Inhibitory Effect Of Agents Selection On Silica-Induced Stimulation In Rat Lung Fibroblast

Silicosis is a chronic lung disease, which is caused by inhalation of silica-containing dusts, leading to pulmonary fibrosis. It is a chronic disorder characterized by the disposition of a large amount of silica proteins components, such as collagens. The pathology is characterized by a large number of alveolar protein secretion, followed by fibrous tissue may be associated with diffuse hyperplasia, the formation of silicon nodules and even small fiber lesions. Fibroblasts play an important role in the fibrogenesis. Free SiO2 dust can activate lung fibroblasts, and a-smooth muscle actin (a-SMA) is highly expressed. Then, the fibroblast turns into myofibroblasts, and a large number of extracellular matrix, especially collagen fibers, are secreted. Therefore lung fibroblast is playing an importanr role in lung fibrogenesis..Therefore, it is an effect way to cue silicosis through inhibit the stimulation of lung fibroblast, and through the inhibition of secretion of extracellular matrix, especially the collagen. The rat lung macrophages and lung fibroblasts in vitro were cultrured. Then the lung fibroblasts were exposed to the macrophages culture supernatant which was stimulated by free silica dust. Hence, the lung fibroblast were stimulated by the AM supernatant. Add the RGD peptide, colchicine, IFNa-1b and imatinib mesylate to the AM supernatant, select the inhibitory effect of agents to lung myofibroblast.Objective:To evaluate the inhibitive effect of RGD peptide, Colchicine, Interferon-a1b, Imatinib mesylate on rat lung fibroblast exposed to silica, to select a useful agent to inhibit the lung myofibroblast.Methods:1 Silica suspension without FBS were used to stimulate AM for 24 hours. Then supernatants were collected and centrifuged to remove silica. The collected medium were used to treat Lung fibroblast for 24 hours as SiO2 group; AM supernatant without silica was used to treat Lung fibroblast for 24 h as normal group. Lung fibroblast was treated by the collected silica supernatant for 24 h with RGD, or colchicine, or Interferon-a1b, or Imatinib mesylate.2 MTT assay was used to screening the cell activity; The expression of the a-SMA, COLⅠand COLⅢwere detected by using real-time PCR(RT-PCR), and ELISA was used to examine the expression of the COL I and COLⅢin cultured supernatant, and BradFord method was used to examine the expression of the total of secreted proteins which was secreted by LF.Results:1 The results show that, the difference of cell activity among the myofibroblasts which were added by RGD peptide, was not statistically significant. The mRNA of a-smooth muscle actin, collagenⅠand collagenⅢwere decreased in cultured supernatant exposed to RGD at all levels, and the values at 100μg/ml and 150μg/ml were statistically different(P<0.05), compared the SiO2 group. The concentration of COLⅠ, COLⅢand total protein were decreased in cultured supernatant exposed to RGD at all levels, and the values at 100μg/ml and 150μg/ml were statistically different(P<0.05), compared the SiO2 group.2 The results show that, the difference of cell activity among the myofibroblasts which were added by colchicine, was statistically significant(P<0.05), and the inhibition rate LC25 was 40μmol/L. The mRNA of a-smooth muscle actin, collagenⅠand collagenⅢwere decreased in cultured supernatant exposed to colchicine at all levels, and the values at 80μmol/L was statistically different(P <0.05), compared the SiO2 group. The concentration of COLⅠand total protein were decreased in cultured supernatant exposed to colchicine at all levels, and the values at 80μmol/L was statistically different(P<0.05), compared the SiO2 group.3 The results show that, the difference of cell activity among the myofibroblasts which were added by Interferon-alb, was statistically significant(P<0.05), and the inhibition rate LC25 was 100 u/ml. The mRNA of a-smooth muscle actin, collagenⅠand collagenⅢwere decreased in cultured supernatant exposed to Interferon-alb at all levels, and the values at 1000u/ml was statistically different(P <0.05), compared the SiO2 group. The concentration of COLⅠ, COLⅢand total protein were decreased in cultured supernatant exposed to Interferon-alb at all levels, and the values at 1000u/ml was statistically different(P<0.05), compared the SiO2 group.4 The results show that, the difference of cell activity among the myofibroblasts which were added by Imatinib mesylate, was statistically significant(P<0.05), and the inhibition rate LC50 was 5μmol/L. The mRNA of a-smooth muscle actin, collagenⅠand collagenⅢwere decreased in cultured supernatant exposed to Imatinib mesylate at all levels, and the values at 5μmol/ml was statistically different(P<0.05), compared the SiO2 group. The concentration of COLⅠand COLⅢwere decreased in cultured supernatant exposed to Imatinib mesylate at all levels, and the values at 5μmol/ml was statistically different(P<0.05), compared the SiO2 group.Conclusions:1 Colchicine, interferon-alb and imatinib mesylate can inhibit the activation of myofibroblasts.2 RGD peptide, Colchicine, Interferonalb and Imatinib mesylate could decrease the levels of COLⅠ, COLⅢand supernatant protein in LFs culture, suggesting inhibition effect on LF stimulated by silica.