Gefitinib Protects From Pulmonary Fibrosis Induced By Intra-tracheal Administration Of Bleomycin In Mice

BackgroundBleomycin is an antitumor antibiotic that was isolated from a strain of strptomyces verticillus in 1966. It has been used successfully to treat a variety of malignancies, including squamous cell carcinoma of the head and neck, cervix and esophagus, germ cell tumors and both Hodgkin and non-Hodgkin lymphoma. The major limitation of bleomycin therapy is the potential for life-threatning interstitial pulmonary fibrosis (also called fibrosing alveolitis) in up to 10 percent of patients receiving the drug. Other, less common forms of lung injury include organizing pneumonia and hypersensitivity pneumonitis.Gefitinib, an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, is an effective treatment for epidermal tumors, including non-small cell lung cancer (NSCLC), and is generally well tolerated.Transforming growth factor and epidermal growth factor stimulate fibroblast proliferation and play an important role in pathogenesis of pulmonary fibrosis. However, some studies reveal that inhibition of epidermal growth factor signal by epidermal growth factor receptor inhibitors (EGFR-TKI) may prevent pulmonary fibrosis.ObjectivesThe aim of this study is:1. By studying the the effect of bleomycin and gefitinib in combination regimens, combination therapy was observed on HUVECs cells.2. To study the effects of gefitinib, epidermal growth factor receptor inhibitor in lung fibrosis model.Methods1.In vivoWe generated a mouse model of lung fibrosis induced by bleomycin to investigate the fibrotic effects of gefitinib. C57BL/6 mice were injected intratraceally with bleomycin or saline, with intragastric administration of gefitinib (100mg/kg body weight) or saline for 3 weeks. After 3 weeks mice were sacrificed and lung tissue were harvested and investigated for pathogenesis.During whole study body weight and other physical activities check every day.Mice lung were also fixed into 10% formaldehyde and did hematoxyline and eosin staining to find out pathogenesis in lung tissue of bleomycin and gefitinib and control group.RNA was extracted from the tissue of mice lungs from all three groups and RNA converted into cDNA and by PCR find out the difference in inflammatory proteins. MPO content was found out in mice lung tissue by standard protocol.2.In vitroCells processing and grouping:Human umbilical vein endothelial cells(HUVECs), The cells treatment with gefitinib, bleomycin and bleomycin+gefitinib respectively. Then the cells were divided into normal control group (PBS), bleomycin group, gefitinib group, bleomycin+gefitinib group. Then detection methods were test with the following:(1) MTT cell proliferation assay affect bleomycin and gefitinib on cell; (2) Cells staining experiments, cell migration assay experimetns and toxic effects on cells;(3) RT-PCR detection bleomycin and gefitnib impact on HUVECs inflammatory cytokines mRNA levels; (4) Western blotting experimental methods to detect NF-κB and cell adhesion molecule (VCAM 1) levels of three proteins.Results1.In vivoAfter 21 days of drug treatment mice were sacrificed and lung tissue harvested and take photograph of mice lung which shows that mice treated with bleomycin without gefitinib are worse than mice treated with gefitinib when compare to control which prove that bleomycin cause severe lung injury in mice and gefitinib treatment reverse that or prevent that injury. The histopathological results showed that bleomycin successfully induced lung fibrosis in mice, and gefitinib prevented lung fibrosis and suppressed the proliferation of fibroblast cells. Mice with gefitinib survived more than mice without gefitinib after intratraceal administration of bleomycin.Results also showed that mice treated with gefitinib have high body weight than mice treated with gefitinib or alone with bleomycin. Body weights of gefitinib treated mice are nearly growth of mice in control group.RT-PCR results showed that inflammatory marker TNF-α, IL-1β and IFNy are more significantly reduced in gefitinib treated mice lung. Bleomycin group showed high level of TNF-α, IL-1β and IFNy mRNA level as compare to the control group while Gefitinib treatment after bleomycin intra-tracheal administration these inflammatory marker reduce their expression.2.In vitroThe treatment determined by MTT assay to detect the gefitinib and bleomycin on HUVECs proliferation and cytotoxicity:(1) Cell proliferation assay:Bleomycin group’s value higher than the normal control group (P<0.05), the OD value of bleomycin+ gefitinib group was significantly lower than bleomycin group. (2) Cytotoxic to cells in each group did not show a significant difference. The experimental results of cytostatic shows bleomycin on HUVECs proliferation was significantly inhibited, and bleomycin+ gefitinib on HUVECs cytostatic effect is not obvious, cytotoxicity and apoptosis also no significant effect (P> 0.05).RT-PCR analysis demonstrated that, (1) Compared with control group,the expression of HUVECs inflammatory cytokine IL-6 mRNA, CXCL-2 mRNA, MCP-1 mRNA in bleomycin group was significantly increased (P<0.05).(2) In bleomycin+gefitinib group, the expression of inflammatory cytokines IL-6 mRNA, CXCL-2 mRNA, IL-8 mRNA was significantly lower than bleomycin group (P<0.05). (3)The expression of MCP-1 mRNA in gefitinib group was significantly higher than the control group (P<0.05),and in bleomycin+gefitinib group was significantly higher than bleomcin group. These results suggest that gefitinib can significantly inhibit the secretion of HUVECs inflammatory cytokines (MCP-1 exception) on basis of bleomycin stimulation.Western blotting to detect bleomycin and gefitinib on protein expression in HUVECs:(1) Compared with the control group, the expression of NF-κB, MAPK8 in gefitinib group was significantly decreased (P<0.05). (2)Compared with the bleomycin group, the expression of NF-κB, MAPK8 in bleomycin+HUVECs was also significantly decreased (P<0.05). These results indicate that bleomycin can can up regulate the expression of HUVECs NF-κB protein, VCAM-1 protein, but this effect can be supressed by gefitinib.ConclusionThese results reveal that gefitinib reduces pulmonary fibrosis by bleomycin in mice and suggest that administration of small molecule EGFR tyrosine kinase inhibitor has potential to prevent pulmonary fibrosis by inhibiting the proliferation of mesenchymal cells, and that targeting tyrosine kinase receptors might be useful for the treatment of pulmonary fibrosis in human.In vitro experiments showed that the treatment by detecting bleomycin and gefitnib on HUVECs.The result of proliferation, cytotoxicity, a variety of cytokines (IL-6,IL-8, MCP-1,CXCL-2), cell adhesion molecules VCAM-1, pathway protein NF-κB, and so on. The result determine that gefitnib can inhibit bleomycin-induced HUVECs proliferation and secretion of various cytokines.