蜂毒素抑制博萊霉素誘導小鼠肺纖維化的機制研究

李莉　危蕾　王眾福　張秀蓮　錢葉長

摘要?目的：觀察蜂毒素對博萊霉素誘導小鼠肺纖維化的干預作用。方法：70只SPF級C57BL/6小鼠被隨機分為正常組10只和模型組、地塞米松組、蜂毒素低劑量組、蜂毒素中劑量組、蜂毒素高劑量組各12只。除正常組外，均采用氣管穿刺注入博萊霉素（BLM）制備肺纖維化小鼠模型。從術后第1天開始，地塞米松組按3 mg/kg的劑量腹腔注射，蜂毒素低、中、高劑量組分別給予5 μg/（kg·d）、10 μg/（kg·d）、20 μg/（kg·d），對照組和模型組給予等體積生理鹽水灌胃，連續2周。分別于第7、第14天處死動物，收集小鼠外周血樣本，通過ELISA方法檢測血清轉化生長因子（TGF-β1）、膠原蛋白I（CollagenI）、膠原蛋白III（CollagenIII）、基質金屬蛋白酶2（MMP2）和基質金屬蛋白酶9（MMP9）的水平。取肺組織進行蘇木精-伊紅（HE）分析，Masson染色和羥脯氨酸（HYP）評估以觀察組織病理學變化和膠原沉積。采用實時熒光定量（Real-ime PCR）法和蛋白兔疫印跡法（Western blot）觀察各組大鼠肺組織TGF-β1、Smad2、Smad3等蛋白和基因的表達變化。結果：與對照組比較，模型組大鼠肺纖維化明顯，HYP、TGF-β1、CollagenI、CollagenI的含量升高（P<0.05），肺組織TGF-β1、Smad2、Smad3蛋白和基因表達升高（P<0.05）;與模型組比較，蜂毒素中高劑量組血清HYP、TGF-β1、CollagenI、CollagenI的含量下降（P<0.05），肺組織TGF-β1、Smad2、Smad3蛋白和基因表達降低，差異有統計學意義（P<0.05）。結論：蜂毒素可以減輕博來霉素誘導的小鼠肺纖維化的程度，其機制可能與抑制TGF-β1/Smads通路有關。

關鍵詞?蜂毒素;抑制;博萊霉素;小鼠;肺纖維化;機制;TGF-β1/Smads通路;羥脯氨酸

Study on the Mechanism of Melittin Inhibiting Pulmonary Fibrosis Induced by Bleomycin in Rats

Li Li，Wei Lei，Wang Zhongfu，Zhang Xiulian，Qian Yechang

（1 Baoshan Branch，Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine，Shanghai 201900，China）

Abstract?Objective：To observe the effects of melittin on inhibiting pulmonary fibrosis induced by bleomycin in rats. Methods：Seventy SPF grade C57BL/6 rats were randomly divided into the normal group（n=10）and the model group，dexamethasone group and melittin low dose group，melittin middle dose group and melittin high dose group（n=12）. In addition to the normal group，a rat model of pulmonary fibrosis was prepared by injecting bleomycin（BLM）into the trachea. From the first day after surgery，the dexamethasone group was intraperitoneally injected at a dose of 3 mg/kg，while the low，medium and high doses of melittin were administered to 5，10，and 20 μg/（kg·d） respectively. The control group and the model group were given an equal volume of saline for 2 weeks. The animals were sacrificed on the 7th and 14th day，respectively，peripheral blood samples of which were collected. ELISA was used to detect serum transforming growth factor（TGF-β1），collagen I（Collagen I），collagen III（Collagen III），and matrix metalloproteinase 2（MMP2）and the level of matrix metalloproteinase 9（MMP9）. Lung tissue was taken for hematoxylin-eosin（HE）analysis，Masson staining and hydroxyproline（HYP）evaluation to observe histopathological changes and collagen deposition. The expressions of TGF-β1，Smad2，Smad3 and other proteins and genes in lung tissue of each group were observed by Real-time PCR and Western blot. Results：Compared with the control group，the pulmonary fibrosis was significantly increased in the model group. The contents of HYP，TGF-β1，Collagen I and Collagen I were increased（P<0.05），and the expression of TGF-β1，Smad2，Smad3 protein and gene in lung tissue was increased（P<0.05）; Compared with the model group，the levels of serum HYP，TGF-β1，Collagen I，CollagenI in the high dose group of melittin decreased（P<0.05），and TGF-β1，Smad2，Smad3 protein in lung tissue and gene expression was decreased（P<0.05）. There was no statistically significant difference in the low-dose group. Conclusion：Melittin can effectively reduce the degeneration of pulmonary fibrosis induced by bleomycin，and its mechanism may be related to the regulation of TGF-β1/Smads pathway.

Key Words?Melittin; Inhibition; Bleomycin; Pulmonary fibrosis; Mechanism; TGF-β1/Smads pathway; Hydroxyproline

中圖分類號：R563;R256文獻標識碼：Adoi：10.3969/j.issn.1673-7202.2019.03.018

肺纖維化（Pulmonary Fibrosis，PF）是一種進行性加重的肺間質疾病，以限制性通氣功能障礙、進行性彌漫性肺間質纖維化為特征，最終可導致呼吸衰竭，預后極差[1]。……