Rapamycin attenuates bleomycin-induced pulmonary fibrosis in rats and the expression of metalloproteinase-9 and tissue inhibitors of metalloproteinase-1 in lung tissue
Authors: Huaping Dai
Chen Wang
Ke Ding
Xiaoguang Jin
Baosen Pang
Xuefeng Xu
Journal: Chinese medical journal (2014)
Discipline: CLINICAL MEDICINE
Keywords: Idiopathic pulmonary fibrosis; Rapamycin; Bleomycin-induced pulmonary fibrosis; Metalloproteinase-9 (MMP-9); Tissue inhibitor of metalloproteinase-1 (TIMP-1)
Affiliations: Department of Respiratory and Critical Care Medicine, Beijing Chao-Yang Hospital, Capital Medical University, Beijing 100020, China; Beijing Key Laboratory of Respiratory and Pulmonary Circulation; Beijing Institute of Respiratory Medicine, Beijing 100020, China., Department of Respiratory and Critical Care Medicine, Beijing Chao-Yang Hospital, Capital Medical University, Beijing 100020, China; Beijing Key Laboratory of Respiratory and Pulmonary Circulation; Beijing Institute of Respiratory Medicine, Beijing 100020, China. Email: ., Beijing Key Laboratory of Respiratory and Pulmonary Circulation; Beijing Institute of Respiratory Medicine, Beijing 100020, China; Beijing Hospital, Beijing 100730, China. Email: .

Abstract:  Background: Idiopathic pulmonary fibrosis (IPF) is the most common and devastating form of interstitial lung disease (ILD) in the clinic. There is no effective therapy except for lung transplantation. Rapamycin is an immunosuppressive drug with potent antifibrotic activity. The purpose of this study was to examine the effects of rapamycin on bleomycin-induced pulmonary fibrosis in rats and the relation to the expression of metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). Methods: Sprague-Dawley rats were treated with intratracheal injection of 0.3 ml of bleomycin (5 mg/kg) in sterile 0.9% saline to make the pulmonary fibrosis model. Rapamycin was given at a dose of 0.5 mg/kg per gavage, beginning one day before bleomycin instillation and once daily until animal sacrifice. Ten rats in each group were sacrificed at 3, 7, 14, 28 and 56 days after bleomycin administration. Alveolitis and pulmonary fibrosis were semi-quantitatively assessed after HE staining and Masson staining under an Olympus BX40 microscope with an IDA-2000 Image Analysis System. Type I and III collagen fibers were identified by Picro-sirius-polarization. Hydroxyproline content in lung tissue was quantified by a colorimetric-based spectrophotometric assay, MMP-9 and TIMP-1 were detected by immunohistochemistry and by realtime quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Results: Bleomycin induced alveolitis and pulmonary fibrosis of rats was inhibited by rapamycin. Significant inhibition of alveolitis and hydroxyproline product were demonstrated when daily administration of rapamycin lasted for at least 14 days. The inhibitory efficacy on pulmonary fibrosis was unremarkable until rapamycin treatment lasted for at least 28 days (P &lt; 0.05). It was also demonstrated that rapamycin treatment reduced the expression of MMP-9 and TIMP-1 in lung tissue that was increased by bleomycin. Conclusion: These results highlight the significance of rapamycin in alleviating alveolitis and pulmonary fibrosis, which is associated with decreased expression of MMP-9 and TIMP-1. 

Retracted: No

PubPeer comments:
 Figure 4 and Figure 6 showed identical b-actin control. In the legend, the authors claimed that b-actin RT-PCR was performed in every PCR reaction so that the b-actin control shouldn t be the same for both experiments. Please explain. ![file] ![file] , 
 We would like to thank Dr. Rubioidesu2019 comments for the identical u03b2-actin band shown in in the bottom panels of Figure 4 and Figure 6. Specifically, the size of u03b2-actin is 750bp (the upper bands in both gels). The 545bp bands indicate MMP9 expression (the lower bands in Figure 4), while the 295bp bands indicate TIMP-1 expression (the lower bands in Figure 6). As shown in both Figure 4 and Figure 6, the results of all samples showed the expression of MMP-9 or TIMP-1 with their u03b2-actin controls in the same gel. Therefore, the u03b2-actin bands shown in both figures are not necessary and confusing. We apologize for this confusion. ,