Antifibrogenic effects of vitamin D derivatives on mouse pancreatic stellate cells

To study the molecular effects of three different D-vitamins, vitamin D2, vitamin D3 and calcipotriol, in pancreatic stellate cells (PSCs).

The three D-vitamins inhibit, with similar efficiencies, activation of PSCs in vitro, but cannot reverse the phenotype once the cells are fully activated.

Quiescent PSCs were isolated from mouse pancreas and activated in vitro by seeding on plastic surfaces. The cells were exposed to D-vitamins as primary cultures (early-activated PSCs) and upon re-culturing (fully-activated cells). Exhibition of vitamin A-containing lipid droplets was visualized by oil-red staining. Expression of α-smooth muscle actin (α-SMA), a marker of PSC activation, was monitored by immunofluorescence and immunoblot analysis. The rate of DNA synthesis was quantified by 5-bromo-2'-deoxyuridine (BrdU) incorporation assays. Real-time PCR was employed to monitor gene expression, and protein levels of interleukin-6 (IL-6) were measured by ELISA. Uptake of proline was determined using 18F-proline.

Sustained culture of originally quiescent PSCs induced cell proliferation, loss of lipid droplets and exhibition of stress fibers, indicating cell activation. When added to PSCs in primary culture, all three D-vitamins diminished expression of α-SMA (to 32%-39% of the level of control cells; P < 0.05) and increased the storage of lipids (scores from 1.97-2.15 on a scale from 0-3; controls: 1.49; P < 0.05). No such effects were observed when Dvitamins were added to fully-activated cells, while incorporation of BrdU remained unaffected under both experimental conditions. Treatment of re-cultured PSCs with Dvitamins was associated with lower expression of IL-6 (-42% to -49%; P < 0.05; also confirmed at the protein level) and increased expression of the vitamin D receptor gene (209%-321% vs controls; P < 0.05). There was no effect of Dvitamins on the expression of transforming growth factor-β1 and collagen type 1 (chain α1). The lowest uptake of proline, a main component of collagen, was observed in calcipotriol-treated PSCs.