Recombinant TSG-6 protein inhibits the growth of capsule fibroblasts in frozen shoulder via suppressing the TGF-ß/Smad2 signal pathway.

ABSTRACT

BACKGROUND: The tumor necrosis factor-stimulated gene-6 (TSG-6) has been confirmed to inhibit inflammation. It is now generally accepted that local inflammatory stimulation around shoulder capsule causes proliferative fibrosis. This study aims to investigate the mechanism of recombinant TSG-6 protein inhibiting the growth of capsule fibroblasts in frozen shoulder via the TGF-ß/Smad2 signal pathway. METHODS: Human frozen shoulder capsule tissue was taken for primary and passage culture, and the 3rd generation fibroblasts from pathological frozen shoulder capsule were treated with different concentrations of recombinant TSG-6 protein, or with TGF-ß1 agonist SRI-011381. Immunoconfocal analysis was used to identify the isolated fibroblasts, and MTT assay, colony formation assay, and flow cytometry were used to detect the viability, proliferation, and apoptosis rate of fibroblast. The contents of fibrosis and inflammation indexes COL1A1, TNF-α, IL-6, and IL-1ß in the cell supernatant were detected using ELISA and then further examined by qRT-PCR. The expression of Bax, Bcl-2, and proteins related to TGF-ß/Smad2 pathway were detected by Western Blot. RESULTS: Compared with the blank control group, fibroblasts intervened with TSG-6 (2 µg and 5 µg) showed significantly decreased viability and proliferation ability and enhanced cell apoptosis, concurrent with the reductions in Bcl-2 expression; COL1A1, TNF-α, IL-6, and IL-1ß levels; and the expression of TGF-ß1 and phosphorylated Smad22, and an increase in Bax expression, while SRI-011381 treatment would reverse the effect of recombinant TSG-6 protein. CONCLUSION: Recombinant TSG-6 protein inhibited the growth of primary fibroblasts from human frozen shoulder capsule by suppressing the TGF-ß/Smad2 signaling pathway.