Lentivirus Overexpressing Soluble ST2 Protein Inhibits Pulmonary Fibrosis In Mice

ObjectiveInterkeukin(IL)-33 is a new member of the IL-1 family, and soluble ST2(s ST2) is a decoy receptor for IL-33. IL-33 mainly functions through IL-33/ST2 signaling pathway. Pulmonary fibrosis diseases is the general term that pulmonary fibrosis cased by a variety of causes, including idiopathic pulmonary fibrosis, hypersensitivity pneumonitis, interstitial pneumonia, sarcoidosis, pneumoconiosis, drugs, and radiation. It is caused by the aggregation of fibroblasts, the extracellular matrix deposition with inflammatory responses and damage, which results to the destruction of lung structures. Currently, there is no effective treatment all over the world. Studies have shown that the levels of s ST2 and IL-33 were elevated in patients with idiopathic pulmonary fibrosis and bleomycin-induced pulmonary fibrosis in mice. Therefore, we will construct lentiviral vector overexpressing soluble ST2 protein to explore the role and mechanism of ST2 on pulmonary fibrosis in mice.Methods1. Full length mice ST2 gene was amplified from the plasmid pc DNA3.1-m ST2 by designed primers. Then the ST2 gene and p LVX-IRES-Zs Green1 plasmid were cleavaged by Xho I and Bam H I restriction enzymes. After that, T4 ligase was used to link the two fragments and then the ligation product was transformed into the competent cells to construct p LVX-ST2-IRES-Zs Green1 lentiviral vector.2. The constructed p LVX-ST2-IRES-Zs Green1 lentiviral vector and three helper plasmids were co-transfected into human embryonic kidney(HEK) 293 T cells to package ST2 lentivirus. Moreover, the titer and activity of the lentivirus were tested.3. ST2 lentivirus was injected intranasally to bleomycin-induced pulmonary fibrosis mouse models. Then the samples were collected to assess the effects of ST2 on pulmonary fibrosis in mice after sacrificed on days 7 and 28 after administration of bleomycin for the first time. HE staining was used to evaluate the inflammation extent. Sirius red staining and hydroxyproline content were applied to detect the changes of lung fibrosis and collagen content in mice, respectively. RT-PCR and ELISA were employed to examine the expression of relevant cytokines and chemokines.Results1. The full length ST2 amplified by PCR was inserted to multiple cloning site of p LVX-IRES-Zs Green1 and p LVX-ST2-IRES-Zs Green1 lentiviral vector was successfully constructed.2. ST2 lentivirus was successfully packaged after the co-transfection of recombinant plasmid p LVX-ST2-IRES-Zs Green1 and helper plasmids into HEK 293 T cells. After concentration, the titer of virus can achieve 1011 TU/m L, and the lentivirus can express ST2 gene in eukaryotic cells.3. Soluble ST2 protein was overexpressed in mice after injection of ST2 lentivirus. ST2 lentivirus treatment alleviated significantly the pulmonary inflammation and fibrosis of mice. The expression of TNF-α m RNA and protein, MMP9 m RNA was increased, but the levels of IL-4, IL-6, and IL-13 m RNA were decreased after administration of ST2 lentivirus on day 7. Morever, MCP-1 and MMP12 m RNA expression was reduced by ST2 on day 28. Additionally, the degree of IFN-γ m RNA was elevated on days 7 and 28.ConclusionST2 lentiviral vector was successfully constructed, and then packaged to lentivirus, which can express ST2 gene in eukaryotic cells. The injection with ST2 lentivirus can overexpress soluble ST2 protein in mice of pulmonary fibrosis. The pulmonary fibrosis was inhibited after administration of ST2 lentivirus, but its mechanism is not clear and needs further study.