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ABSTRACT: The transforming growth factor (TGF)-β/Smad pathway plays a key role in keloid development. We have previously demonstrated that compound Astragalus and Salvia miltiorrhiza extract (CASE) inhibits liver fibrosis and reduces invasion capacity of HepG2 cells by mediating the TGF-β/Smad pathway. We therefore hypothesize that CASE may also exert antifibrotic effects in keloids by mediating the TGF-β/Smad pathway.
To investigate the effects of CASE on cell proliferation, invasion and collagen synthesis in keloid fibroblasts, and to explore the effects of CASE on the TGF-β/Smad signal pathway in order to elucidate its mechanisms of action.
The inhibitory effects of CASE on keloid fibroblasts were evaluated. Cell proliferation was studied by MTT assay; cell invasion was observed utilizing Transwell invasion chambers; and collagen synthesis in keloid fibroblasts was measured by (3) H-proline incorporation assay. Expression of proteins induced by TGF-β1 and their intracellular localization in keloid fibroblasts were investigated by Western blot and immunofluorescence, respectively. Plasminogen activator inhibitor-1 (PAI-1) transcriptional activity was measured by real-time reverse transcription-polymerase chain reaction.
CASE significantly inhibited cell proliferation induced by newborn bovine serum as well as collagen synthesis and cell invasion induced by TGF-β1 in keloid fibroblasts, while it showed weak effects on normal fibroblasts. The phosphorylation of Smad2/3 was markedly reduced by CASE treatment, while CASE exhibited stronger inhibitory effects on linker region phosphorylation (pSmad2L and pSmad3L) compared with effects on C-terminal region phosphorylation (pSmad2C and pSmad3C). In addition, CASE blocked formation of Smad2/3/4 complexes and their nuclear translocation, but upregulated Smad7 expression in a dose-dependent manner. PAI-1 mRNA and protein levels were also suppressed by CASE treatment.
These results suggest that CASE exhibits inhibitory effects on cell proliferation, invasion and collagen synthesis in keloid fibroblasts, and its mechanisms of action may involve the TGF-β/Smad pathway.
British Journal of Dermatology 10/2011; 166(3):564-74. · 3.76 Impact Factor
ChemInform 01/2010; 30(4).
ChemInform 01/2010; 29(26).
ChemInform 01/2010; 28(41).
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ABSTRACT: Keloids are recognized as benign tumours characterized by fibroblastic proliferation and accumulation of extracellular matrix, especially collagen deposition. The transforming growth factor (TGF)-beta(1)/Smad pathway plays an important role in keloid pathogenesis; however the underlying mechanisms are not fully understood.
To define further the mechanisms of TGF-beta(1)/Smad signal transduction mediated by mitogen-activated protein kinases (MAPKs), including the extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 pathways, in keloid fibroblasts.
In the absence or presence of three MAPK (ERK, JNK and p38)-specific inhibitors, keloid fibroblasts were stimulated with exogenous TGF-beta(1) to activate Smad signalling. Smad protein expression was measured by immunoprecipitation/immunoblotting and immunofluorescence; plasminogen activator inhibitor (PAI)-1 transcriptional activity was measured by real-time reverse transcriptase-polymerase chain reaction analysis.
TGF-beta(1) induced Smad2/3 phosphorylation at both the C-terminal and the linker region, thus promoting formation of the Smad2/3/4 complex and nuclear translocation, and PAI-1 mRNA expression in keloid fibroblasts; in addition, TGF-beta(1) decreased inhibitory Smad7 expression. Meanwhile, the p38 inhibitor significantly inhibited Smad2/3 phosphorylation, especially at the linker region, and furthermore blocked Smad2/3/4 complex formation, and thus decreased PAI-1 mRNA expression; decreased Smad7 expression induced by TGF-beta(1) was also reversed by the p38 inhibitor. The ERK and JNK inhibitors interrupted Smad2/3/4 complex translocation into the nucleus and consequently decreased PAI-1 mRNA expression.
These results suggested that the ERK, JNK and p38 pathways mediate TGF-beta(1)/Smad signal transduction and might be considered as specific targets of drug therapy for keloids.
British Journal of Dermatology 09/2009; 162(3):538-46. · 3.76 Impact Factor