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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]

• The circadian clock in the intervertebral disc (IVD) rhythmically controls key genes involved in extracellular matrix (ECM) homeostasis. • Deletion of the local IVD clock in the Col2a1- Bmal1 knockout mice leads to profound age-related changes of ECM microarchitecture and an endochondral ossification-like phenotype in the IVD. • Circadian time series RNAseq analysis reveals a loss of circadian patterns in the transcriptome of Bmal1 KO IVDs, and an unexpected emergence of 12 h ultradian rhythms in gene expression, including FOXO transcription factors. • Consistent with an up-regulation of FOXO1 mRNA and protein levels in Bmal1 KO IVDs, inhibition of FOXO1 in IVD cells suppresses their osteogenic differentiation. • Our data highlight the importance of the local BMAL1-driven molecular clock mechanism in the maintenance of cell fate and ECM homeostasis of the IVD. The circadian clock in mammals temporally coordinates physiological and behavioural processes to anticipate daily rhythmic changes in their environment. Chronic disruption to circadian rhythms (e.g., through ageing or shift work) is thought to contribute to a multitude of diseases, including degeneration of the musculoskeletal system. The intervertebral disc (IVD) in the spine contains circadian clocks which control ∼6% of the transcriptome in a rhythmic manner, including key genes involved in extracellular matrix (ECM) homeostasis. However, it remains largely unknown to what extent the local IVD molecular clock is required to drive rhythmic gene transcription and IVD physiology. In this work, we identified profound age-related changes of ECM microarchitecture and an endochondral ossification-like phenotype in the annulus fibrosus (AF) region of the IVD in the Col2a1 - Bmal1 knockout mice. Circadian time series RNA-Seq of the whole IVD in Bmal1 knockout revealed loss of circadian patterns in gene expression, with an unexpected emergence of 12 h ultradian rhythms, including FOXO transcription factors. Further RNA sequencing of the AF tissue identified region-specific changes in gene expression, evidencing a loss of AF phenotype markers and a dysregulation of ECM and FOXO pathways in Bmal1 knockout mice. Consistent with an up-regulation of FOXO1 mRNA and protein levels in Bmal1 knockout IVDs, inhibition of FOXO1 in AF cells suppressed their osteogenic differentiation. Collectively, these data highlight the importance of the local molecular clock mechanism in the maintenance of the cell fate and ECM homeostasis of the IVD. Further studies may identify potential new molecular targets for alleviating IVD degeneration. [ABSTRACT FROM AUTHOR]

Female night‐workers get exposed to frequent light shifts, hence have altered circadian rhythm and are at high risk of endometrial cancer; the underlying mechanism however is still not clear. We, therefore examined the effect of long light exposure (16L:8D, LD1) and regular shift (8 h) in long nighttime (LD2) on endometrial changes of female golden hamsters. Morphometric analysis, scanning electron microscopy imaging, alcian blue staining, and cytological nuclear atypia of endometrial stromal cells confirmed the incidence of endometrial adenocarcinoma in LD2 exposed hamsters. But, less severe pathomorphological alterations were noted in uterus of LD1 exposed hamsters. Altered Aanat and Bmal1 mRNA, melatonin rhythm, downregulation of important marker gene of adenocarcinoma like Akt, 14‐3‐3, and PR protein expression and upregulation PKCα, pAkt‐S473 and vascular epithelial growth factor (VEGF) were observed in LD2 exposed hamsters suggesting the endometrial adenocarcinoma. Further, our western blot analysis supported the immunohistochemical localization of PR, PKCα, and VEGF in uterine tissues along low progesterone. Overall, our data indicates that light shift and long light exposure potentially induced endometrioid adenocarcinoma via activation of PKC‐α/Akt pathway in female hamsters. Therefore, duration of light is essential for female normal uterine function. [ABSTRACT FROM AUTHOR]

During the aging process, the reduced osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) results in decreased bone formation, which contributes to senile osteoporosis. Previous studies have confirmed that interrupted circadian rhythm plays an indispensable role in age-related disease. However, the mechanism underlying the impaired osteogenic differentiation of BM-MSCs during aging and its relationship with interrupted circadian rhythm remains unclear. In this study, we confirmed that the circadian rhythm was interrupted in aging mouse skeletal systems. The level of the core rhythm component BMAL1 but not that of CLOCK in the osteoblast lineage was decreased in senile osteoporotic specimens from both human and mouse. BMAL1 targeted TTK as a circadian-controlled gene to phosphorylate MDM2 and regulate H2Bub1 level, while H2Bub1 in turn regulated the expression of BMAL1. The osteogenic capacity of BM-MSCs was maintained by a positive loop formed by BMAL1-TTK-MDM2-H2Bub1. Furthermore, we demonstrated that using bone-targeting recombinant adeno-associated virus 9 (rAAV9) to enhance Bmal1 or Ttk might have a therapeutic effect on senile osteoporosis and delays bone repair in aging mice. In summary, our study indicated that targeting the BMAL1-TTK-MDM2-H2Bub1 axis via bone-targeting rAAV9 might be a promising strategy for the treatment of senile osteoporosis.

Circadian rhythms, the natural cycles of physical, mental, and behavioral changes that follow a roughly 24-hour cycle, are known to have a profound effect on the human body. Light plays an important role in the regulation of circadian rhythm in human body. When light from the outside enters the eyes, cones, rods, and specialized retinal ganglion cells receive the light signal and transmit it to the suprachiasmatic nucleus of the hypothalamus. The central rhythm oscillator of the suprachiasmatic nucleus regulates the rhythm oscillator of tissues all over the body. Circadian rhythms, the natural cycles of physical, mental, and behavioral changes that follow a roughly 24-hour cycle, are known to have a profound effect on the human body. As the largest organ in the human body, skin plays an important role in the peripheral circadian rhythm regulation system. Like photoreceptor cells in the retina, melanocytes express opsins. Studies show that melanocytes in the skin are also sensitive to light, allowing the skin to “see” light even without the eyes. Upon receiving light signals, melanocytes in the skin release hormones that maintain homeostasis. This process is called “photoneuroendocrinology”, which supports the health effects of light exposure. However, inappropriate light exposure, such as prolonged work in dark environments or exposure to artificial light at night, can disrupt circadian rhythms. Such disruptions are linked to a variety of health issues, emphasizing the need for proper light management in daily life. Conversely, harnessing light’s beneficial effects through phototherapy is gaining attention as an adjunctive treatment modality. Despite these advancements, the field of circadian rhythm research still faces several unresolved issues and emerging challenges. One of the most exciting prospects is the use of the skin’s photosensitivity to treat diseases. This approach could revolutionize how we think about and manage various health conditions, leveraging the skin’s unique ability to respond to light for therapeutic purposes. As research continues to unravel the complexities of circadian rhythms and their impact on health, the potential for innovative treatments and improved wellbeing is immense.

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.

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.

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.

Background & Aims: Circadian rhythms play significant roles in immune responses, and many inflammatory processes in liver diseases are associated with malfunctioning molecular clocks. However, the significance of the circadian clock in autoimmune hepatitis (AIH), which is characterised by immune-mediated hepatocyte destruction and extensive inflammatory cytokine production, remains unclear. Methods: We tested the difference in susceptibility to the immune-mediated liver injury induced by concanavalin A (ConA) at various time points throughout a day in mice and analysed the effects of global, hepatocyte, or myeloid cell deletion of the core clock gene, Bmal1 (basic helix–loop–helix ARNT-like 1), on liver injury and inflammatory responses. Multiple molecular biology techniques and mice with macrophage-specific knockdown of Junb, a Bmal1 target gene, were used to investigate the involvement of Junb in the circadian control of ConA-induced hepatitis. Results: The susceptibility to ConA-induced liver injury is highly dependent on the timing of ConA injection. The treatment at Zeitgeber time 0 (lights on) triggers the highest mortality as well as the severest liver injury and inflammatory responses. Further study revealed that this timing effect was driven by macrophage, but not hepatocyte, Bmal1. Mechanistically, Bmal1 controls the diurnal variation of ConA-induced hepatitis by directly regulating the circadian transcription of Junb and promoting M1 macrophage activation. Inhibition of Junb in macrophages blunts the administration time-dependent effect of ConA and attenuates liver injury. Moreover, we demonstrated that Junb promotes macrophage inflammation by regulating AKT and extracellular signal-regulated kinase (ERK) signalling pathways. Conclusions: Our findings uncover a critical role of the Bmal1–Junb–AKT/ERK axis in the circadian control of ConA-induced hepatitis and provide new insights into the prevention and treatment of AIH. Impact and Implications: This study unveils a critical role of the Bmal1–Junb–AKT/ERK axis in the circadian control of ConA-induced liver injury, providing new insights into the prevention and treatment of immune-mediated hepatitis, including autoimmune hepatitis (AIH). The findings have scientific implications as they enhance our understanding of the circadian regulation of immune responses in liver diseases. Furthermore, clinically, this research offers opportunities for optimising treatment strategies in immune-mediated hepatitis by considering the timing of therapeutic interventions.

It is common for diabetic kidney disease (DKD) to be complicated by abnormal blood glucose, blood lipids, and blood pressure rhythms. Thus, it is essential to examine diagnostic and treatment plans from the perspective of circadian disruption. This brief review discusses the clinical relevance of circadian rhythms in DKD and how the core clock gene encoding brain and muscle arnt-like protein 1 (BMAL1) functions owing to the importance of circadian rhythm disruption processes, including the excretion of urinary protein and irregular blood pressure, which occur in DKD. Exploring Bmal1 and its potential mechanisms and signaling pathways in DKD following contact with Sirt1 and NF-κB is novel and important. Finally, potential pharmacological and behavioral intervention strategies for DKD circadian rhythm disturbance are outlined. This review aids in unveiling novel, potential molecular targets for DKD based on circadian rhythms.

Aims The circadian clock is an internal biological timer that co-ordinates physiology and gene expression with the 24-h solar day. Circadian clock perturbations have been associated to vascular dysfunctions in mammals, and a function of the circadian clock in angiogenesis has been suggested. However, the functional role of the circadian clock in endothelial cells (ECs) and in the regulation of angiogenesis is widely unexplored. Methods and results Here, we used both in vivo and in vitro approaches to demonstrate that ECs possess an endogenous molecular clock and show robust circadian oscillations of core clock genes. By impairing the EC-specific function of the circadian clock transcriptional activator basic helix-loop-helix ARNT like 1 (BMAL1) in vivo , we detect angiogenesis defects in mouse neonatal vascular tissues, as well as in adult tumour angiogenic settings. We then investigate the function of circadian clock machinery in cultured EC and show evidence that BMAL and circadian locomotor output cycles protein kaput knock-down impair EC cell cycle progression. By using an RNA- and chromatin immunoprecipitation sequencing genome-wide approaches, we identified that BMAL1 binds the promoters of CCNA1 and CDK1 genes and controls their expression in ECs. Conclusion(s) Our findings show that EC display a robust circadian clock and that BMAL1 regulates EC physiology in both developmental and pathological contexts. Genetic alteration of BMAL1 can affect angiogenesis in vivo and in vitro settings. [ABSTRACT FROM AUTHOR]

Understanding the physiology of human-induced pluripotent stem cells (iPSCs) is necessary for directed differentiation, mimicking embryonic development, and regenerative medicine applications. Pluripotent stem cells (PSCs) exhibit unique abilities such as self-renewal and pluripotency, but they lack some functions that are associated with normal somatic cells. One such function is the circadian oscillation of clock genes; however, whether or not PSCs demonstrate this capability remains unclear. In this study, the reason why circadian rhythm does not oscillate in human iPSCs was examined. This phenomenon may be due to the transcriptional repression of clock genes resulting from the hypermethylation of histone H3 at lysine 27 (H3K27), or it may be due to the low levels of brain and muscle ARNT-like 1 (BMAL1) protein. Therefore, BMAL1-overexpressing cells were generated and pre-treated with GSK126, an inhibitor of enhancer of zest homologue 2 (EZH2), which is a methyltransferase of H3K27 and a component of polycomb repressive complex 2. Consequently, a significant circadian rhythm following endogenous BMAL1, period 2 (PER2), and other clock gene expression was induced by these two factors, suggesting a candidate mechanism for the lack of rhythmicity of clock gene expression in iPSCs. [ABSTRACT FROM AUTHOR]

Increasing data suggest a crucial role of circadian rhythm in regulating metabolic and neurological diseases, and Bmal1 is regarded as a key regulator of circadian transcription. The aim of this study is to investigate the role of Bmal1 in the disruption of circadian rhythm and neuropsychiatric injuries in type 2 diabetes mellitus (T2DM). A T2DM model was induced by the combination of high-fat-diet (HFD) and streptozotocin (STZ) in vivo or HT-22 cells challenged with palmitic-acid (PA) in vitro. The glucolipid metabolism indicators, behavioral performance, and expression of synaptic plasticity proteins and circadian rhythm-related proteins were detected. These changes were also observed after interference of Bmal1 expression via overexpressed plasmid or small interfering RNAs in vitro. The results showed that HFD/STZ could induce T2DM-like glycolipid metabolic turmoil and abnormal neuropsychiatric behaviors in mice, as indicated by the increased concentrations of fasting blood-glucose (FBG), HbA1c and lipids, the impaired glucose tolerance, and the decreased preference index of novel object or novel arm in the novel object recognition test (NOR) and Y-maze test (Y-maze). Consistently, the protein expression of synaptic plasticity proteins and circadian rhythm-related proteins and the positive fluorescence intensity of MT1B and Bmal1 were decreased in the hippocampus of HFD/STZ-induced mice or PA-challenged HT-22 cells. Furthermore, overexpression of Bmal1 could improve the PA-induced lipid metabolic dysfunction and increase the decreased expressions of synaptic plasticity proteins and circadian rhythm-related proteins, and vice versa. These results suggested a crucial role of Bmal1 in T2DM-related glycolipid metabolic disorder and neuropsychiatric injury, which mechanism might be involved in the regulation of synaptic plasticity and circadian rhythms. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is a major leukemia with high mortality. Ferroptosis is an important regulator of cancers. However, the role of ferroptosis and its regulatory mechanisms in AML remain largely unknown. In this study, we reported elevated brain and muscle ARNT‐Like protein‐1 (Bmal1) expression in AML patients and cell lines, and its upregulation indicated the poor survival of patients. The correlation analysis showed that Bmal1 expression was closely correlated with cytogenetics and the French–American–British subtypes, but was not correlated with age, gender and white blood cells. RSL3 reduced Bmal1 expression in HL‐60 and NB4 cells. Malondialdehyde, total iron, Fe2+, glutathione and lipid peroxidation were examined to evaluate ferroptosis. Overexpression of Bmal1 repressed RSL3‐induced ferroptosis in AML cells. Bmal1 recruited Enhancer of zeste homolog 2 (EZH2) to the Early B cell factor 3 (EBF3) promoter and enhanced its methylation, thus suppressing EBF3 expression. Moreover, the knockdown of Bmal1 sensitized AML cells to RSL3‐induced ferroptosis, and it was counteracted by EBF3 knockdown. Furthermore, EBF3 bound to the Arachidonate 15‐pipoxygenase (ALOX15) promoter to enhance its expression, and overexpression of EBF3 enhanced RSL3‐induced ferroptosis dependent on ALOX5. We established a subcutaneous AML xenograft tumor model and reported that knockdown of Bmal1 and overexpression of EBF3 restrained AML growth by promoting ALOX15‐mediated ferroptosis in vivo. Collectively, Bmal1 inhibits RSL3‐induced ferroptosis by promoting EZH2‐mediated EBF3 methylation and suppressing the expression of EBF3 and ALOX15, thus accelerating AML. [ABSTRACT FROM AUTHOR]

While the suprachiasmatic nucleus (SCN) controls 24-h rhythms in breathing, including minute ventilation (VE), the mechanisms by which the SCN drives these daily changes are not well understood. Moreover, the extent to which the circadian clock regulates hypercapnic and hypoxic ventilatory chemoreflexes is unknown. We hypothesized that the SCN regulates daily breathing and chemoreflex rhythms by synchronizing the molecular circadian clock of cells. We used whole-body plethysmography to assess ventilatory function in transgenic BMAL1 knockout (KO) mice to determine the role of the molecular clock in regulating daily rhythms in ventilation and chemoreflex. Unlike their wild-type littermates, BMAL1 KO mice exhibited a blunted daily rhythm in VE and failed to demonstrate daily variation in the hypoxic ventilatory response (HVR) or hypercapnic ventilatory response (HCVR). To determine if the observed phenotype was mediated by the molecular clock of key respiratory cells, we then assessed ventilatory rhythms in BMAL1fl/fl; Phox2bCre/+ mice, which lack BMAL1 in all Phox2b-expressing chemoreceptor cells (hereafter called BKOP). BKOP mice lacked daily variation in HVR, similar to BMAL1 KO mice. However, unlike BMAL1 KO mice, BKOP mice exhibited circadian variations in VE and HCVR comparable to controls. These data indicate that the SCN regulates daily rhythms in VE, HVR, and HCVR, in part, through the synchronization of the molecular clock. Moreover, the molecular clock of Phox2b-expressing cells is specifically necessary for daily variation in the hypoxic chemoreflex. These findings suggest that disruption of circadian biology may undermine respiratory homeostasis, which, in turn, may have clinical implications for respiratory disease. Graphical Abstract [ABSTRACT FROM AUTHOR]

Danshen has been widely used for the treatment of central nervous system diseases. We investigated the effect of dihydroisotanshinone I (DT), a compound extracted from Danshen, as well as the corresponding mechanisms in an in vitro-based 6-OHDA-induced Parkinson's disease (PD) model. SH-SY5Y human neuroblastoma cell lines were pretreated with 6-hydroxydopamine (6-OHDA) and challenged with DT. Subsequently, the cell viability and levels of reactive oxygen species (ROS) and caspase-3 were analyzed. The effect of DT on the 6-OHDA-treated SH-SY5Y cells and the expression of the core circadian clock genes were measured using a real-time quantitative polymerase chain reaction. Our results indicated that DT attenuated the 6-OHDA-induced cell death in the SH-SY5Y cells and suppressed ROS and caspase-3. Moreover, DT reversed both the RNA and protein levels of BMAL1 and SIRT1 in the 6-OHDA-treated SH-SY5Y cells. Additionally, the SIRT1 inhibitor attenuated the effect of DT on BMAL1 and reduced the cell viability. The DT and SIRT1 activators activated SIRT1 and BMAL1, and then reduced the death of the SH-SY5Y cells damaged by 6-OHDA. SIRT1 silencing was enhanced by DT and resulted in a BMAL1 downregulation and a reduction in cell viability. In conclusion, our investigation suggested that DT reduces cell apoptosis, including an antioxidative effect due to a reduction in ROS, and regulates the circadian genes by enhancing SIRT1 and suppressing BMAL1. DT may possess novel therapeutic potential for PD in the future, but further in vivo studies are still needed. [ABSTRACT FROM AUTHOR]

Physiological activities of the body exhibit an obvious biological rhythm. At the core of the circadian rhythm, BMAL1 is the only clock gene whose deletion leads to abnormal physiological functions. However, whether intermittent heat stress influences cardiovascular function by altering the circadian rhythm of clock genes has not been reported. This study aimed to investigate whether intermittent heat stress induces autophagy and apoptosis, and the effects of BMAL1 on thoracic aortic autophagy and apoptosis. An intermittent heat stress model was established in vitro , and western blotting and immunofluorescence were used to detect the expression of autophagy, apoptosis, the AMPK/mTOR/ULK1 pathway, and BMAL1. After BMAL1 silencing, RT-qPCR was performed to detect the expression levels of autophagy and apoptosis-related genes. Our results suggest that heat stress induces autophagy and apoptosis in RTAECs. In addition, intermittent heat stress increased the phosphorylation of AMPK and ULK1, but reduced the phosphorylation of mTOR, AMPK inhibitor Compound C reversed the phosphorylation of AMPK, mTOR, and ULK1, and Beclin1 and LC3-II/LC3-I were downregulated. Furthermore, BMAL1 expression was elevated in vitro and shBMAL1 decreased autophagy and apoptosis. We revealed that intermittent heat stress induces autophagy and apoptosis, and that BMAL1 may be involved in the occurrence of autophagy and apoptosis. • Intermittent heat stress increased the expression of BMAL1, autophagy and apoptosis in RTAECs. • AMPK/mTOR/ULK1 pathway involved in the autophagy occurrence. • BMAL1 plays a role in autophagy and apoptosis in RTAECs under heat stress. [ABSTRACT FROM AUTHOR]

Sleep and circadian disruption are associated with an increased risk of metabolic disease, including obesity and diabetes. Mounting evidence indicates that misaligned and/or non-functional clock proteins in peripheral tissues critically contribute to the presentation of metabolic disease. Many of the foundational studies which led to this conclusion have focused on specific tissues such as the adipose, pancreas, muscle, and liver. While these studies have greatly advanced the field, the use of anatomical markers to manipulate tissue-specific molecular clocks may not be representative of the circadian disruption that occurs within the clinical population. In this manuscript, we argue that investigators can gain a better understanding of the consequences of sleep and circadian disruption by targeting groups of cells with a functional relationship, even if those cells go beyond anatomical boundaries. This approach is especially important when considering metabolic outcomes which rely on endocrine signaling molecules, such as leptin, that have multiple sites of action. Through the review of several studies, as well as our own work, this article reframes peripheral clock disruption from a functional approach. We additionally present new evidence that disruption of the molecular clock within all cells expressing the leptin receptor affects leptin sensitivity in a time-dependent manner. Taken together, this perspective aims to provide new insight into the mechanisms leading to metabolic disease associated with circadian disruption and various sleep disorders. [ABSTRACT FROM AUTHOR]

Circadian misalignment occurs with age, jet lag, and shift work, leading to maladaptive health outcomes including cardiovascular diseases. Despite the strong link between circadian disruption and heart disease, the cardiac circadian clock is poorly understood, prohibiting identification of therapies to restore the broken clock. Exercise is the most cardioprotective intervention identified to date and has been suggested to reset the circadian clock in other peripheral tissues. Here, we tested the hypothesis that conditional deletion of core circadian gene Bmal1 would disrupt cardiac circadian rhythm and function and that this disruption would be ameliorated by exercise. To test this hypothesis, we generated a transgenic mouse with spatial and temporal deletion of Bmal1 only in adult cardiac myocytes (Bmal1 cardiac knockout [cKO]). Bmal1 cKO mice demonstrated cardiac hypertrophy and fibrosis concomitant with impaired systolic function. This pathological cardiac remodeling was not rescued by wheel running. While the molecular mechanisms responsible for the profound cardiac remodeling are unclear, it does not appear to involve activation of the mammalian target of rapamycin (mTOR) signaling or changes in metabolic gene expression. Interestingly, cardiac deletion of Bmal1 disrupted systemic rhythms as evidenced by changes in the onset and phasing of activity in relationship to the light/dark cycle and by decreased periodogram power as measured by core temperature, suggesting cardiac clocks can regulate systemic circadian output. Together, we suggest a critical role for cardiac Bmal1 in regulating both cardiac and systemic circadian rhythm and function. Ongoing experiments will determine how disruption of the circadian clock causes cardiac remodeling in an effort to identify therapeutics to attenuate the maladaptive outcomes of a broken cardiac circadian clock. [ABSTRACT FROM AUTHOR]

Nobiletin (NOB), a naturally occurring small-molecule compound abundant in citrus peels, has displayed potential lipid-lowering and circadian-enhancing properties in preclinical studies. However, the requirement of specific clock genes for the beneficial effects of NOB is not well understood. In the current study, mice with a liver-specific deletion of the core clock component, Bmal1—Bmal1LKO—were fed a high-fat diet (HFD) ad libitum for eight weeks, while NOB (200 mg/kg) was administered by daily oral gavage from the fifth week and throughout the last four weeks. NOB decreased liver triglyceride (TG) alongside the decreasing mRNA levels of de novo lipogenesis (DNL) genes in both Bmal1flox/flox and Bmal1LKO mice. NOB increased serum very low-density lipoprotein (VLDL) levels in Bmal1LKO mice, which was consistent with higher liver Shp and lower Mttp mRNA expression levels, the key genes that facilitate VLDL assembly and secretion. NOB decreased liver and serum cholesterol levels in the Bmal1flox/flox mice, consistent with lower Hmgcr and higher Cyp7a1, Cyp8b1, Gata4 and Abcg5 mRNA levels in the liver. In contrast, in the Bmal1LKO mice, NOB increased Hmgcr mRNA levels and had no effect on the above-mentioned genes related to bile acid synthesis and cholesterol excretion, which might contribute to the elevation of liver and serum cholesterol levels in NOB-treated Bmal1LKO mice. NOB inhibited hepatic DNL and decreased liver TG levels in HFD-fed mice independently of liver Bmal1, whereas liver-specific Bmal1 depletion reversed the beneficial effects of NOB on liver cholesterol homeostasis. The complex interactions between NOB, the circadian clock and lipid metabolism in the liver warrant further research. [ABSTRACT FROM AUTHOR]

Rationale: Mechanical ventilation (MV) is life-saving but may evoke ventilator-induced lung injury (VILI). Objectives: To explore how the circadian clock modulates severity of murine VILI via the core clock component BMAL1 (basic helix-loop-helix ARNT like 1) in myeloid cells. Methods: Myeloid cell BMAL1-deficient (LysM (lysozyme 2 promoter/enhancer driving cre recombinase expression)Bmal1−/−) or wild-type control (LysMBmal1+/+) mice were subjected to 4 hours MV (34 ml/kg body weight) to induce lung injury. Ventilation was initiated at dawn or dusk or in complete darkness (circadian time [CT] 0 or CT12) to determine diurnal and circadian effects. Lung injury was quantified by lung function, pulmonary permeability, blood gas analysis, neutrophil recruitment, inflammatory markers, and histology. Neutrophil activation and oxidative burst were analyzed ex vivo. Measurements and Main Results: In diurnal experiments, mice ventilated at dawn exhibited higher permeability and neutrophil recruitment compared with dusk. Experiments at CT showed deterioration of pulmonary function, worsening of oxygenation, and increased mortality at CT0 compared with CT12. Wild-type neutrophils isolated at dawn showed higher activation and reactive oxygen species production compared with dusk, whereas these day–night differences were dampened in LysMBmal1−/− neutrophils. In LysMBmal1−/− mice, circadian variations in VILI severity were dampened and VILI-induced mortality at CT0 was reduced compared with LysMBmal1+/+ mice. Conclusions: Inflammatory response and lung barrier dysfunction upon MV exhibit diurnal variations, regulated by the circadian clock. LysMBmal1−/− mice are less susceptible to ventilation-induced pathology and lack circadian variation of severity compared with LysMBmal1+/+ mice. Our data suggest that the internal clock in myeloid cells is an important modulator of VILI. [ABSTRACT FROM AUTHOR]

The circadian clock coordinates a variety of immune responses with signals from the external environment to promote survival. We investigated the potential reciprocal relationship between the circadian clock and skin inflammation. We treated mice topically with the Toll-like receptor 7 (TLR7) agonist imiquimod (IMQ) to activate IFN-sensitive gene (ISG) pathways and induce psoriasiform inflammation. IMQ transiently altered core clock gene expression, an effect mirrored in human patient psoriatic lesions. In mouse skin 1 d after IMQ treatment, ISGs, including the key ISG transcription factor IFN regulatory factor 7 (Irf7), were more highly induced after treatment during the day than the night. Nuclear localization of phosphorylated-IRF7 was most prominently time-of-day dependent in epidermal leukocytes, suggesting that these cell types play an important role in the diurnal ISG response to IMQ. Mice lacking Bmal1 systemically had exacerbated and arrhythmic ISG/Irf7 expression after IMQ. Furthermore, daytime-restricted feeding, which affects the phase of the skin circadian clock, reverses the diurnal rhythm of IMQ-induced ISG expression in the skin. These results suggest a role for the circadian clock, driven by BMAL1, as a negative regulator of the ISG response, and highlight the finding that feeding time can modulate the skin immune response. Since the IFN response is essential for the antiviral and antitumor effects of TLR activation, these findings are consistent with the time-of-day-dependent variability in the ability to fight microbial pathogens and tumor initiation and offer support for the use of chronotherapy for their treatment.

Abstract Background Fine particulate matter (PM2.5) is associated with increased incidence and severity of asthma. PM2.5 exposure disrupts airway epithelial cells, which elicits and sustains PM2.5-induced airway inflammation and remodeling. However, the mechanisms underlying development and exacerbation of PM2.5-induced asthma were still poorly understood. The aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) is a major circadian clock transcriptional activator that is also extensively expressed in peripheral tissues and plays a crucial role in organ and tissue metabolism. Results In this study, we found PM2.5 aggravated airway remodeling in mouse chronic asthma, and exacerbated asthma manifestation in mouse acute asthma. Next, low BMAL1 expression was found to be crucial for airway remodeling in PM2.5-challenged asthmatic mice. Subsequently, we confirmed that BMAL1 could bind and promote ubiquitination of p53, which can regulate p53 degradation and block its increase under normal conditions. However, PM2.5-induced BMAL1 inhibition resulted in up-regulation of p53 protein in bronchial epithelial cells, then increased-p53 promoted autophagy. Autophagy in bronchial epithelial cells mediated collagen-I synthesis as well as airway remodeling in asthma. Conclusions Taken together, our results suggest that BMAL1/p53-mediated bronchial epithelial cell autophagy contributes to PM2.5-aggravated asthma. This study highlights the functional importance of BMAL1-dependent p53 regulation during asthma, and provides a novel mechanistic insight into the therapeutic mechanisms of BMAL1. Graphic abstract Video Abstract

Abstract Aging is associated with loss of circadian immune responses and circadian gene transcription in peripheral macrophages. Microglia, the resident macrophages of the brain, also show diurnal rhythmicity in regulating local immune responses and synaptic remodeling. To investigate the interaction between aging and microglial circadian rhythmicity, we examined mice deficient in the core clock transcription factor, BMAL1. Aging Cd11b cre ;Bmal lox/lox mice demonstrated accelerated cognitive decline in association with suppressed hippocampal long-term potentiation and increases in immature dendritic spines. C1q deposition at synapses and synaptic engulfment were significantly decreased in aging Bmal1-deficient microglia, suggesting that BMAL1 plays a role in regulating synaptic pruning in aging. In addition to accelerated age-associated hippocampal deficits, Cd11b cre ;Bmal lox/lox mice also showed deficits in the sleep–wake cycle with increased wakefulness across light and dark phases. These results highlight an essential role of microglial BMAL1 in maintenance of synapse homeostasis in the aging brain.

Abstract Background Cancer metastasis is still a life threat to patients with colorectal cancer (CRC). Brain and muscle ARNT‐like protein 1 (BMAL1) is an important biological proteins that can regulate the behavior of cancer cells and their response to chemotherapy. However, the role of BMAL1 in the tumorigenic phenotype of CRC remains unclear. Here, we aim to investigate the functional role and mechanisms of BMAL1 in CRC. Methods The mRNA expression of BMAL1 was studied using the Cancer Genome Atlas (TCGA) databases. The protein level in clinical tissues was confirmed by immunohistochemistry (IHC). The effects of BMAL1 on the epithelial‐to‐mesenchymal transition (EMT) and proliferation of CRC cell lines (including BMAL1 overexpressed or silencing cells) were studied by Transwell, wound healing, CCK‐8 and colony formation experiments. A series of experiments were conducted to demonstrate the mechanisms of BMAL1 regulating EMT and cancer proliferation in vitro and in vivo. Results We found that BMAL1 expression was closely related to the poor prognosis of CRC. BMAL1 overexpression promoted cell proliferation and migration. Mechanistically, we found that BMAL1 may activate the epithelial‐to‐mesenchymal transition (EMT) pathway and induce the β‐catenin release further promotes the expression of oncogene c‐Myc and the migration of colorectal cells by activating MAPK pathway. However, BMAL1 silencing achieved the opposite effect. In addition, blocking MAPK‐signaling pathway with specific inhibitors of ERK1/2 and JNK can also downregulate the expressions of c‐Myc in vitro. Taken together, these results suggested that the BMAL1/ c‐Myc‐signaling pathway may regulate the metastasis of CRC through the JNK/ERK1/2 MAPK‐dependent pathway. Conclusions Our study showed that BMAL1 promotes CRC metastasis through MAPK‐c‐Myc pathway. These results deepen our understanding of the relationship between BMAL1 and tumorigenic phenotypes, which may become a promising therapeutic target for BMAL1 overexpressing CRC.

Circadian rhythms are natural rhythms that widely exist in all creatures, and regulate the processes and physiological functions of various biochemical reactions. The circadian clock is critical for cancer occurrence and progression. Its function is regulated by metabolic activities, and the expression and transcription of various genes. This review summarizes the composition of the circadian clock; the biological basis for its function; its relationship with, and mechanisms in, cancer; its various functions in different cancers; the effects of anti-tumor treatment; and potential therapeutic targets. Research in this area is expected to advance understanding of circadian locomotor output cycles kaput (CLOCK) and brain and muscle ARNT-like protein 1 (BMAL1) in tumor diseases, and contribute to the development of new anti-tumor treatment strategies.

Abstract Bmal1 (Brain and muscle arnt-like, or Arntl) is a bHLH/PAS domain transcription factor central to the transcription/translation feedback loop of the biologic clock. Although Bmal1 is well-established as a major regulator of circadian rhythm, a growing number of studies in recent years have shown that dysfunction of Bmal1 underlies a variety of psychiatric, neurodegenerative-like, and endocrine metabolism-related disorders, as well as potential oncogenic roles. In this review, we systematically summarized Bmal1 expression in different brain regions, its neurological functions related or not to circadian rhythm and biological clock, and pathological phenotypes arising from Bmal1 knockout. This review also discusses oscillation and rhythmicity, especially in the suprachiasmatic nucleus, and provides perspective on future progress in Bmal1 research.

Introduction: Chronic hyperglycemia is associated with an increase in cellular damage due to oxidative stress in pancreatic tissue. The effect of exercise in different phases of the circadian cycle on protecting pancreatic tissue from oxidative stress in diabetic patients is unknown. The aim of this study was to investigate the effect of eight weeks of endurance training in light and dark phases of circadian rhythm on the oxidative stress index in pancreas of diabetic mice. Methods: In this study, 18 mice from the Naval Medical Research Institute (26±3.22 gr) were selected and after inducing diabetes through high-fat food and Streptozotocin injection (20 mg/kg), randomly divided into 6 groups: light phase healthy control, dark phase healthy control, light phase diabetic control, dark phase diabetic control, light phase diabetic training and dark phase diabetic training. The endurance training protocol (50-60 %Vmax) was performed 5 d/w for 8 weeks. After anesthesia, blood samples and pancreatic tissues were removed. Insulin resistance markers, oxidative stress index and expression of Brain and Muscle ARNT-Like1 protein expression were measured in pancreas of diabetic rats. Data were analyzed by one way analysis of variance at the significance level of p

An emerging role for the circadian clock in autophagy and lysosome function has opened new avenues for exploration in the field of neurodegeneration. The daily rhythms of circadian clock proteins may coordinate gene expression programs involved not only in daily rhythms but in many cellular processes. In the brain, astrocytes are critical for sensing and responding to extracellular cues to support neurons. The core clock protein BMAL1 serves as the primary positive circadian transcriptional regulator and its depletion in astrocytes not only disrupts circadian function but also leads to a unique cell-autonomous activation phenotype. We report here that astrocyte-specific deletion of Bmal1 influences endolysosome function, autophagy, and protein degradation dynamics. In vitro, Bmal1-deficient astrocytes exhibit increased endocytosis, lysosome-dependent protein cleavage, and accumulation of LAMP1- and RAB7-positive organelles. In vivo, astrocyte-specific Bmal1 knockout (aKO) brains show accumulation of autophagosome-like structures within astrocytes by electron microscopy. Transcriptional analysis of isolated astrocytes from young and aged Bmal1 aKO mice indicates broad dysregulation of pathways involved in lysosome function which occur independently of TFEB activation. Since a clear link has been established between neurodegeneration and endolysosome dysfunction over the course of aging, this work implicates BMAL1 as a key regulator of these crucial astrocyte functions in health and disease [ABSTRACT FROM AUTHOR]

Nicotine has shown the toxic effects on male reproductive system, and testicular damage is associated with ferroptosis, which is a non-apoptotic regulated cell death driven by iron-dependent lipid peroxidation. However, the role of nicotine on ferroptosis of testicular cells is largely elusive. In the present study, we showed that nicotine destroyed blood-testis barrier (BTB) by interfering with the circadian rhythm of BTB-related factors (ZO-1, N-Cad, Occludin and CX-43) and induced ferroptosis, as reflected via increased clock-control levels of lipid peroxide and decreased ferritin and GPX4, which involved in the circadian. Inhibition of ferroptosis with Fer-1 alleviated nicotine-induced injury of BTB and impaired sperm in vivo. Mechanically, we uncover that the core molecular clock protein, Bmal1, regulates the expression of Nrf2 via direct E-box binding to its promoter to regulate its activity, and nicotine decreases the transcription of Nrf2 through Bmal1 and inactivates Nrf2 pathway and its downstream antioxidant gene, which leads to the imbalance of redox state and ROS accumulation. Intriguingly, nicotine induced lipid peroxidation and subsequent ferroptosis by Bmal1-mediated Nrf2. In conclusion, our study reveals a clear role for the molecular clock in controlling Nrf2 in testis to mediate the ferroptosis induced by nicotine. These findings provide a potential mechanism to prevent smoking and/or cigarette smoke-induced male reproductive injury. [Display omitted] • Nicotine interfered with the rhythmic expression of BTB-related proteins, leading to BTB injury. • Nicotine induced BTB damage by causing the ferroptosis in the testis. • Nicotine decreases the transcription of Nrf2 through Bmal1 and inactivates Nrf2 pathway and its downstream antioxidant gene. • Nicotine contributed to ferroptosis via inactivation of the Bmal1/Nrf2 pathway. [ABSTRACT FROM AUTHOR]

Bone mass declines with age and its maintenance is tightly linked to osteoblasts (crucial bone-building cells). Although disruption of the peripheral circadian clock is involved in various pathologies including aging-related diseases, evidence regarding how the peripheral clock regulates bone mass remains elusive. In the present study, we aimed to elucidate the effects of Bmal1 (the key activator of the peripheral circadian clock system) knockdown by lentivirus-mediated shRNA on osteoblast differentiation and its related mechanisms. We found that the expression of osteogenic markers, alkaline phosphatase activity, and mineralization were decreased, whereas apoptosis and inflammatory response were increased in Bmal1 knockdown osteoblasts. In addition, Bmal1 knockdown promoted ERK and JNK phosphorylation, as well as mTOR activity, whereas mTOR inhibition by rapamycin abrogated Bmal1 knockdown-mediated effects on osteoblast differentiation and mineralization capacity. Remarkably, Bmal1 knockdown in osteoblasts inhibited GSK3β/β-catenin signaling with decreased β-catenin expression and GSK-3β phosphorylation at serine 9, while GSK3β inhibition with TDZD-8, but not WNT3a or SKL2001, rescued Bmal1 knockdown-induced defects in osteoblast differentiation. Moreover, rapamycin partly nullified the suppression of Bmal1 knockdown on β-catenin expression and GSK-3β phosphorylation. Collectively, overall data indicated that circadian gene Bmal1 regulated osteoblast differentiation and inflammatory response in an mTOR/GSK3β/β-catenin-dependent manner, and thereby may contribute to the mineralization process and bone modeling/remodeling. [ABSTRACT FROM AUTHOR]

Hepatic ischemia-reperfusion injury (HIRI) significantly contributes to liver dysfunction following liver transplantation and hepatectomy. However, the role of the celiac ganglion (CG) in HIRI remains unclear. Adeno-associated virus was used to silence Bmal1 expression in the CG of twelve beagles that were randomly assigned to the Bmal1 knockdown group (KO-Bmal1) and the control group. After four weeks, a canine HIRI model was established, and CG, liver tissue, and serum samples were collected for analysis. The virus significantly downregulated Bmal1 expression in the CG. Immunofluorescence staining confirmed a lower proportion of c-fos+ and NGF+ neurons in TH+ cells in the KO-Bmal1 group than in the control group. The KO-Bmal1 group exhibited lower Suzuki scores and serum ALT and AST levels than the control group. Bmal1 knockdown significantly reduced liver fat reserve, hepatocyte apoptosis, and liver fibrosis, and it increased liver glycogen accumulation. We also observed that Bmal1 downregulation inhibited the hepatic neurotransmitter norepinephrine, neuropeptide Y levels, and sympathetic nerve activity in HIRI. Finally, we confirmed that decreased Bmal1 expression in CG reduces TNF-α, IL-1β, and MDA levels and increases GSH levels in the liver. The downregulation of Bmal1 expression in CG suppresses neural activity and improves hepatocyte injury in the beagle model after HIRI. [ABSTRACT FROM AUTHOR]

Asthma often presents with a daily rhythm; however, the underlying mechanisms remain unclear. Circadian rhythm genes have been proposed to regulate inflammation and mucin expression. Here, ovalbumin (OVA)-induced mice and serum shock human bronchial epidermal cells (16HBE) were used in in vivo and in vitro models, respectively. We constructed a brain and muscle ARNT-like 1 (BMAL1) knockdown 16HBE cell line to analyze the effects of rhythmic fluctuations on mucin expression. Serum immunoglobulin E (IgE) and circadian rhythm genes in asthmatic mice showed rhythmic fluctuation amplitude. Mucin (MUC) 1 and MUC5AC expression was increased in the lung tissue of the asthmatic mice. MUC1 expression was negatively correlated with that of the circadian rhythm genes, particularly BMAL1 (r = −0.546, P = 0.006). There was also a negative correlation between BMAL1 and MUC1 expression (r = −0.507, P = 0.002) in the serum shock 16HBE cells. BMAL1 knockdown negated the rhythmic fluctuation amplitude of MUC1 expression and upregulated MUC1 expression in the 16HBE cells. These results indicate that the key circadian rhythm gene, BMAL1 , causes periodic changes in airway MUC1 expression in OVA-induced asthmatic mice. Targeting BMAL1 to regulate periodic changes in MUC1 expression may, therefore, improve asthma treatments. • Rhythm gene disorders in OVA asthmatic mice manifested by an increased amplitude of transcriptional oscillations. • MUC1 was accompanied by rhythmic oscillations in asthma due to BMAL1 regulation. • The serum IgE levels observed a rhythmic fluctuation in the OVA-challenged mice. [ABSTRACT FROM AUTHOR]

Circadian dysfunction is a common non-motor symptom in Parkinson's disease (PD). The potential influence of aggravated α-synuclein (SNCA) on circadian disruption remains unclear. SNCAA53T-overexpressing transgenic mice (SNCAA53T mice) and wild-type (WT) littermates were used in this study. The energy metabolism cage test showed differences in 24-h activity pattern between SNCAA53T and WT mice. When compared with the age-matched littermates, brain and muscle ARNT-like 1 (BMAL1) was downregulated in SNCAA53T mice. BMAL1 was downregulated in PC12 cells overexpressing SNCA. Degradation of BMAL1 protein remained unchanged after overexpression of SNCA, while its mRNA level decreased. miRNA (miR)-155 was upregulated by overexpression of SNCA, and downregulation of BMAL1 was partially reversed by transfection with miR-155 inhibitor. Our findings demonstrated that overexpression of SNCA induced biorhythm disruption and downregulated BMAL1 expression through decreasing stability of BMAL1 mRNA via miR-155. [ABSTRACT FROM AUTHOR]

Artepillin C (APC), a cAMP-response element−binding (CREB)/CREB regulated transcription coactivator 2 (CRTC2) inhibitor isolated from Brazilian green propolis, can ameliorate metabolic syndrome in obese mice. Because the sensitivity and responsiveness of the body to the drug depend on the time of day and the circadian clock alignment, the optimal administration time of APC for desired efficacy in treating metabolic syndrome remains unclear. In this study, APC (20 mg/kg) or the vehicle was intraperitoneally injected into obese mice once daily for one or three weeks. The results of the insulin tolerance test, pyruvate tolerance test, and histological and biochemical assays showed that APC could improve whole−body glucose homeostasis and decrease hepatic lipid synthesis following a circadian rhythm. Further exploration of the underlying mechanism revealed that APC may disturb the diurnal oscillations of the expression of brain and muscle ARNT−like protein (BMAL1) in primary hepatocytes and the livers of the study subjects. Moreover, APC could inhibit hepatic BMAL1 expression by blocking the CREB/CRTC2 transcription complex. BMAL1 overexpression in primary hepatocytes or the livers of db/db mice antagonized the inhibitory effect of APC on hepatic lipid metabolism. In conclusion, the chronotherapy of APC may relieve metabolic syndrome in obese mice, and the mechanism behind APC−mediated time−of−day effects on metabolic syndrome were unveiled, thereby providing a foundation for optimized APC treatment from a mechanistic perspective. [ABSTRACT FROM AUTHOR]

Periodically oscillating biological processes, such as circadian rhythms, are carefully concerted events that are only beginning to be understood in the context of tissue pathology and organismal health, as well as the molecular mechanisms underlying these interactions. Recent reports indicate that light can independently entrain peripheral circadian clocks, challenging the currently prevalent hierarchical model. Despite the recent progress that has been made, a comprehensive overview of these periodic processes in skin is lacking in the literature. In this review, molecular circadian clock machinery and the factors that govern it have been highlighted. Circadian rhythm is closely linked to immunological processes and skin homeostasis, and its desynchrony can be linked to the perturbation of the skin. The interplay between circadian rhythm and annual, seasonal oscillations, as well as the impact of these periodic events on the skin, is described. Finally, the changes that occur in the skin over a lifespan are presented. This work encourages further research into the oscillating biological processes occurring in the skin and lays the foundation for future strategies to combat the adverse effects of desynchrony, which would likely have implications in other tissues influenced by periodic oscillatory processes. [ABSTRACT FROM AUTHOR]

Deoxynivalenol (DON) is widely emerging in various grain crops, milk, and wine products, which can trigger different toxic effects on humans and animals by inhalation or ingestion. It also imposes a considerable financial loss on the agriculture and food industry each year. Previous studies have reported acute and chronic toxicity of DON in liver, and liver is not only the main detoxification organ for DON but also the circadian clock oscillator directly or indirectly regulates critical physiologically hepatic functions under different physiological and pathological conditions. However, researches on the association of circadian rhythm in DON-induced liver damage are limited. In the present study, mice were divided into four groups (CON, DON, Bmal1OE, and Bmal1OE + DON) and AAV8 was used to activate (Bmal1) expression in liver. Then mice were gavaged with 5 mg/kg bw/day DON or saline at different time points (ZT24 = 0, 4, 8, 12, 16, and 20 h) in 1 day and were sacrificed 30 min after oral gavage. The inflammatory cytokines, signal transducers, and activators of transcription Janus kinase/signal transducers and activator of transcription 3 (JAKs/STAT3) pathway and bile acids levels were detected by enzyme-linked immunosorbent assay (ELISA), western blotting, and target metabolomics, respectively. The DON group showed significantly elevated interleukin-1β (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α) levels (P < 0.05 for both) and impaired liver function with rhythm disturbances compared to the CON and Bmal1OE groups. At the molecular level, expressions of some circadian clock proteins were significantly downregulated (P < 0.05 for both) and JAKs/STAT3 pathway was activated during DON exposure, accompanied by indicated circadian rhythm disturbance and inflammatory damage. Importantly, Bmal1 overexpression attenuated DON-induced liver damage, while related hepatic bile acids such as cholic acid (CA) showed a decreasing trend in the DON group compared with the CON group. Our study demonstrates a novel finding that Bmal1 plays a critical role in attenuating liver damage by inhibiting inflammatory levels and maintaining bile acids levels under the DON condition. Therefore, Bmal1 may also be a potential molecular target for reducing the hepatotoxic effects of DON in future studies. [ABSTRACT FROM AUTHOR]

Sex differences in alcohol use and abuse are pervasive and carry important implications for the prevention and treatment of alcohol use disorder (AUD), yet insight into underlying sexually dimorphic mechanisms is limited. Growing experimental and clinical evidence points to an important influence of circadian rhythms and circadian clock genes in the control of alcohol drinking behavior and AUD. Sex differences in the expression of circadian rhythms and in the molecular circadian clock that drive these rhythms have been reported in humans and animals. While studying the role of striatal circadian clock gene expression in the control of affective and goal-directed behaviors, we uncovered a novel sexually dimorphic function of the clock genes Bmal1 and Per2 in the control of voluntary alcohol consumption in mice, which may contribute to sex differences in alcohol drinking behavior. In this mini review, we briefly discuss relevant literature on AUD, circadian rhythms and clock genes, and on sex differences in these domains, and describe our own findings on clock genes as sexually dimorphic regulators of alcohol drinking behavior in mice. [ABSTRACT FROM AUTHOR]

The circadian clock is a timekeeping system for numerous biological rhythms that contribute to the regulation of numerous homeostatic processes in humans. Disruption of circadian rhythms influences physiology and behavior and is associated with adverse health outcomes, especially cancer. However, the underlying molecular mechanisms of circadian disruption-associated cancer initiation and development remain unclear. It is essential to construct good circadian disruption models to uncover and validate the detailed molecular clock framework of circadian disruption in cancer development and progression. Mouse models are the most widely used in circadian studies due to their relatively small size, fast reproduction cycle, easy genome manipulation, and economic practicality. Here, we reviewed the current mouse models of circadian disruption, including suprachiasmatic nuclei destruction, genetic engineering, light disruption, sleep deprivation, and other lifestyle factors in our understanding of the crosstalk between circadian rhythms and oncogenic signaling, as well as the molecular mechanisms of circadian disruption that promotes cancer growth. We focused on the discoveries made with the nocturnal mouse, diurnal human being, and cell culture and provided several circadian rhythm-based cancer therapeutic strategies. [ABSTRACT FROM AUTHOR]

Brain and muscle arnt-like protein 1 (Bmal1) is a crucial transcription factor, regulating circadian rhythm and involved in multiple heart diseases. However, it is unknown whether Bmal1 promotes diabetic cardiomyopathy (DCM) pathogenesis. The objective of this investigation was to ascertain the vital role of Bmal1 in the progression of DCM. Mice with T2D and H9c2 cardiomyoblasts exposed to high glucose and palmitic acid (HGHP) were used. Cardiomyocyte-specific knockout mouse of Bmal1 (CKB) was also generated, and cardiac Bmal1 was overexpressed in type 2 diabetes (T2D) mice using an adeno-associated virus. Bmal1 gene recombinant adenovirus was used to either knockdown or overexpress in H9c2 cardiomyoblasts. Bmal1 expression was significantly altered in diabetic mice hearts. Bmal1 downregulation in CKB and T2D mice heart accelerated cardiac hypertrophy and diastolic dysfunction, while Bmal1 overexpression ameliorated these pathological changes in DCM mice. Furthermore, DCM mice had significant mitochondrial ultrastructural defects, reactive oxygen species accumulation, and apoptosis, which could be alleviated by overexpressing Bmal1. In H9c2 cardiomyoblasts, genetic downregulation of Bmal1 or HGHP markedly decreased the binding of Bcl2 to IP3R, thus increasing Ca2+ release to mitochondria through mitochondria-associated endoplasmic reticulum membranes. Importantly, chromatin immunoprecipitation revealed Bmal1 could bind directly to the Bcl2 gene promoter region. Bmal1 overexpression augmented the Bmal1/Bcl2 binding, enhancing the inhibition of Bcl2 on IP3R activity, thus alleviating mitochondrial Ca2+ overload and subsequent cell apoptosis. These results show that Bmal1 is involved in the DCM development through Bcl2/IP3R-mediated mitochondria Ca2+ overload. Therapy targeting the circadian clock (Bmal1) can treat DCM.

Coronavirus disease 2019 (COVID-19), is known as one of the most known challenge worldwide. Numerous studies have tried to introduce different mechanisms involved in the pathophysiology of COVID-19 and efforts in this field are also ongoing. The presence of SARS-CoV-2 RNA in feces of COVID-19 patients along with a variety of gastrointestinal symptoms may show a significant association between gut microbiota and SARS-CoV-2 infection. However, the exact mechanism indicating how SARS-CoV-2 and gut flora influence each other remains unknown. This paper aims to introduce a possible molecular mechanism based on recent findings on the association between circadian rhythm and gut flora in COVID-19 patients to express a new insight into the probable mechanism of melatonin in protection against SARS-CoV-2 infection.

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.

Bmal1 is the only single circadian clock gene that is essential for rhythmic gene expression in the mammalian circadian timing system. Genetic approaches targeting Bmal1 expression have been used to further assess its role in the circadian clock and to test for behavioral effects of clock disruption. In particular, disruptions in circadian clock function have been implicated in human mood disorders, and clock gene manipulation in mice may provide valuable models for studying depression-like behavior. In this review, we explore various approaches to manipulating Bmal1 in mouse models and review their effects on the brain's master circadian pacemaker, on circadian rhythmicity in other brain regions, and on circadian and mood-related behavior.

Mammals rely on a network of circadian clocks to control daily systemic metabolism and physiology. The central pacemaker in the suprachiasmatic nucleus (SCN) is considered hierarchically dominant over peripheral clocks, whose degree of independence, or tissue-level autonomy, has never been ascertained in vivo. Using arrhythmic Bmal1-null mice, we generated animals with reconstituted circadian expression of BMAL1 exclusively in the liver (Liver-RE). High-throughput transcriptomics and metabolomics show that the liver has independent circadian functions specific for metabolic processes such as the NAD+ salvage pathway and glycogen turnover. However, although BMAL1 occupies chromatin at most genomic targets in Liver-RE mice, circadian expression is restricted to ∼10% of normally rhythmic transcripts. Finally, rhythmic clock gene expression is lost in Liver-RE mice under constant darkness. Hence, full circadian function in the liver depends on signals emanating from other clocks, and light contributes to tissue-autonomous clock function.

In rodents, the preovulatory LH surge is temporally gated, but the timing cue is unknown. Estrogen primes neurons in the anteroventral periventricular nucleus (AVPV) to secrete kisspeptin, which potently activates GnRH neurons to release GnRH, eliciting a surge of LH to induce ovulation. Deletion of the circadian clock gene Bmal1 results in infertility. Previous studies have found that Bmal1 knockout (KO) females do not display an LH surge at any time of day. We sought to determine whether neuroendocrine disruption contributes to the absence of the LH surge. Because Kiss1 expression in the AVPV is critical for regulating ovulation, we hypothesized that this population is disrupted in Bmal1 KO females. However, we found an appropriate rise in AVPV Kiss1 and Fos mRNA at the time of lights out in ovariectomized estrogen-treated animals, despite the absence of a measureable increase in LH. Furthermore, Bmal1 KO females have significantly increased LH response to kiss-10 administration, although the LH response to GnRH was unchanged. We then created Kiss1- and GnRH-specific Bmal1 KO mice to examine whether Bmal1 expression is necessary within either kisspeptin or GnRH neurons. We detected no significant differences in any measured reproductive parameter. Our results indicate that disruption of the hypothalamic regulation of fertility in the Bmal1 KO females is not dependent on endogenous clocks within either the GnRH or kisspeptin neurons.