CircRNA hsa_circ_0001944 Regulates FXR/TLR4 Pathway and Ferroptosis in NiONP-Induced Collagen Formation

Study Background and Research Question

Nickel oxide nanoparticles (NiONPs) are increasingly prevalent in industrial environments, raising concerns about their hepatotoxicity and fibrogenic potential. Prior research establishes that NiONPs can induce liver fibrosis by promoting inflammation and the activation of hepatic stellate cells (HSCs), specifically through enhanced collagen type I alpha 1 chain (COL1A1) synthesis. However, the precise molecular mechanisms mediating this effect—particularly the interplay between non-coding RNAs, nuclear receptor signaling, and ferroptosis—remain insufficiently understood. The reference study (Zhou et al., 2025) addresses this gap by interrogating the role of the circular RNA hsa_circ_0001944 in modulating the farnesoid X receptor (FXR)/toll-like receptor 4 (TLR4) pathway and ferroptosis during NiONP-induced collagen deposition in the human HSC line LX-2.

Key Innovation from the Reference Study

The principal innovation of Zhou et al. lies in uncovering a regulatory axis where hsa_circ_0001944 acts upstream of FXR, influencing both TLR4 signaling and ferroptosis—a form of iron-dependent cell death increasingly recognized in fibrogenesis. The study demonstrates, for the first time, that overexpression of hsa_circ_0001944 restores FXR expression, attenuates TLR4 activity, enhances ferroptotic features, and ultimately reduces collagen accumulation in LX-2 cells exposed to NiONPs. This establishes a previously uncharacterized circRNA–FXR–TLR4–ferroptosis axis as a mechanistic contributor to nanoparticle-induced fibrotic responses.

Methods and Experimental Design Insights

The experimental framework involves both in vivo and in vitro models:

• Animal studies to confirm that NiONPs promote liver fibrosis and alter FXR/TLR4 expression and ferroptosis markers in rat livers.
• In vitro collagen deposition assays using LX-2 cells to dissect molecular mechanisms, including qPCR and immunoblotting for gene/protein expression.
• Pharmacological manipulation of key pathways: FXR agonist (GW4064), TLR4 inhibitor (TAK-242), and ferroptosis inducer (Erastin) were applied individually or in combination with NiONPs to parse pathway dependencies.
• Bioinformatic prediction and gain-of-function experiments to establish hsa_circ_0001944 as an upstream regulator of FXR.

The use of GW4064 as a selective non-steroidal FXR agonist enabled precise interrogation of FXR’s role in the context of NiONP-mediated stress, while TAK-242 and Erastin allowed for complementary modulation of TLR4 and ferroptosis, respectively.

Core Findings and Why They Matter

Zhou et al. report several interconnected findings (reference study):

• Exposure to NiONPs decreases FXR and hsa_circ_0001944 expression, while increasing TLR4 expression and markers of collagen synthesis in both rat liver and LX-2 cells.
• Activation of FXR using GW4064 reduces TLR4 expression, elevates ferroptosis features (e.g., increased ROS and MDA, reduced GPX4 and GSH), and alleviates collagen deposition in LX-2 cells.
• Inhibition of TLR4 with TAK-242 also diminishes collagen accumulation, apparently through a ferroptosis-dependent mechanism.
• Overexpression of hsa_circ_0001944 in LX-2 cells restores FXR levels, suppresses TLR4, augments ferroptosis, and mitigates NiONP-induced collagen formation.

These results converge to support a model in which hsa_circ_0001944 is a regulatory node that modulates FXR signaling, leading to suppression of TLR4-mediated pro-fibrotic activity and enhanced ferroptosis, thereby limiting collagen deposition. This mechanistic clarity deepens our understanding of how nuclear receptor and innate immune pathways integrate with cell death programs during nanoparticle-induced liver injury.

Comparison with Existing Internal Articles

The reference study’s findings resonate with prior reviews of FXR signaling in metabolic and fibrotic disease models. Internal resources, such as GW4064: Selective Non-Steroidal FXR Agonist for Metabolic... and GW4064: Non-Steroidal FXR Agonist for Metabolic Pathway Research, emphasize GW4064’s role as a tool compound for dissecting FXR-driven metabolic and fibrotic pathways. Notably, these internal articles discuss the utility of GW4064 for specific interrogation of FXR signaling in cell-based and animal models, and highlight its value in mapping downstream effects on cholesterol and triglyceride regulation, as well as the bile acid metabolism pathway. However, the Zhou et al. paper extends beyond traditional metabolic studies by integrating the FXR/TLR4 axis with ferroptosis and circular RNA regulation, thus bridging metabolic and innate immune signaling domains in the context of nanoparticle toxicity. This nuanced mechanistic insight complements the workflow recommendations and troubleshooting guides available in the internal articles, which focus on optimizing FXR activation in metabolic research and fibrosis studies.

Limitations and Transferability

While the study provides compelling evidence for a circRNA–FXR–TLR4–ferroptosis axis in LX-2 cells and rat liver, several considerations limit the immediate generalizability of the findings:

• The main mechanistic experiments were conducted in vitro using the LX-2 cell line; primary HSCs or in vivo validation in additional models would strengthen the translational relevance.
• The study focuses on a single circular RNA, and the broader landscape of circRNAs modulating FXR or ferroptosis in fibrosis remains to be mapped.
• Potential off-target effects of pathway modulators (including GW4064 and TAK-242) cannot be fully excluded, though the use of pharmacological controls mitigates this concern.
• The clinical significance of targeting this axis for fibrosis therapy awaits further preclinical development and safety evaluations.

Nevertheless, the research provides a robust proof of concept for using FXR activation and circRNA modulation to interrogate fibrogenic pathways in response to environmental nanoparticles.

Protocol Parameters

• FXR activation using GW4064: In LX-2 cell models, apply GW4064 at concentrations consistent with published EC₅₀ values (e.g., 90 nM in human FXR-transfected cells), adjusting for solubility and cell viability as indicated in the product information.
• NiONP exposure: Dose and duration should be optimized to induce measurable collagen deposition and stress responses without confounding cytotoxicity, as described in the reference study.
• Pathway modulation: Use TAK-242 for TLR4 inhibition and Erastin for ferroptosis induction in combination or sequentially with GW4064 to dissect pathway interdependencies.
• Gene overexpression: Employ lentiviral or plasmid constructs for hsa_circ_0001944 overexpression, validating transfection efficiency and target gene modulation prior to downstream assays.
• Readouts: Assess collagen deposition (e.g., COL1A1 protein), FXR/TLR4 expression, and ferroptosis markers (GPX4, ROS, GSH, MDA) by qPCR, Western blot, and biochemical assays.

Research Support Resources

For researchers aiming to reproduce or extend these workflows, GW4064 (SKU B1527) is available as a potent, selective non-steroidal FXR agonist suitable for cell-based and biochemical studies of FXR activation in metabolic and fibrotic contexts. As reviewed in both the reference study and internal articles, GW4064’s well-characterized pharmacology allows precise interrogation of the FXR signaling pathway, though users should be mindful of its solubility constraints and light sensitivity. For protocol optimization and troubleshooting, consult the recent internal reviews for additional guidance on best practices and experimental design.