Effect Of N-acetyl-seryl-aspartyl-lysyl-proline On The Proliferation And Collagen Synthesis In Cultured Rat Pulmonary Fibroblasts Through JNK Signal Transduction Mediated By TGF-β1

Pulmonary fibrosis is a chronic lung disease caused by a variety of reasons and characterized in persistent lung injury, repair, remodeling of the organizational structure and extra cellular matrix (mainly collagen typeⅠand typeⅢ) deposition associated with inflammatory response in lung. Recently,study hotspot focuses on the role of cytokines in forming and development of pulmonary fibrosis, including transforming growth factor-β, platelet derived growth factor, connective tissue growth factor, insulin-like growth factor-1 and so on. TGF-βis one of more extensive and indepth study cytokines, including TGF-β1, TGF-β2 and TGF-β3 three forms,which are basically the same biological characteristics. However,TGF-β1 is the most important among them. TGF-β1 can effectively stimulate fibroblast cell division, proliferation and extracellular matrix synthesis and secretion.TGF-βsignal transduction includes two main pathways,Smads protein family and mitogen activated protein kinase (MAPK) pathway. There are four subfamilies in MAPK including signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase (P38) and extracellular signal regulated kinase (ERK5). In which,JNK pathway plays a pivotal role in various physiological and pathological events. In recent years, studies have confirmed that JNK signal transduction involved in forming and development of fibrosis in the heart, liver, brain and other organs.N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a physiological hemoregulatory inhibitor. Recent study found that AcSDKP could inhibit the interstitial cell proliferation and collagen deposition in kidney and heart, and weakened cardiac and renal fibrosis. In previous experiments,we have confirmed that AcSDKP has an effect on anti-fibrosis because it could inhibit the expression of TGF-β1 and synthesis of collagen in the lung tissue in rat with silicosis. However,we don't know that AcSDKP whether plays a role in pulmonary fibrosis through regulation of the signal transduction pathways. This study will investigated whether TGF-β1 can induced pulmonary fibroblast proliferation and collagen synthesis through activation of JNK pathways, as well as AcSDKP whether can inhibit pulmonary fibroblast proliferation and collagen synthesis by blocking activation of this pathway.Objectives1. To explore the role in proliferation and collagen synthesis of pulmonary fibroblasts through activation of JNK pathway mediated by TGF-β1.2. To investigate the effect of AcSDKP on inhibiting proliferation and collagen synthesis of pulmonary fibroblast through regulation of JNK pathway mediated by TGF-β1.3. To provide some theory and experimental data in mechanism of AcSDKP anti-fibrosis in lung.Methods1. Pulmonary fibroblasts from newborn Wistar rat were isolated in primary generation and fourth generation of cells was used for experiments.2. The proliferation of pulmonary fibroblasts was detected by MTT assay.3. The localization and distribution of phosphorylation-JNK were observed by confocal laser scanning microscopy.4. The expressions of JNK protein,phosphorylation-JNK protein,c-jun protein,typeⅠand typeⅢcollagen protein in pulmonary fibroblasts were measured with western blot analysis and immunocytochemistry.5. Experimental Groups①0.4% serum group(Control): 0.4% serum in DMEM as a basis medium.②TGF-β1 stimulating group(TGF-β1): TGF-β1 (5μg/L) in basis medium incubated cells for 40 min, to observe the effect of TGF-β1 on stimulating proliferation and collagen synthesis in pulmonary fibroblasts.③SP600125 treating group(SP600125): SP600125 (10nmol/L) in basis medium pre-incubated cells for 45 minutes, then added TGF-β1(5μg/L)in medium and incubated cells for 40 min,to observe the effect of SP600125 on inhibiting proliferation and collagen synthesis and activation of JNK pathway in pulmonary fibroblasts mediated by TGF-β1.④AcSDKP treating group(AcSDKP): AcSDKP (10-8 mol/L) in basis medium pre-incubated cells for 45 minutes,then added TGF-β1(5μg/L)in medium and incubated cells for 40 min,to observe the effect of AcSDKP on inhibiting proliferation and collagen synthesis and activation of JNK pathway in pulmonary fibroblasts mediated by TGF-β1.6. Results were analyzed statistically by single-factor analysis of variance.Results1. Results of MTT assay showed that 1×105/ml density was optimum growth density for pulmonary fibroblasts. The strongest stimulation effect of TGF-β1 in cell proliferation was at 5μg /L in 0.4% serum culture conditions. SP600125(10nmol/L) and AcSDKP(10-8 mol/L) inhibited pulmonary fibroblast proliferation induced by TGF-β1.2. Results of confocal laser scanning microscopy displayed that expression of phosphorylation-JNK labed fluorescence was located more in the cytoplasm in control group. Compared with control group, expression of phosphorylation-JNK decreased in the cytoplasm and increased in nuclei in the TGF-β1 stimulating group. The ratio of nuclei to cytoplasm of phosphorylation-JNK fluorescence density increased in TGF-β1 groups. Compared with the TGF-β1 stimulating group, the fluorescence density in cytoplasm increased and the ratio of nuclei to cytoplasm of fluorescence density decreased in AcSDKP treating group and SP600125 treating group.3. Compared with control group, the expression of phosphorylation-JNK, c-jun and typeⅠand typeⅢcollagen protein increased significantly in TGF-β1 group with western blot analysis. Compared with TGF-β1 group, the expression of phosphorylation-JNK,c-jun and typeⅠand typeⅢcollagen protein decreased in AcSDKP treating group and SP600125 treating group, while the protein levels of JNK were not changed in four groups.ConclusionTGF-β1 could promote pulmonary fibroblast proliferation and typeⅠand typeⅢcollagen expression by activation of JNK pathway. However, AcSDKP could inhibit the proliferation and typeⅠand typeⅢcollagen expression of pulmonary fibroblasts through blocking activation of JNK pathway mediated by TGF-β1.