Resetting the fibrotic liver clock: NR1D1 couples Hedyotis diffusa to the HIF-1/urea-cycle-ammonia axis and stellate cell activation

Abstract

Hepatic fibrosis is a pivotal and reversible stage of chronic liver disease, yet approved antifibrotic therapies remain scarce. In World Journal of Gastroenterology, Xia et al reframes fibrosis as disordered circadian-hypoxia-metabolic crosstalk and highlights NR1D1-centered reprogramming of the HIF-1/urea-cycle-ammonia axis as a testable antifibrotic strategy. After identifying six Hedyotis diffusa injection (HDI)-derived constituents detectable in blood and liver, the authors integrate network pharmacology, Gene Expression Omnibus mining, and liver proteomics to nominate circadian rhythm regulation, hypoxia-inducible factor 1 signaling, and urea-cycle/ammonia metabolism as convergent hubs. In a carbon tetrachloride-induced mouse model, HDI reduces collagen deposition and α-smooth muscle actin expression and improves fibrosis-associated serum indices. Zeitgeber-time sampling indicates disrupted clock gene profiles in fibrotic liver and partial restoration after HDI, with prominent NR1D1 recovery. HDI also reduces hepatic HIF-1α abundance, restores carbamoyl phosphate synthetase 1 activity, and lowers hepatic ammonia. AAV9-mediated NR1D1 knockdown blunts these biochemical and histologic improvements, supporting target dependency. In vitro, ammonium chloride activates LX2 stellate cells and induces mitochondrial hyperfusion, consistent with local ammonia stress amplifying fibrogenic activation. Translational priorities include identifying the active HDI constituents and confirming direct NR1D1 engagement, validating efficacy across etiologic fibrosis models, and assessing ammonia/urea-cycle markers for patient selection, monitoring, and phase-aligned dosing.

Key Words: Circadian rhythm; NR1D1; HIF-1 signaling; Urea cycle; Hepatic stellate cell activation; Hedyotis diffusa injection

Core Tip: Hepatic fibrosis is a pivotal and potentially reversible stage of chronic liver disease, yet approved antifibrotic therapies remain scarce. Xia et al propose that Hedyotis diffusa injection mitigates fibrogenesis by engaging the core clock gene NR1D1, with two downstream branches—suppression of HIF-1 signaling and restoration of urea-cycle/ammonia homeostasis—converging on reduced hepatic stellate cell activation and collagen deposition. This work connects circadian regulation with hypoxia responses and nitrogen metabolism, addressing a key mechanistic gap in liver scarring. By highlighting a druggable clock node and clinically accessible metabolic readouts, it may inform clock-guided antifibrotic development and biomarker-based staging and response monitoring.

This editorial refers to “Integrative study reveals NR1D1 mediates Hedyotis diffusa’s antifibrosis via hypoxia inducible factor-1/ammonia axis” Xia et al, 2026; https://doi.org/10.3748/wjg.v32.i10.115334.

---

INTRODUCTION

Hepatic fibrosis is a central stage of chronic liver injury, and hepatic stellate cell (HSC) activation is a key biological event that drives fibrotic scarring[1,2]. Recent proof-of-concept studies show that disruption to circadian rhythm primes HSCs towards an accelerated fibrotic response[3,4], reinforcing the therapeutic rationale for circadian-oriented antifibrotic strategies. Despite major advances in the understanding of fibrosis pathogenesis, approved therapies that directly reverse liver fibrosis are still lacking[5]. In the recent issue of World Journal of Gastroenterology, Xia et al[6] build on this landscape by defining fibrosis as a manifestation of disordered circadian-hypoxia-metabolic crosstalk and by proposing the core clock-system gene NR1D1 as a druggable regulatory node. Through targeting this node, Hedyotis diffusa injection (HDI) is suggested to reduce HIF-1 abundance, limit hepatic ammonia accumulation, and thereby mitigate fibrogenesis. By linking clock resetting to HIF-1 regulation and urea-cycle-ammonia homeostasis, Xia et al's work[6] fills a critical mechanistic gap in fibrosis and offers a blueprint for deeper mechanistic studies, clock-guided drug development, and ammonia-metabolite-based staging and response monitoring. Here, we evaluate the work in terms of conceptual coherence and scientific impact and highlight priorities for follow-up studies.

THE ROLE OF HDI IN ALLEVIATING HEPATIC FIBROSIS

Xia et al[6] used liquid chromatography-mass spectrometry to detect six major HDI-derived constituents in blood and liver. Network pharmacology, Gene Expression Omnibus analyses, and liver proteomics highlighted circadian rhythm regulation, HIF-1 signaling, and urea-cycle and ammonia metabolism as the main pathways associated with HDI and fibrosis. In a carbon tetrachloride-induced mouse fibrosis model, HDI improved liver histology, reduced collagen deposition on fibrosis staining, and decreased α-smooth muscle actin (α-SMA) expression, with parallel improvements in liver injury and fibrosis-related serum indices. Zeitgeber-time sampling showed disrupted clock-gene rhythms in fibrotic liver and a clear restoration after HDI, with NR1D1 showing the most prominent recovery. Western blotting and biochemical assays showed that HDI lowered hepatic HIF-1α protein, increased carbamoyl phosphate synthetase 1 (CPS1) activity, and reduced hepatic ammonia levels. AAV9-mediated NR1D1 knockdown and overexpression showed an inverse relationship between NR1D1 and HIF-1α protein levels and consistent changes in CPS1 activity and urea/ammonia readouts. NR1D1 knockdown also weakened the histologic and biochemical improvements produced by HDI. In LX2 cells, ammonium chloride increased α-SMA and induced mitochondrial hyperfusion, supporting ammonia load as a factor that can promote stellate cell activation. These main findings and their proposed mechanistic relationships are summarized in Figure 1.

Figure 1 Schematic summary of the main findings reported by Xia et al[6]. In a CCl₄-induced mouse model of liver fibrosis, Hedyotis diffusa injection (HDI) was administered intraperitoneally. Six constituents capable of migrating to the liver were identified, including coumarin, geniposidic acid, quercetin, kaempferol, rutin, and emodin. HDI treatment was associated with increased hepatic expression of the core circadian regulator NR1D1. Functional studies indicated that the antifibrotic effects of HDI were NR1D1-dependent and were accompanied by decreased HIF-1α protein levels, increased CPS1 activity, and reduced hepatic ammonia levels. Reduction of ammonia burden was also associated with decreased mitochondrial fusion and attenuation of hepatic stellate cell (HSC) activation. However, a causal role of mitochondrial dynamics in HSC activation was not directly demonstrated. Collectively, these findings support a model in which NR1D1 upregulation is linked to suppression of HIF-1α signaling and restoration of CPS1-associated urea cycle function, leading to reduced ammonia levels and diminished HSC activation, thereby contributing to the alleviation of liver fibrosis. In addition, the authors proposed a putative crosstalk between hypoxia signaling and ammonia metabolism, whereby elevated ammonia may stabilize HIF-1α, while HIF-1α may suppress CPS1 and urea cycle function, potentially forming a positive feedback loop. HDI: Hedyotis diffusa injection; HSC: Hepatic stellate cell; α-SMA: α-smooth muscle actin. Created in BioRender (Supplementary material).

Xia et al[6] provides a clear mechanistic framework that links circadian regulation with hypoxia signaling and nitrogen metabolism in fibrogenesis, and it offers a valuable starting point for clock-guided antifibrotic development and ammonia-metabolite-based staging and response monitoring. The value of this study on HDI lies not only in highlighting NR1D1 as a therapeutically significant node in liver fibrosis, but also in offering a clearer paradigm for studying the mechanisms of traditional Chinese medicine (TCM) components. Compared with prior TCM mechanistic studies that use network pharmacology to predict numerous targets and then validate a selected pathway, this work shifts the focus from isolated, single-molecule changes to adjustments in functional metabolic modules, and it outlines a more complete mechanistic cascade with potential feedback loops. The multi-target nature of TCM may be better framed as the synergistic correction of multiple stress-response and metabolic modules, rather than the strong inhibition of a single pathway[7], which better fits the network biology of chronic fibrosis. Moreover, this study suggests that greater attention should be paid to the time dimension, a factor that has often been overlooked in TCM mechanistic research. In traditional practice, TCM prescriptions often consider dosing time[8], which may relate to the rhythmicity of the key targets highlighted here. Clinically, these findings motivate a mechanism-informed view of efficacy differences. HDI may show greater benefit in patients with prominent impairments in ammonia handling or circadian disruption, although this hypothesis still requires clinical validation.

NR1D1 AND HEPATIC FIBROGENESIS: BEYOND A ONE-DIRECTIONAL PROGRAM FOR HIF-1α SIGNALING AND AMMONIA METABOLISM

NR1D1 is an important regulator within the core circadian machinery. A growing number of circadian clock components and regulators have been shown to influence liver fibrosis progression through diverse mechanisms, as summarized in Table 1[3,9-13]. Substantial evidence supports that HIF-1α activation and ammonia accumulation contribute to hepatic fibrogenesis[14,15], whereas the directionality of NR1D1 in regulating ammonia metabolism and fibrosis remains debated. Prior studies have implicated NR1D1 in liver fibrosis, but its effects appear highly compartment-specific across both subcellular localization and metabolic state. Nuclear NR1D1 activation suppresses HSC proliferation and fibrogenic transcriptional programs[16]. In contrast, fibrogenic injury is associated with cytoplasmic accumulation of NR1D1 in activated stellate cells, where it aligns with contractile machinery and promotes a myofibroblast-like, profibrotic state[17]. A similar compartment-specific mechanism may underlie divergent observations in nitrogen metabolism: NR1D1 has been reported to repress C/EBPα-driven transcription of urea-cycle genes such as Arg1, Cps1, and Otc, thereby limiting ammonia clearance under basal conditions[18], whereas Xia et al[6] observed that NR1D1 upregulation coincided with restored CPS1 activity and reduced hepatic ammonia accumulation in fibrotic liver. Together, these findings raise the possibility that NR1D1’s net impact may vary with cellular compartment, cell type, circadian phase, and disease-altered metabolic flux, rather than following a fixed, one-directional program.

Table 1 Circadian clock components and regulators implicated in liver fibrosis.

Circadian component/regulator	Model/context	Main fibrosis-related finding	Key pathways/mechanistic features	Ref.
PER1	Schistosoma japonicum-infected mouse model; primary mouse HSCs; JS-1 and LX-2 cells	PER1 restrains hepatic stellate cell activation in schistosomiasis-associated liver fibrosis. PER1 silencing increases fibrogenic gene expression and largely abolishes the antifibrotic effect in HSCs	PER1 functions as an antifibrotic clock gene in HSCs and mediates suppression of HSC activation, with reduced COL1A1, α-SMA, and TIMP1 expression; its expression is directly induced by glucocorticoid receptor signaling	Tang et al[9], 2025
NR1D1	CCl4-induced mouse liver fibrosis model; TGF-β1-treated LX-2 cells	NR1D1 is downregulated in fibrotic liver and activated HSCs. Its activation attenuates collagen deposition, HSC activation, and fibrogenic gene expression, whereas its inhibition exacerbates fibrosis	NR1D1 acts as an upstream negative regulator of the NLRP3 inflammasome; suppresses NLRP3, ASC, caspase-1, and downstream IL-1β/IL-18 signaling; reduces α-SMA, COL-1, and TGF-β1 expression, thereby limiting HSC activation and inflammatory fibrogenesis	Wang et al[10], 2025
NR1D1	CCl4-induced mouse liver fibrosis model; rhythm-disrupted mouse models; primary mouse HSCs and LX-2 cells	NR1D1 disruption disturbs the circadian clock in HSCs, promotes persistent HSC activation, and increases susceptibility to aggravated fibrosis in mice, whereas NR1D1 overexpression alleviates fibrosis	NR1D1 deficiency reduces DRP1 S616 phosphorylation, weakens mitochondrial fission, increases mitochondrial fusion and mtDNA release, activates cGAS inflammatory signaling, and enhances the profibrotic inflammatory microenvironment	Chen et al[11], 2023
CLOCK (ClockΔ19 mutant)	CCl4-induced mouse liver fibrosis model; primary HSCs	Circadian disruption caused by CLOCKΔ19 reprograms qHSCs into a primed profibrotic state. This transcriptional and chromatin-level shift predisposes HSCs to accelerated activation and worsened fibrosis after injury	The CLOCK regulome is linked to maintenance of HSC quiescence; hepatic fibrosis/HSC activation, ECM organization, and RhoGDI signaling are upregulated, whereas FXR/RXR, PXR/RXR, PPARα-related metabolic programs, fatty acid oxidation, and retinoid metabolism are downregulated	Jokl et al[3], 2023
BMAL1	CCl4-induced mouse liver fibrosis model; primary mouse HSCs; TGF-β1-treated LX-2 cells	BMAL1 is downregulated in fibrotic liver and activated HSCs. BMAL1 overexpression suppresses glycolysis, inhibits HSC proliferation and phenotypic transformation, and attenuates liver fibrosis	BMAL1 inhibits HSC glycolytic reprogramming through the IDH1/α-KG axis, reducing HK2 and PKM2 expression, lactate production, and ECAR, thereby decreasing α-SMA and COL1A1 expression and ECM deposition	Xu et al[12], 2022
PER2	CCl4-induced mouse liver fibrosis model; fibrosis regression model; HSC-T6 cells	Loss of PER2 aggravates CCl4-induced liver fibrosis, increases HSC activation, and impairs fibrosis resolution. PER2 overexpression promotes HSC apoptosis and suppresses HSC proliferation	PER2 promotes activated HSC apoptosis via CHOP-dependent upregulation of TRAIL-R2/DR5; mPer2 deficiency is associated with increased TGF-β1, TNF-α, COL1A1, TIMP1, and TIMP2, enhanced α-SMA expression, and reduced fibrosis regression	Chen et al[13], 2010

HSC: Hepatic stellate cell; α-SMA: α-smooth muscle actin.

As noted by Xia et al[6], the specific route by which NR1D1 regulates HIF-1α remains undefined, and the literature directly addressing NR1D1-HIF-1α regulation is still limited. However, two core positive regulators in the circadian network, BMAL1 and NR1F1, have been reported to modulate HIF-1 activity[19]. In particular, NR1F1 can physically associate with HIF-1α through its DNA-binding domain and enhance HIF-1α-dependent transcriptional activation[20]. Mechanistic insights from BMAL1- and NR1F1-mediated control of HIF-1 signaling may help frame testable models for how NR1D1 could interface with the HIF-1 axis. Moreover, when HIF-1α is knocked down or genetically ablated, the oxygen-driven rhythmic induction of NR1D1 is lost, with NR1D1 remaining at persistently low levels and showing a markedly dampened oscillatory pattern[21]. These findings suggest that the relationship between NR1D1 and HIF-1α may be more complex.

BIOLOGICAL AND TRANSLATIONAL IMPLICATIONS: FROM CIRCADIAN-HYPOXIA-METABOLIC COUPLING TO NR1D1-TARGETED INTERVENTION

Daily oscillations in tissue oxygenation occur in vivo, and physiological oxygen cycles are sufficient to synchronize cellular clocks[21]. Hypoxia can slow the circadian period and dampen oscillation amplitude[22]. Multiple clock components, in turn, have also been shown to directly regulate hypoxia signaling pathways[23], while hypoxia-driven metabolic by-products such as lactate can disrupt cellular rhythmicity[24]. This bidirectional coupling offers a useful systems-level framework for fibrotic liver, where microenvironmental hypoxia and metabolic rewiring may destabilize rhythmic homeostasis, and where restoring clock output could simultaneously reshape hypoxia responses and metabolic capacity. Consistently, CLOCK-dependent circadian regulation has been linked to hypoxia signaling and metabolic rewiring in the liver, with CLOCK disruption increasing HIF1α-CD36-mediated fatty acid uptake and associating with progression across the nonalcoholic fatty liver disease spectrum[25].

Beyond the liver, a conserved BMAL1-HIF2α transcriptional partnership is observed across organs-driving diurnal susceptibility to myocardial injury in the heart[26] and sustaining HIF2α chromatin engagement and downstream gene programs in the kidney[27]. Furthermore, studies have reported the coupling phenomenon between hypoxia and circadian rhythms in intervertebral disc, skeletal muscle, cornea, and lung-related models, suggesting that this interaction has a certain degree of cross-organ consistency[28-31].

Building on this growing interest in the circadian-hypoxia-metabolic axis across diseases, the novelty of Xia et al[6] lies not only in applying this framework to hepatic fibrosis but also in highlighting NR1D1 as a tractable therapeutic entry point. Overall, Xia et al[6] add value in two clear ways. First, NR1D1 is a ligand-responsive nuclear receptor, so it is easier to target with drugs than many other clock genes discussed in liver fibrosis[32]. The study also detects six HDI-derived compounds in blood and liver, providing practical candidates to pinpoint active ingredients and develop more defined NR1D1-directed therapies. Second, the work broadens circadian-oriented antifibrotic thinking by moving beyond “clock correction” and linking circadian regulation to hypoxia responses and nitrogen handling, with metabolic readouts that can be measured and followed.

RESEARCH DEFICIENCIES AND EXISTING CONSTRAINTS

First, it is still unclear how well these findings generalize across different causes of liver fibrosis. In clinical practice, first-line management is etiology-specific: Antiviral therapy is central for viral hepatitis, sustained alcohol abstinence is key for alcohol-associated liver disease, and metabolic risk control remains the cornerstone for non-alcoholic steatohepatitis. Therefore, testing HDI in more than one disease setting would strengthen its translational relevance. Multi-etiology models can also clarify the main mode of action. If HDI shows a clearly stronger benefit in an ammoniagenic diet model, this pattern would support the proposed “HDI-NR1D1-ammonia” axis as a key driver of its antifibrotic activity and would justify deeper mechanistic validation in that setting. By contrast, if HDI achieves broadly similar efficacy across fibrosis models of different etiologies, this would suggest that additional pathways may contribute to its benefit, potentially including routes that directly restrain collagen production or myofibroblast activation.

Second, several mechanistic links remain insufficiently defined in the current study. The causal chain from HDI to NR1D1 upregulation is not mapped to a specific compound or minimal active combination, and it remains unclear whether NR1D1 engagement is direct at the receptor level or indirect through upstream signaling. Downstream, the routes connecting NR1D1 to HIF-1α regulation and to restoration of urea-cycle function, particularly CPS1 activity, are also not mechanistically resolved. Moving forward, priority work should pinpoint the active HDI constituents that are necessary and sufficient to induce NR1D1, demonstrate target engagement with receptor-focused assays, and then delineate the intervening molecular steps from NR1D1 to HIF-1α control and to urea-cycle enzyme activation using cell-type-resolved perturbation and pathway-specific readouts.

Third, ammonia-related metabolites are a promising functional readout, but they need to be tested as workable biomarkers for patient stratification and response monitoring. Ammonia has emerging support as a pathogenic factor in fatty liver disease and fibrosis, yet translating this indicator into a clinic-facing tool requires defining which analytes are robust, how they relate to fibrosis stage, and whether they change with treatment in a manner that tracks histologic or elastography endpoints. In addition to blood ammonia, more specific readouts of urea-cycle function, such as serial CPS1 activity or the urinary ornithine-to-citrulline ratio, could be considered for pharmacodynamic monitoring. A practical next step would be targeted metabolomics of the urea cycle and ammonia-handling pathway in well-phenotyped cohorts, paired with longitudinal sampling during antifibrotic intervention studies.

Fourth, can HDI truly reconstruct circadian rhythms? The reported restoration of rhythmic oscillations in the expression of some clock genes, based on measurements at only two time points, seems insufficient to establish this. Higher-resolution assessments could strengthen the evidence, for example, by using dense 24-hour time-series sampling, PER2: LUCIFERASE circadian reporter clock reporters, or longitudinal in vivo monitoring of peripheral clocks. If HDI is proposed to act through clock restoration, testing whether its antifibrotic benefit is markedly reduced or lost under constant light exposure or other rhythm-disrupting conditions would better support clock recovery as a key driver rather than an accompanying phenomenon. In addition, the broader chronopharmacology literature in metabolic liver disease suggests that timing can influence both efficacy and toxicity[33,34]. Dosing time, sampling time, and the therapeutic window should be compared systematically to determine whether the intervention is phase-sensitive and to identify an optimizable regimen.

CONCLUSION

Hepatic fibrosis is a pivotal stage of chronic liver disease that can progress to cirrhosis and hepatocellular carcinoma. Xia et al[6] suggest that HDI alleviates fibrogenesis by targeting a circadian-hypoxia-metabolic axis centered on NR1D1, linking clock regulation to reduced HIF-1α abundance, restored urea-cycle capacity, and lower ammonia burden. Although the work is constrained by issues such as model breadth and incomplete definition of active constituents and direct mechanisms, an NR1D1-focused strategy remains attractive given its druggability and the availability of measurable metabolic readouts. Future studies should validate efficacy across etiologically distinct fibrosis models, pinpoint the active HDI components and NR1D1-downstream pathways, and test ammonia-related metabolites for patient stratification and treatment-response monitoring. Building on chronopharmacology precedents discussed above, circadian phase should be treated as an explicit design variable, with dosing and sampling aligned accordingly to optimize benefit.

ACKNOWLEDGEMENTS

We thank the reviewers for their comments that helped to improve the manuscript.