| Objects:Silicosis is a common occupational respiratory disease in the workers exposed to dust around the world. In China, the number of cases has increased rapidly due to the expansive growth of industry and the absence of available methods to prevent dust. By the end of2010, China recorded676,541cases of pneumonoconiosis totally (approximately10,000new cases per year), with half of the cases being silicosis. Silica is a potent fibrogenic agent that induces apoptosis in alveolar macrophages (AMs). After exposure to silica, AMs are activated and mediate chronic inflammation, resulting in the release of a variety of enzymes and inflammatory cytokines, such as transforming growth factor (TGF-β). TGF-β promotes extracellular matrix (ECM) accumulation by up-regulating collagen and fibronectin gene expression, and inhibits matrix degradation by decreasing secretion of proteases and increasing secretion of protease inhibitors. TGF-P has been implicated in the pathogenesis of both experimental pulmonary fibrosis and human disease. It is also an important inducer of myofibroblast differentiation characterized by the expression of a-smooth muscle actin (α-SMA). Myofibroblasts synthesize large amounts of ECM proteins and play a central role in fibrotic disorders. Myofibroblast differentiation is regulated by serum response factor (SRF) mediated by TGF-β in lung.N-acetyl-seryl-aspartyl-lysylproline (Ac-SDKP) is a natural anti-inflammatory and anti-fibrotic peptide. It is hydrolyzed exclusively by angiotensin converting enzyme (ACE), and its plasma concentration is increased substantially by ACE inhibitors. Previously, we and others have found that Ac-SDKP inhibits organ fibrosis, such as heart, renal and lung fibrosis. Along these lines, we previously reported that Ac-SDKP can inhibit pulmonary fibrosis in rats with SiO2-induced silicosis by inhibiting chronic inflammation, TGF-β1production, and TGF-β1induced pulmonary fibroblast proliferation and collagen synthesis.Here we examined whether an anti-fibrotic agent, N-acetyl-seryl-asparty-lysylproline (Ac-SDKP), can regulate induction of TGF-β and its receptor and myofibroblast differentiation as a potential key component of its anti-fibrotic mechanism in vivo and in vitro. To explore the pathophysiologic mechanisms of silicosis in depth, and find targets of Ac-SDKP, proteomics strategy was performed to screen for dynamic proteomics profile of control4/8w, silicosis4/8w and Ac-SDKP antifibrotic/prevent treatment group.Methods:Rat pulmonary fibroblasts were cultured in vitro and divided to4groups:1) control;2) TGF-β1;3) TGF-β1+LY364947;4) TGF-β1+Ac-SDKP. For in vivo studies, six groups of animals were utilized:1) control4w;2) control8w;3) silicotic model4w;4) silicotic model8w;5) Ac-SDKP post-treatment;6) Ac-SDKP pre-treatment. SiO2powders were douched in the trachea of rat to make the silicotic model. Myofibroblast differentiation was measured by examining expression of the myofibroblast differentiation marker, a-SMA, as well as expression of serum response factor (SRF), a key regulator of myofibroblast differentiation. Total collagen, collagen type I and III, TGF-β1, TGF-β receptor I and II were also assessed by Western blot or Realtime PCR.Two-dimensional gel electrophoresis and stained by colloidal Coomassie brilliant blue method. The gels were scanned with Lab Scan III system and analyzed by Imagemaster software6.0. The determinant criterion of differential protein was statistical significance of the volume%of the same protein spot between the two groups (i.e. P value was less than0.05which obtained by One-way ANOVA), and difference of volume%exceeding1.3fold compared with NC group. The differentially expressed proteins were subject to MALDI-TOF-MS and Mascot search engine analysis to get the molecular weight and β1value of the protein. Results:1Regulatory role of Ac-SDKP in TGF-β receptor type I and II in cultured rat pulmonary fibroblasts induced by TGF-β1TGF-β1treatment of cultured pulmonary fibroblasts resulted in up-regulation of the TGF-β receptor type I and II by1.7and1.8fold, respectively compared to non-induced cells. Furthermore, treatment with LY364947, a TGF-β receptor inhibitor, attenuated the up-regulation effect induced by TGF-β1. Finally, pre-treatment with the anti-inflammatory agent, Ac-SDKP induced a similar decrease of the TGF-β receptor type I and II expression to76.83%and84.92%, respectively.2Regulatory role of Ac-SDKP in expression of SRF and a-SMA in cultured rat pulmonary fibroblasts induced by TGF-β1The effects of TGF-β1and Ac-SDKP on myofibroblast differentiation were examined by accessing their effects upon the myofibroblast differentiation marker, a-SMA, and upon serum response factor (SRF), a key factor that regulates myofibroblast differentiation. TGF-β1induced a marked change in fibroblast morphology after48hours of treatment, with a larger cell body and a spingde-shaped, parallel or overlaped actin fiber markerd by a-SMA as visualized by microscopy, and had altered cytoskeletons characterized by dramatically increased a-SMA formation. Pre-treatment with LY364947and Ac-SDKP was able to significantly attenuate these changes. Western blot analysis further confirmed the immunocytochemistry results by showing that Ac-SDKP and LY364947pretreatment induced a significant reduction of a-SMA to51.54%and68.08%of that in the TGF-β1treated group, respectively. Likewise, Western blot analysis confirmed a strong up-regulation of SRF expression in TGF-β1treated fibroblasts as compared to control cells, and that this effect was significantly inhibited by pretreatment with either LY364947or Ac-SDKP. Expressions of SRF and a-SMA mRNA in fibroblasts induced by TGF-(31were examined by Realtime PCR. Significant increasing of SRF and a-SMA mRNA levels in TGF-β1treated fibroblasts as compared to uninduced cells, and that up-regulation induced by TGF-β1was significantly inhibited by pretreatment with either LY364947or Ac-SDKP.3Regulatory role of Ac-SDKP in expression of protein and mRNA of collagen type I and type III in cultured rat pulmonary fibroblasts induced by TGF-βWe next examined expression of collagen type I and III after TGF-p induction to elucidate the effect of Ac-SDKP on extracellular matrix (ECM) synthesis. Following induction by TGF-β1, collagen type I and III expression was significantly increased compared to the control group. In contrast, pretreatment with Ac-SDKP resulted in significant attenuation of collagen type I and III expression. In addition, LY364947also significantly reduced expression of collagen type I and III in cultured rat pulmonary fibroblasts induced by TGF-β1. Likewise, Realtime PCR analysis also shown a strong up-regulation of collagen type I and III mRNA levels in TGF-β1treated fibroblasts as compared to control cells, and that this effect was significantly inhibited by pretreatment with either LY364947or Ac-SDKP.4Regulatory role of Ac-SDKP in expression of TGF-β1、 SRF、 a-SMA and collagen type I and type III in silicotic model of ratWe next sought to determine whether Ac-SDKP can regulate myofibroblast differentiation in a pathologically relevant in vivo model of silicosis. Specifically, we examined whether Ac-SDKP could suppress expression of TGF-β1and its receptors, SRF, a-SMA and ECM in an in vivo silicotic model described previously by our group and others. The expression of collagen levels in the silicotic model (4w and8w) was higher than that in control group (4w and8w), as assessed by hydroxyproline assay and Western blot. Interestingly, compare to silicosis8w group, in Ac-SDKP post-treatment animals, silicotic nodules were less than that in silicosis model, and total collagen, collagen type I and III were reduced to84.08%,59.79%, and65.15%, respectively. Ac-SDKP pre-treatment was able to significantly attenuate the silicosis-induced increases ECM deposition in the lung, and total collagen, collagen type I and III were reduced to81.85%,55.67%, and47.72%, respectively. The plasma concentration of TGF-β1was also elevated in silicosis model (4w and8w) and inhibited by Ac-SDKP either post-treated or pre-treated. The expressions of TGF-β1in silicotic model4w increased by2.09folds, respectively compared with the control4w group; and increased by2.62folds in silicotic8w group compare with control8w group. The up-regulation of TGF-(31observed in silicotic model8w was significantly reversed by Ac-SDKP post-treatment by62.26%of silicotic model8w, respectively. Ac-SDKP pretreatment decreased the expression of TGF-β1by38.68%of silicotic model8w.Induction of interstitial myofibroblasts was assessed by immunohistochemical detection of a-SMA and SRF in the silicotic model. As shown in Fig.5-6, positive staining for a-SMA and SRF were seen in vascular vessels and trachea smooth muscle cells, but not in the interstitial space in the control group. Positive expressions of a-SMA and SRF were observed in silicotic nodules and interstitial fibrotic regions in silicosis model. Of significant interest, Ac-SDKP post-and pre-treatment markedly reduced the appearance of a-SMA and SRF in the silicotic nodules and interstitial fibrotic area. Western blot analysis further confirmed immunohistochemical results by showing that silica (4w and8w) increases the expressions of a-SMA by3.11and3.13folds compared with control group (4w and8w), and that Ac-SDKP post-treatment and pre-treatment significantly ameliorated the expression of a-SMA in rat lung with silicosis to66.96%and63.69%, respectively. Similar to the expression of a-SMA, the up-regulation of SRF observed in silicotic model8w group can be attenuated to54.02%and53.63%in Ac-SDKP post-and pre-treatment, compare with silicotic8w group respectively.5Regulatory role of Ac-SDKP in differential proteins in rat with silicosis by proteomicsCompared with control4w group, three soluble protein spots were up regulated, and4soluble protein spots were down-regulated in silicosis4w group. Compared with control8w group, two soluble protein spots were up regulated, and4soluble protein spots were down-regulated in silicosis4w group. In Ac-SDKP post-treatment treatrment group, two soluble protein spots were up-regulated and2soluble protein spots were down-regulated compared with silicosis8w group. And in Ac-SDKP pretreatment group, one soluble protein spots were up-regulated and2soluble protein spots were down-regulated compared with silicosis8w group.Compared with control4w group, fourteen insoluble protein spots were up regulated, and7insoluble protein spots were down-regulated in silicosis4w group. Compared with control8w group, fourteen insoluble protein spots were up regulated, and8insoluble protein spots were down-regulated in silicosis4w group. In Ac-SDKP post-treatment treatrment group, three insoluble protein spots were up-regulated and4insoluble protein spots were down-regulated compared with silicosis8w group. And in Ac-SDKP pretreatment group, seven insoluble protein spots were up-regulated and8insoluble protein spots were down-regulated compared with silicosis8w group.Conclusions:1The results of the present study suggest a novel mechanism of action for Ac-SDKP’s anti-silicotic effect, which involves attenuation of TGF-β1and its receptors, SRF expression, collagen deposition and myofibroblast differentiation in vivo and in vitro.2In the present study, we screened the critical regulatory proteins related to silicotic formation and the anti-silicotic effect of Ac-SDKP, which might play an important role in proliferation, apoptosis, inflammation, epithelial-mesenchymal transitions and signal transduction in silicosis. These results provided some significant evidences to explore the mechanisms of silicosis and find the new target of anti-silicotic effect of Ac-SDKP. |
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