Published online Jan 14, 2018. doi: 10.3748/wjg.v24.i2.170
Peer-review started: October 10, 2017
First decision: October 30, 2017
Revised: November 15, 2017
Accepted: November 27, 2017
Article in press: November 27, 2017
Published online: January 14, 2018
To study the molecular effects of three different D-vitamins, vitamin D2, vitamin D3 and calcipotriol, in pancreatic stellate cells (PSCs).
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.
The three D-vitamins inhibit, with similar efficiencies, activation of PSCs in vitro, but cannot reverse the phenotype once the cells are fully activated.
Core tip: Modulation of the stroma response by vitamin D has been suggested as a concept to treat chronic pancreatitis and pancreatic cancer. Here we show that three derivatives, vitamin D2, vitamin D3 and calcipotriol, with similar efficiencies prevented pancreatic stellate cell (PSC) activation in vitro. Once the cells were fully activated, vitamin D failed to induce a reversal of the myofibroblastic phenotype, but still exerted antifibrotic effects by diminishing the uptake of proline and secretion of interleukin-6, an autocrine mediator of PSC activation. Our findings encourage further studies on the potential of vitamin D derivatives as antifibrotic drugs.