Abstract: OBJECTIVE To explore whether the reduction of collagen expression in mouse cardiac fibroblasts (CFs) transfected with eukaryotic vector pcDNA3.1(+)-TGF-β1 directly associated with inhibition of TGF-β1/Smad signaling pathway.METHODS The mouse CFs were transfected with eukaryotic vector pcDNA3.1(+)-TGF-β1,and the TGF-β1-transfected CFs were then treated with different concentrations of osthole for 24 h in the presence or absence of pretreatment with TGF-β1 receptor Ⅰ inhibitor SB431542.The supernatant TGF-β1 level was determined by ELISA method,and the intracellular α-SMA,collagen Ⅰ,collagen Ⅲ,Smad2/3,p-Smad2/3 and Smad4 and Smad7 protein expressions were determined by Western blotting.RESULTS After transfection of CFs with eukaryotic vector pcDNA3.1(+)-TGF-β1,the release of TGF-β1 was increased,indicating that the cell model of TGF-β1-overexpression was established.Following treatment of TGF-β1-overexpressed CFs with osthole 5-20 μmol·L^-1 for 24 h,the TGF-β1,α-SMA,collagen Ⅰ,collagen Ⅲ,p-Smad2/3 and Smad4 protein expressions were decreased,while Smad7 protein expressions were increased.After pretreatment with TGF-β1 receptor Ⅰ inhibitor SB431542 for 2 h,the inhibitory effects of osthole on α-SMA,collagen Ⅰ,collagen Ⅲ,Smad2/3,p-Smad2/3 and Smad4 protein expressions were further enhanced.CONCLUSION Osthole can downregulate the expressions of α-SMA and collagen proteins by directly inhibiting TGF-β1 production and the Smad pathway,which might be one of its anti-fibrotic mechanisms.