Inter-individual variations in circadian misalignment-induced NAFLD pathophysiology in mice.

Pathological consequences of circadian misalignment, such as shift work, show considerable individual differences, but the lack of mechanistic understanding hinders precision prevention to prevent and mitigate disease symptoms. Here, we employed an integrative approach involving physiological, transcriptional, and histological phenotypes to examine inter-individual differences in pre-symptomatic pathological progression, preceding irreversible disease onset, in wild-type mice exposed to chronic jet-lag (CJL). We observed that CJL markedly increased the prevalence of hepatic steatosis with pronounced inter-individual differences. Stratification of individual mice based on CJL-induced hepatic transcriptomic signature, validated by histopathological analysis, pinpoints dysregulation of lipid metabolism. Moreover, the period and power of intrinsic behavioral rhythms were found to significantly correlate with CJL-induced gene signatures. Together, our results suggest circadian rhythm robustness of the animals contributes to inter-individual variations in pathogenesis of circadian misalignment-induced diseases and raise the possibility that these physiological indicators may be available for predictive hallmarks of circadian rhythm disorders.

The BMAL1/HIF2A heterodimer modulates circadian variations of myocardial injury.

Acute myocardial infarction stands as a prominent cause of morbidity and mortality worldwide1-6. Clinical studies have demonstrated that the severity of cardiac injury following myocardial infarction exhibits a circadian pattern, with larger infarct sizes and poorer outcomes in patients experiencing morning onset myocardial infarctions7-14. However, the molecular mechanisms that govern circadian variations of myocardial injury remain unclear. Here, we show that BMAL114-20, a core circadian transcription factor, orchestrates diurnal variability in myocardial injury. Unexpectedly, BMAL1 modulates circadian-dependent cardiac injury by forming a transcriptionally active heterodimer with a non-canonical partner, hypoxia-inducible factor 2 alpha (HIF2A)6,21-23, in a diurnal manner. Substantiating this finding, we determined the cryo-EM structure of the BMAL1/HIF2A/DNA complex, revealing a previously unknown capacity for structural rearrangement within BMAL1, which enables the crosstalk between circadian rhythms and hypoxia signaling. Furthermore, we identified amphiregulin (AREG) as a rhythmic transcriptional target of the BMAL1/HIF2A heterodimer, critical for regulating circadian variations of myocardial injury. Finally, pharmacologically targeting the BMAL1/HIF2A-AREG pathway provides effective cardioprotection, with maximum efficacy when aligned with the pathway's circadian trough. Our findings not only uncover a novel mechanism governing the circadian variations of myocardial injury but also pave the way for innovative circadian-based treatment strategies, potentially shifting current treatment paradigms for myocardial infarction.

Efficacy of Supplemental Ultrasound-Guided Pericapsular Nerve Group (PENG) Block Combined with Lateral Femoral Cutaneous Nerve Block in Patients Receiving Local Infiltration Analgesia after Hip Fracture Surgery: A Prospective Randomized Controlled Trial.

Background and Objectives: Local infiltration analgesia (LIA) represents a potential approach to reducing pain in patients undergoing total hip arthroplasty (THA). The pericapsular nerve group (PENG) block also provides adequate analgesia for fractures and THA. As most hip surgeries use a lateral incision, affecting the cutaneous supply by branches of the lateral femoral cutaneous nerve (LFCN), the LFCN block can contribute to postoperative analgesia. However, no studies have investigated the effectiveness of supplemental PENG block combined with LFCN block in patients undergoing LIA after hip fracture surgery. Our study aimed to assess the effectiveness of PENG combined with LFCN block following hip fracture surgery in patients who underwent LIA. Materials and Methods: Forty-six patients were randomly assigned to LIA or PENG + LFCN + LIA groups. The primary outcome was the pain score at rest and during movement at 2, 6, 12, 24, and 48 h postoperatively. The total opioid dose for postoperative analgesia was also measured at the same time points. Secondary outcomes included postoperative cognitive function assessment. Results: The median pain scores at rest and during movement were lower in the PENG + LFCN + LIA group throughout the study periods compared to the LIA group, except at 2 h (at rest) and 48 h (during movement) after surgery. The total fentanyl dose was lower in the PENG + LFCN + LIA group at all time points after surgery when compared to the LIA group. Postoperative delirium incidence and the median abbreviated mental test scores were not significantly different between the two groups. Conclusions: The combination of PENG and LFCN blocks may contribute to enhanced recovery for patients undergoing LIA after hip fracture surgery. However, further well-controlled research is necessary to determine the effectiveness of supplemental PENG combined with LFCN block in addressing cognitive deficits in these patients.

Regulation of headache response and transcriptomic network by the trigeminal ganglion clock.

OBJECTIVE: To characterize the circadian features of the trigeminal ganglion in a mouse model of headache. BACKGROUND: Several headache disorders, such as migraine and cluster headache, are known to exhibit distinct circadian rhythms of attacks. The circadian basis for these rhythmic pain responses, however, remains poorly understood. METHODS: We examined trigeminal ganglion ex vivo and single-cell cultures from Per2::LucSV reporter mice and performed immunohistochemistry. Circadian behavior and transcriptomics were investigated using a novel combination of trigeminovascular and circadian models: a nitroglycerin mouse headache model with mechanical thresholds measured every 6 h, and trigeminal ganglion RNA sequencing measured every 4 h for 24 h. Finally, we performed pharmacogenomic analysis of gene targets for migraine, cluster headache, and trigeminal neuralgia treatments as well as trigeminal ganglion neuropeptides; this information was cross-referenced with our cycling genes from RNA sequencing data to identify potential targets for chronotherapy. RESULTS: The trigeminal ganglion demonstrates strong circadian rhythms in both ex vivo and single-cell cultures, with core circadian proteins found in both neuronal and non-neuronal cells. Using our novel behavioral model, we showed that nitroglycerin-treated mice display circadian rhythms of pain sensitivity which were abolished in arrhythmic Per1/2 double knockout mice. Furthermore, RNA-sequencing analysis of the trigeminal ganglion revealed 466 genes that displayed circadian oscillations in the control group, including core clock genes and clock-regulated pain neurotransmitters. In the nitroglycerin group, we observed a profound circadian reprogramming of gene expression, as 331 of circadian genes in the control group lost rhythm and another 584 genes gained rhythm. Finally, pharmacogenetics analysis identified 10 genes in our trigeminal ganglion circadian transcriptome that encode target proteins of current medications used to treat migraine, cluster headache, or trigeminal neuralgia. CONCLUSION: Our study unveiled robust circadian rhythms in the trigeminal ganglion at the behavioral, transcriptomic, and pharmacogenetic levels. These results support a fundamental role of the clock in pain pathophysiology. PLAIN LANGUAGE SUMMARY: Several headache diseases, such as migraine and cluster headache, have headaches that occur at the same time each day. We learned that the trigeminal ganglion, an important pain structure in several headache diseases, has a 24-hour cycle that might be related to this daily cycle of headaches. Our genetic analysis suggests that some medications may be more effective in treating migraine and cluster headache when taken at specific times of the day.

The Effect of Dexmedetomidine on the Mini-Cog Score and High-Mobility Group Box 1 Levels in Elderly Patients with Postoperative Neurocognitive Disorders Undergoing Orthopedic Surgery.

Dexmedetomidine prevents postoperative cognitive dysfunction by inhibiting high-mobility group box 1 (HMGB1), which acts as an inflammatory marker. This study investigated the HMGB1 levels and the cognitive function using a Mini-Cog© score in elderly patients undergoing orthopedic surgery with dexmedetomidine infusion. In total, 128 patients aged ≥ 65 years were analyzed. The patients received saline in the control group and dexmedetomidine in the dexmedetomidine group until the end of surgery. Blood sampling and the Mini-Cog© test were performed before the surgery and on postoperative days 1 and 3. The primary outcomes were the effect of dexmedetomidine on the HMGB1 levels and the Mini-Cog© score in terms of postoperative cognitive function. The Mini-Cog© score over time differed significantly between the groups (p = 0.008), with an increase in the dexmedetomidine group. The postoperative HMGB1 levels increased over time in both groups; however, there was no significant difference between the groups (p = 0.969). The probability of perioperative neurocognitive disorders decreased by 0.48 times as the Mini-Cog© score on postoperative day 3 increased by 1 point. Intraoperative dexmedetomidine has shown an increase in the postoperative Mini-Cog© score. Thus, the Mini-Cog© score is a potential tool for evaluating cognitive function in elderly patients.

The Circadian Nobiletin-ROR Axis Suppresses Adipogenic Differentiation and IκBα/NF-κB Signaling in Adipocytes.

Obesity is a known risk factor for metabolic diseases and is often associated with chronic inflammation in adipose tissue. We previously identified the polyethoxylated flavonoid Nobiletin (NOB) as a circadian clock modulator that directly binds to and activates the ROR receptors in the core oscillator, markedly improving metabolic fitness in obese mice. Here, we show that NOB enhanced the oscillation of core clock genes in differentiated 3T3-L1 adipocytes, including ROR target genes such as Bmal1, Cry1, Dec1, and Dec2. NOB inhibited lipid accumulation in 3T3-L1 and SVF cells, concomitant with the dysregulated circadian expression of adipogenic differentiation-related genes including Cebpb, Pparg, Lpl, Scd1, and Fas. Importantly, RORα/RORγ double knockdown in 3T3-L1 cells (Ror DKD) significantly attenuated the effects of NOB on circadian gene expression and lipid accumulation. Furthermore, whereas NOB upregulated the expression of IκBα, a target of RORs, to inhibit NF-κB activation and proinflammatory cytokine expression, Ror DKD cells exhibited a heightened activation of the NF-κB pathway, further indicating a requisite role of RORs for NOB efficacy in adipocytes. Together, these results highlight a significant regulatory function of the NOB-ROR axis in the circadian expression of clock and clock-controlled genes in adipocytes, thereby governing adipogenic differentiation, lipogenesis, and inflammation.

Efficacy of scrambler therapy in patients with painful diabetic peripheral neuropathy: A single-arm, prospective, pilot study.

BACKGROUND: A variety of medications are available to manage painful diabetic peripheral neuropathy (DPN), but the proper treatment remains challenging. Accordingly, various neuromodulation modalities have been used. However, no prospective clinical trials have evaluated the use of scrambler therapy (ST) in painful DPN. This study aimed to explore the long-term effects of ST in managing painful DPN. METHODS: The patients received 10 consecutive STs of 45 minutes every 1 to 2 days. The primary outcome was pain score. We measured the visual analog scale (VAS) pain scores at baseline, during ST, immediately after ST, and at 1, 2, 3, and 6 months after ST. The secondary outcomes were Michigan Neuropathy Screening Instrument (MNSI), Semmes-Weinstein monofilament test, and Leeds Assessment of Neuropathic Symptoms and Signs pain scores, which were measured at baseline, immediately after ST, and at 1, 2, 3, and 6 months after ST. RESULTS: VAS scores showed significant improvement at the 8th, 9th, and 10th sessions during ST and 1 month after ST. The MNSI self-report component score was decreased 1 month after the ST. However, all other outcomes did not show significant differences compared to the baseline. CONCLUSION: ST may have short-term effects and limited long-term effects on painful DPN.

Circadian Features of Cluster Headache and Migraine: A Systematic Review, Meta-analysis, and Genetic Analysis.

BACKGROUND AND OBJECTIVES: Cluster headache and migraine have circadian features at multiple levels (cellular, systems, and behavioral). A thorough understanding of their circadian features informs their pathophysiologies. METHODS: A librarian created search criteria in MEDLINE Ovid, Embase, PsycINFO, Web of Science, and Cochrane Library. Two physicians independently performed the remainder of the systematic review/meta-analysis using Preferred Reporting Items for Systematic Review and Meta-Analyses guidelines. Separate from the systematic review/meta-analysis, we performed a genetic analysis for genes with a circadian pattern of expression (clock-controlled genes or CCGs) by cross-referencing genome-wide association studies (GWASs) of headache, a nonhuman primate study of CCGs in a variety of tissues, and recent reviews of brain areas relevant in headache disorders. Altogether, this allowed us to catalog circadian features at the behavioral level (circadian timing, time of day, time of year, and chronotype), systems level (relevant brain areas where CCGs are active, melatonin and corticosteroid levels), and cellular level (core circadian genes and CCGs). RESULTS: For the systematic review and meta-analysis, 1,513 studies were found, and 72 met the inclusion criteria; for the genetic analysis, we found 16 GWASs, 1 nonhuman primate study, and 16 imaging reviews. For cluster headache behavior, meta-analyses showed a circadian pattern of attacks in 70.5% (3,490/4,953) of participants across 16 studies, with a clear circadian peak between 21:00 and 03:00 and circannual peaks in spring and autumn. Chronotype was highly variable across studies. At the systems level, lower melatonin and higher cortisol levels were reported in cluster headache participants. At the cellular level, cluster headache was associated with core circadian genes CLOCK and REV-ERBα, and 5 of the 9 cluster headache susceptibility genes were CCGs. For migraine behavior, meta-analyses showed a circadian pattern of attacks in 50.1% (2,698/5,385) of participants across 8 studies, with a clear circadian trough between 23:00 and 07:00 and a broad circannual peak between April and October. Chronotype was highly variable across studies. At the systems level, urinary melatonin levels were lower in participants with migraine and even lower during an attack. At the cellular level, migraine was associated with core circadian genes CK1δ and RORα, and 110 of the 168 migraine susceptibility genes were CCGs. DISCUSSION: Cluster headache and migraine are highly circadian at multiple levels, reinforcing the importance of the hypothalamus. This review provides a pathophysiologic foundation for circadian-targeted research into these disorders. TRIAL REGISTRATION INFORMATION: The study was registered with PROSPERO (registration number CRD42021234238).

The Polymethoxyflavone Sudachitin Modulates the Circadian Clock and Improves Liver Physiology.

SCOPE: Polymethoxylated flavones (PMFs) are a group of natural compounds known to display a wide array of beneficial effects to promote physiological fitness. Recent studies reveal circadian clocks as an important cellular mechanism mediating preventive efficacy of the major PMF Nobiletin against metabolic disorders. Sudachitin is a PMF enriched in Citrus sudachi, and its functions and mechanism of action are poorly understood. METHODS AND RESULTS: Using circadian reporter cells, it shows that Sudachitin modulates circadian amplitude and period of Bmal1 promoter-driven reporter rhythms, and real-time qPCR analysis shows that Sudachitin alters expression of core clock genes, notably Bmal1, at both transcript and protein levels. Mass-spec analysis reveals systemic exposure in vivo. In mice fed with high-fat diet with or without Sudachitin, it observes increased nighttime activity and daytime sleep, accompanied by significant metabolic improvements in a circadian time-dependent manner, including respiratory quotient, blood lipid and glucose profiles, and liver physiology. Focusing on liver, RNA-sequencing and metabolomic analyses reveal prevalent diurnal alteration in both gene expression and metabolite accumulation. CONCLUSION: This study elucidates Sudachitin as a new clock-modulating PMF with beneficial effects to improve diurnal metabolic homeostasis and liver physiology, suggesting the circadian clock as a fundamental mechanism to safeguard physiological well-being.

Reliability of submaximal stimulation for the train-of-four test using acceleromyography and electromyography with individualized stimulation currents.

PURPOSE: The supramaximal stimulation (SMS) of the TOF test causes uncomfortable sensations in patients. We aimed to determine whether the submaximal stimulation would be reliable in TOF tests with reduced painful sensation. METHODS: The accelomyography (AMG) and electromyography (EMG) monitor was applied at each arm and general anesthesia was induced and maintained by total intravenous anesthesia. At extubation, we conducted TOF test three times at each of four different currents: SMS, 70% SMS, 50% SMS, and 30% SMS. The same procedure was performed in the postanesthesia care unit (PACU) only with EMG, and the pain scores on the numerical rating scale (NRS) during the tests were recorded. RESULTS: A total of 36 patients were enrolled. At extubation, TOF ratios with SMS in AMG and EMG were 112.0 ± 13.1% and 93.7 ± 8.9%, respectively. There were no significant differences in TOF ratios between the SMS and lower stimulation intensities. However, 30% and 50% SMS showed significantly higher rates of the unmeasurable results of tests in the PACU. In terms of the stimulation pain, NRS showed a downward pattern as the current decreased and was significantly lower at 50% and 30% SMS than the NRS at SMS. CONCLUSION: The TOF test with submaximal stimulation is still reliable and can reduce stimulation pain. Considering the importance of the TOF results in determining extubation, the authors suggest the minimal current for the TOF test as 70% SMS.

Circadian regulation of cardiac muscle function and protein degradation.

The circadian clock plays a fundamental role in physiology. In particular, the heart is a target organ where the clock orchestrates various aspects of cardiac function. At the molecular level, the clock machinery governs daily rhythms of gene expression. Such circadian regulation is in tune with the dynamic nature of heart structure and function, and provides the foundation for chronotherapeutic applications in cardiovascular diseases. In comparison, a regulatory role of the clock in cardiac protein degradation is poorly documented. Sarcomere is the structural and functional unit responsible for cardiac muscle contraction, and sarcomere components are closely regulated by protein folding and proteolysis. Emerging evidence supports a role of the circadian clock in governing sarcomere integrity and function. Particularly, recent studies uncovered a circadian regulation of a core sarcomere component TCAP. It is possible that circadian regulation of the cardiac muscle protein turnover is a key regulatory mechanism underlying cardiac remodeling in response to physiological and environmental stimuli. While the detailed regulatory mechanisms and the molecular links to cardiac (patho)physiology remain to be further studied, therapeutic strategies targeting circadian control in the heart may markedly enhance intervention outcomes against cardiovascular disease.

The circadian E3 ligase FBXL21 regulates myoblast differentiation and sarcomere architecture via MYOZ1 ubiquitination and NFAT signaling.

Numerous molecular and physiological processes in the skeletal muscle undergo circadian time-dependent oscillations in accordance with daily activity/rest cycles. The circadian regulatory mechanisms underlying these cyclic processes, especially at the post-transcriptional level, are not well defined. Previously, we reported that the circadian E3 ligase FBXL21 mediates rhythmic degradation of the sarcomere protein TCAP in conjunction with GSK-3ß, and Psttm mice harboring an Fbxl21 hypomorph allele show reduced muscle fiber diameter and impaired muscle function. To further elucidate the regulatory function of FBXL21 in skeletal muscle, we investigated another sarcomere protein, Myozenin1 (MYOZ1), that we identified as an FBXL21-binding protein from yeast 2-hybrid screening. We show that FBXL21 binding to MYOZ1 led to ubiquitination-mediated proteasomal degradation. GSK-3ß co-expression and inhibition were found to accelerate and decelerate FBXL21-mediated MYOZ1 degradation, respectively. Previously, MYOZ1 has been shown to inhibit calcineurin/NFAT signaling important for muscle differentiation. In accordance, Fbxl21 KO and MyoZ1 KO in C2C12 cells impaired and enhanced myogenic differentiation respectively compared with control C2C12 cells, concomitant with distinct effects on NFAT nuclear localization and NFAT target gene expression. Importantly, in Psttm mice, both the levels and diurnal rhythm of NFAT2 nuclear localization were significantly diminished relative to wild-type mice, and circadian expression of NFAT target genes associated with muscle differentiation was also markedly dampened. Furthermore, Psttm mice exhibited significant disruption of sarcomere structure with a considerable excess of MYOZ1 accumulation in the Z-line. Taken together, our study illustrates a pivotal role of FBXL21 in sarcomere structure and muscle differentiation by regulating MYOZ1 degradation and NFAT2 signaling.

Circadian stabilization loop: the regulatory hub and therapeutic target promoting circadian resilience and physiological health.

The circadian clock is a fundamental biological mechanism that orchestrates essential cellular and physiological processes to optimize fitness and health. The basic functional unit is the cell-autonomous oscillator, consisting of intersecting negative feedback loops. Whereas the core loop is primarily responsible for rhythm generation, auxiliary loops, most notably the secondary or stabilization loop, play pivotal roles to confer temporal precision and molecular robustness. The stabilization loop contains opposing nuclear receptor subfamilies REV-ERBs and retinoic acid receptor-related orphan receptors (RORs), competing to modulate rhythmic expression of the basic helix-loop-helix ARNT like 1 ( Bmal1) genes in the core loop as well as other clock-controlled genes. Therefore, REV-ERBs and RORs are strategically located to interface the oscillator and the global transcriptomic network, promoting cellular homeostasis and physiological fitness throughout lifespan. Disruption of REV-ERB and ROR functions has been linked with diseases and aging, and pharmacological manipulation of these factors has shown promise in various mouse disease models. Nobiletin is a natural compound that directly binds to and activates RORα/γ, modulating circadian rhythms, and shows robust in vivo efficacies to combat clock-associated pathophysiologies and age-related decline. Results from several studies demonstrate an inverse relation between nobiletin efficacy and clock functional state, where nobiletin elicits little effect in young and healthy mice with growing efficacy as the clock is perturbed by environmental and genetic challenges. This mode of action is consistent with the function of the stabilization loop to promote circadian and physiological resilience. Future studies should further investigate the function and mechanism of REV-ERBs and RORs, and test strategies targeting these factors against disease and aging.

Hepatic circadian and differentiation factors control liver susceptibility for fatty liver disease and tumorigenesis.

Hepatocellular carcinoma (HCC) is a leading cause of cancer deaths, and the most common primary liver malignancy to present in the clinic. With the exception of liver transplant, treatment options for advanced HCC are limited, but improved tumor stratification could open the door to new treatment options. Previously, we demonstrated that the circadian regulator Aryl Hydrocarbon-Like Receptor Like 1 (ARNTL, or Bmal1) and the liver-enriched nuclear factor 4 alpha (HNF4α) are robustly co-expressed in healthy liver but incompatible in the context of HCC. Faulty circadian expression of HNF4α- either by isoform switching, or loss of expression- results in an increased risk for HCC, while BMAL1 gain-of-function in HNF4α-positive HCC results in apoptosis and tumor regression. We hypothesize that the transcriptional programs of HNF4α and BMAL1 are antagonistic in liver disease and HCC. Here, we study this antagonism by generating a mouse model with inducible loss of hepatic HNF4α and BMAL1 expression. The results reveal that simultaneous loss of HNF4α and BMAL1 is protective against fatty liver and HCC in carcinogen-induced liver injury and in the "STAM" model of liver disease. Furthermore, our results suggest that targeting Bmal1 expression in the absence of HNF4α inhibits HCC growth and progression. Specifically, pharmacological suppression of Bmal1 in HNF4α-deficient, BMAL1-positive HCC with REV-ERB agonist SR9009 impairs tumor cell proliferation and migration in a REV-ERB-dependent manner, while having no effect on healthy hepatocytes. Collectively, our results suggest that stratification of HCC based on HNF4α and BMAL1 expression may provide a new perspective on HCC properties and potential targeted therapeutics.

Comparison of radiation exposure to physicians between anteroposterior and lateral real-time fluoroscopy when performing lumbar transforaminal epidural steroid injections: A randomized controlled trial.

BACKGROUND: Lumbar transforaminal epidural steroid injections are used widely to alleviate low back radicular pain, but it requires real-time fluoroscopy, which can increase the risk of radiation exposure. Anteroposterior or lateral real-time fluoroscopy can be used during lumbar transforaminal epidural steroid injections, but there have been no comparative studies on the exposure of physicians to radiation from anteroposterior or lateral real-time fluoroscopy. The aim of this study was to compare the cumulative radiation exposure to each body part of the physician according to the method of real-time fluoroscopy when performing lumbar transforaminal epidural steroid injections. METHODS: A single physician performed lumbar transforaminal epidural steroid injections, and 2 groups of patients were formed based on the method used: group A (anteroposterior real-time fluoroscopy) and group L (lateral real-time fluoroscopy). Dosimeters were placed outside the chest, inside the chest, outside the thyroid collar, inside the thyroid collar, outside the groin, inside the groin, outside the lead gloves, and left rim of the glasses. RESULTS: A total of 200 lumbar transforaminal epidural steroid injections were analyzed, and the radiation exposure was measured by cumulative dose equivalents in mSv. The dose equivalents were lower at every level in group A compared with group L except for outside the groin. CONCLUSIONS: The cumulative radiation exposure at all the measurement sites was lower for anteroposterior real-time fluoroscopy compared with lateral real-time fluoroscopy when performing lumbar transforaminal epidural steroid injections, except for outside the groin.

ROR activation by Nobiletin enhances antitumor efficacy via suppression of IκB/NF-κB signaling in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease characterized by poor response to standard therapies and therefore unfavorable clinical outcomes. Better understanding of TNBC and new therapeutic strategies are urgently needed. ROR nuclear receptors are multifunctional transcription factors with important roles in circadian pathways and other processes including immunity and tumorigenesis. Nobiletin (NOB) is a natural compound known to display anticancer effects, and our previous studies showed that NOB activates RORs to enhance circadian rhythms and promote physiological fitness in mice. Here, we identified several TNBC cell lines being sensitive to NOB, by itself or in combination. Cell and xenograft experiments showed that NOB significantly inhibited TNBC cell proliferation and motility in vitro and in vivo. ROR loss- and gain-of-function studies showed concordant effects of the NOB-ROR axis on MDA-MB-231 cell growth. Mechanistically, we found that NOB activates ROR binding to the ROR response elements (RRE) of the IκBα promoter, and NOB strongly inhibited p65 nuclear translocation. Consistent with transcriptomic analysis indicating cancer and NF-κB signaling as major pathways altered by NOB, p65-inducible expression abolished NOB effects, illustrating a requisite role of NF-κB suppression mediating the anti-TNBC effect of NOB. Finally, in vivo mouse xenograft studies showed that NOB enhanced the antitumor efficacy in mammary fat pad implanted TNBC, as a single agent or in combination with the chemotherapy agent Docetaxel. Together, our study highlights an anti-TNBC mechanism of ROR-NOB via suppression of NF-κB signaling, suggesting novel preventive and chemotherapeutic strategies against this devastating disease.

The clock modulator Nobiletin mitigates astrogliosis-associated neuroinflammation and disease hallmarks in an Alzheimer's disease model.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder, and there is a pressing need to identify disease-modifying factors and devise interventional strategies. The circadian clock, our intrinsic biological timer, orchestrates various cellular and physiological processes including gene expression, sleep, and neuroinflammation; conversely, circadian dysfunctions are closely associated with and/or contribute to AD hallmarks. We previously reported that the natural compound Nobiletin (NOB) is a clock-enhancing modulator that promotes physiological health and healthy aging. In the current study, we treated the double transgenic AD model mice, APP/PS1, with NOB-containing diets. NOB significantly alleviated ß-amyloid burden in both the hippocampus and the cortex, and exhibited a trend to improve cognitive function in these mice. While several systemic parameters for circadian wheel-running activity, sleep, and metabolism were unchanged, NOB treatment showed a marked effect on the expression of clock and clock-controlled AD gene expression in the cortex. In accordance, cortical proteomic profiling demonstrated circadian time-dependent restoration of the protein landscape in APP/PS1 mice treated with NOB. More importantly, we found a potent efficacy of NOB to inhibit proinflammatory cytokine gene expression and inflammasome formation in the cortex, and immunostaining further revealed a specific effect to diminish astrogliosis, but not microgliosis, by NOB in APP/PS1 mice. Together, these results underscore beneficial effects of a clock modulator to mitigate pathological and cognitive hallmarks of AD, and suggest a possible mechanism via suppressing astrogliosis-associated neuroinflammation.

Effects of the Clock Modulator Nobiletin on Circadian Rhythms and Pathophysiology in Female Mice of an Alzheimer's Disease Model.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder and the most common cause of dementia. Various pathogenic mechanisms have been proposed to contribute to disease progression, and recent research provided evidence linking dysregulated circadian rhythms/sleep and energy metabolism with AD. Previously, we found that the natural compound Nobiletin (NOB) can directly activate circadian cellular oscillators to promote metabolic health in disease models and healthy aging in naturally aged mice. In the current study, using the amyloid-ß AD model APP/PS1, we investigated circadian, metabolic and amyloid characteristics of female mice and the effects of NOB. Female APP/PS1 mice showed reduced sleep bout duration, and NOB treatment exhibited a trend to improve it. While glucose tolerance was unchanged, female APP/PS1 mice displayed exaggerated oxygen consumption and CO2 production, which was mitigated by NOB. Likewise, cold tolerance in APP/PS1 was impaired relative to WT, and interestingly was markedly enhanced in NOB-treated APP/PS1 mice. Although circadian behavioral rhythms were largely unchanged, real-time qPCR analysis revealed altered expression of several core clock genes by NOB in the cerebral cortex, notably Bmal1, Npas2, and Rora. Moreover, NOB was also able to activate various clock-controlled metabolic genes involved in insulin signaling and mitochondrial function, including Igf1, Glut1, Insr, Irs1, Ucp2, and Ucp4. Finally, we observed that NOB attenuated the expression of several AD related genes including App, Bace1, and ApoE, reduced APP protein levels, and strongly ameliorated Aß pathology in the cortex. Collectively, these results reveal novel genotype differences and importantly beneficial effects of a natural clock-enhancing compound in biological rhythms and related pathophysiology, suggesting the circadian clock as a modifiable target for AD.

Clock-Modulating Activities of the Anti-Arrhythmic Drug Moricizine.

Dysregulated circadian functions contribute to various diseases, including cardiovascular disease. Much progress has been made on chronotherapeutic applications of drugs against cardiovascular disease (CVD); however, the direct effects of various medications on the circadian system are not well characterized. We previously conducted high-throughput chemical screening for clock modulators and identified an off-patent anti-arrhythmic drug, moricizine, as a clock-period lengthening compound. In Per2:LucSV reporter fibroblast cells, we showed that under both dexamethasone and forskolin synchronization, moricizine was able to increase the circadian period length, with greater effects seen with the former. Titration studies revealed a dose-dependent effect of moricizine to lengthen the period. In contrast, flecainide, another Class I anti-arrhythmic, showed no effects on circadian reporter rhythms. Real-time qPCR analysis in fibroblast cells treated with moricizine revealed significant circadian time- and/or treatment-dependent expression changes in core clock genes, consistent with the above period-lengthening effects. Several clock-controlled cardiac channel genes also displayed altered expression patterns. Using tissue explant culture, we showed that moricizine was able to significantly prolong the period length of circadian reporter rhythms in atrial ex vivo cultures. Using wild-type C57BL/6J mice, moricizine treatment was found to promote sleep, alter circadian gene expression in the heart, and show a slight trend of increasing free-running periods. Together, these observations demonstrate novel clock-modulating activities of moricizine, particularly the period-lengthening effects on cellular oscillators, which may have clinical relevance against heart diseases.