Sonic Hedgehog promotes fibroblast-like synoviocytes proliferation via modulating the mitogen-acti-vated protein kinase/extracellular signal-regulated kinase signaling pathway in rheumatoid arthritis

Objective To study the effect of mitogen-activated protein kinas/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway on cell proliferation modulated by Sonic Hedgehog (Shh) signaling in fibroblast-like synoviocytes (FLS) isolated from patients with active rheumatoid arthritis (RA). Methods The synovial tissue were collected by the synovial arthroscopic debridement or arthroscopic syn-ovectomy of RA patients with active disease activity [disease activity score(DAS)28≥3.2]. The RA-FLS were primarily cultured by the explanted culture, and then were treated with Shh agonist purmorphamine, inhibitor cyclopamine or MAPK/ERK signaling pathway inhibitor U0126, respectively. Western blotting was used to examine the phosphorylation level of ERK 1/2 (p-ERK1/2), which was the critical protein of MAPK/ERK signaling. The cell proliferation activity was detected using cell proliferation and cytotoxicity kit-8 (CCK8), and the cell proliferation rate was detected using a flow cytometry. Analysis of variance and Kruskal-Wallis H(K) test were used for statistical analysis. Results Compared with the control group, purmorphamine transiently increased p-ERK1/2 protein at the concentration of 1 μmol/L, and the peak activations of p-ERK1/2 took place at 15 min (P<0.01). Cyclopamine and U0126 decreased the expression of p-ERK1/2 protein(P<0.01). After the RA-FLS treated with purmorphmine (1 μmol/L) for 48 hours, the cell proliferation activity was (114±4)% and the percentage of S phase cells was (8.39±0.60)%, which was significantly higher than those of the control group (100±0) % (P<0.01) and (3.29±0.69) % (P<0.01). After treated with cyclopamine (10 μmol/L) for 48 hours, the cell proliferation activity of RA-FLS was (89±1)% (P<0.05) and the percentage of S phase cells was (1.53±0.22)% (P<0.05). When co-treated with purmorphamine (1 μmol/L) and U0126 (10 μmol/L) , the cell proliferative activity was (89±2)% (P<0.05) and the percentage of S phase cells was (1.07±0.25)% (P<0.05). Conclusion Shh may promote proliferation of RA-FLS via modulating MAPK/ERK signaling, which in turn contributes to hyperplasia of synovium and ultimately leading to RA. Key words: Arthritis, rheumatoid; Sonic Hedgehog; Mitogen-activated protein kinase kinases; Cell proliferation