| Keloid is recognized as a fibrotic disease characterized by accumulation of extracellular matrix (ECM), especially the deposition of collagen. Current studies believe that transforming growth factor beta 1 (TGF-β1) is the principal factor for keloid by promoting fibroblasts proliferation and producing collagen which leads to ECM accumulation and fibrosis. In recent years, some studies have shown that TGF-β1 /Smad signaling pathway plays an important role in keloid pathogenesis, but many of them only focus on C terminal by TβRI pathway. We have reported that TGF-β1 could induce phosphorylation of C-terminal of Smad in KFs, and found that TGF-β1 could also highly induce phosphorylation of linking regions of Smad, and confirmed the linking- regions of Smad is the key point in keloid.Compound Astragalus and Salvia miltiorrhiza extracts (CASE) is proved to exert anti-hepaticfibrosis and inhibit proliferation of HepG2 cells through medating the TGF-β1/Smad signaling pathway. To investigate the effect of CASE on proliferation, synthesis of collagen and invasion ability in KFs induced by TGF-β1, and to study the effect of CASE on phosphorylation at linker region in KFs induced by TGF-β1, those methods such as MTT assay method, 3H-Proline incorporation method, transwell chamber assay and immunofluorescence staining were used and the results are as follows:1 Inhibiting effect of CASE on KFs proliferation induced by serumNBS (10%) increased KFs cell proliferation, and the elevated cell proliferation was markedly inhibited by CASE(15～60 mg·L-1) treatment in a dose-dependent manner and IC50 was 18.90 mg·L-1.2 Inhibiting effect of CASE on collagen synthesis in KFsTGF-β1(40 pmol·L-1) and 15% NBS could both stimulate synthesis of collagen. CASE (3.75～60 mg·L-1)could inhibit synthesis of collagen in KFs induced by TGF-β1 in a dose-dependent manner, and CASE (7.5～60 mg·L-1)could also inhibit the synthesis of collagen induced by NBS in a dose-dependent manner. IC50 of CASE for inhibition of collagen synthesis induced by NBS and TGF-β1 are 6.49 mg·L-1 and 4.20 mg·L-1.3 Inhibiting effect of CASE on invasion ability in KFsTGF-β1(40 pmol·L-1) and 15% NBS could both significantly strengthen the invasion ability of KFs. CASE(7.5～30 mg·L-1) could significantly inhibit the invasion ability of KFs induced by TGF-β1 , and CASE((7.5～60 mg·L-1)also inhibit the invasion ability of KFs induced by NBS. And CASE and TGF-β1 have no significant effect on invasion ability of NFs. IC50 of CASE to invasion ability of KFs induced by NBS and TGF-β1 are 19.70 mg·L-1 and 12.37 mg·L-14 Inhibiting effect of CASE on proteinum expression of pSmad2L and pSmad3L in KFs induced by TGF-β1The results tested by immunofluorescence staining method showed that: TGF-β1(40 pmol·L- 1) could significantly increase proteinum expression of pSmad2L and pSmad3L, and stimulate translocation into nucleus of pSmad2L and pSmad3L; CASE (7.5～30 mg·L-1) significantly inhibit phosphorylated protein increase of pSmad2L and pSmad3L in KFs induced by TGF-β1, and inhibit translocation into nucleus of pSmad2L and pSmad3L. TGF-β1 and CASE have no significant effect on protein phosphorylation of Smad2/3 in NFs. In summary, CASE could significantly inhibit proliferation, synthesis of collagen, invasion ability, and phosphorylation of Smad2/3 at linking region in KFs induced by TGF-β1. Therefore, CASE might play a role in anti-keloid through interfering TGF-β1/Smad signaling pathway in vitro.
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