Effect Of AcSDKP On The Collagen Synthesis Through Regulating P38MAPK Pathway Mediated By TGF-β1 In Cultured Rat Pulmonary Fibroblasts

Silicosis which is characterized by pulmonary fibrosis is a common occupational respiratory disease in the workers exposed chronically to silica.Silicosis is caused by breathing in lots of liberation SiO2,which is a occupational disease that extensive nodosity fibrosis is formed in fibrosis. The pathological features of silicosis are the formation of silicotic nodules and diffuse pulmonary interstitial fibrosis.The features of pathology are that appear distinctive focus of infection which forms silicotic nodule and diffuse interstitial fibrosis. Although there had already been lots of study and research towards the mechanism, prevention and therapy of silicosis, the precise mechanism especially how to prevent and treat its formation and development is not elucidated so far. Although we do lots of things to study and research the pathogenesis, prevention and cure of it for so many years, the exact pathogenesis isn't distinct. The nodus of our research is how to prevent and cure Silicosis formation and progression. So the research on the mechanism, prevention and therapy of silicosis of pathogenesis and prevention are always the hotspot in the field of occupational disease intensive and hot topic. It had been reported that many cytokines play an important role in the formation and development of silicosis by composing a complex molecular net of signal transduction. Recently, study hotspot focuses on all kinds of cytokines play an important role in the fibrosis forming, which form a complex network of cells and extracellular signal transduction molecules chain reaction systems. TGF-β1 plays an important role in this net and has been deeply researched. In these cytokines, TGF-β1 conduct one of intense cytokines which is researched most deeply. TGF-βis It's a group of polypeptide growth factors which has many biological functions and can adjust the growth, proliferation, differentiation and immunological function of cells. It is also an intensive fibrosis mediator which participates in the formation and development of lung fibrosis with its type I and type II receptors. It induces pulmonary fibrosis, and transforming growth factor-βtypeⅠreceptor(TGF-βRⅠ)and transforming growth factor-βtypeⅡreceptor(TGF-βRⅡ) participate the signal transmission of TGF-β1. A mass of data show that TGF-βcan induce the transduction of signal from extracellular to intracellular till it combine with its cell membrane receptor. While the activity and expression of these receptors can affect the biological functions of TGF-β. There are many research have implicated that TGF-β1 is activated by linking membrance receptor,then mediate transmission of cellular signal from ectocellular to intracellular. The Biological function and function of TGF-βare also directly influenced by the activity and quantity of its receptor production and synthesis.Mitongen activited protein kinase (MAPK) is a kind of Serine/ Threonine protein kinase which includes four components extracellular signal-regulated(ERK), P38 mitogn-activated protein kinase (P38 MAPK), c-jun amino-terminal kinase/stress-activated protein kinase (JNK/SAPK), and Big MAP kinasel(BMK1)(BMK1).Mitongen activited protein kinase(MAPK) are protein kinase of Serine/ Threonine in the cells. They include four members: extracellular signal-regulated(ERK), P38 mitogn-activated protein kinase (P38 MAPK), c-jun amino-terminal kinase/stress-activated protein kinase (JNK/SAPK), Big MAP kinasel(BMK1)(BMK1). The signal transduction pathways through mitongen activited protein kinase(MAPK) are huge, complex and subtle lattice which make the signal transmit from extracellular stimuli to the intracellular, and regulate a series of complex biological functions. Recent discovery indicates that P38 MAPK induced mediated by TGF-β1 maybe correlate with the forming of fibrosis.N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a tetrapeptide which was found finded in marrow of foetus cattle by Frindel et.al in 1971s. They first considered it as They researched it was a physiological hemoregulatory inhibitor and might have some effect on could regulating haematogenesis. While along with deeply research, we find it has widely anti-fibrotic effects for example inhibiting the collagen deposition and fibroblasts proliferation in heart and kidney. In previous experiments, we have confirmed that AcSDKP has an effect on anti-fibrosis for because it can could inhibit the synthesis of collagen in lung tissue in rat with silicosis. However, the mechanism of AcSDKP on inhibiting silicosis is not clear so far.The purpose of our study is to observe the effect of AcSDKP on the differentiation of lung fibroblast and synthesis of collagen by regulating TGF-β1 induced P38MAPK signal transduction pathway. Then group for more mechanisms of AcSDKP on the prevention and therapy of silicosis.The purpose of our study is to observe AcSDKP's influence on expression of TGF-β1 mediated P38 MAPK signal transduction pathway in lung fibroblast proliferation and collagen synthesis, in order to provide some basis for exploring a new way of inhibiting silicosis pulmonary fibrosis.Objectives1 To study and investigate the function of TGF-β1 and its receptors induced P38 MAPK signal transduction pathway in the proliferation of lung fibroblasts and the synthesis of collagen. Lung fibroblast proliferation and collagen synthesis.2 To study and investigate whether AcSDKP can develop its effect of inhibiting the proliferation of lung fibroblast and the synthesis of collagen by regulating TGF-β1 and its receptors induced P38 MAPK signal transduction pathway.To study and investigate the regulation of AcSDKP on the TGF-β1 / TGF-βreceproe-P38 MAPK signal transduction pathway in lung fibroblast proliferation and collagen synthesis. 3 To illuminate the mechanism of AcSDKP on inhibiting silicosis and provide some theory and experimental base.Methods1 Pulmonary fibroblasts from newborn Wistar rat were isolated in primary generation and fourth generation of cells was used for experiments.2 Experimental Groups:⑴Control: 0.4% serum in DMEM as a basis medium.⑵TGF-β1 stimulating group(TGF-β): TGF-β1 (5 ng/ml) in basis medium incubated cells for 45 min;⑶TGF-βreceptor inhibition treating(LY364947) group(LY): TGF-βreceptor inhibition (59 nmol/L) in basis medium pre-incubated cells for3 hours, then added TGF-β1(5 ng/ml)in medium and incubated cells for 45 min;⑷Inhibition of P38 MAPK pathway treating(SB203580) group(SB): inhibition of P38 MAPK pathway (10 nmol/L) in basis medium pre-incubated cells for 45 minutes, then added TGF-β1(5 ng/ml)in medium and incubated cells for 45 min;⑸AcSDKP treating group(Ac): AcSDKP (10-8 mol/L) in basis medium pre-incubated cells for 45 minutes, then added TGF-β1(5 ng/ml)in medium and incubated cells for 45 min;3 The proliferation of pulmonary fibroblasts was detected by MTT assay.4 The expression of TGF-βRⅠ, TGF-βRⅡ,P38 MAPK, phospho-P38 -MAPK , c-myc, collagen typeⅠand typeⅢin pulmonary fibroblasts was measured by western blot.5 The expression of phospho-P38 MAPK in pulmonary fibroblasts was measured by immuocytochemistry and confocal laser scanning microscopy.6 Using Real Time-PCR to measure the mRNA of TGF-βRⅠand TGF-β- RⅡ.7 Results were analyzed statistically by single-factor analysis of variance.It haved statistical significance when P was less than 0.05. Results:1 Results of MTT assay showed that compared with the control group, TGF-β1 could promote the proliferation of pulmonary fibroblasts, while LY364947, SB203580 and AcSDKP could inhibit the proliferation of pulmonary fibroblasts.2 Results of RT-PCR showed that compared with the control group, TGF-β1 could promote the mRNA expressions of TGF-βRⅠ, TGF-βRⅡvisibly, while LY364947 and AcSDKP could inhibit they expressed .but the inhibition of SB203580 in the mRNA expressions of TGF-βRⅠ, TGF-βRⅡwas not visible .3 Compared with control group, the expression of TGF-βRⅠ, TGF-βRⅡ, phospho-P38 MAPK, c-myc ,typeⅠand typeⅢcollagen protein increased significantly in TGF-β1 group with western blot analysis. Compared with TGF-β1 group, the expression of TGF-βRⅠ, TGF-βRⅡ, phospho-P38 MAPK, c-myc ,typeⅠand typeⅢcollagen protein decreased in LY364947 treating group and AcSDKP treating group, while SB203580 treating group the protein levels of phospho-P38 MAPK, c-myc ,typeⅠand typeⅢcollagen reduced visibly ,but TGF-βRⅠand TGF-βRⅡprotein levels don't reduced visibly ;P38- MAPK protein were not changed in five groups.4 Results of immunocytochemistry assay showed that compared with control group, the expression phospho-P38 MAPK protein increased significantly in TGF-β1 group. Compared with TGF-β1 group, the expression of phospho-P38 MAPK protein decreased in LY364947 treating group , SB203580 treating group and AcSDKP treating group.5 Results of confocal laser scanning microscopy displayed that expression of phospho-P38 MAPK labed fluorescence was located few in the cytoplasm and more in cytoblast in control group. Compared with TGF-β1 group, expression of phospho-P38 MAPK increased in the cytoplasm and increased in nuclei in the LY364947,SB203580and AcSDKP group. ConclusionTGF-β1 can induce P38 MAPK signal transduction pathway by combining with its receptors, then result in the proliferation of fibroblast and the synthesis of collagen.TGF-β1 could mediate P38 MAPK signal transduction pathway through its receptors, and then induce lung fibroblast proliferation and collagen synthesis. AcSDKP can block the TGF-β1–induced P38 MAPK pathway, and then inhibit the proliferation of lung fibroblast and the expressions of collagen type I and collagen type III. The effect may have some relate to the anti-fibrotic function of AcSDKP.AcSDKP could block TGF-β1–induced P38 MAPK pathway, then inhibit the proliferation and the expression of type I , type III collagen,which AcSDKP could play possibly an important role in anti-silicotic fibrosis.