Role of Osteopontin in Diet-Induced Brown Gallstone Formation in Rats
Gallstone disease (GSD) is one of the most common biliary disorders, affecting 10% to 20% of the global adult population. It imposes significant financial burdens on individuals, families, and society due to its associated complications. GSD is influenced by genetic, metabolic, and demographic factors, including age and gender. Gallstones are classified into cholesterol, pigment, and mixed types based on their chemical composition. While cholesterol gallstones have been extensively studied, the pathogenesis of pigment gallstones, particularly those occurring in the intrahepatic bile duct, remains poorly understood. Pigment gallstones are further divided into black and brown subtypes, each with distinct chemical compositions and formation mechanisms. This study focuses on the role of osteopontin (OPN) in the pathogenesis of brown pigment gallstones in a diet-induced rat model.
Experimental Design and Methods
The study utilized male Sprague-Dawley rats, aged 6–8 weeks, which were housed in a controlled environment. The rats were divided into two groups: one fed a standard chow diet (CD) and the other fed a lithogenic diet (LD) for 10 consecutive weeks. The LD was designed to induce gallstone formation by altering bile composition. To investigate the role of OPN, a blocking experiment was conducted using an OPN antibody (OPN-Ab), with non-specific IgG (NS-IgG) as a control. Biochemical analyses were performed to measure bile components, including bile acids (BA), total cholesterol, total bilirubin, conjugated bilirubin, unconjugated bilirubin, and phospholipids. Additionally, the expression levels of OPN, tumor necrosis factor-alpha (TNF-α), and cholesterol 7 alpha-hydroxylase (CYP7A1) were analyzed in liver tissues. In vitro experiments were conducted using rat primary liver macrophages and BRL liver cells to further explore the mechanisms involving OPN, TNF-α, and CYP7A1.
Results
Gallstone Formation and Bile Composition
The incidence of gallstones was significantly higher in LD-fed rats (80%) compared to CD-fed rats (10%). Fourier transform infrared spectroscopy (FTIR) confirmed that the gallstones formed in LD-fed rats were brown pigment stones, characterized by specific absorption bands corresponding to bilirubin. Histopathological examination revealed structural abnormalities in the liver tissues of LD-fed rats, including blurred hepatic plate-like structures, lymph node infiltration, and ballooning degeneration of hepatocytes. These changes were accompanied by a significant increase in macrophage infiltration. Biochemical analyses showed that LD-fed rats had significantly reduced BA and phospholipid levels in both bile and liver tissues, along with increased levels of total bilirubin and unconjugated bilirubin. These alterations in bile composition mirrored those observed in patients with pigment gallstones, suggesting that the LD-induced rat model is a suitable representation of human gallstone disease.
Expression of OPN, TNF-α, and CYP7A1
Immunohistochemical and western blot analyses revealed that OPN expression was significantly upregulated in the liver tissues of LD-fed rats compared to CD-fed rats. Similarly, TNF-α expression was markedly increased in LD-fed rats, while CYP7A1 mRNA and protein levels were significantly downregulated. Correlation analyses indicated a positive relationship between OPN and TNF-α expression and a negative correlation between OPN and CYP7A1 expression. These findings suggest that OPN and TNF-α play crucial roles in the pathogenesis of brown pigment gallstones, potentially through the suppression of CYP7A1, a key enzyme in BA biosynthesis.
Blocking Experiment with OPN-Ab
To further investigate the role of OPN, a blocking experiment was conducted using OPN-Ab. Pretreatment with OPN-Ab significantly reduced the incidence of gallstone formation in LD-fed rats from 85% to 25%. Additionally, OPN-Ab treatment downregulated TNF-α expression and upregulated CYP7A1 expression in liver tissues. BA content in bile and liver tissues also increased following OPN-Ab treatment. These results indicate that OPN promotes gallstone formation by enhancing TNF-α production and suppressing CYP7A1 expression, leading to reduced BA synthesis.
In Vitro Experiments
In vitro experiments were performed to validate the mechanisms observed in vivo. Rat liver macrophages were treated with recombinant rat OPN (rrOPN) to assess its effect on TNF-α production. The results showed that rrOPN significantly increased TNF-α levels in macrophage culture supernatants. This effect was suppressed by pretreatment with CD44 and integrin receptor blockers, indicating that OPN promotes TNF-α production through these receptors. Furthermore, BRL liver cells were treated with recombinant rat TNF-α (rrTNF-α) to evaluate its impact on CYP7A1 expression. The results demonstrated that TNF-α suppressed CYP7A1 mRNA and protein expression in a dose- and time-dependent manner. Additionally, TNF-α treatment reduced BA synthesis in liver cells, further supporting the role of TNF-α in inhibiting CYP7A1 and disrupting BA metabolism.
Discussion
The findings of this study provide novel insights into the pathogenesis of brown pigment gallstones, highlighting the critical role of OPN in this process. OPN was found to promote gallstone formation by enhancing TNF-α production in hepatic macrophages, which in turn suppressed CYP7A1 expression and reduced BA synthesis. This cascade of events leads to alterations in bile composition, including reduced BA and phospholipid levels and increased unconjugated bilirubin, creating a favorable environment for gallstone formation.
The LD-induced rat model used in this study effectively replicated the biochemical and pathological changes observed in human pigment gallstone disease. The significant reduction in BA and phospholipid levels, along with the increase in unconjugated bilirubin, mirrors the bile composition alterations seen in patients with pigment gallstones. These findings suggest that OPN and TNF-α are key mediators in the development of brown pigment gallstones, offering potential targets for therapeutic intervention.
The blocking experiment with OPN-Ab further confirmed the role of OPN in gallstone formation. By inhibiting OPN function, gallstone incidence was significantly reduced, and BA synthesis was restored. This suggests that targeting OPN could be a viable strategy for preventing or treating pigment gallstones. Additionally, the in vitro experiments provided mechanistic evidence that OPN promotes TNF-α production through CD44 and integrin receptors, and that TNF-α suppresses CYP7A1 expression and BA synthesis in liver cells.
Conclusion
This study demonstrates that OPN plays a pivotal role in the pathogenesis of brown pigment gallstones by promoting TNF-α production and suppressing CYP7A1 expression, leading to altered bile composition and gallstone formation. The findings provide a deeper understanding of the molecular mechanisms underlying pigment gallstone disease and highlight potential therapeutic targets. Future research should focus on exploring the genetic and signaling pathways involved in this process to develop effective treatments for pigment gallstones.
doi.org/10.1097/CM9.0000000000001519
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