Thromboxane A2 Receptor Contributes to the Activation of Rat Pancreatic Stellate Cells Induced by 8-epi-Prostaglandin F2α
Chronic pancreatitis (CP) is a progressive inflammatory disease characterized by irreversible damage to the pancreatic tissue, leading to exocrine insufficiency and diabetes. A key pathological feature of CP is pancreatic fibrosis, which results from the excessive deposition of extracellular matrix (ECM). Pancreatic stellate cells (PSCs) are the primary source of ECM in the pancreas, and their activation is critical in the development of pancreatic fibrosis. Understanding the molecular mechanisms underlying PSC activation is essential for developing effective treatments for CP.
Oxidative stress has been increasingly recognized as a significant contributor to the activation of PSCs in CP. Oxidative stress triggers pancreatic inflammation and fibrogenesis through various signaling pathways. One of the key markers of oxidative stress is 8-epi-prostaglandin F2α (8-epi-PGF2α), a member of the F2-isoprostane family. 8-epi-PGF2α has been shown to promote hepatic stellate cell activation and collagen production, leading to liver fibrosis. However, its role in pancreatic fibrosis and PSC activation remains poorly understood.
The thromboxane A2 receptor (TxA2r) is a G-protein-coupled receptor that has been implicated in various pathological processes, including fibrosis. Previous studies have shown that TxA2r is overexpressed in activated PSCs following pancreatic injury in rats. However, the role of TxA2r in PSC activation induced by oxidative stress, particularly by 8-epi-PGF2α, has not been fully explored. This study aimed to investigate the role of TxA2r in the activation of PSCs induced by 8-epi-PGF2α and to explore the potential of TxA2r as a therapeutic target for pancreatic fibrosis.
Methods
The study was conducted using male Sprague-Dawley rats. PSCs were isolated from the pancreas using a combination of enzymatic digestion and density gradient centrifugation. The isolated PSCs were cultured in Iscove modified Dulbecco medium (IMDM) supplemented with fetal bovine serum and antibiotics. The cells were either maintained in a quiescent state or activated by culturing for 48 hours.
The expression of TxA2r in both quiescent and activated PSCs was detected using immunocytochemistry and immunoblot analysis. For immunocytochemistry, activated PSCs were fixed and incubated with a polyclonal antibody against TxA2r, followed by visualization using an EliVision Plus kit. Immunoblot analysis was performed to quantify TxA2r protein levels in PSCs. The protein samples were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with antibodies against TxA2r and β-actin.
To investigate the role of TxA2r in PSC activation, PSCs were treated with different concentrations of 8-epi-PGF2α (10⁻⁶, 10⁻⁷, and 10⁻⁸ mol/L) for 48 hours. The mRNA levels of α-smooth muscle actin (α-SMA) and collagen I, markers of PSC activation, were quantified using real-time polymerase chain reaction (RT-PCR). The effect of TxA2r inhibition on PSC activation was examined by treating PSCs with SQ29548, a specific TxA2r antagonist, at concentrations of 10⁻⁴, 10⁻⁶, and 10⁻⁷ mol/L for 48 hours. The mRNA levels of α-SMA and collagen I were again measured using RT-PCR.
To further explore the interaction between 8-epi-PGF2α and TxA2r, PSCs were pre-treated with SQ29548 (10⁻⁴ mol/L) for 2 hours, followed by treatment with 8-epi-PGF2α (10⁻⁷ mol/L) for 48 hours. The mRNA levels of α-SMA and collagen I were quantified to assess the effect of TxA2r inhibition on 8-epi-PGF2α-induced PSC activation.
Results
The expression of TxA2r was significantly up-regulated in activated PSCs compared to quiescent PSCs, as demonstrated by both immunocytochemistry and immunoblot analysis. Immunocytochemistry showed that TxA2r was localized to the cell surface and perinuclear cytoplasm in PSCs. Co-localization studies using immunofluorescence double staining confirmed that TxA2r was expressed in both quiescent and activated PSCs, with higher expression levels in the latter.
Treatment of PSCs with 8-epi-PGF2α at different concentrations significantly increased the mRNA levels of α-SMA and collagen I, indicating that 8-epi-PGF2α promotes PSC activation. The highest increase in α-SMA mRNA levels was observed at a concentration of 10⁻⁷ mol/L 8-epi-PGF2α. In contrast, treatment with SQ29548, the TxA2r antagonist, significantly reduced the mRNA levels of α-SMA and collagen I in a dose-dependent manner. The most pronounced reduction in α-SMA mRNA levels was observed at a concentration of 10⁻⁴ mol/L SQ29548.
Pre-treatment of PSCs with SQ29548 (10⁻⁴ mol/L) followed by treatment with 8-epi-PGF2α (10⁻⁷ mol/L) resulted in significantly lower mRNA levels of α-SMA and collagen I compared to the control group. This finding suggests that TxA2r plays a crucial role in mediating the activation of PSCs induced by 8-epi-PGF2α.
Discussion
The results of this study demonstrate that 8-epi-PGF2α promotes the activation of PSCs, as evidenced by the increased expression of α-SMA and collagen I. This finding is consistent with previous studies showing that oxidative stress, mediated by 8-epi-PGF2α, plays a role in the activation of hepatic stellate cells and the development of liver fibrosis. The study also provides evidence that TxA2r is involved in the activation of PSCs induced by 8-epi-PGF2α, as inhibition of TxA2r with SQ29548 significantly reduced the expression of α-SMA and collagen I.
The up-regulation of TxA2r in activated PSCs suggests that this receptor may be a key mediator of PSC activation in response to oxidative stress. The study’s findings are in line with previous research showing that TxA2r is overexpressed in PSCs following pancreatic injury and that its expression is correlated with the expression of α-SMA, a marker of PSC activation. The results also suggest that TxA2r may be a potential therapeutic target for the treatment of pancreatic fibrosis.
The study has several limitations. First, the experiments were conducted in vitro, and the findings may not fully reflect the complex in vivo environment of the pancreas. Second, the study focused on the role of TxA2r in PSC activation induced by 8-epi-PGF2α, but other pathways and receptors may also be involved in this process. Future studies should investigate the role of TxA2r in animal models of CP to confirm its potential as a therapeutic target.
In conclusion, this study provides evidence that 8-epi-PGF2α promotes the activation of PSCs through the up-regulation of TxA2r. The findings suggest that TxA2r may be a potential target for the treatment of pancreatic fibrosis. Further research is needed to explore the therapeutic potential of TxA2r inhibition in the context of CP and to investigate the role of other pathways in PSC activation.
doi.org/10.1097/CM9.0000000000000838
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