银杏叶提取物对肺纤维化FIZZ1／NOTCH信号通路的影响

Ginkgo biloba extract effects on FIZZ1/NOTCH signal pathway in pulmonary fibrosis

目的研究博来霉素致肺纤维化（PF）动物模型中银杏叶提取物（GBE）对FIZZ1／NOTCH1信号通路的影响，探索中药银杏叶治疗肺纤维化的作用机制。方法将25只昆明鼠按随机数字表法随机分为5组：对照组、模型组、GBE组、GBE-NOTCH1-shRNA组、空白质粒组，每组5只。除对照组外其余各组注入博来霉素（5mg／kg），对照组注入生理盐水（1．25mE／ks）制造阴性对照。空白质粒组于造模第2天进行空质粒尾静脉快速注射（3mL／kg）。GBE组于造模当天开始每天用GBE溶液灌胃[0．6g／（kg·d）]，GBE—NOTCH1-shRNA组在每天GBE灌胃基础上于造模第2天尾静脉注射NOTCH1-shRNA质粒（3mL／kg）。于第28天处死动物，取左肺组织行HE染色观察肺泡炎和肺纤维化程度。取右肺组织Western及RT—PCR方法检测FIZZ1、NOTCH1及α-SMA表达。结果模型组及空白质粒组肺泡炎及纤维化程度较重，GBE组炎症程度显著减轻，纤维组织大量减少，GBE—NOTCH1-shRNA组较GBE组肺泡炎及纤维化程度有所加重；Western结果显示，模型组FIZZ1、NOTCH1、α-SMA表达最高（P〈0．05），空白质粒组α-SMA表达与模型组比较差异无统计学意义（P〉0．05），GBE组NOTCH1、α-SMA表达较模型组下降（P〈0．05），GBE—NOTCH1-shRNA组α-SMA表达较GBE组显著上升（P〈0．05）；RT—PCR结果显示，模型组及空白质粒组NOTCH1、α-SMA表达较对照组显著增高（P〈0．05），GBE—NOTCH1-shRNA组α-SMA表达较GBE组显著上升（P〈0．05）。结论FIZZ1能通过NOTCH信号通路的激活促进成纤维细胞向肌成纤维细胞的转化增殖，导致OL—SMA分泌增多，加重肺纤维的发生发展。GBE能减轻肺纤维化程度，其机制可能通过阻断NOTCH信号通路来抑制FIZZ1诱导α-SMA表达。

Objective To investigate the effect of Chinese ginkgo biloba extract （GBE） on NOTCH1 signaling pathway in bleomycin-induced pulmonary fibrosis in animal models, to explore medicine mechanisms of GBE in treatment of pulmonary fibrosis. Methods Twenty-five Kunming mice were randomly divided into 5 groups： control group, model group, GBE group, GBE-NOTCH 1-shRNA group, negative plasmids group （n = 5）. Apart from the control group, other groups were induced by using the method of intratracheal instillation of Neomycin （5 mg/kg）, the control group was treated by intratracheal instillation of saline （1.25 mL/kg） to obtain the negative control. Mice in GBE group were administered with GBE solution by gavage [0.6 g/（kg·d）] daily; mice in GBE-NOTCHI-shRNA group were administered by tail vein injection NOTCH1 - shRNA plasmids （3 mL/kg）. On the day of 28th all mice were sacririced and observed expression and FIZZ1, NOTCH1 and α-SMA in lung tissue by RT-PCR and Western blotting. Results The severity of pulmonary fibrosis was the heaviest in model group and negative plasraids group. The severity of pulmonary fibrosis in GBE group was significantly reduced compared with model group. The severity of pulmonary fibrosis and alveolar inflammation in GBE-NOTCH 1-shRNA group increased compared with GBE group. Western blotting showed the expression of FIZZ1, NOTCH1 and α-SMA in the model group was significantly higher than the control group （P 〈 0.05）. The expression of NOTCH1 and α-SMA in GBE group was significantly reduced compared with the model group （P 〈 0.05 ）. α-SMA protein levels in GBE-NOTCH 1-shRNA increased compared with GBE group （P 〈 0.05 ）. The result of RT-PCR was similar to that of Western blotting. Conclusion The NOTCH signaling pathway is involved in fibroblast transit process. GBE relieves pulmonary fibrosis by inhibiting the activation of NOTCH signaling pathway.