Effects of cholesterol on the phenotype of rabbit bile duct fibroblasts

Abstract

Aim: To investigate how cholesterol (Ch) can affect the phenotype of bile duct fibroblasts of New Zealand rabbits.

Methods: 16 rabbits were divided randomly into two groups: the control group and the experiment group. The rabbits in experiment group were fed with hypercholesterol diet for 8 weeks. Bile duct was dissociated from rabbits and prepared for transmission electron microscopy. The purified bile duct fibroblasts were cultured and divided randomly into three groups: control group, Ch middle concentration group (0.6 g/L), Ch high concentration group (1.2 g/L). After incubated for 72 h, the fibroblasts were made into specimens for transmission electron microscopy. The expression of alpha-actin in bile duct fibroblasts was measured by means of laser scanning confocal microscopy.

Results: With the transmission electron microscopy, the normal bile duct fibroblasts were shuttle-shaped, and there were abundant rough endoplasmic reticulums (RER), but few mitochondria or microfilaments in cytoplasm. This is the typical phenotype of fibroblasts. Bile duct fibroblasts of hypercholesterolemic rabbits were observed. by the transmission electron microscopy Rough endoplasmic reticulums were significantly reduced, with a lot of microfilament bundles or stress fibers appeared in cytoplasm, especially under plasma membrane. Dense bodies were scattered within these bundles. Macula densas and discontinuous sarcolemma were found under plasma membrane. It suggested that the bile duct fibroblasts of hypercholesterolemic rabbits presented the phenotype of smooth muscle cell. The cultured bile duct fibroblasts also had typical phenotype of fibroblasts. After stimulated by middle concentration cholesterol (0.6 g/L) for 72 h, there appeared lots of microfilaments in cytoplasm, but without dense body, macula densa and discontinuous sarcolemma. Observed with confocal microscopy, there were many regular bundles of microfilaments in fibroblasts treated with middle concentration ch (0.6 g/L) and the expression of alpha-actin was significantly increased. The average fluorescence value of middle concentration group was 1 628+/-189 (P<0.01 vs control group). Microfilaments and the expression of alpha-actin were greatly decreased in fibroblasts of high concentration group (1.2 g/L). The average fluorescence value of high concentration group was 1 427+/-153 (P<0.05 vs middle concentration group). There were a lower expression of alpha-actin and few microfilaments in bile duct fibroblasts of control group with an average fluorescence value of 1 224+/-138.

Conclusion: Cholesterol can make bile duct fibroblasts have the phenotypic characteristics of smooth muscle cell both in vitro and in vivo and this effect is more significant in vivo. The effect is probably associated with some other factors besides cholesterol.

Figure 1

Bile duct fibroblasts of normal rabbit presented shuttle-shaped with abundant rough endoplasmic reticulums (RER), but few mitochondria and microfilament in cytoplasm.

Figure 2

The bile duct fibroblasts of hypercholesterolemic rabbits showed the phenotype of smooth muscle cell. Rough endoplasmic reticulums (RER) was significantly reduced, but a lot of microfilament bundles or stress fibers appeared in cytoplasm. Dense bodies were scattered within these bundles. Macula densas and discontinuous sarcolemma were found under splasma membrane.

Figure 3

Cultured normal rabbit bile duct fibroblasts characterized typical phenotype of fibroblasts with abundant rough endoplasmic reticulums (RER) and golgi bodies, but few mitochondria and microfilament.

Figure 4

After stimulated by middle concentration cholesterol (0.6 g/L) for 72 h, the cultured bile duct fibroblasts showed some phenotype of fibroblasts. and there appeared lots of microfilament in cytoplasm, but without dense body. macula densa and discontinuous sarcolemma.

Figure 5

Effects of middle concentration cholesterol (0.6 g/L) on the cultured bile duct fibroblasts. There were many regular bundles of microfilament that were located along cytoplasmic extension.

Figure 6

Effects of cholesterol on the α-actin expression of rabbit bile duct fibroblasts. Note ^aP < 0.05, versus control and high concentration group (1.2 g/L cholesterol).