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Circadian clocks, biochemical oscillators that are regulated by environmental time cues including the day/night cycle, have a central function in the majority of biological processes. The disruption of the circadian clock can alter breast biology negatively and may promote the development of breast tumors. The expression status of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) were used to classify breast cancer into different molecular subtypes such as triple-negative breast cancer (TNBC). Receptor status-dependent expression of circadian clock genes have been previously studied in breast cancer using relatively small sample sizes in a particular population. Here, using TCGA-BRCA data (n=1119), we found that the expressions of CRY1, PER1, PER2, PER3, BMAL1, CLOCK, RORA, RORB, RORC, NR1D1, NR1D2, and FBXL3 were higher in ER+ breast cancer cells compared with those of ER− status. Similarly, we showed that transcript levels of CRY2, PER1, PER2, PER3, BMAL1, RORA, RORB, RORC, NR1D1, NR1D2, and FBXL3 were higher in PR+ breast cancer cells than in PR− breast cancer cells. We report that the expressions of CRY2, PER1, BMAL1, and RORA were lower, and the expression of NR1D1 was higher, in HER2+ breast cancer cells compared with HER2− breast cancer cells. Moreover, we studied these receptor status-dependent changes in the expressions of circadian clock genes also based on the race and age of breast cancer patients. Lastly, we found that the expressions of CRY2, PER1, PER2, PER3, and CLOCK were higher in non-TNBC than in TNBC, which has the worst prognosis among subtypes. We note that our findings are not always parallel to the observations reported in previous studies with smaller sample sizes performed in different populations and organisms. Our study suggests that receptor status in breast cancer (thus, subtype of breast cancer) might be more important than previously shown in terms of its influence on the expression of circadian clock genes and on the disruption of the circadian clock, and that ER or PR might be important regulators of breast cancer chronobiology that should be taken into account in personalized chronotherapies. [ABSTRACT FROM AUTHOR]

The interplay between circadian rhythms and epilepsy has gained increasing attention. The suprachiasmatic nucleus (SCN), which acts as the master circadian pacemaker, regulates physiological and behavioral rhythms through its complex neural networks. However, the exact role of the SCN and its Bmal1 gene in the development of epilepsy remains unclear. In this study, we utilized a lithium–pilocarpine model to induce epilepsy in mice and simulated circadian disturbances by creating lesions in the SCN and specifically knocking out the Bmal1 gene in the SCN neurons. We observed that the pilocarpine‐induced epileptic mice experienced increased daytime seizure frequency, irregular oscillations in core body temperature, and circadian gene alterations in both the SCN and the hippocampus. Additionally, there was enhanced activation of GABAergic projections from the SCN to the hippocampus. Notably, SCN lesions intensified seizure activity, concomitant with hippocampal neuronal damage and GABAergic signaling impairment. Further analyses using the Gene Expression Omnibus database and gene set enrichment analysis indicated reduced Bmal1 expression in patients with medial temporal lobe epilepsy, potentially affecting GABA receptor pathways. Targeted deletion of Bmal1 in SCN neurons exacerbated seizures and pathology in epilepsy, as well as diminished hippocampal GABAergic efficacy. These results underscore the crucial role of the SCN in modulating circadian rhythms and GABAergic function in the hippocampus, aggravating the severity of seizures. This study provides significant insights into how circadian rhythm disturbances can influence neuronal dysfunction and epilepsy, highlighting the therapeutic potential of targeting SCN and the Bmal1 gene within it in epilepsy management. [ABSTRACT FROM AUTHOR]

Physiological processes within the human body are regulated in approximately 24‐h cycles known as circadian rhythms, serving to adapt to environmental changes. Bone rhythms play pivotal roles in bone development, metabolism, mineralization, and remodeling processes. Bone rhythms exhibit cell specificity, and different cells in bone display various expressions of clock genes. Multiple environmental factors, including light, feeding, exercise, and temperature, affect bone diurnal rhythms through the sympathetic nervous system and various hormones. Disruptions in bone diurnal rhythms contribute to the onset of skeletal disorders such as osteoporosis, osteoarthritis and skeletal hypoplasia. Conversely, these bone diseases can be effectively treated when aimed at the circadian clock in bone cells, including the rhythmic expressions of clock genes and drug targets. In this review, we describe the unique circadian rhythms in physiological activities of various bone cells. Then we summarize the factors synchronizing the diurnal rhythms of bone with the underlying mechanisms. Based on the review, we aim to build an overall understanding of the diurnal rhythms in bone and summarize the new preventive and therapeutic strategies for bone disorders. [ABSTRACT FROM AUTHOR]

The hypothalamic–pituitary–gonadal axis (HPG) is the key neuroendocrine axis involved in reproductive regulation. Brain and muscle ARNT‐like protein 1 (Bmal1) participates in regulating the metabolism of various endocrine hormones. However, the regulation of Bmal1 on HPG and female fertility is unclear. This study aims to explore the regulation of female reproduction by Bmal1 via the HPG axis in mice. Bmal1‐knockout (Ko) mice were generated using the CRISPR/Cas9 technology. The structure, function, and estrous cycle of ovarian in Bmal1 Ko female mice were measured. The key genes and proteins of the HPG axis involved in regulating female reproduction were examined through transcriptome analysis and then verified by RT‐PCR, immunohistochemistry, and western blot. Furthermore, the fertility of female mice was detected after intervening prolactin (PRL) and progesterone (Pg) in Bmal1 ko mice. The number of offspring and ovarian weight were significantly lower in Bmal1‐Ko mice than in wild‐type (Wt) mice. In Bmal1‐Ko mice, ovarian cells were arranged loosely and irregularly, and the total number of follicles was significantly reduced. No corpus luteum was found in the ovaries. Vaginal smears revealed that Bmal1‐Ko mice had an irregular estrus cycle. In Bmal1‐Ko mice, Star expression was decreased, PRL and luteinizing hormone (LH) levels were increased, and dopamine (DA) and Pg levels were decreased. Inhibition of PRL partially recovered the estrous cycle, corpus luteum formation, and Star expression in the ovaries. Pg supplementation promoted embryo implantation in Bmal1‐Ko female mice. Bmal1 Ko increases serum PRL levels in female mice likely by reducing DA levels, thus affecting luteal formation, resulting in decreased Star expression and Pg production, hindering female reproduction. Inhibition of PRL or restoration of Pg can partially restore reproductive capacity in female Bmal1‐Ko mice. Thus, Bmal1 may regulate female reproduction via the HPG axis in mice, suggesting that Bmal1 is a potential target to treat female infertility. [ABSTRACT FROM AUTHOR]

The master circadian clock, located in the suprachiasmatic nuclei (SCN), organizes the daily rhythm in minute ventilation (V ̇ E). However, the extent that the daily rhythm in V ̇ E is secondary to SCN-imposed O2 and CO2 cycles (i.e., metabolic rate) or driven by other clock mechanisms remains unknown. Here, we experimentally shifted metabolic rate using time-restricted feeding (without affecting light-induced synchronization of the SCN) to determine the influence of metabolic rate in orchestrating the daily V ̇ E rhythm. Mice eating predominantly at night exhibited robust daily rhythms in O2 consumption (V ̇ O2), CO2 production (V ̇ CO2), and V ̇ E with similar peak times (approximately ZT18) that were consistent with SCN organization. However, feeding mice exclusively during the day separated the relative timing of metabolic and ventilatory rhythms, resulting in an approximately 8.5-h advance in V ̇ CO2 and a disruption of the V ̇ E rhythm, suggesting opposing circadian and metabolic influences on V ̇ E. To determine if the molecular clock of cells involved in the neural control of breathing contributes to the daily V_ E rhythm, we examined V ̇ E in mice lacking BMAL1 in Phox2b-expressing respiratory cells (i.e., BKOP mice). The ventilatory and metabolic rhythms of predominantly night-fed BKOP mice did not differ from wild-type mice. However, in contrast to wild-type mice, exclusive day feeding of BKOP mice led to an unfettered daily V̇ E rhythm with a peak time aligning closely with the daily V ̇ CO2 rhythm. Taken together, these results indicate that both daily V ̇ CO2 changes and intrinsic circadian time-keeping within Phox2b respiratory cells are predominant orchestrators of the daily rhythm in ventilation. [ABSTRACT FROM AUTHOR]

Supplemental O2 (hyperoxia) is a critical intervention for premature infants (<34 weeks) but consequently is associated with development of bronchial airway hyperreactivity (AHR) and asthma. Clinical practice shifted toward the use of moderate hyperoxia (<60% O2), but risk for subsequent airway disease remains. In mouse models of moderate hyperoxia, neonatal mice have increased AHR with effects on airway smooth muscle (ASM), a cell type involved in airway tone, bronchodilation, and remodeling. Understanding mechanisms by which moderate O2 during the perinatal period initiates sustained airway changes is critical to drive therapeutic advancements toward treating airway diseases. We propose that cellular clock factor BMAL1 is functionally important in developing mouse airways. In adult mice, cellular clocks target pathways highly relevant to asthma pathophysiology and Bmal1 deletion increases inflammatory response, worsens lung function, and impacts survival outcomes. Our understanding of BMAL1 in the developing lung is limited, but our previous findings show functional relevance of clocks in human fetal ASM exposed to O2. Here, we characterize Bmal1 in our established mouse neonatal hyperoxia model. Our data show that Bmal1 KO deleteriously impacts the developing lung in the context of O2 and these data highlight the importance of neonatal sex in understanding airway disease. [ABSTRACT FROM AUTHOR]

Ting Liu,1,2 Luxu Wang,1– 3 Tuo Shi,4 Hongrui Liu,1,2 Bo Liu,5 Jie Guo,1,2 Minqi Li1,2,5 1Department of Bone Metabolism, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Research Center of Dental Materials and Oral Tissue Regeneration & Shandong Provincial Clinical Research Center for Oral Diseases, Jinan, People’s Republic of China; 2Center of Osteoporosis and Bone Mineral Research, Shandong University, Jinan, People’s Republic of China; 3School of Stomatology, Jinzhou Medical University, Jinzhou, People’s Republic of China; 4School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, People’s Republic of China; 5School of Clinical Medicine, Jining Medical University, Jining, People’s Republic of ChinaCorrespondence: Bo Liu, School of Clinical Medicine, Jining Medical University, Jining, 272067, People’s Republic of China, Tel +86-0537-6051782, Email liubo7230@mail.jnmc.edu.cn Jie Guo, Department of Bone Metabolism, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Research Center of Dental Materials and Oral Tissue Regeneration & Shandong Provincial Clinical Research Center for Oral Diseases, Jinan, Shandong, 250012, People’s Republic of China, Tel +86-0531-88382923, Email kqgj@sdu.edu.cnPurpose: Bone loss is a common complication of type 2 diabetes mellitus (T2DM). Circadian rhythms play a significant role in T2DM and bone remodeling. Eldecalcitol (ED-71), a novel active vitamin D analog, has shown promise in ameliorating T2DM. We aimed to investigate whether the circadian rhythm coregulator BMAL1 mediates the anti-osteoporotic effect of ED-71 in T2DM and its associated mechanisms.Methods: A T2DM mouse model was established using high-fat diet (HDF) and streptozotocin (STZ) injection, and blood glucose levels were monitored weekly. HE staining, Masson staining, and Micro-CT were performed to assess the changes in bone mass. IHC staining and IF staining were used to detect osteoblast status and BMAL1 expression and RT-qPCR was applied to detect the change of oxidative stress factors. In vitro, high glucose (HG) stimulation was used to simulate the cell environment in T2DM. RT-qPCR, Western blot, IF, ALP staining and AR staining were used to detect osteogenic differentiation and SIRT1/GSK3β signaling pathway. DCFH-DA staining was used to detect reactive oxygen species (ROS) levels.Results: ED-71 increased bone mass and promoted osteogenesis in T2DM mice. Moreover, ED-71 inhibited oxidative stress and promoted BMAL1 expression in osteoblasts The addition of STL1267, an agonist of the BMAL1 transcriptional repressor protein REV-ERB, reversed the inhibitory effect of ED-71 on oxidative stress and the promotional effect on osteogenic differentiation. In addition, ED-71 facilitated SIRT1 expression and reduced GSK3β activity. The inhibition of SIRT1 with EX527 partially attenuated ED-71’s effects, whereas the GSK3β inhibitor LiCl further enhanced ED-71’s positive effects on BMAL1 expression.Conclusion: ED-71 ameliorates bone loss in T2DM by upregulating the circadian rhythm coregulator BMAL1 and promoting osteogenesis through inhibition of oxidative stress. The SIRT1/GSK3β signaling pathway is involved in the regulation of BMAL1.Keywords: eldecalcitol, type 2 diabetic mellitus, osteoblast, BMAL1, SIRT1/GSK3β signaling pathway

Background and purpose: Drug disposition undergoes significant alteration in patients with inflammatory bowel disease (IBD), yet circadian time‐dependency of these changes remains largely unexplored. In this study, we aimed to determine the temporal effects of experimental colitis on drug disposition and toxicity. Experimental Approach: RNA‐sequencing was used to screen genes relevant to colitis induced by dextran sodium sulfate in mice. Liver microsomes and pharmacokinetic analysis were used to analyze the activity of key enzymes. Dual luciferase assays and chromatin immunoprecipitation (ChIP) were employed to elucidate regulatory mechanisms. Key Results: RNA sequencing analysis revealed that colitis markedly influenced expression of cytochrome P450 (CYP) enzymes. Specifically, a substantial down‐regulation of CYP1A2 and CYP2E1 was observed in livers of mice with colitis at Zeitgeber Time 8 (ZT8), with no significant changes detected at ZT20. At ZT8, the altered expression corresponded to diminished metabolism and enhanced incidence of hepato‐cardiac toxicity of theophylline, a substrate specifically metabolized by these enzymes. A combination of assays, integrating liver‐specific Bmal1 knockout and targeted activation of BMAL1 showed that dysregulation in CYP1A2 and CYP2E1 during colitis was attributable to perturbed BMAL1 functionality. Luciferase reporter and ChIP assays collectively substantiated the role of BMAL1 in regulating Cyp1a2 and Cyp2e1 transcription through its binding affinity to E‐box‐like sites. Conclusion and implication: Our findings establish a strong link between colitis and chronopharmacology, shedding light on how IBD affects drug disposition and toxicity over time. This research provides a theoretical foundation for optimizing drug dosage in patients with IBD. [ABSTRACT FROM AUTHOR]

Abstract Background Zinc finger SWIM-type containing 4 (ZSWIM4) is a zinc finger protein with its function largely uncharacterized. In this study, we aimed to investigate the role of ZSWIM4 in gastrointestinal stromal tumors (GISTs). Results We found that ZSWIM4 expression is inhibited by the predominantly mutated protein KIT in GISTs, while conversely, ZSWIM4 inhibits KIT expression and downstream signaling. Consistent with the observation, ZSWIM4 inhibited GIST cell survival and proliferation in vitro. RNA sequencing of GISTs from KITV558A/WT mice and KITV558A/WT/ZSWIM4−/− mice showed that loss of ZSWIM4 expression increases the expression of circadian clock pathway member BMAL1 which contributes to GIST cell survival and proliferation. In addition, we found that KIT signaling increases the distribution of ZSWIM4 in the nucleus of GIST cells, and which is important for its inhibition of KIT and BMAL1. In agreement with the results in vitro, the in vivo studies showed that ZSWIM4 deficiency increases the tumorigenesis of GISTs in KITV558A/WT mice. Conclusions Taken together, our results revealed that the entry of ZSWIM4 to the nucleus is important for its inhibition of KIT and BMAL1, ultimately attenuating GIST tumorigenesis. The results provide a novel insight in the understanding of signal transduction in GISTs and lay strong theoretical basis for the advancement of GIST treatment.

Abstract Background Sleep deprivation (SD) is a common public health problem that contributes to various physiological disorders and increases the risk of ocular diseases. However, whether sleep loss can damage corneal endothelial function remains unclear. This study aimed to determine the effect and possible mechanism of SD on the corneal endothelium. Methods Male C57BL/6J mice were subjected to establish SD models. After 10 days, quantitative RT-PCR (qRT-PCR) and western blot or immunostaining for the expression levels of zonula occludens-1 (ZO-1), ATPase Na+/K + transporting subunit alpha 1 (Atp1a1), and core clock genes in the corneal endothelium were evaluated. Reactive oxygen species staining and mitochondrial abundance characterized the mitochondrial function. The regulatory role of Bmal1 was confirmed by specifically knocking down or overexpressing basic helix-loop-helix ARNT like 1 protein (Bmal1) in vivo. In vitro, a mitochondrial stress test was conducted on cultured human corneal endothelial cells upon Bmal1 knockdown. Results SD damaged the barrier and pump functions of mouse corneal endothelium, accompanied by mitochondrial dysfunction. Interestingly, SD dramatically downregulated the core clock gene Bmal1 expression level. Bmal1 knockdown disrupted corneal endothelial function, while overexpression of Bmal1 ameliorated the dysfunction induced by SD. Mitochondrial bioenergetic deficiency mediated by Bmal1 was an underlying mechanism for SD induced corneal endothelial dysfunction. Conclusion The downregulation of Bmal1 expression caused by SD led to corneal endothelial dysfunction via impairing mitochondrial bioenergetics. Our findings offered insight into how SD impairs the physiological function of the corneal endothelium and expanded the understanding of sleep loss leading to ocular diseases.

Background: Zinc finger SWIM-type containing 4 (ZSWIM4) is a zinc finger protein with its function largely uncharacterized. In this study, we aimed to investigate the role of ZSWIM4 in gastrointestinal stromal tumors (GISTs). Results: We found that ZSWIM4 expression is inhibited by the predominantly mutated protein KIT in GISTs, while conversely, ZSWIM4 inhibits KIT expression and downstream signaling. Consistent with the observation, ZSWIM4 inhibited GIST cell survival and proliferation in vitro. RNA sequencing of GISTs from KITV558A/WT mice and KITV558A/WT/ZSWIM4−/− mice showed that loss of ZSWIM4 expression increases the expression of circadian clock pathway member BMAL1 which contributes to GIST cell survival and proliferation. In addition, we found that KIT signaling increases the distribution of ZSWIM4 in the nucleus of GIST cells, and which is important for its inhibition of KIT and BMAL1. In agreement with the results in vitro, the in vivo studies showed that ZSWIM4 deficiency increases the tumorigenesis of GISTs in KITV558A/WT mice. Conclusions: Taken together, our results revealed that the entry of ZSWIM4 to the nucleus is important for its inhibition of KIT and BMAL1, ultimately attenuating GIST tumorigenesis. The results provide a novel insight in the understanding of signal transduction in GISTs and lay strong theoretical basis for the advancement of GIST treatment. [ABSTRACT FROM AUTHOR]

Background: Circadian rhythm disruption (CRD) is thought to increase the risk of inflammatory bowel disease. The deletion of Bmal1, a core transcription factor, leads to a complete loss of the circadian rhythm and exacerbates the severity of dextran sodium sulfate (DSS)-induced colitis in mice. However, the underlying mechanisms by which CRD and Bmal1 mediate IBD are still unclear. Methods: We used a CRD mouse model, a mouse colitis model, and an in vitro model of colonic epithelial cell monolayers. We also knocked down and overexpressed Bmal1 in Caco-2 cells by transfecting lentivirus in vitro. The collected colon tissue and treated cells were assessed and analyzed using immunohistochemistry, immunofluorescence staining, quantitative reverse transcription-polymerase chain reaction, western blot, flow cytometry, transmission electron microscopy, and terminal deoxynucleotidyl transferasemediated dUTP nick-end labelling staining. Results: We found that CRD mice with downregulated Bmal1 expression were more sensitive to DSS-induced colitis and had more severely impaired intestinal barrier function than wild-type mice. Bmal1-/- mice exhibited more severe colitis, accompanied by decreased tight junction protein levels and increased apoptosis of intestinal epithelial cells compared with wild-type mice, which were alleviated by using the autophagy agonist rapamycin. Bmal1 overexpression attenuated Lipopolysaccharide-induced apoptosis of intestinal epithelial cells and impaired intestinal epithelial cells barrier function in vitro, while inhibition of autophagy reversed this protective effect. Conclusion: This study suggests that CRD leads to the downregulation of Bmal1 expression in the colon, which may exacerbate DSS-induced colitis in mice, and that Bmal1 may serve as a novel target for treating inflammatory bowel disease. [ABSTRACT FROM AUTHOR]

Background: Sleep deprivation (SD) is a common public health problem that contributes to various physiological disorders and increases the risk of ocular diseases. However, whether sleep loss can damage corneal endothelial function remains unclear. This study aimed to determine the effect and possible mechanism of SD on the corneal endothelium. Methods: Male C57BL/6J mice were subjected to establish SD models. After 10 days, quantitative RT-PCR (qRT-PCR) and western blot or immunostaining for the expression levels of zonula occludens-1 (ZO-1), ATPase Na+/K + transporting subunit alpha 1 (Atp1a1), and core clock genes in the corneal endothelium were evaluated. Reactive oxygen species staining and mitochondrial abundance characterized the mitochondrial function. The regulatory role of Bmal1 was confirmed by specifically knocking down or overexpressing basic helix-loop-helix ARNT like 1 protein (Bmal1) in vivo. In vitro, a mitochondrial stress test was conducted on cultured human corneal endothelial cells upon Bmal1 knockdown. Results: SD damaged the barrier and pump functions of mouse corneal endothelium, accompanied by mitochondrial dysfunction. Interestingly, SD dramatically downregulated the core clock gene Bmal1 expression level. Bmal1 knockdown disrupted corneal endothelial function, while overexpression of Bmal1 ameliorated the dysfunction induced by SD. Mitochondrial bioenergetic deficiency mediated by Bmal1 was an underlying mechanism for SD induced corneal endothelial dysfunction. Conclusion: The downregulation of Bmal1 expression caused by SD led to corneal endothelial dysfunction via impairing mitochondrial bioenergetics. Our findings offered insight into how SD impairs the physiological function of the corneal endothelium and expanded the understanding of sleep loss leading to ocular diseases. [ABSTRACT FROM AUTHOR]

The disruption of circadian rhythms (CRs) has been linked to metabolic disorders, yet the role of hepatic BMAL1, a key circadian regulator, in the whole-body metabolism and the associated lipid metabolic phenotype in the liver remains unclear. Bmal1 floxed (Bmal1f/f) and hepatocyte-specific Bmal1 knockout (Bmal1hep−/−) C57BL/6J mice underwent a regular feeding regimen. Hepatic CR, lipid content, mitochondrial function, and systemic metabolism were assessed at zeitgeber time (ZT) 0 and ZT12. Relevant molecules were examined to elucidate the metabolic phenotype. Hepatocyte-specific knockout of Bmal1 disrupted the expression of rhythmic genes in the liver. Bmal1hep−/− mice exhibited decreased hepatic TG content at ZT0, primarily due to enhanced lipolysis, reduced lipogenesis, and diminished lipid uptake. The β-oxidation function of liver mitochondria decreased at both ZT0 and ZT12. Our findings on the metabolic profile and associated hepatic lipid metabolism in the absence of Bmal1 in hepatocytes provides new insights into metabolic syndromes from the perspective of liver CR disturbances. [ABSTRACT FROM AUTHOR]

Adult bones are continuously remodeled by the balance between bone resorption by osteoclasts and subsequent bone formation by osteoblasts. Many studies have provided molecular evidence that bone remodeling is under the control of circadian rhythms. Circadian fluctuations have been reported in the serum and urine levels of bone turnover markers, such as digested collagen fragments and bone alkaline phosphatase. Additionally, the expressions of over a quarter of all transcripts in bones show circadian rhythmicity, including the genes encoding master transcription factors for osteoblastogenesis and osteoclastogenesis, osteogenic cytokines, and signaling pathway proteins. Serum levels of calcium, phosphate, parathyroid hormone, and calcitonin also display circadian rhythmicity. Finally, osteoblast- and osteoclast-specific knockout mice targeting the core circadian regulator gene Bmal1 show disrupted bone remodeling, although the results have not always been consistent. Despite these studies, however, establishing a direct link between circadian rhythms and bone remodeling in vivo remains a major challenge. It is nearly impossible to repeatedly collect bone materials from human subjects while following circadian changes. In addition, the differences in circadian gene regulation between diurnal humans and nocturnal mice, the main model organism, remain unclear. Filling the knowledge gap in the circadian regulation of bone remodeling could reveal novel regulatory mechanisms underlying many bone disorders including osteoporosis, genetic diseases, and fracture healing. This is also an important question for the basic understanding of how cell differentiation progresses under the influence of cyclically fluctuating environments. [ABSTRACT FROM AUTHOR]

Purpose: Acute myeloid leukemia (AML) is a refractory hematologic malignancy that poses a serious threat to human health. Exploring alternative therapeutic strategies capable of inducing alternative modes of cell death, such as ferroptosis, holds great promise as a viable and effective intervention. Methods: We analyzed online database data and collected clinical samples to verify the expression and function of BMAL1 in AML. We conducted experiments on AML cell proliferation, cell cycle, ferroptosis, and chemotherapy resistance by overexpressing/knocking down BMAL1 and using assays such as MDA detection and BODIPY 581/591 C11 staining. We validated the transcriptional regulation of HMGB1 by BMAL1 through ChIP assay, luciferase assay, RNA level detection, and western blotting. Finally, we confirmed the results of our cell experiments at the animal level. Results: BMAL1 up-regulation is an observed phenomenon in AML patients. Furthermore, there existed a strong correlation between elevated levels of BMAL1 expression and inferior prognosis in individuals with AML. We found that knocking down BMAL1 inhibited AML cell growth by blocking the cell cycle. Conversely, overexpressing BMAL1 promoted AML cell proliferation. Moreover, our research results revealed that BMAL1 inhibited ferroptosis in AML cells through BMAL1-HMGB1-GPX4 pathway. Finally, knocking down BMAL1 can enhance the efficacy of certain first-line cancer therapeutic drugs, including venetoclax, dasatinib, and sorafenib. Conclusion: Our research results suggest that BMAL1 plays a crucial regulatory role in AML cell proliferation, drug resistance, and ferroptosis. BMAL1 could be a potential important therapeutic target for AML. [ABSTRACT FROM AUTHOR]

Obesity and overweight are common and complex conditions influenced by multiple genetic and environmental factors. Several genetic variants located in the genes involved in clock systems and fat taste perception can affect metabolic health. In particular, the polymorphisms in CLOCK and BMAL1 genes were reported to be significantly related to cardiovascular disease, metabolic syndrome, sleep reduction, and evening preference. Moreover, genetic variants in the CD36 gene have been shown to be involved in lipid metabolism, regulation of fat intake, and body weight regulation. The aim of this study is to evaluate, for the first time, the association between variants in some candidate genes (namely, BMAL1 rs7950226 (G>A), CLOCK rs1801260 (A>G), CLOCK rs4864548 (G>A), CLOCK rs3736544 (G>A), CD36 rs1984112 (A>G), CD36 rs1761667 (G>A)) and overweight/obesity (OB) in pregnant women. A total of 163 normal-weight (NW) and 128 OB participants were included. A significant correlation was observed between A-allele in CLOCK rs4864548 and an increased risk of obesity (OR: 1.97; 95% CI 1.22–3.10, p = 0.005). In addition, we found that subjects carrying the haplotype of rs1801260-A, rs4864548-A, and rs3736544-G are likely to be overweight or obese (OR 1.47, 95% CI 1.03–2.09, p = 0.030), compared with those with other haplotypes. Moreover, a significant relation was observed between third-trimester lipid parameters and genetic variants—namely, CD36 rs1984112, CD36 rs1761667, BMAL1 rs7950226, and CLOCK rs1801260. A multivariate logistic regression model revealed that CLOCK rs4864548 A-allele carriage was a strong risk factor for obesity (OR 2.05, 95% CI 1.07–3.93, p = 0.029); on the other hand, greater adherence to Mediterranean diet (OR 0.80, 95% CI 0.65–0.98, p = 0.038) and higher HDL levels (OR 0.96, 95% CI 0.94–0.99, p = 0.021) were related to a reduced risk of obesity. Interestingly, an association between maternal CLOCK rs4864548 and neonatal birthweight was detected (p = 0.025). These data suggest a potential role of the polymorphisms in clock systems and in fat taste perception in both susceptibility to overweight/obesity and influencing the related metabolic traits in pregnant women. [ABSTRACT FROM AUTHOR]

Physiology is regulated by interconnected cell and tissue circadian clocks. Disruption of the rhythms generated by the concerted activity of these clocks is associated with metabolic disease. Here we tested the interactions between clocks in two critical components of organismal metabolism, liver and skeletal muscle, by rescuing clock function either in each organ separately or in both organs simultaneously in otherwise clock-less mice. Experiments showed that individual clocks are partially sufficient for tissue glucose metabolism, yet the connections between both tissue clocks coupled to daily feeding rhythms support systemic glucose tolerance. This synergy relies in part on local transcriptional control of the glucose machinery, feeding-responsive signals such as insulin, and metabolic cycles that connect the muscle and liver. We posit that spatiotemporal mechanisms of muscle and liver play an essential role in the maintenance of systemic glucose homeostasis and that disrupting this diurnal coordination can contribute to metabolic disease.

Brain Muscle ARNT-Like Protein 1 (BMAL1) suppresses oxidative stress in brain injury during surgery. Epigallocatechin-3-gallate (EGCG), a monomer in green tea, has been identified as an antioxidant and a potential agonist for BMAL1. In this work, the mechanism by which BMAL1 is regulated was investigated, as well as the therapeutic effect of EGCG on surgically injured rats. The pathological environment after brain injury during surgery was simulated by excising the right frontal lobe of rats. Rats received an intraperitoneal injection of EGCG immediately after surgery. Neurological scores and cerebral edema were recorded after surgery. Fluoro-Jade C staining, TUNEL staining, western blot, and lipid peroxidation analyses were conducted 3 days later. Here we show that the endogenous BMAL1 level decreased after brain injury. Postoperative administration of EGCG up-regulated the content of BMAL1 around the cerebral cortex, reduced the oxidative stress level, reduced neuronal apoptosis and the number of degenerated neurons, alleviated cerebral edema, and improved neurological scores in rats. This suggests that BMAL1 is an effective target for treating surgical brain injury, as well as that EGCG may be a promising agent for alleviating postoperative brain injury.

Maintaining proper circadian rhythms is essential for coordinating biological functions in mammals. This study investigates the effects of daily arrhythmicity using Bmal1-knockout (KO) mice as a model, aiming to understand behavioural and motivational implications. By employing a new mathematical analysis based on entropy divergence, we identified disrupted intricate activity patterns in mice derived by the complete absence of BMAL1 and quantified the difference regarding the activity oscillation’s complexity. Changes in locomotor activity coincided with disturbances in circadian gene expression patterns. Additionally, we found a dysregulated gene expression profile particularly in brain nuclei like the ventral striatum, impacting genes related to reward and motivation. Further investigation revealed that arrhythmic mice exhibited heightened motivation for food and water rewards, indicating a link between circadian disruptions and the reward system. This research sheds light on how circadian clock alterations impact the gene expression regulating the reward system and how this, in turn, can lead to altered seeking behaviour and motivation for natural rewards. In summary, the present study contributes to our understanding of how reward processing is under the regulation of circadian clock machinery.

Abstract Supplemental O2 (hyperoxia) is a critical intervention for premature infants (

Background and Aims: Sleep is predicted as a key modulator of cognition, but the underlying mechanisms are poorly understood. In this study, we investigated the effects of melatonin on chronic rapid eye movement sleep deprivation (CRSD)‐induced cognitive impairment and circadian dysfunction in rat models. Methods: Thirty‐six Sprague‐Dawley male rats were divided into three groups: CRSD with saline treatment, CRSD with chronic melatonin injection (20 mg/kg/day), and non‐sleep‐deprived control. The cognitive behavioral tests as well as the expression of clocks and HDAC3 were evaluated in all groups. Results: CRSD significantly reduced recognition index in novel object location, increased escape latency and distance traveling in Morris water maze while melatonin treatment attenuated CRSD‐induced hippocampal‐dependent spatial learning and memory deficits. Furthermore, the mRNAs of brain and muscle aryl hydrocarbon receptor nuclear translocator‐like 1(Bmal1) and circadian locomotor output cycles kaput (Clock) were globally down‐regulated by CRSD with constant intrinsic oscillation in both hippocampus and peripheral blood. The protein levels of hippocampal Bmal1, Clock, and HDAC3 were also remarkably down‐regulated following CRSD. Melatonin treatment reversed CRSD‐induced alterations of Bmal1/Clock and HDAC3 on both mRNA levels and protein levels. Conclusions: Our data indicate that melatonin treatment attenuates CRSD‐induced cognitive impairment via regulating HDAC3‐Bmal1/Clock interaction. These findings explore a broader understanding of the relationship between sleep and cognition and provide a potential new therapeutic target for cognitive impairment. [ABSTRACT FROM AUTHOR]

The circadian clock throughout the day organizes the activity of neural stem cells (NSCs) in the dentate gyrus (DG) of adult hippocampus temporally. However, it is still unclear whether and how circadian signals from the niches contribute to daily rhythmic variation of NSC activation. Here, we show that norepinephrinergic (NEergic) projections from the locus coeruleus (LC), a brain arousal system, innervate into adult DG, where daily rhythmic release of norepinephrine (NE) from the LC NEergic neurons controlled circadian variation of NSC activation through ß3-adrenoceptors. Disrupted circadian rhythmicity by acute sleep deprivation leads to transient NSC overactivation and NSC pool exhaustion over time, which is effectively ameliorated by the inhibition of the LC NEergic neuronal activity or ß3-adrenoceptors-mediated signaling. Finally, we demonstrate that NE/ß3-adrenoceptors-mediated signaling regulates NSC activation through molecular clock BMAL1. Therefore, our study unravels that adult NSCs precisely coordinate circadian neural circuit and intrinsic molecular circadian clock to adapt their cellular behavior across the day. [ABSTRACT FROM AUTHOR]

Background: The core clock gene brain and muscle ARNT like-1 (Bmal1) is involved in the regulation of bone tissue aging. However, current studies are mostly limited to the establishment of the association between Bmal1 and bone senescence, without in-depth exploration of its main upstream and downstream regulatory mechanisms. Methods: The luciferase reporter assay, RT-qPCR and Western blotting were performed to detect the interaction between miR-155-5p and Bmal1. The effects of miR-155-5p and Bmal1 on the aging and osteogenic differentiation ability of mouse bone marrow mesenchymal stem cells (BMSCs) were investigated by cell counting kit-8 (CCK-8) assay, flow cytometry, β-gal staining, alkaline phosphatase quantitative assay and alizarin red staining in vitro. The potential molecular mechanism was identified by ChIP-Seq, RNA-seq database analysis and immunofluorescence staining. Results: The expression of Bmal1 declined with age, while the miR-155-5p was increased. miR-155-5p and Bmal1 repressed each other's expression, and miR-155-5p targeted the Bmal1. Besides, miR-155-5p inhibited the proliferation and osteogenic differentiation of BMSCs, promoted cell apoptosis and senescence, inhibited the expression and nuclear translocation of YAP and TAZ. However, Bmal1 facilitated the osteogenic differentiation and suppressed the aging of BMSCs, meanwhile inactivated the Hippo pathway. Moreover, YAP inhibitors abrogated the positive regulation of aging and osteogenic differentiation in BMSCs by miR-155-5p and Bmal1. Conclusion: In mouse BMSCs, miR-155-5p and Bmal1 regulated the aging and osteogenic differentiation ability of BMSCs mainly through the Hippo signaling pathway. Our findings provide new insights for the interventions in bone aging. [ABSTRACT FROM AUTHOR]

The circadian clock is generated by a molecular timekeeping mechanism coordinating daily oscillations of physiology and behaviors in mammals. In the mammalian circadian clockwork, basic helix–loop–helix ARNT‐like protein 1 (BMAL1) is a core circadian component whose defects lead to circadian disruption and elicit behavioral arrhythmicity. To identify previously unknown regulators for circadian clocks, we searched for genes influencing BMAL1 protein level by using a CRISPR/Cas9‐based genome‐wide knockout library. As a result, we found that the deubiquitinase ubiquitin carboxyl‐terminal hydrolase 1 (USP1) positively affects BMAL1 protein abundance. Overexpression of wild‐type USP1, but not a deubiquitinase‐inactive mutant USP1, upregulated BMAL1 protein level, whereas genetic ablation of USP1 downregulated BMAL1 protein level in U2OS cells. Furthermore, treatment with USP1 inhibitors led to significant downregulation of BMAL1 protein in U2OS cells as well as mouse tissues. Subsequently, genetic ablation or pharmacological inhibition of USP1 resulted in reduced mRNA levels of a panel of clock genes and disrupted circadian rhythms in U2OS cells. Mechanistically, USP1 was able to de‐ubiquitinate BMAL1 and inhibit the proteasomal degradation of BMAL1. Interestingly, the expression of Usp1 was much higher than the other two deubiquitinases of BMAL1 (Usp2 and Usp9X) in the mouse heart, implying a tissue‐specific function of USP1 in the regulation of BMAL1 stability. Our work thus identifies deubiquitinase USP1 as a previously unknown regulator of the mammalian circadian clock and highlights the potential of genome‐wide CRISPR screens in the identification of regulators for the circadian clock. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of BMAL1 on H2O2-induced cardiomyocyte injury through NRF2-regulated ROS/NLRP3 inflammasome pathway. Methods H9c2 cells and H9c2 cells with stable over-expressed BMAL1 were cultured and divided into the control group, the H2O2 group, the BMAL1-OE group, the BMAL1-OE+H2O2 group, the BMAL1-OE+ML385 group and the BMAL1-OE+ML385+H2O2 group. All groups were pre-intervened with corresponding inhibitors, and then treated with 0.2 mmol/L H2O2, except for the control group and the BMAL1-OE group. After the intervention, CCK-8 assay was used to measure cell viability, fluorescent probe DCFH-DA was used to measure ROS generation and Western blot assay was used to detect BMAL1, NRF2 and NLRP3 protein expressions. ELISA was used to determine IL-1β release. Results Compared with the control group, the cell viability was decreased, ROS generation was increased, BMAL1 and NRF2 protein expressions were decreased, NLRP3 expression and IL-1β release were increased in the H2O2 group (P＜0.05). Compared with the H2O2 group, the cell viability was increased, ROS generation was decreased, BMAL1-OE and NRF2 protein expressions were increased, NLRP3 expression and IL-1β release were decreased in the BMAL1-OE+H2O2 group (P＜0.05). Compared with the BMAL1-OE+H2O2 group, the cell viability was decreased, ROS generation was increased, NLRP3 expression and IL-1β release were increased in the BMAL1-OE+ML385+H2O2 group (P＜0.05). Conclusion BMAL1 attenuates H2O2-induced H9c2 cardiomyocyte injury, and its mechanism may be related to the regulation of ROS/NLRP3 inflammasome pathway through NRF2. [ABSTRACT FROM AUTHOR]

In children, teenagers, and young adults, environmental factors and genetic modifications have contributed to the development of obesity. There is a close relationship between obesity and circadian rhythm. To understand the role of CLOCK and BMAL1 in obesity, we analyzed the methylation status of CLOCK and BMAL1 in obese and control subjects. In this paper, we analyzed the methylation status of the CLOCK and BMAL1 genes by using MS-HRM in a total of 55 obese and 54 control subjects. In our study, we demonstrated that the level of fasting glucose and the level of HDL-cholesterol were associated with CLOCK methylation in obesity. We also showed a significant association between BMAL1 gene methylation and waist and hip circumference in obese subjects. This is the first study that shows the methylation of BMAL1 is associated with the obese phenotype. However, we could not show a direct association between CLOCK methylation and the obese phenotype. In this paper, a novel epigenetic interaction between circadian clock genes and obesity was demonstrated. [ABSTRACT FROM AUTHOR]

Circadian misalignment (CM) caused by shift work can increase the risk of mood impairment. However, the pathological mechanisms underlying these deficits remain unclear. In the present study, we used long‐term variable photoperiod (L‐VP) in wild‐type mice to better simulate real‐life shift patterns and study its effects on the prefrontal cortex (PFC) and hippocampus, which are closely related to mood function. The results showed that exposure to L‐VP altered the activity/rest rhythms of mice, by eliciting phase delay and decreased amplitude of the rhythms. Mice with CM developed anxiety and depression‐like manifestations and the number of mature oligodendrocytes (OL) was reduced in the medial prefrontal cortex and hippocampal CA1 regions. Mood impairment and OL reduction worsened with increased exposure time to L‐VP, while normal photoperiod restoration had no effect. Mechanistically, we identified upregulation of Bmal1 in the PFC and hippocampal regions of CM mice at night, when genes related to mature OL and myelination should be highly expressed. CM mice exhibited significant inhibition of the protein kinase B (AKT)/mTOR signaling pathway, which is directly associated to OL differentiation and maturation. Furthermore, we demonstrated in the OL precursor cell line Oli‐Neu that overexpression of Bmal1 inhibits AKT/mTOR pathway and reduces the expression of genes OL differentiation. In conclusion, BMAL1 might play a critical role in CM, providing strong research evidence for BMAL1 as a potential target for CM therapy. [ABSTRACT FROM AUTHOR]

Sarcopenia is an age‐related disease characterized by a reduction in muscle mass, strength, and function and, therefore, a deterioration in skeletal muscle health and frailty. Although the cause of sarcopenia is still unknown and, thus, there is no treatment, increasing evidence suggests that chronodisruption, particularly alterations in Bmal1 clock gene, can lead to those deficits culminating in sarcopenia. To gain insight into the cause and mechanism of sarcopenia and the protective effect of a therapeutic intervention with exercise and/or melatonin, the gastrocnemius muscles of male and female skeletal muscle‐specific and inducible Bmal1 knockout mice (iMS‐Bmal1−/−) were examined by phenotypic tests and light and electron microscopy. Our results revealed a disruption of the normal activity/rest rhythm, a drop in skeletal muscle function and mass, and increased frailty in male and female iMS‐Bmal1−/− animals compared to controls. A reduction in muscle fiber size and increased collagenous tissue were also detected, accompanied by reduced mitochondrial oxidative capacity and a compensatory shift towards a more oxidative fiber type. Electron microscopy further supports mitochondrial impairment in mutant mice. Melatonin and exercise ameliorated the damage caused by loss of Bmal1 in mutant mice, except for mitochondrial damage, which was worsened by the latter. Thus, iMS‐Bmal1−/− mice let us to identify Bmal1 deficiency as the responsible for the appearance of sarcopenia in the gastrocnemius muscle. Moreover, the results support the exercise and melatonin as therapeutic tools to counteract sarcopenia, by a mechanism that does not require the presence of Bmal1. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor 21 (FGF21) is expressed in several organs, including the liver, adipose tissue, and cardiovascular system, and plays an important role in cross-talk with other organs by binding to specific FGF receptors and their co-receptors. FGF21 represents a potential target for the treatment of obesity, type 2 diabetes mellitus, and non-alcoholic steatohepatitis (NASH). The production of FGF21 in skeletal muscle was recently suggested to be beneficial for metabolic health through its autocrine and paracrine effects. However, the regulatory mechanisms of FGF21 in skeletal muscle remain unclear. In the present study, we showed that berberine regulated FGF21 production in C2C12 myotubes in a dose-dependent manner. We also examined the effects of A-674563, a selective Akt1 inhibitor, on the berberine-mediated regulation of FGF21 expression in C2C12 myotubes. Berberine significantly increased the secretion of FGF21 in C2C12 myotubes, while A-674563 attenuated this effect. Moreover, a pre-treatment with A-674563 effectively suppressed berberine-induced increases in Bmal1 expression in C2C12 myotubes, indicating that the up-regulation of Bmal1 after the berberine treatment was dependent on Akt1. Additionally, berberine-induced increases in FGF21 secretion were significantly attenuated in C2C12 cells transfected with Bmal1 siRNA, indicating the contribution of the core clock transcription factor BMAL1 to Akt-regulated FGF21 in response to berberine. Collectively, these results indicate that berberine regulates the expression of FGF21 through the Akt1 pathway in C2C12 myotubes. Moreover, the core clock gene Bmal1 may participate in the control of the myokine FGF21. Berberine stimulated Akt1-dependent FGF21 expression in C2C12 myotubes. The up-regulation of FGF21 through the modulation of PI3K/AKT1/BMAL1 in response to berberine may be involved in the regulation of cellular function (such as Glut1 expression) by acting in an autocrine and/or paracrine manner in skeletal muscle. [ABSTRACT FROM AUTHOR]

Abstract Objectives Growing evidence demonstrates a relationship between epilepsy and the circadian system. However, relatively little is known about circadian function in disease states, such as epilepsy. This study aimed to characterize brain and peripheral core circadian clock gene expression in rat models of genetic and acquired epilepsy. Methods For the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) study, we used 40 GAERS and 40 non‐epileptic control (NEC) rats. For the kainic acid status epilepticus (KASE) study, we used 40 KASE and 40 sham rats. Rats were housed in a 7 am:7 pm light–dark cycle. Hypothalamus, hippocampus, liver, and small intestine samples were collected every 3 h throughout the light period. We then assessed core diurnal clock gene expression of per1, cry1, clock, and bmal1. Results In the GAERS rats, all tissues exhibited significant changes in clock gene expression (P 0.05) compared with shams. Significance These results indicate marked diurnal disruption to core circadian clock gene expression in rats with both generalized and focal chronic epilepsy. This could contribute to epileptic symptomology and implicate the circadian system as a viable target for future treatments.

ABSTRACTPerception and production of second-level temporal intervals are critical in several behavioral and cognitive processes, including adaptive anticipation, motor control, and social communication. These processes are impaired in several neurological and psychological disorders, such as Parkinson’s disease and attention-deficit hyperactivity disorder. Although evidence indicates that second-level interval timing exhibit circadian patterns, it remains unclear whether the core clock machinery controls the circadian pattern of interval timing. To investigate the role of core clock molecules in interval timing capacity, we devised a behavioral assay called the interval timing task to examine prospective motor interval timing ability. In this task, the mouse produces two separate nose pokes in a pretrained second-level interval to obtain a sucrose solution as a reward. We discovered that interval perception in wild-type mice displayed a circadian pattern, with the best performance observed during the late active phase. To investigate whether the core molecular clock is involved in the circadian control of interval timing, we employed Bmal1 knockout mice (BKO) in the interval timing task. The interval production of BKO did not display any difference between early and late active phase, without reaching the optimal interval production level observed in wild-type. In summary, we report that the core clock gene Bmal1 is required for the optimal performance of prospective motor timing typically observed during the late part of the active period.

Aims: Currently, the effect of drug treatment for osteoporosis is relatively poor, and the side effects are numerous and serious. Melatonin is a potential drug to improve bone mass in postmenopausal women. Unfortunately, the mechanism by which melatonin improves bone metabolism remains unclear. The aim of this study was to further investigate the potential mechanism of melatonin in the treatment of osteoporosis. Methods: The effects of melatonin on mitochondrial apoptosis protein, bmal1 gene, and related pathway proteins of RAW264.7 (mouse mononuclear macrophage leukaemia cells) were analyzed by western blot. Cell Counting Kit-8 was used to evaluate the effect of melatonin on cell viability. Flow cytometry was used to evaluate the effect of melatonin on the apoptosis of RAW264.7 cells and mitochondrial membrane potential. A reactive oxygen species (ROS) detection kit was used to evaluate the level of ROS in osteoclast precursors. We used bmal1-small interfering RNAs (siRNAs) to downregulate the Bmal1 gene. We established a postmenopausal mouse model and verified the effect of melatonin on the bone mass of postmenopausal osteoporosis in mice via micro-CT. Bmal1 lentiviral activation particles were used to establish an in vitro model of overexpression of the bmal1 gene. Results: Melatonin promoted apoptosis of RAW264.7 cells and increased the expression of BMAL1 to inhibit the activation of ROS and phosphorylation of mitogen-activated protein kinase (MAPK)-p38. Silencing the bmal1 gene weakened the above effects of melatonin. After that, we used dehydrocorydaline (DHC) to enhance the activation of MAPK-p38, and the effects of melatonin on reducing ROS levels and promoting apoptosis of RAW264.7 cells were also blocked. Then, we constructed a mouse model of postmenopausal osteoporosis and administered melatonin. The results showed that melatonin improves bone loss in ovariectomized mice. Finally, we established a model of overexpression of the bmal1 gene, and these results suggest that the bmal1 gene can regulate ROS activity and change the level of the MAPK-p38 signalling pathway. Conclusion: Our study confirmed that melatonin promotes the apoptosis of RAW264.7 cells through BMAL1/ROS/MAPK-p38, and revealed the therapeutic effect and mechanism of melatonin in postmenopausal osteoporosis. This finding enriches BMAL1 as a potential target for the treatment of osteoporosis and the pathogenesis of postmenopausal osteoporosis. Cite this article: Bone Joint Res 2023;12(11):677–690.

Alcohol abuse has recently become a serious health concern worldwide, and the incidence of alcoholic liver disease (ALD) is rapidly increasing with high morbidity and mortality. Ferroptosis is a newly recognized form of regulated cell death caused by the iron-dependent accumulation of lipid peroxidation. Here we showed that the circadian clock protein BMAL1 in hepatocytes is both necessary and sufficient to protect against ALD by mitigating ferroptosis. Upon exposure to alcohol (5 % Lieber-DeCarli liquid alcohol diet for 10 days before binged alcohol with 5 g/kg body weight in vivo, 300 mmol/L for 12 h in vitro, respectively), the content of iron, reactive oxygen species (ROS) and malondialdehyde (MDA) was boosted significantly while glutathione (GSH) was decreased that mainly based on the downregulated protein expression of ferritin heavy chain (FTH), ferroportin (FPN), heme oxygenase1(HO-1) and anti-cystine/glutamate antiporter (SLC7A11), while these changes could be abolished by ferroptosis inhibitor Ferrostatin-1[Fer-1 (5 mg/kg body weight for 10 days in vivo, 10 μmol/L for 2 h in vitro, respectively)]. Further study indicated that the alcohol could activate the protein expression of brain and muscle arnt-like protein-1 (BMAL1) which exerts a protective effect against ferroptosis through promoting nuclear factor erythroid 2-related factor 2 (Nrf2) translocation into nuclear and subsequently stimulating its downstream proteins FTH, FPN, glutathione peroxidase 4 activity (GPX4), HO-1, SLC7A11, while knockdown of BMAL1 and Nrf2 by RNA interference further downregulated the expression of these protein and thus promoting ferroptosis in response to alcohol. Collectively, our results unveiled that the protective action of BMAL1 during alcohol challenge depends on its ability to activate Nrf2-ARE antiferroptosis pathway and targeting hepatic BMAL1 to dampen hepatic ferroptosis signaling may have therapeutic potential for ALD.

Sarcopenia is an age-related condition that involves a progressive decline in muscle mass and function, leading to increased risk of falls, frailty, and mortality. Although the exact mechanisms are not fully understood, aging-related processes like inflammation, oxidative stress, reduced mitochondrial capacity, and cell apoptosis contribute to this decline. Disruption of the circadian system with age may initiate these pathways in skeletal muscle, preceding the onset of sarcopenia. At present, there is no pharmacological treatment for sarcopenia, only resistance exercise and proper nutrition may delay its onset. Melatonin, derived from tryptophan, emerges as an exceptional candidate for treating sarcopenia due to its chronobiotic, antioxidant, and anti-inflammatory properties. Its impact on mitochondria and organelle, where it is synthesized and crucial in aging skeletal muscle, further highlights its potential. In this review, we discuss the influence of clock genes in muscular aging, with special reference to peripheral clock genes in the skeletal muscle, as well as their relationship with melatonin, which is proposed as a potential therapy against sarcopenia. [ABSTRACT FROM AUTHOR]

Peripheral circadian clocks control cell proliferation and survival, but little is known about their role and regulation in megakaryocytic cells. N-methyl-D-aspartate receptor (NMDAR) regulates the central clock in the brain. The purpose of this study was to determine whether NMDAR regulates the megakaryocytic cell clock and whether the megakaryocytic clock regulates cell proliferation and cell death. We found that both the Meg-01 megakaryocytic cell line and native murine megakaryocytes expressed circadian clock genes. Megakaryocyte-directed deletion of Grin1 in mice caused significant disruption of the circadian rhythm pathway at the transcriptional level and increased expression of BMAL1 at the protein level. Similarly, both pharmacological (MK-801) and genetic (GRIN-/-) inhibition of NMDAR in Meg-01 cells in vitro resulted in widespread changes in clock gene expression including increased expression of BMAL1, the core clock transcription factor. BMAL1 overexpression reduced Meg-01 cell proliferation and altered the time-dependent expression of the cell cycle regulators MYC and WEE1, whereas BMAL1 knockdown led to increased cell death in Meg-01-GRIN1-/- cells. Our results demonstrate that NMDAR regulates the circadian clock in megakaryocytic cells and that the circadian clock component BMAL1 contributes to the control of Meg-01 cell proliferation and survival. [ABSTRACT FROM AUTHOR]

Background: Today, modern lifestyles and disrupted sleep patterns cause circadian clock rhythm impairments that are associated with altered leptin levels, which subsequently affect a wide range of physiological processes and have significant health burdens on societies. Nevertheless, there has been no systematic review of circadian clock genes and proteins, leptin, and related signaling pathways. Methods: Accordingly, we systematically reviewed circadian clock proteins, leptin, and molecular mechanisms between them by searching Pubmed, Scopus, ProQuest, Web of Sciences, and Google Scholar until September 2022. After considering the inclusion and exclusion criteria, 20 animal studies were selected. The risk of bias was assessed in each study. Results: The results clarified the reciprocal interconnected relationship between circadian clock genes and leptin. Circadian clock genes regulate leptin expression and signaling via different mechanisms, such as CLOCK-BMAL1 heterodimers, which increase the expression of PPARs. PPARs induce the expression of C/EBPα, a key factor in upregulating leptin expression. CLOCK-BMAL1 also induces the expression of Per1 and Rev-erb genes. PER1 activates mTORC1 and mTORC1 enhances the expression of C/EBPα. In addition, REV-ERBs activate the leptin signaling pathway. Also, leptin controls the expression of circadian clock genes by triggering the AMPK and ERK/MAPK signaling pathways, which regulate the activity of PPARs. Moreover, the roles of these molecular mechanisms are elucidated in different physiological processes and organs. Conclusions: Crosstalk between circadian clock genes and leptin and their affecting elements should be considered in the selection of new therapeutic targets for related disorders, especially obesity and metabolic impairments. [ABSTRACT FROM AUTHOR]

Hypertension is a progressive metabolic disease characterized by circadian regulation of lipid metabolism disorder. Identifying specific lipid components and maintaining circadian homeostasis of lipid metabolism might be a promising therapeutic strategy for hypertension. Isorhynchophylline (IRP) can regulate lipid metabolism; however, the underlying mechanism of IRP in improving lipid metabolism rhythm disorder is still unclear. The lipid circadian biomarkers and abnormal metabolic pathways intervened by IRP were investigated using diurnal lipidomic research methods. The 24‐h circadian changes in mRNA and protein expression levels of circadian genes, including Bmal1, Clock, Cry1, Cry2, Per1, and Per2, and lipid metabolism‐related factors (PPARα and LPL) were determined using RT‐PCR and western blot analyses, respectively. The underlying mechanisms were intensively investigated by inhibiting Bmal1. Molecular docking and drug affinity responsive target stability analyses were performed to assess the binding affinity of IRP and Bmal1. IRP treatment could effectively improve 24‐h blood pressure, ameliorate the lipid metabolic rhythm disorder, reverse the expression levels of circadian rhythm genes, and regulate lipid metabolism‐related genes (PPARα and LPL) by mediating Bmal1. This study highlighted the potential effects of IRP in maintaining the circadian homeostasis of lipid metabolism and the treatment of hypertension. [ABSTRACT FROM AUTHOR]

Objectives: Growing evidence demonstrates a relationship between epilepsy and the circadian system. However, relatively little is known about circadian function in disease states, such as epilepsy. This study aimed to characterize brain and peripheral core circadian clock gene expression in rat models of genetic and acquired epilepsy. Methods: For the Genetic Absence Epilepsy Rats from Strasbourg (GAERS) study, we used 40 GAERS and 40 non‐epileptic control (NEC) rats. For the kainic acid status epilepticus (KASE) study, we used 40 KASE and 40 sham rats. Rats were housed in a 7 am:7 pm light–dark cycle. Hypothalamus, hippocampus, liver, and small intestine samples were collected every 3 h throughout the light period. We then assessed core diurnal clock gene expression of per1, cry1, clock, and bmal1. Results: In the GAERS rats, all tissues exhibited significant changes in clock gene expression (P < 0.05) when compared to NEC. In the KASE rats, there were fewer effects of the epileptic condition in the hypothalamus, hippocampus, or small intestine (P > 0.05) compared with shams. Significance: These results indicate marked diurnal disruption to core circadian clock gene expression in rats with both generalized and focal chronic epilepsy. This could contribute to epileptic symptomology and implicate the circadian system as a viable target for future treatments. [ABSTRACT FROM AUTHOR]

BACKGROUND: Temporomandibular joint osteoarthritis is a common disease in stomatology and its pathogenesis is complex. Recently, it has been found that the decreased expression of clock gene Bmal1 can lead to osteoarthritis-like pathological changes of the temporomandibular joint in mice, but the mechanism of Bmal1 in temporomandibular joint osteoarthritis remains unclear. OBJECTIVE: To summarize the possible mechanism of clock gene Bmal1 in the development of temporomandibular joint osteoarthritis, so as to provide new insights into the pathogenesis of temporomandibular joint osteoarthritis. METHODS: Articles published from inception to 2022 were retrieved in PubMed, Web of Science, and CNKI by the keywords of “TMJOA, osteoarthritis, TMD, condylar cartilage, Bmal1, circadian, circadian clock, sleep” in English and Chinese. Fifty articles were finally selected for this review according to the criteria of inclusion and exclusion. RESULTS AND CONCLUSION: Circadian rhythm disturbance caused by sleep deprivation can lead to osteoarthritis-like pathological changes of the temporomandibular joint in mice, suggesting that circadian rhythm disturbance is a new risk factor for temporomandibular joint osteoarthritis. As a core clock factor of circadian rhythm, Bmal1 plays an important role in maintaining cartilage homeostasis of the temporomandibular joint. When Bmal1 expression is decreased, anabolism of the articular cartilage is decreased, catabolism is increased, condylar cartilage is degraded, and temporomandibular joint osteoarthritis is induced. Bmal1 overexpression can alleviate the pathological symptoms of osteoarthritis. Bmal1 can also affect chondrogenesis and endochondral ossification of the condyle cartilage through downstream signaling pathways, resulting in dysplasia of the condyle and mandible, which provides an anatomical basis for the occurrence and development of temporomandibular joint osteoarthritis. At the same time, Bmal1 can promote aging and obesity by regulating oxidative stress and fat metabolism, thereby increasing the susceptibility of temporomandibular joint osteoarthritis. Therefore, the clock gene Bmal1 may have direct and/or indirect effects on the occurrence and development of temporomandibular joint osteoarthritis. [ABSTRACT FROM AUTHOR]

Obesity and overweight represent a growing health problem worldwide. Genes regulating the intake and metabolism of different nutrients can positively or negatively influence the efficacy of nutritional interventions against obesity and its complications. The aim of this study was to assess changes in anthropometric and clinical parameters and the adherence to a Mediterranean diet (MedDiet) over time in relation to nutrigenetic variants in overweight or obese subjects affected by Type 2 Diabetes (T2D) or dysglycemia, who were included in a nutritional program. A total of 23 subjects were included in this study. Clinical parameters, physical activity levels, and the adherence to a MedDiet were evaluated at baseline, at 6 (T6), and at 12 months (T12) during and after a diet/lifestyle intervention. In a single blood sample from each subject, rs1984112 (A>G) and rs1761667 (G>A) in CD36; rs7950226 (G>A) in BMAL1; and rs1801260 (A>G), rs4864548 (A>G), and rs3736544 (G>A) in CLOCK were genotyped with Real-Time PCR. Significant associations were observed between CD36 rs1761667 and weight (p = 0.025), hip circumference (p = 0.042), triglycerides (p = 0.047), and HbA1c (p = 0.012) at baseline. Moreover, the genotype AA in CD36 rs1761667 was significantly associated with a lower BMI when compared to G carriers at baseline, at T6, and also at T12. In addition, subjects with the AA genotype at CD36 rs1984112 had significantly lower levels of HbA1c (p = 0.027) than the GG and AG genotypes at baseline. These results show that variants in CD36 can have an impact on anthropometric and clinical parameters in overweight or obese subjects affected by T2D or dysglycemia, and that it might influence the success of the diet/lifestyle intervention. [ABSTRACT FROM AUTHOR]

Objective: This study aims to investigate the role of BMAL1 in promoting angiogenesis in cerebral glioma among athletes by regulating vascular endothelial growth factor (VEGF) and angiopoietin-2 (ANG2). Methods: Human brain tumor cells, representative of gliomas often observed in athletes, were categorized into three groups: blank, control, and BMAL1-treated research group. Cell growth and apoptosis were analyzed using the MTT assay and flow cytometry, respectively. VEGF and ANG2 expressions, along with microvessel density (MVD), were assessed via immunohistochemistry. The Spearman rank test evaluated the correlation between VEGF and ANG2 levels. Further, groups A, B, and C (representing different pathological grades of human glioma tissues) and BMAL1-treated groups (D, E, and F) were compared to analyze BMAL1's role in modulating VEGF and ANG2. Results: The control group showed significantly lower cell survival and higher apoptosis rates compared to the blank group (P<0.05). Higher glioma grades correlated positively with increased apoptosis rates (P<0.05). VEGF, ANG2, and MVD expressions were significantly higher in the control group than in the blank group (P<0.05), with notable differences observed between the study and control groups (P<0.05). A positive correlation was found between glioma grade, MVD count, and the expressions of VEGF and ANG2 (P<0.05). Groups D, E, and F demonstrated significant differences in VEGF and ANG2 expressions, with group F exhibiting the highest levels (P<0.05), indicating BMAL1's regulatory effect on these angiogenic factors. Conclusion: The study confirms that BMAL1 regulates VEGF and ANG2 expression, promoting angiogenesis in cerebral glioma, a condition relevant to athletes due to their unique physiological stresses. This highlights the potential of targeting BMAL1 in developing therapeutic strategies for glioma angiogenesis in athletic populations, providing a novel approach to manage this challenging aspect of cerebral glioma treatment. [ABSTRACT FROM AUTHOR]

Understanding of the relationship between the circadian molecular clock and allergic diseases continues to be studied and may be of practical importance for further selection of optimal treatment. The aim of this study was to evaluate the degree of mRNA expression of the circadian molecular clock genes bmal1 and per1 in buccal epithelial cells and to investigate the efficacy of protocol treatment in children with seasonal allergic rhinitis, taking into account the time dependence. Material and methods. The study included 20 patients aged 6 to 17 years with a confirmed diagnosis of seasonal allergic rhinitis (SAR). The control group included 7 healthy children of the same age with no history of allergic or chronic diseases. All patients with SAR were divided into 2 groups of 10 patients each, who received the protocol therapy in the morning (group 1) and in the evening (group 2) for 4 weeks. The expression of circadian molecular clock genes was studied by real-time polymerase chain reaction (real-time PCR) method by sampling the buccal epithelium of the children's oral cavity in the morning (8:00 am) and in the evening (8:00 pm), with a repeat examination of the samples 1 month after treatment. Statistical analysis was performed using the GraphPad Prism 5.00 computer program (GraphPad Software, Inc., San Diego, CA, USA). The research design was discussed and approved at the meeting of the medical and ethical commission of the Ukrainian Medical and Stomatological Academy, protocol No. 188 dated 11/25/2020, where permission to conduct medical and biological research was issued. The research design was discussed and approved at the meeting of the Medical and Ethical Committee of the Ukrainian Academy of Medicine and Dentistry, Protocol No. 188 dated 25.11.2000, where permission to conduct medical and biological research was granted. The study was conducted in accordance with the research plan of the Pediatrics Department no. 2 of Poltava State Medical University «Optimization of early diagnosis, treatment and prognosis of the most common childhood diseases», state registration number 0122U001876 (years of implementation 2021-2026) and research work - Research Institute of genetic and immunological bases of development of pathology and pharmacogenetics of PDMU «Development of methods of treatment and prevention of development of pulmonary fibrosis by activation of PPAR-gamma receptors», state registration number 0122U201686 (years of implementation 2023-2025). Results. Morning gene expression in healthy children showed statistically higher levels of per1 (0.780 ± 0.070) compared to bmal1 (0.293 ± 0.074; p<0.001), whereas in the evening the level of the latter (0.697 ± 0.130) was significantly higher than per1 (0.370 ± 0.044; p<0.05). Comparing the levels of per1 in this group, the expression of this gene was higher in the morning (0.780 ± 0.070) than in the evening (0.370 ± 0.044; p<0.001), while the expression of bmal1 was higher at 20:00 (0.697 ± 0.130 vs. 0.293 ± 0.074; p<0.05). In contrast, patients with SAR had a significantly higher level of bmal1 gene mRNA expression (1.036±0.161 vs. 0.293±0.074 in healthy children (p<0.05). Examination of gene expression in children with SAR in the evening revealed that bmal1 values showed similar levels of expression in all participants (0.775 ± 0.163 vs. 0.697 ± 0.130; p>0.05), while the mRNA level of the per1 gene was statistically higher in patients with rhinitis (1.293 ± 0.186; p<0.01). Conclusions. The results obtained indicate a dysregulation of the peripheral molecular circadian clock in the buccal epithelium of patients with SAR. The use of the protocol treatment in children with SAR in the evening demonstrated a better restoration of the expression of the positive and negative regulatory link of the molecular circadian clock. [ABSTRACT FROM AUTHOR]

Objective 　We aimed to investigate the inhibitory effect of serum containing Huangqi Jianzhong Decoction on isoproterenol (ISO) induced lipolysis and browning in 3T3-L1 adipocytes and its relationship with the regulation of the cyclic adenosine monophosphate ( cAMP) / protein kinase A (PKA) / cyclic adenosine monophosphate response element binding protein (CREB) signaling pathway by brain and muscle aromatic hydrocarbon receptor nuclear transcription factor like protein-1 (BMAL1). Methods　Blank serum and Huangqi Jianzhong Decoction drug-containing serum were prepared. 3T3-L1 adipocytes were cultured in vitro and induced to differentiate and mature. They were divided into the control group, the blank group, the 2. 5% drug-containing serum group, the 5% drug-containing serum group, and the 10% drug-containing serum group. 10 mmol/ L ISO was given to all groups except for the control group to establish the hypertipolysis and browning model. Oil red O staining and enzyme-linked immunosorbent assay (ELISA) were used to detect the lipid droplet positive area ratio and the content of hydrolyzed products. Real-time PCR and Western blotting were conducted to detect the expression of lipolysis- and browning-related genes and proteins, respectively, to select the optimal volume fraction of drug-containing serum for the lentivirus transfection experiment. Mature adipocytes were transfected with si-BMAL1 to silence BMAL1. Control cells were transfected with si-NC. Then the cells were divided into the si-NC group, the containing serum si-NC group, the si-BMAL1 group, and the drug-containing serum si-BMAL1 group. After incubation with ISO, the lipid droplet positive area ratio in adipocytes was detected by oil red O staining. The contents of triglyceride (TG) and cAMP in adipocytes and free fatty acid (FFA) in medium were detected by ELISA. The protein expression levels of PKA and CREB were determined by Western blotting. The mRNA expression levels of hormone-sensitive lipase ( HSL), triglyceride lipase (ATGL), uncoupling protein 1 (UCP1), and T-box transcription factor 1 (TBX1) were determined by real-time PCR. Results　Compared with the 2. 5% and 5% drug-containing serum groups, the contents of cAMP in adipocytes and FFA in culture medium were decreased (P<0. 05), the content of TG was increased (P<0. 05), the protein expression of PKA was decreased (P<0. 05), and the mRNA expression levels of HSL and ATGL were decreased (P<0. 05) in the 10% drug-containing serum group. The relative protein expression of BMAL1 was significantly lower in 3T3-L1 adipocytes transfected with si-BMAL1 than in the si-NC group (P<0. 05), indicating that BMAL1 was successfully silenced. Compared with the si-NC group, the lipid droplet positive area ratio and TG content were decreased in the si-BMAL1 group (P<0. 05), while the FFA content in culture medium, cAMP content in adipocytes, and PKA and CREB protein expression levels were increased (P<0. 05), and the mRNA expression levels of HSL, ATGL, UCP1, and TBX1 were also increased (P<0. 05). Compared with the si-BMAL1 group, the lipid droplet positive area ratio and TG content were increased in the drug- containing serum si-BMAL1 group (P<0. 05), while the content of FFA in culture medium, the content of cAMP in adipocytes, and the protein expression levels of PKA and CREB were significantly decreased (P<0. 05) and the mRNA expression levels of HSL, ATGL, UCP1, and TBX1 were also decreased (P<0. 05). Conclusion　Drug-containing serum Huangqi Jianzhong Decoction can inhibit ISO-induced lipolysis and browning in 3T3-L1 adipocytes, the mechanism of action may be related to the regulation of the cAMP / PKA/ CREB signaling pathway by BMAL1. [ABSTRACT FROM AUTHOR]

Pharmacological intervention of circadian rhythms is a potentially useful approach for ameliorating various health problems caused by disturbed circadian rhythms including sleep disorder and metabolic diseases. To find compounds that affect circadian rhythms, we screened mushroom extracts using mouse cells expressing the luciferase gene under the control of the mouse Bmal1 promoter. The culture filtrate extract from the basidiomycete Cyclocybe erebia enhanced the oscillation of bioluminescence caused by the expression of the luciferase gene and prolonged the period of bioluminescence. Bioassay-guided fractionation of the extract resulted in purification of compounds 1 and 2. Spectroscopic analyses along with single-crystal X-ray diffraction analysis, revealed that these compounds were diterpenoids with a unique skeleton and a fused ring system comprising 3-, 7-, and 5-membered rings. Compounds 1 and 2 were named cyclocircadins A and B, respectively. These findings suggested that natural diterpenoids could be a source of compounds with the activity affecting circadian rhythms. [Display omitted] • An extract of edible mushroom Cyclocybe erebia modulated circadian rhythms. • Two novel diterpenoids, cyclocircadins A and B, were identified. • The carbon skeleton of the compounds has not been reported from fungi kingdom. • The compounds enhanced and prolonged the periodic expression of the rhythm gene. [ABSTRACT FROM AUTHOR]

Background: L-Theanine, a nonproteinogenic amino acid derived from green tea, is being recognized as an anti-cancer candidate. However, it’s roles in the development of cancer chemoresistance is still unknown and the molecular mechanism is urgently to be explored. Methods: The effects of L-Theanine on lung cancer chemoresistance were validated by Cell Counting Kit-8 (CCK-8) assay, transwell assay, and in vitro tumor spheroid formation assay; the expression of proteins was detected by using polymerase chain reaction (PCR) and western blotting. RNA-sequencing (RNA-seq) and bioinformatics analysis were used to identify differentially expressed genes induced by L-Theanine. BMAL1 knockdown and overexpression were constructed by using a lentivirus-mediated transfection system. Results: L-Theanine improved the chemoresistance to cis-diamminedichloroplatinum (DDP) and inhibited stemness of DDP-resistant lung cancer cells but not non-resistant lung cancer cells. The results from RNA-seq analysis showed that STAT3/NOTCH1 pathway was a potential dominant signaling involved in L-Theanine improving the chemoresistance in DDP-resistant lung cancer. Mechanistically, L-Theanine impeded migration and stemness activation of DDP-resistant lung cancer cells via regulating the expression of STAT3/NOTCH1/BMAL1 signaling-induced stemness markers as well as inhibiting the expression levels of drug resistance-related genes. In addition, a combination of L-Theanine and Stat3 blockade synergistically improved the chemoresistance in DDP-resistant lung cancer. Conclusion: L-Theanine improves the chemoresistance by regulating STAT3/NOTCH1/BMAL1 signaling, reducing stemness, and inhibiting the migration of DDP-resistant lung cancer cells. The finding might provide some evidence for therapeutic options in overcoming the chemoresistance in cancers, including lung cancer.