Osteopontin and integrin are involved in cholesterol gallstone formation

Abstract

Background: This study aimed to investigate the role of osteopontin and its receptor, integrin αv, in gallstone formation using human tissue specimens and a guinea pig lithogenic model.

Material/methods: The nucleation role of osteopontin was determined in patients' and normal gallbladder bile samples in vitro. Normal gallbladder was the control, and gallstone gallbladders were divided into group I (with normal epithelia) and group II (with degenerated epithelia) based on pathology change. Immunostaining, mRNA and protein expressions of osteopontin and integrin αv were analyzed. The animals were randomly divided into a lithogenic diet group and a normal diet group; the osteopontin mRNA expression in gallbladder and liver and osteopontin concentrations were determined.

Results: Osteopontin prolonged nucleation time and inhibited the pro-nucleating role induced by calcium in human bile in vitro. Immunostaining for osteopontin and integrin αv in human gallbladder tissues showed a higher reactivity in Group I than control group and Group II. The immunostaining in Group II was weaker than control group; similar results were observed for mRNA and protein expression of osteopontin and integrin αv. In the animal assay, the mRNA expression and concentration of osteopontin in gallbladder and liver gradually increased at initial stages and decreased in later stages. The concentrations of osteopontin in bile and serum of guinea pig showed similar trends.

Conclusions: Our results suggest that osteopontin is involved in cholesterol gallstone formation, and the role of osteopontin might correlate with integrin αv and calcium.

Figure 1

OPN and integrin αv immunostaining in experimental and control gallbladder wall: In normal gallbladder, the focal and diffused immunohistochemical staining of OPN (A) and integrin αv (B) were examined in cytoplasm of epithelial cell and smooth muscle cell. In experimental group I, intensive and strong OPN immunostaining was found in epithelial cell (C), the diffuse and strong membranous integrin αv staining was found in epithelial cytoplasm (D). In group II, immunostaining for OPN was relative weaker and diffused (E). Meanwhile, immunostaining for integrin αv was lower expression in these specimens (F). OPN and integrin αv, ×20.

Figure 2

The mRNA expression of OPN in human gallbladder. Compared with control, the mRNA expression of OPN and integrin αv was increased in Group I (A). The mRNA expression of OPN and integrin αv markedly decreased in specimens of group II (B). M: marker; A: lane 1–3, control group; lane 4–6, experimental group. B: lane 1, 3, 5, control group; lane 2, 4, 6, experimental group.

Figure 3

OPN protein expression in human gallbladder: Compared with control, the protein expression of OPN and integrin αv was significantly increased in Group I (A) but markedly decreased in Group II (B). 1: control group; 2: experimental group.

Figure 4

The mRNA expression of OPN in gallbladder and liver of guinea pigs: In lithogenic group, the highest level expression were found at week 6 (A). Moreover, the mRNA expression of OPN was detected in liver at week 6 only (B). In the control group, the OPN mRNA expression was weak throughout the study.

Figure 5

OPN content in gallbladder bile and blood of guinea pig: (A). Compared with control, the highest content of OPN was detected at week 6 (P=0.004); however, at weeks 1, 2, and 4, the OPN contents increased but without statistic difference (P>0.05). At weeks 8 and 10, the OPN contents decreased gradually. The OPN contents in animal blood exhibited similar results (B). The highest value of OPN was seen at week 4 (P=0.002 vs. control).