Department of Medicine II, Division of Gastroenterology, Rostock University Medical Center, Rostock 18057, Germany.
Oscar-Langendorff-Institute of Physiology, Rostock University Medical Center, Rostock 18057, Germany.
World J Gastroenterol. 2018 Jan 14;24(2):170-178. doi: 10.3748/wjg.v24.i2.170.
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 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 F-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; < 0.05) and increased the storage of lipids (scores from 1.97-2.15 on a scale from 0-3; controls: 1.49; < 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%; < 0.05; also confirmed at the protein level) and increased expression of the vitamin D receptor gene (209%-321% controls; < 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 , but cannot reverse the phenotype once the cells are fully activated.
研究三种不同的维生素 D，维生素 D2、维生素 D3 和卡泊三醇，对胰腺星状细胞（PSC）的分子作用。
从小鼠胰腺中分离出静止的 PSCs，并通过接种在塑料表面使其激活。将细胞作为原代培养物（早期激活的 PSCs）和再培养（完全激活的细胞）暴露于 D 维生素中。用油红染色可视化含有维生素 A 的脂滴的表达。通过免疫荧光和免疫印迹分析监测α-平滑肌肌动蛋白（α-SMA）的表达，α-SMA 是 PSC 激活的标志物。通过 5-溴-2'-脱氧尿苷（BrdU）掺入测定法定量测定 DNA 合成率。采用实时 PCR 监测基因表达，采用 ELISA 法测定白细胞介素 6（IL-6）的蛋白水平。使用 F-脯氨酸测定脯氨酸的摄取。
持续培养原本静止的 PSCs 诱导细胞增殖、脂质滴丢失和应激纤维表达，表明细胞激活。当添加到原代培养的 PSCs 中时，所有三种 D 维生素均降低了 α-SMA 的表达（至对照细胞水平的 32%-39%；<0.05）并增加了脂质的储存（评分范围为 0-3 的 1.97-2.15；对照：1.49；<0.05）。当 D 维生素添加到完全激活的细胞时，未观察到这种作用，而在两种实验条件下 BrdU 的掺入均不受影响。用 D 维生素处理再培养的 PSCs 与 IL-6 的表达降低（-42%至-49%；<0.05；也在蛋白水平上得到证实）和维生素 D 受体基因的表达增加（对照的 209%-321%；<0.05）相关。D 维生素对转化生长因子-β1 和胶原类型 1（链α1）的表达没有影响。在卡泊三醇处理的 PSCs 中观察到脯氨酸的摄取最低，脯氨酸是胶原蛋白的主要成分。
三种 D 维生素以相似的效率抑制 PSCs 的激活，但一旦细胞完全激活，就不能逆转表型。
