组NOTCH1、α-SMA蛋白含量均较模型组明显降低[NOTCHl（A值，×10^3）：GBE组为70．54±2．46，DAPT组为67．38±2．35；α-SMA（A值，×10^3）：GBE组为68．95±3．46，DAPT组为61．63±1．99，均P〈0．05]；WesternBlot结果显示，模型组NOTCH1、α-SMA的蛋白表达水平较对照组增高；GBE组和DAPT组NOTCH1、α-SMA的蛋白表达水平较模型组明显降低。结论NOTCH信号通路激活参与了肺纤维化中FB向肌成纤维细胞（MFb）表型转化过程，中药GBE通过抑制NOTCH信号通路的激活达到减轻肺纤维化的作用。
Objective To study the impact of NOTCH signaling pathway on fibroblasts （FB） polarization in mice models with pulmonary fibrosis induced by bleomycin （BLM） and to explore the mechanisms of Chinese medicine Ginkgo biloba extracts （GBE） in treatment of pulmonary fibrosis. Methods Tweenty Kunming mice were randomly divided into four groups： control group, model group, γsecretase inhibiting agent DAPT group, GBE group （each n = 5）. Apart from the control group, 5 mg/kg BLM was injected via an intratracheal route in other groups to replicate the mice models of pulmonary fibrosis. The control group was treated by intratracheal instillation of normal saline （1.25 mL/kg） to obtain the negative control. On the day of modeling in GBE group, GBE solution （0.6 g· kg^-1· d^-1） began to be administered by gavage daily, while in DAPT group, on the next day after modeling, the DAPT solution （5 mg· kg^-1· d^-1） began to be administered by gavage, once every 2 days. On the 28th day, all the mice were sacrificed, and the left lung tissue was taken for hematoxylin and eosin （HE） staining to observe alveolitis and pulmonary fibrosis; the protein expressions of NOTCH1 and α-smooth muscle actin （α-SMA） in lung tissue were measured by immunohistochemical staining and Western Blot. Results The severity of pulmonary fibrosis in model group was the most serious, manifesting increase of alveolar septum width, alveolar continuously damaged, alveolar diameter and area size not uniform, marked inflammatory cell infiltration at periphery of terminal small airway, at the periphery of blood vessels obvious inflammatory cell infiltration also found and in the mean time accompanied by fibrosis formation relatively prominent at the peripheral area of bronchus, focal fibrosis also seen at the peripheral local area. The severity of pulmonary fibrosis in GBE and DAPT groups was signifcantly attenuated compared with that in the model group. Immunohistochemistry showed the protein expressions of NOTCH1 and α -SMA in the model group were significantly higher than those in the control group [NOTCH1 （A value, × 10^3）： 129.21 ± 1.28 vs. 59.60 ± 8.25; α -SMA （A value,× 10^3）： 108.23 ±21.73 vs. 55.42 ± 9.23, both P 〈 0.05]; the expressions of NOTCH1 and α -SMA in GBE group and DAPT group were significantly reduced eompared with those in the model group [NOTCH1 （A value, × 10^3）： 70.54 ±2.46 in GBE group, 67.38 ±2.35 in DAPT group;α -SMA （A value, × 10^3）： 68.95 ± 3.46 in GBE group, 61.63 ± 1.99 in DAPT group, all P 〈 0.05]. Western Blot results showed NOTCH1 and α-SMA protein levels were increased in the model group compared with those in the control group; the expressions of NOTCH1 and α-SMA in GBE group and DAPT group were decreased compared with those in the model group. Conclusion The activation of NOTCH signaling pathway participates in the phenotypic transformation of pulmonary fibrosis from FB to myofibroblast （MFb）, the Chinese medicine GBE attenuates the pulmonary fibrosis via inhibiting the activation of Notch signaling pathway.
出处
《中国中西医结合急救杂志》
CAS
北大核心
2016年第4期417-420,共4页
Chinese Journal of Integrated Traditional and Western Medicine in Intensive and Critical Care
基金
湖北省武汉市卫计委专项基金资助项目（WZ14D09）
