Exploring the Impact of the Gut Microbiota/REV-ERBα/NF-κB Axis on the Circadian Rhythmicity of Gout Flares from a Chronobiological Perspective

Gout

Gout is a metabolic disorder characterized by recurrent inflammatory flares, primarily stemming from disturbances in purine metabolism that lead to the accumulation of monosodium urate (MSU) crystals in and around joints.¹ This condition results in a spectrum of clinical manifestations, including acute and chronic arthritis, the formation of soft tissue tophi (gout stones), kidney stones, and urate nephropathy.^2,3 While the specific pathogenesis of gout is not yet fully elucidated, elevated serum urate concentration, known as hyperuricemia, is recognized as a significant risk factor for the development of the disease.⁴ MSU crystals are widely acknowledged as potent endogenous danger signals that activate the innate immune system, thereby triggering an inflammatory response in gout.⁵ The affected joints of gout patients are marked by the presence of a variety of pro-inflammatory cytokines and chemokines, including interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), and IL-18. Among these, IL-1β is considered a pivotal mediator in the inflammatory cascade induced by MSU crystals.⁶ The global prevalence of gout has significantly increased over the past few decades, closely associated with obesity, metabolic syndrome, an aging population, and changes in dietary patterns (such as increased consumption of high-fructose beverages and red meat). From 1990 to 2019, the number of gout patients worldwide increased from 22 million (95% UI 17.52–27.37) to 53 million (95% UI 43.38–66.34), with an incidence growth rate of 63.44% (95% UI 57.33–68.78%).⁷ In addition to its impact on health, gout also imposes significant economic burdens, including increased healthcare costs.⁸ Moreover, gout is significantly associated with multiple comorbidities, such as kidney disease⁹ and cardiovascular disease,¹⁰ which place a heavy burden on the population. Frequent flares of gout can also lead to decreased quality of life and even productivity loss. In 2020, the global age-standardized incidence rate of years lived with disability due to gout was 20.5 (14.4–28.2) per 100,000 individuals.¹¹ Despite advances in medical treatment, the global burden of gout continues to increase. Therefore, it is urgent to develop targeted interventions to address this growing health concern.

Circadian Rhythm

In mammals, the circadian clock functions as a complex network of transcription factors that drive the rhythmic expression of clock genes through a precisely regulated transcription-translation feedback loop operating over a 24-hour cycle. Specifically, the transcriptional activators CLOCK and BMAL1 regulate the expression of repressor proteins, including the period (PER1, PER2, PER3) and cryptochrome (CRY1 and CRY2) families, thereby modulating CLOCK/BMAL1-dependent gene transcription.¹² Retinoid-related orphan receptors (RORs) and REV-ERBs are both regulated by CLOCK/BMAL1 but can also exert feedback control over CLOCK/BMAL1 expression.¹³ Specifically, REV-ERBα and RORα alternately regulate the expression of genes containing RORE elements, including BMAL1 and NFIL3.¹⁴ The biological clock is present in nearly all tissues and is synchronized with the ambient photoperiod through neuronal and hormonal signals from the brain’s central clock.¹⁵

Furthermore, Pan et al¹⁶ have explored the regulation of circadian clocks in phospholipid metabolism and metabolic disorders. Similar to many other physiological processes, the immune response is also governed by circadian clock networks. Both innate and adaptive immunity components within the immune system exhibit discernible circadian oscillations.¹⁷ Key immune cells, such as macrophages and natural killer cells, display circadian rhythms driven by cellular autonomy mediated by the circadian clock.¹⁸ In summary, the transcriptional oscillation of circadian clock genes generates a crucial circadian rhythm that plays an integral role in regulating human metabolism and immune response, thereby being essential for maintaining environmental homeostasis in the human body.

The Circadian Rhythm of Gout Attacks

Recent studies¹⁹ have established a clear link between circadian rhythms and the onset of gout, demonstrating a 3-fold higher likelihood of acute gout flares occurring nocturnally or matutinally (during transitions from rest to activity) compared to diurnal episodes.²⁰ Furthermore, Oh et al²¹ showed that individuals engaged in shift work experience disruptions in their endogenous circadian oscillations, resulting in elevated serum urate levels and increased susceptibility to gout flares. Choi et al²⁰ proposed that localized dehydration around the joints, caused by the supine position during sleep, may elevate the risk of gout attacks during nighttime and early morning. Another intriguing hypothesis suggests that sleep apnea may trigger gout flares by enhancing nucleotide turnover due to hypoxia, leading to increased production of purines that are metabolized into UA. Additionally, evidence indicates a positive correlation between symptoms of obstructive sleep apnea and the incidence of gout.²² The circadian rhythmicity of gout attacks also suggests the potential value of chronotherapy for the treatment of gout. Additionally, it has been reported that methotrexate chronotherapy is associated with significant improvements in RA activity scores.²³ Similar effects have also been observed in polymyalgia rheumatica and gout,²⁴ although the existing literature remains limited. Interestingly, in a multicenter, randomized, double-blind trial involving 288 patients with active rheumatoid arthritis over a 12-week period,²⁵ it was found that the use of extended-release prednisone at night was more effective in alleviating morning stiffness than the use of conventional immediate-release prednisone in the morning. Our study results may have similar implications for the optimal timing of various anti-gout therapeutic measures. These findings suggest several potential biological mechanisms that may underlie the circadian pattern of gout attacks.

The Significance of NLRP3 Inflammatories as the Core of the Body’s Inflammatory Response in Inducing the Onset of Gout

The NLRP3 inflammasome is widely recognized as a pivotal factor in the pathogenesis of gout²⁶ and is expressed in a diverse array of immune cells, including T lymphocytes, B lymphocytes, granulocytes, macrophages, and antigen-presenting cells. The NLRP3 inflammasome is a multiprotein complex composed of the sensor protein NLRP3, the apoptosis-associated speck-like protein (ASC), and the effector protein caspase-1.²⁷ The activation of the NLRP3 inflammasome is widely recognized to require two distinct signal transduction processes: priming and activation.²⁶ During the priming phase, nuclear factor kappa-B (NF-κB) is activated through engagement of pattern recognition receptors, such as Toll-like receptors (TLRs), by pathogen-associated molecular patterns (PAMPs) like lipopolysaccharides. This interaction leads to the transcriptional activation of the NLRP3 inflammasome gene via the TLR4/NF-κB signaling pathway. During the activation phase, the assembly and activation of the NLRP3 inflammasome are completed through processes such as K⁺ efflux,²⁸ Ca²⁺ mobilization,²⁹ release of reactive oxygen species (ROS),³⁰ and lysosomal rupture.³¹ These processes lead to the secretion of various inflammatory factors, such as IL-1β and IL-6.

Gout is an autoinflammatory disease characterized by the NLRP3 inflammasome-mediated production of pro-inflammatory cytokines in response to the deposition of MSU crystals in the joints and surrounding tissues. MSU crystals function as DAMPs, activating TLR4 and the NLRP3 inflammasome in a manner similar to PAMPs.²⁶ This activation results in the release of cytokines such as IL-1β, IL-18, and IL-6. IL-1β, in particular, binds to its receptor on endothelial cells and macrophages within the joint, initiating downstream signaling and gene activation, thereby perpetuating NF-κB activation and exacerbating the inflammatory response. Moreover, dysregulation of the NLRP3 signaling cascade has been implicated in a variety of inflammatory, autoimmune, and metabolic conditions, including gout, inflammatory bowel disease, Sjögren’s syndrome, diabetes, and atherosclerosis.^32–35

NF-κB

NF-κB Underlies the Interregulation of the Circadian Clock and Inflammation

NF-κB, as a transcriptional activator of NLRP3, is implicated in the development of numerous inflammatory diseases and mediates immune responses, such as in rheumatoid arthritis and inflammatory bowel disease. In recent years, studies have shown that various pro-inflammatory factors can affect the function of the biological clock. For instance, inflammation induced by TNF-α and endotoxin³⁶ or lipopolysaccharide(LPS)³⁷ leads to abnormal expression of circadian clock genes. Conversely, inflammatory responses mediated by circadian clock genes can exhibit diurnal oscillations, as observed in hepatitis B,³⁸ periodontitis,³⁹ and gouty arthritis.⁴⁰ Notably, Shen et al found that NF-κB perturbations alter circadian oscillations in cells, tissues, and mice, suggesting that NF-κB plays an essential role in directly regulating the circadian clock and highlighting the interregulation between circadian rhythms and inflammatory pathways.⁴¹ Further studies revealed that the inflammatory signaling transcription factor NF-κB antagonizes the transcription of CLOCK/BMAL1 target genes by directly binding to the promoters of the core clock repressors PER and CRY, resulting in highly specific transcriptional inhibition of core clock genes.⁴² This NF-κB-mediated transcriptional inhibition of clock feedback loops is emphasized as the cause of circadian rhythm disturbances in response to inflammation. Importantly, the NLRP3 inflammasome pathway exhibits a circadian rhythm due to the oscillatory nature of NLRP3 transcription and expression. In summary, the basal level of NF-κB expression is critical for the regulation of circadian homeostasis and inflammation.

REV-ERBα Regulates the Circadian Rhythm Expression of NLRP3 Inflammasome by Inhibiting NF-κB Signaling and NLRP3 Gene Transcription

REV-ERBα, encoded by the gene NR1D1 (nuclear receptor subfamily 1, group D, member 1), is a core component of the circadian clock and functions as a transcription factor that negatively regulates NLRP3 in macrophages. This regulatory activity suppresses the expression of genes involved in diverse physiological processes, including circadian rhythm, inflammation, and metabolism, thereby emerging as a central regulator linking metabolic homeostasis, immune response, and circadian rhythmicity. Zhao et al demonstrated that activation of REV-ERBα mitigates the LPS-induced inflammatory response in human endometrial stromal cells by inhibiting TLR4-regulated NF-κB.⁴³ Similarly, Wang et al showed through in vivo and in vitro experiments in mice that REV-ERBα attenuates colitis by inhibiting NF-κB and Nlrp3 transcription via inactivation of the NLRP3 inflammasome.⁴⁴

Furthermore, additional studies have explored the molecular mechanisms underlying the inhibition of the NF-κB signaling pathway by REV-ERBα. Specifically, it has been shown that REV-ERBα reduces LPS-induced neuroinflammation in mouse microglia by inhibiting the phosphorylation and degradation of the NF-κB inhibitor IκBα and by blocking the nuclear translocation of the NF-κB subunit p65.⁴⁵ Subsequent investigations by Pourcet et al revealed that the mRNA expression of NLRP3 inflammasome components oscillates daily in peritoneal mouse macrophages under the control of REV-ERBα, with peak expression occurring during the active phase, which corresponds to the lowest expression level of REV-ERBα mRNA.⁴⁶

Platr4 Regulates the Circadian Rhythm of the NLRP3 Inflammasome by Inhibiting the NF-κB Signaling Pathway

In recent years, Lin et al⁴⁷ experimentally identified a novel molecular mechanism underlying the regulation of inflammation through the biological clock, mediated by the long non-coding RNA (lncRNA) Platr4. Platr4 is an oscillating lncRNA regulated by the circadian clock component REV-ERBα. In their study, Platr4-deficient mice exhibited significantly reduced Nlrp3 inflammasome activity and disrupted circadian rhythms in the liver, rendering them more susceptible to experimental steatohepatitis. As a repressor of NF-κB, Platr4 inhibits the activation of the NLRP3 inflammasome in macrophages by suppressing NF-κB-dependent transcription of inflammasome components, including NLRP3 and ASC. Thus, Platr4 is crucial for driving the diurnal oscillations of Nlrp3 inflammasome expression and activity. Based on these findings, Platr4 appears to function as an integrator of the circadian clock and inflammation through the NF-κB/NLRP3 inflammasome axis. However, there is currently no evidence indicating a direct impact of Platr4 on the circadian rhythm of gout attacks, and further investigation into this potential mechanism is warranted.

The Potential Role of Gut Microbiota Imbalance in the Circadian Rhythm of Gout Attacks

Gut Microbiota

The gut microbiota is a complex and diverse community of viruses, fungi, and bacteria that inhabit the gastrointestinal tract, playing essential roles in food digestion and maintaining host homeostasis. Often referred to as gut bacteria, the gut microbiota primarily consists of up to 1000 bacterial species.⁴⁸ The gut microbiota is predominantly composed of four phyla: Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria, with the former two being the most abundant. Moreover, the byproducts (such as LPS) and metabolites (such as short-chain fatty acids [SCFAs], tryptophan [Trp], and bile acid metabolites) of the gut microbiota play crucial roles in modulating immune cell function and inflammatory responses. Recent studies have provided compelling evidence highlighting the key roles of the gut microbiota in human metabolism, immune regulation, and defense against pathogenic microorganisms. Consequently, disturbances in the gut microbiota have been implicated in a range of metabolic and immune disorders, including obesity,⁴⁹ type 2 diabetes,⁵⁰ inflammatory bowel disease,⁵¹ gout,⁵² and others.

Gut Microbiota and Gout are Closely Related in Metabolic Relationship

Gout is directly linked to elevated levels of UA, which can result from disrupted purine metabolism, reduced UA excretion, or increased UA production. UA is the end product of purine metabolism. Purine nucleotides are hydrolyzed to form adenine and guanine, which are then deaminated to produce xanthine. Xanthine is subsequently oxidized by xanthine oxidase (XOD) to generate UA. In healthy individuals, UA is excreted through two primary pathways: approximately 25% via the gastrointestinal tract and 75% via the kidneys. This gastrointestinal excretion pathway may lead to alterations in the composition of the gut microbiota due to exposure to UA. Previous studies have identified that patients with gout indeed experience an imbalance in the gut microbiota,⁵³ and Lin et al⁵⁴ developed a classification model based on bacterial genera that are significantly enriched in healthy individuals compared to those with gout. The results showed that, through receiver operating characteristic (ROC) analysis, the average area under the curve (AUC) was as high as 0.973. Given the significant correlation between the gut microbiota and the development of gout, a gout-specific gut microbiota profile may serve as a diagnostic marker. Therefore, the imbalance in the gut microbiota characteristic of gout could potentially become a non-invasive diagnostic tool for gout. This suggests that it offers a promising avenue for future clinical interventions.

The gut microbiota can modulate UA levels and inflammatory signaling pathways through its bacteria, derivatives, metabolites, and other metabolic activities, thereby impacting the onset of gout. Specifically, the gut microbiota may regulate UA levels by influencing purine metabolism. For instance, certain strains of gut bacteria, such as Lactobacillus casei PA-3, have been shown to absorb and utilize purines, thereby reducing the intestinal absorption of dietary purines and lowering serum UA levels.⁵⁵ Secondly, LPS, a byproduct of the gut microbiota, is a key component of the cell wall of Gram-negative bacteria. Dysbiosis of the gut microbiota can increase intestinal permeability, facilitating the translocation of LPS into the human circulatory system. This process can induce metabolic endotoxemia and trigger inflammatory pathways. Ramos et al⁵⁶ observed that chronic inflammation resulting from elevated serum LPS levels is frequently associated with increased XOD activity. A recent study reported that Phascolarctobacterium and Bacteroides are enriched in patients with gout, and identified a “core microbiota” of three Bacteroides species in the gout group.⁵⁷ Lactobacillus gasseri has been shown to reduce purine levels in the gut, with its fermentation products exhibiting urate-lowering effects.⁵⁸ Additionally, Lactobacillus reuteri TSR332 and Lactobacillus fermentum TSF331 can control the level of UA by degrading purine.⁵⁹

In addition to modulating UA levels, the gut microbiota can also regulate the activation of the NLRP3 inflammasome, thereby impacting the onset of gout. For example, Bacteroides fragilis exerts a negative regulatory effect on the NLRP3-mediated inflammatory signaling pathway, inhibiting macrophage activation and the secretion of pro-inflammatory mediators such as IL-18 and IL-1β, thereby reducing intestinal inflammation.⁶⁰ Lactic acid bacteria mitigate the generation of ROS by restoring aberrant mitochondrial membrane potential, thereby suppressing NLRP3 activation.⁶¹ Conversely, SCFAs produced through gut microbiota metabolism exert a dual impact on the initiation and progression of inflammation. Vieira et al⁶² found that SCFAs are essential for inflammasome assembly and IL-1β production. Among these, acetate plays a pivotal role in inflammasome assembly in response to MSU crystals. The production of ROS induced by acetate in immune cells may represent a potential mechanism for regulating the NLRP3 inflammasome, suggesting that acetate contributes to the pathogenesis of gout. However, certain SCFAs may exert protective effects against inflammation. For example, butyrate⁶³ is associated with increased expression of IκBα, which inhibits the phosphorylation and nuclear translocation of NF-κB p65, as well as the expression of downstream inflammatory cytokines such as MCP-1 and IL-1β. Interestingly, there is also a close relationship between tryptophan metabolism and gout. Mahbub et al⁶⁴ observed significant alterations in the plasma amino acid profile of gout patients by analyzing free amino acids in their plasma. Specifically, elevated levels of alanine, isoleucine, leucine, phenylalanine, tryptophan, and valine were found to be significantly positively correlated with gout (P < 0.005–0.05), while glycine and serine showed significant negative correlations with gout.

Effects of Gut Microbiota on the Circadian Rhythm of Gout Attacks

Nuclear factor interleukin 3 (NFIL3) is an immunomodulatory transcription factor predominantly expressed in natural killer cells, B lymphocytes, T lymphocytes, monocytes, and other immune cells. It plays a pivotal role in the pathogenesis of autoimmune diseases.⁶⁵ Wang et al⁶⁶ demonstrated that the gut microbiota modulates host body composition by influencing the circadian transcription factor NFIL3. Nfil3 transcription exhibits diurnal oscillations in intestinal epithelial cells, and the amplitude of these circadian fluctuations is regulated by the microbiota through Group 3 innate lymphoid cells, STAT3, and the epithelial circadian clock. Consequently, NFIL3 is considered a crucial molecular bridge connecting the microbiome, the circadian clock, and host metabolism. The expression of NFIL3 is directly regulated by the core clock transcriptional repressor REV-ERBα. In T cells⁶⁷ and hepatocytes,⁶⁸ REV-ERBα binds to conserved sequences in the NFIL3 gene loci, suppressing transcription and resulting in rhythmic diurnal patterns of NFIL3 expression. This implies that REV-ERBα governs the circadian rhythm of NFIL3 expression, and that the microbiome may induce NFIL3 expression by inhibiting REV-ERBα. Moreover, empirical evidence has shown that NFIL3 can potentiate NF-κB signaling by inhibiting IκBα.⁶⁹ As a transcriptional activator of NLRP3, NF-κB triggers aberrant activation of the NLRP3 inflammasome, initiating an inflammatory cascade that may contribute to the pathogenesis of gout. Therefore, when the gut microbiota is dysfunctional, the circadian clock gene REV-ERBα suppresses the diurnal oscillation of NFIL3 expression. This leads to aberrant activation of the NF-κB signaling pathway and upregulation of transcription for various inflammatory factors, such as IL-1β, IL-6, and TNF-α, thereby exacerbating the inflammatory response in gout patients. This mechanism may underlie the circadian pattern of gout attacks.

Based on current research, it is hypothesized that the gut microbiota may influence the rhythmic expression of XOD by modulating bile acid metabolism, thereby causing circadian variations in UA levels and potentially triggering gout flares. The gut microbiota is involved in bile acid biotransformation through processes such as deconjugation, dehydroxylation, and reconjugation of bile acids.⁷⁰ Additionally, bile acid synthesis exhibits a robust circadian rhythm,⁷¹ which is crucial for maintaining metabolic homeostasis. Conversely, XOD, a rate-limiting enzyme in the terminal phase of purine nucleotide degradation, plays a key role in UA metabolism. The diurnal regulation of XOD activity can contribute to rhythmic fluctuations in serum UA levels. Kanemitsu et al⁷² demonstrated that periodic modulation of bile acids can regulate the circadian fluctuations of UA by modulating XOD via the orphan nuclear receptor PPAR-α. This mechanism may represent an endogenous pathway by which the gut microbiota influences circadian gout attacks through metabolic regulation.

Exploring Chronobiological Therapeutic Approaches Based on the Gut Microbiota/REV-ERBα/NF-κB Axis

The gut microbiota and the circadian clock coordinate to establish homeostasis, which generates diurnal rhythms in gut innate immunity.⁷³ Based on the aforementioned theories, restoring the homeostasis of the gut microbiota and circadian rhythms to lower UA levels may emerge as a potential therapeutic strategy for gout, such as probiotics combined with prebiotic diets and fecal microbiota transplantation. Dietary fibers, prebiotics, and probiotics that can be metabolized by gastrointestinal microbiota are generally considered dietary strategies for modulating the microbiota.⁷⁴ Zhao et al⁷⁵ investigated the role of the gut microbiota in gout management following probiotic supplementation and found that Lactobacillus fermentum GR-3 significantly reduced UA levels and inflammation in individuals at high risk for gout. Feng et al⁷⁶ discovered that prebiotic diets, such as β-carotene and green tea powder, lowered serum UA and pro-inflammatory cytokine levels, improved the gut microbiota profile, and reduced the metabolism of purines and pyrimidines, thereby alleviating joint swelling and pain in gout mice and mitigating acute gout flares. Numerous studies have demonstrated that the gut microbiota of gout patients significantly differs in abundance from that of healthy individuals.^52,53 Washed Microbiota Transplantation (WMT) leverages this principle by transplanting the fecal microbiota from healthy individuals, after purification and separation, into the gastrointestinal tract of patients.⁷⁷ Its mechanism of action is strongly associated with the restoration of gut microbiota homeostasis,⁷⁸ and it has been effectively applied in the treatment of many diseases.⁷⁹ Xie et al⁸⁰ found that WMT can effectively reduce serum UA levels and improve gout symptoms in patients with gout, and also helps to restore impaired intestinal barrier function. Since the homeostasis of the gut microbiota is intrinsically linked to the balance of circadian rhythms, restoring the homeostasis of the gut microbiota and circadian rhythms may represent an emerging therapeutic strategy for gout, deserving further in-depth investigation.

Considering that the diurnal variation in the severity of inflammatory diseases may be related to the circadian regulator REV-ERBα, an increasing body of evidence supports the targeting and activation of REV-ERBα as a promising approach for managing inflammation.^44,46,81 Once activated, REV-ERBα may exert anti-inflammatory effects by inhibiting the NF-κB signaling pathway,⁴⁵ downregulating Nlrp3 inflammasome activity,⁴⁴ and directly modulating immune-related genes (eg, IL-1β, TLR4, IL-6).^82,83 Although the pharmacological effects of a series of REV-ERBα agonists (such as heme, GSK4112, SR9009, etc). have been demonstrated in animal studies (preclinical research), progress in translating these findings into clinical trials has been limited.⁸⁴ Therefore, REV-ERBα agonists hold potential as a new targeted therapeutic strategy for gout in future clinical applications.

Regarding the mechanism underlying the peak incidence of gout flares at night, some researchers have speculated⁸⁵ that the lower body temperature from night to early morning may increase the risk of urate crystal formation, thereby triggering gout attacks. This study suggests that elevated UA levels are associated with nocturnal acute gout attacks, and the diurnal variation in UA levels may directly influence the circadian differences in acute gout flares. Therefore, based on the circadian rhythmicity of gout flares, administering sustained-release urate-lowering and anti-inflammatory medications at night or before bedtime may offer a novel chronotherapy approach for the clinical treatment of gout. However, robust clinical research evidence is currently lacking.

Conclusion

Based on our observations and summary of the literature, we propose that REV-ERBα serves as a key link between the gut microbiota and the NF-κB pathway. The NF-κB signaling pathway, as the core inflammatory pathway in gout, exhibits strong circadian rhythmicity under the regulation of circadian clock genes such as REV-ERBα. The gut microbiota can also rhythmically activate the NF-κB inflammatory signaling pathway by influencing the expression of REV-ERBα and can alter its own population abundance, diversity, metabolic pathways, and metabolites,⁶³ thereby inducing circadian oscillations in UA levels and triggering the rhythmic occurrence of gout flares. In summary, the Gut microbiota/REV-ERBα/NF-κB axis (Figure 1) may represent a potential biological mechanism underlying the circadian pattern of gout attacks. Additionally, Platr4 and NFIL3 could emerge as therapeutic targets within this pathway, suggesting a promising future direction for gout treatment. Therefore, a chronobiology-based therapeutic approach (such as adjusting medication timing, dietary interventions to modulate the gut microbiota, and targeted pharmacological agents) targeting the gut microbiota/REV-ERBα/NF-κB axis holds promise as a safer and more effective new avenue for the treatment of gout. Similarly, a chronotherapy-based treatment paradigm may also be applicable to other inflammatory diseases, with potentially significant clinical implications. However, the conclusions drawn in this paper lack compelling experimental and clinical validation. Future research should focus on designing high-quality randomized controlled trials with double-blinding to verify these findings, thereby more effectively guiding clinical practice and medication use.

Figure 1 Gut microbiota/REV-ERBα/NF-κB axis. Derivatives (such as LPS) and metabolites (such as SCFAs) of the gut microbiota influence the NF-κB signaling pathway by modulating the priming and activation steps of the NLRP3 inflammasome. On one hand, after LPS is recognized and bound by TLR4, IKKα, IKKβ, and NEMO are activated within the cell under the induction of TRIF. This leads to the phosphorylation and subsequent degradation of IκBα. NF-κB itself and its p65/RelA subunit then translocate to the nucleus, where they participate in the synthesis of the NLRP3 inflammasome and various inflammatory factors. On the other hand, SCFAs enter cells via GPR43. Butyrate inhibits the phosphorylation and nuclear translocation of NF-κB p65, thereby blocking the NF-κB signaling pathway. In contrast, acetate induces the production of ROS, which promotes the activation of the NLRP3 inflammasome. The transcription and expression of Platr4 and NFIL3 oscillate diurnally under the regulation of REV-ERBα, thereby influencing the NF-κB signaling pathway. The former inhibits the transcription of NLRP3 inflammasome components to prevent the release of inflammatory factors, while the latter enhances the activity of the NF-κB signaling pathway by inhibiting the transcription of IκBα.