| Objective:To study the effects of collagen binding domain-vascular endothelial growth factor onbleomycin-induced pulmonary fibrosis in mice and to explore its mechanism.Methods:1.Construction of mouse model of pulmonary fibrosis: C57BL/6mice were randomlydivided into Sham group and pulmonary fibrosis model group which were reproduced byintratracheal instillation of BLM. At2,4,16weeks after BLM treatment, the mice weresacrificed. The lung tissue was obtained and stained with HE and MASSON forhistopathological examination, which was evaluated by the Ashcroft score. The expressionof α-smooth muscle actin (α-SMA), Ⅰcollagen (COL-Ⅰ) and transforming growthfactor-β1(TGF-β1) were analyzed by immunohistochemistry, and compared byimmunostaining scores.2.The intervention of CBD-VEGF on BLM-induced pulmonary fibrosis in mice:C57BL/6mice were randomly divided into Sham group, BLM+NS group andBLM+VEGF group. Except the Sham group, the other two groups were injected of BLM.After BLM treatment, BLM+VEGF group were immediately intervened withCBD-VEGF-His-tag (2ug/100ul/body) by intratracheal instillation. The Sham group andBLM+NS group were received equal volume saline. Part of mice in BLM+NS group andBLM+VEGF group were sacrificed at6hours after CBD-VEGF administrated. Theexpression of His-tag was analyzed by immunohistochemistry. The remaining mice weresacrificed at week2,4and16after intervention. The mice lung function was detected before being sacrificed at week4and16. The lung tissue was obtained and stained withHE and MASSON for histopathological examination, which was evaluated by the Ashcroftscore. The expression of α-SMA, COL-Ⅰ and TGF-β1were analyzed byimmunohistochemistry, and compared by immunostaining scores. The micro vessel densitywas assessed by CD31immunohistochemical staining.Results:Compared with Sham group, the HE and MASSON staining of Model group showedBLM treatment significantly induced distortion of the lung structure and collagendeposition in the lungs and the Ashcroft score was significant higher (P<0.001). Theexpression of α-SMA and COL-Ⅰwhich were analyzed by immunohistochemistry andimmunostaining scores were significantly enhanced respectively (P<0.001，P<0.01). Theexpression of TGF-β1was increased at week2and4, but there was a decrease tendency atweek16, although there were no difference among the three time points. The Ashcroftscore and the expression of COL-Ⅰ among Model groups showed no significant differenceat the different time points. At week16, the expression of α-SMA was lower than that ofweek4.Compared with BLM+NS group, BLM+VEGF group showed that the expression ofHis-tag was increased（P<0.001）; and it was more obvious on the distortion of the lungstructure and collagen deposition. The Ashcroft score and the expression of TGF-β1andα-SMA were higher at week2(P<0.05，P<0.01，P<0.01）; the expression of COL-Ⅰwasincreased at week4(P<0.01). The lung function showed no significant difference. At week16, MVD showed the number of vessels were significantly increased in BLM+VEGFgroup（P<0.001）.Among the BLM+VEGF groups, the Ashcroft score and the expression of TGF-β1were significantly decreased at week4and16than at week2; accordingly, the expressionof α-SMA and COL-Ⅰwere also significantly decreased at week16than at week2. Conclusion：Intratracheal instillation of BLM can successfully construct mouse model ofpulmonary fibrosis. This kind of model is stable and there is no apparent reverse changeduring16weeks. CBD-VEGF can be effectively delivered to the lung tissue byintratracheal instillation, and it can stimulate the expression of TGF-β1and α-SMA, whichcould strengthen the collagen deposition, ultimately increase pulmonary fibrosis.CBD-VEGF can promote the angiogenesis of lung tissue, which maybe play a importantrole in the repairment of pulmonary fibrosis. |
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