Abstract:

Objective To study transcriptional characteristics of type Ⅲ procollagen gene in systemic scleroderma （SS）-derived fibroblast clones and their regulation by Radix Salviae miltiorrhizae（RSM）. Methods Eight fibroblast clones with different collagen-producing capacity were previously obtained from patients with SS and normal human controls. Recombinant plasmids containing different deletions of the human alpha 1 chain of type 3 procollagen （COL3A1） gene promoter were constructed, and transiently transfected into the fibroblast clones. Dual-luciferase reporter assay system was used to evaluate the activities of these recombinants in the fibroblast clones and to select a proximal transcriptional regulatory sequence. Then, the fibroblast clones were transfected with the plasmid containing the selected regulatory sequence （phCOLH30.1） followed by the treatment with RSM injection （1 g/L） and active monomers of RSM, including salvianolic acid B （5 mg/L）, tanshinone ⅡA（5 mg/L）, danshensu （20 mg/L） and protocatechuic aldehyde （5 mg/L）, for 48 hours. The transfected fibroblast clones receiving no drug treatment served as the water-soluble control, and those treated with only dimethyl sulfoxide as the lipid-soluble control. Subsequently, the fibroblasts were lysed and subjected to the quantification of cellular proteins and determination of luciferase activity. The activity of recombinant promoters was compared by t test for the selection of proximal transcriptional regulatory sequence, and the activity of phCOLH30.1 by two-way analysis of variance in the RSM-interfering test （if there was interaction, one-way analysis of variance was conducted; and if there was no interaction, the main effect was tested after the removal of interaction item）. Results Of the 6 recombinants, the recombinant containing COL3A1 proximal promoter from -96 bp to +16 bp （phCOLH30.1） showed the highest transcriptional activity in nearly all of the fibroblast clones, and the activity was positively correlated with the collagen-producing capacity of fibroblast clones. Compared with the water-soluble control, RSM injection significantly downregulated the activity of phCOLH30.1 in fibroblast clones with high and low collagen-producing capacity from patients with SS （2.261 ± 0.619 vs. 3.879 ± 0.309, 1.462 ± 0.291 vs. 2.150 ± 0.262, both P < 0.01） and normal human controls （1.681 ± 0.263 vs. 3.039 ± 0.271, 1.121 ± 0.361 vs. 2.223 ± 0.247, both P < 0.01）, salvianolic acid B decreased the phCOLH30.1 activity in SS-derived high collagen-producing fibroblast clones （2.309 ± 0.524, P ＜ 0.01） and in the normal control fibroblast clones with high and low collagen-producing capacity （2.126 ± 0.320 and 1.976 ± 0.362, both P < 0.05）. Tanshinone ⅡA only downregulated the phCOLH30.1 activity in SS-derived high collagen-producing fibroblast clones compared with the lipid-soluble control （2.975 ± 0.666 vs 5.379 ± 0.238, P ＜ 0.01）. Neither danshensu nor protocatechuic aldehyde showed inhibitory effects on phCOLH30.1 activity in SS-derived or normal control fibroblast clones. Conclusions The type Ⅲ procollagen gene is activated at the transcriptional level in high collagen-producing fibroblast clones from patients with SS, and the activation could be suppressed by RSM injection, salvianolic acid B and tanshinone ⅡA.

Key words: Salviae miltiorrhizae