Study Objectives Circadian rhythms regulate sleep-wake cycles and modulate cognitive functions over a 24-h period. Following sleep loss, certain cognitive performance partially rebounds in the early evening, a phenomenon known as circadian rescue. Yet, the magnitude and domain specificity of circadian rescue remain poorly understood. Here, we integrate experimental and meta-analytic approaches to differential contributions of circadian and homeostatic processes to cognitive rescue following sleep deprivation.Methods In study 1, 54 healthy adults remained awake for 35 consecutive hours while repeatedly completing the Psychomotor Vigilance Task, the Digit Symbol Substitution Test, and the Karolinska Sleepiness Scale. Performance dynamics were modeled using the two-process framework of sleep regulation. In study 2, a meta-analysis of published data contextualized these findings across protocols.Results Results reveal domain-specific circadian recovery rates of 33.0%-52.1% for Psychomotor Vigilance Task, 45.7% for Digit Symbol Substitution Test, and 23.5% for Karolinska Sleepiness Scale, indicating that subjective sleepiness is predominantly driven by homeostatic load, whereas objective cognitive performance retains significant circadian modulation under conditions of acute homeostatic pressure.Conclusions These findings clarify how circadian and homeostatic drives interact to shape cognitive task performance and subjective sleepiness outcomes under sleep loss, with practical implications for optimizing performance in fatigue-prone environments.

In our previous report, PERK/NRF2/CX43/StAR/progesterone pathway activation in ovarian granulosa cells was shown to mediate cold-induced female reproductive disorders. However, how PERK is activated by low temperature remained unclear. In the present study, we found that the circadian protein E4BP4 was significantly upregulated in ovarian granulosa cells following exposure to cold or isoproterenol (ISO), a non-selective β-adrenergic receptor agonist that pharmacologically activates β-adrenergic signaling, a key component of the cold stress response. Mechanistically, E4BP4 interacted with PERK and was required for PERK activation and subsequent NRF2/CX43/StAR signaling, leading to increased progesterone secretion. Interestingly, NRF2 also acted as a transcriptional activator of E4BP4 under ISO treatment, and blocking PERK or NRF2 expression attenuated ISO-induced E4BP4 accumulation, suggesting a positive feedback loop involving PERK/NRF2/E4BP4. Collectively, these findings identify E4BP4 as a cold-responsive circadian protein that interacts with PERK and may contribute to cold-induced reproductive disorders via a bidirectional E4BP4-PERK feedback loop.

PURPOSE:Dry eye disease with ocular surface inflammation leads to corneal epithelial cell damage. This study aims to investigate the role of activating transcription factor 3 (ATF3) in hyperosmotic stress (HS)-induced damage in human corneal epithelial (HCE) cells and to identify new targets for dry eye disease treatment. METHODS:HCE cells were treated with isotonic or hypertonic (312 and 500 mOsM) culture media for 24 hours and added by 94 mM of NaCl to achieve hyperosmotic conditions. After siRNA-ATF3 transfection, the expression of ATF3, LncRNA nuclear-enriched abundant transcript 1 (NEAT1), human antigen R (HuR), and toll-like receptor 4 (TLR4) were detected using qRT-PCR and western blot assays. Cell proliferation was analyzed by the CCK-8 assay. LDH, ROS, TNF-α, IL-1β, and IL-6 levels were measured. Cell apoptosis was measured by flow cytometry. ATF3 enrichment on the NEAT1 promoter was analyzed. The binding of ATF3 to the NEAT1 promoter and HuR to NEAT1 and TLR4 was analyzed. TLR4 mRNA stability was measured. Overexpression of NEAT1 or TLR4 combined with ATF3 knockdown was performed to verify the mechanism. RESULTS:HS induced LDH release, ROS production, apoptosis, and inflammation in HCE cells and upregulated ATF3 expression. Knockdown of ATF3 alleviated above cell damage. ATF3 promoted NEAT1 expression, and NEAT1 enhanced the stability of TLR4 mRNA by binding to HuR. Overexpression of NEAT1 or TLR4 partially reversed the protective effect of ATF3 knockdown on HS-induced HCE cell damage. CONCLUSIONS:ATF3 promotes HS-induced damage in HCE cells by increasing TLR4 expression through upregulating NEAT1 expression.

Lack of sleep is a common problem in current society, which can induce various brain dysfunctions. Neuroinflammation is a typical reaction caused by sleep deficit and is considered as a common basis for various neurological disorders and cognitive impairments, but the related mechanisms have not been fully clarified. The circadian clock protein plays a critical role in maintaining physiological homeostasis, including sleep/wake cycles. Circadian disorders induced by sleep deficit might contribute to the development of neuroinflammation. In the current study, we observed that sleep deprivation (SD) induced elevated expression of High-mobility group box 1 (HMGB1), one of the most important mediators of neuroinflammation, in the cortical microglia and cerebrospinal fluids. Moreover, acetylation-dependent nuclear export of HMGB1 was involved in up-regulation and secretion of HMGB1 after sleep deprivation. Further studies indicated that sleep deprivation induced an increase in the expression of acetyltransferase p300 and a decrease in the expression of deacetylase SIRT1, which synergistically enhanced the acetylation level of HMGB1 in the cortical microglial cells, thereby triggered the nuclear export and secretion of HMGB1. Most importantly, circadian clock protein PER2 constitutively interacted with p300 and inhibited its expression in the microglial cells, which can be interrupted by PER2 downregulation upon sleep deprivation, leading to the increased expression of p300 and acetylation and secretion of HMGB1. The truncated PER2 mutant without p300 binding ability lost its ability to regulate p300 expression, indicating that PER2 functioned as a co-suppressor of p300 in regulating acetylation and expression of HMGB1. Taken together, data in this study reveal a new mechanism by which PER2 is involved in controlling HMGB1 dependent neuroinflammation induced by sleep deprivation. Maintaining PER2 levels or blocking HMGB1 acetylation in the cortex might be prospective for preventing sleep deprivation-induced neuroinflammation and the related adverse reactions in the brain.

This study aims to explore the role of enhancer of zeste homolog 2 (EZH2)-mediated histone methylation in basic fibroblast growth factor (bFGF)-induced angiogenesis of human umbilical vein endothelial cells (HUVECs). EZH2, vascular endothelial growth factor A (VEGFA), miR-340-5p, and nuclear factor-erythroid 2-related factor 2 (NRF2) expressions in bFGF-induced HUVECs were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. After transfection of EZH2 siRNA, NRF2 siRNA, or miR-340-5p inhibitor, cell migration and angiopoiesis were assessed by Transwell and tube formation assays. Chromatin immunoprecipitation (ChIP) was performed to analyze the enrichment of EZH2 or trimethylated H3 lysine 27 (H3K27me3) on NRF2 promoter. The binding between NRF2 and miR-340-5p was verified by ChIP and dual-luciferase assay. EZH2 was highly expressed while miR-340-5p and NRF2 were poorly expressed in bFGF-induced HUVECs. Silence of EZH2 restrained HUVEC migration, and reduced the number of branches and tube length. Mechanically, EZH2 enhances the enrichment of H3K27me3 on the NRF2 promoter, thereby repressing NRF2 expression and further leading to transcriptional repression of miR-340-5p. In conclusion, EZH2 inhibits the NRF2/miR-340-5p axis and promotes bFGF-induced angiogenesis of HUVECs by increasing the H3K27me3 modification on the NRF2 promoter.

Sleep is critical for maintaining overall health. Insufficient sleep duration and poor sleep quality are associated with various physical and mental health risks and chronic diseases. To date, plenty of epidemiological research has shown that sleep disorders are associated with the risk of obesity, which is usually featured by the expansion of adipose tissue. However, the underlying mechanism of increased fat accumulation upon sleep disorders remains unclear. Here we demonstrated that sleep deprivation (SD) caused triglycerides (TG) accumulation in the visceral white adipose tissue (vWAT), accompanied by a remarkable decrease in the expression of adipose triglyceride lipase (ATGL) and other two rate-limiting lipolytic enzymes. Due to the key role of ATGL in initiating and controlling lipolysis, we focused on investigating the signaling pathway leading to attenuated ATGL expression in vWAT upon SD in the following study. We observed that ATGL downregulation resulted from the suppression of ATGL transcription, which was mediated by the reduction of the transcriptional factor FOXO1 and its upstream regulator SIRT1 expression in vWAT after SD. Furthermore, impairment of SIRT1/FOXO1/ATGL pathway activation and lipolysis induced by SIRT1 inhibitor EX527 in the 3 T3-L1 adipocytes were efficiently rescued by the SIRT1 activator resveratrol. Most notably, resveratrol administration in SD mice revitalized the SIRT1/FOXO1/ATGL pathway activation and lipid mobilization in vWAT. These findings suggest that targeting the SIRT1/FOXO1/ATGL pathway may offer a promising strategy to mitigate fat accumulation in vWAT and reduce obesity risk associated with sleep disorders.

Ambient air temperature is a key factor affecting human health. Female reproductive disorders are representative health risk events under low temperature. However, the mechanism involving in cold-induced female reproductive disorders remains largely unknown. Female mice were intermittently exposed to cold conditions (4 °C) to address the health risk of low temperature on female reproductive system. Primary granulosa cells (GCs) were prepared and cultured under low temperature (35 °C) or exposed to β3-adrenoreceptor agonist, isoproterenol, to mimic the condition of cold exposure. Western-blot, RT-PCR, co-IP, ELISA, pharmacological inhibition or siRNA-mediated knockdown of target gene were performed to investigate the possible role of hormones, gap conjunction proteins, and ER stress sensor protein in regulating female reproductive disorders under cold exposure. Cold exposure induced estrous cycle disorder and follicular dysplasia in female mice, accompanying with abnormal upregulation of progesterone and its synthetic rate-limiting enzyme, StAR, in the ovarian granulosa cells. Under the same conditions, an increase in connexin 43 (CX43) expressions in the GCs was also observed, which contributed to elevated progesterone levels in the ovary. Moreover, ER stress sensor protein, PERK, was activated in the ovarian GCs after cold exposure, leading to the upregulation of downstream NRF2-dependent CX43 transcription and aberrant increase in progesterone synthesis. Most importantly, blocking PERK expression in vivo significantly inhibited NRF2/CX43/StAR/progesterone pathway activation in the ovary and efficiently rescued the prolongation of estrous cycle and the increase in follicular atresia of the female mice induced by cold stress. We have elucidated the mechanism of ovarian PERK/NRF2/CX43/StAR/progesterone pathway activation in mediating female reproductive disorder under cold exposure. Targeting PERK might be helpful for maintaining female reproductive health under cold conditions.

Well -designed nocturnal light environment could boost performance on the cognitive tasks and promote sleep quality after light exposure. We optimized and fabricated a four -channel mixed white light with peak wavelengths of 429, 523, 591, and 621 nm. Comparing with common white light emitting diode (LED) (5798 K, 212.7 lx), the mixed white light has lower correlated color temperature (CCT) (2799 K), higher illuminance (356.2 lx), similar melanopic illuminance, and better color fidelity. We conducted experiments on 14 healthy young subjects (7 males and 7 females; age 18-25 years) to investigate the effects of nocturnal light environments on the cognitive performances and sleep quality. In consistent with the alpha-opic flux model, the mixed white light with higher illuminance shrinks the pupil size and has less melanopic flux, which results in more melatonin for subjects. More melatonin before sleep benefits the sleep quality by decreasing arousal times and improving sleep continuity. The higher illuminance light environment also promotes the inhibition ability and working memory. Moreover, it presents better color discrimination and less visual fatigue. Therefore, the lower CCT with higher illuminance nocturnal light environment effectively promotes both cognitive performances in the evening and the following sleep quality.

Chronic stress enhances the risk for psychiatric disorders and induces depression and cognitive impairment. Gamma oscillations are essential for neurocircuit function, emotion, and cognition. However, the influence of gamma entrainment by sensory stimuli on specific aspects of chronic stress-induced responses remains unclear. Mice were subjected to corticosterone (CORT) administration and chronic restraint stress (CRS) for weeks, followed by rhythmic gamma frequency light flickering exposure. Local field potentials (LFPs) were recorded from the V1, CA1, and PFC regions to verify the light flicker on gamma oscillations. Behavioral tests were used to examine stress-related and memory-related behaviors. Golgi staining was performed to observe changes in spine morphology. Synaptosomes were isolated to determine the expression of synapse-related proteins through immunoblotting. RNA sequencing (RNA-seq) was applied to explore specific changes in the transcriptome. Immunofluorescence staining, real-time quantitative polymerase chain reaction (qPCR), and ELISA were used to evaluate microglial activation and cytokine levels. In this study, we demonstrated that rhythmic 40 Hz LF attenuated stress-related behavior and cognitive impairments by ameliorating the microstructural alterations in spine morphology and increasing the expression of GluN2A and GluA1 in chronically stressed mice. Transcriptome analysis revealed that significantly downregulated genes in LF-exposed CRS mice were enriched in neuroimmune-related signaling pathways. Rhythmic 40 Hz LF exposure significantly decreased the number of Iba1-positive microglia in the PFC and hippocampus, and the expression levels of the M1 markers of microglia iNOS and CD68 were reduced significantly in CRS mice. In addition, 40 Hz LF exposure suppressed the secretion of cytokines IL-12, which could regulate the production of IFN-gamma gamma and IL-10 in stressed mice. Our results demonstrate that exposure to rhythmic 40 Hz LF induces the neuroimmune response and downregulation of neuroinflammation with attenuated stress-related behaviors and cognitive function in CRS-induced mice. Our findings highlight the importance of sensory-evoked gamma entrainment as a potential therapeutic strategy for stress-related disorders treatment. Abbreviations: CORT, Chronic corticosterone treatment; CRS, Chronic restraint stress; IACUC, Institutional Animal Care and Use Committee; LF, light flickers; FST, Forced swim test; NSFT, Novelty-suppressed feeding test; SPT, Sucrose preference test; NSFT, Novelty-suppressed feeding; qPCR, Quantitative real-time polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; PBS, phosphate-buffered saline; PBS-T, phosphate-buffered saline plus 0.1% Tween 20; PVDF, polyvinylidene fluoride; GFAP, Glial fibrillary acidic protein; DAPI, 4 ',6-Diamid- ' ,6-Diamid- ino-2-phenylindole; Iba1, Ionized calcium- binding adaptor molecule 1; iNOS, Inducible nitric oxide synthase; IL-10, Interleukin-10; IL6, Interleukin 6; IL-1 (3, Interleukin 1(3; (3 ; IL-12, Interleukin 12; TNF-alpha, Tumor necrosis factor alpha; IFN-gamma, gamma, Interferon-gamma; TLR6 and 9, Toll-like Receptor 6 and 9.

Sleep deprivation (SD) weakens the immune system and leads to increased susceptibility to infectious or inflammatory diseases. However, it is still unclear how SD affects humoral immunity. In the present study, sleep disturbance was conducted using an sleep deprivation instrument, and the bacterial endotoxin lipopolysaccharide (LPS) was used to activate the immune response. It was found that SD-pretreatment reduced LPS-induced IgG2b+ B cells and IgG2b isotype antibody production in lymphocytes of spleen. And, SD-pretreatment decreased the proportion of CD4+T cells, production of CD4+T cells derived TGF-β1 and its contribution in helping IgG2b production. Additionally, BMAL1 and CLOCK were selectively up-regulated in lymphocytes after SD. Importantly, BMAL1 and CLOCK deficiency contributed to TGF-β1 expression and production of IgG2b+ B cells. Thus, our results provide a novel insight to explain the involvement of BMAL1 and CLOCK under SD stress condition, and their roles in inhibiting TGF-β1 expression and contributing to reduction of LPS induced IgG2b production.

Abstract Objective To assess serum 25-hydroxyvitamin D3 (25(OH)D3), fibroblast growth factor 23 (FGF23), and C1q/tumor necrosis factor-related protein-3 (CTRP3) levels in nondialysis chronic kidney disease (CKD) patients and their relationship with coronary artery calcification (CAC). Methods One hundred and twenty-eight patients diagnosed with CKD were selected and all underwent cardiac computed tomography. CAC was assessed using the Agatston score, and coronary artery calcification score (CACs) >10 was identified as CAC. The differences in serum 25(OH)D3, FGF23, and CTRP3 levels between the CAC and non-CAC groups were analyzed. Their correlation with CACs was assessed by Spearman’s analysis, and logistic regression analysis was used to find risk factors for CAC. Results Compared to the non-CAC group, the CAC group was older (64.21 ± 9.68 years), with a higher percentage of hypertension (93.10%) and diabetes (63.80%) and higher levels of serum CTRP3 [1079.20 (644.4–1567.2) ng/mL]. However, there was no significant difference in serum 25(OH)D3 and FGF23 between these two groups. The high level CTRP3 group had a higher prevalence of CAC (61.5%). Logistic regression results showed that age, diabetes, decreased 25(OH)D3 (odds ratio (OR) = 0.95, p = .030) and high levels of CTRP3 (OR = 3.19, p = .022) were risk factors for CAC in nondialysis CKD patients. Conclusions Serum CTRP3 levels progressively increased with the progression of kidney disease, while 25(OH)D3 levels progressively decreased. Decreased 25(OH)D3 and high levels of CTRP3 are associated with CAC in patients with nondialysis CKD.

Dyslipidemia and inflammation have great roles in the development of diabetic nephropathy (DN). Oleanolic acid (OA) is a natural triterpenoid that possesses multiple pharmacological properties including anti-oxidation, anti-inflammatory and hypoglycemia. In the present study, the effects of OA on diabetic kidney disease (DKD) and its underlying mechanisms were investigated in DKD rats. Twenty-five of a total thirty-five male Sprague-Dawley (SD) rats were used to establish for Type 2 diabetes mellitus (T2DM) model by high-fat diet combined with streptozotocin (STZ). Then rats were randomly assigned into four group: control group (n = 10), T2DM group (n = 9), OA (50 mg/kg) group (n = 7), OA (100 mg/kg) group (n = 8). Rats were sacrificed at the end of 18 weeks after feeding by intraperitoneal injection of pentobarbital sodium. Body weight (BW), fasting blood glucose (FBG), kidney weight (KW), serum lipid, 24-h urinary microalbumin (UMA), serum creatinine (Scr) and uric acid (UA) were measured. Histopathological changes were observed by PAS staining and electron microscope. The expressions of nephrin, CD68, Collagen-IV, AMPK, p-AMPK, PGC-1α, TLR4, NF-κB and TGF-β1 in kidney were also detected by immunohistochemistry or western blot. OA significantly decreased the levels of FBG, kidney index (KI), serum lipid levels, 24 h UMA, Scr, UA in diabetic rats. Additionally, OA obviously attenuated renal lipid accumulation and renal structure abnormalities in diabetic rats. Furthermore, the expression levels of nephrin, p-AMPK/AMPK, PGC-1α were elevated, while CD68, Collagen-IV, TLR4, NF-κB and TGF-β1 expressions were decreased in renal tissues of OA treated diabetic rats. OA showed dose-independent. OA can alleviate renal injury in diabetic rats through improving lipid metabolism and inflammation via AMPK/PGC-1α and TLR4/NF-κB signaling pathway.

目的 探讨睡眠剥夺(SD)对骨骼肌组织炎性因子表达的影响及相关的分子机制.方法 将体重180～200 g的雄性大鼠随机分为对照组和实验组(SD 72 h).收集各组大鼠骨骼肌组织,利用RT-PCR和Western印迹检测炎性分子白细胞介素-1β(IL-1β)、IL-15、肿瘤坏死因子-α(TNF-α)及炎性反应有关蛋白X-box结合蛋白1(XBP1)、肌醇需求蛋白1α(IRE1α)的表达水平.体外培养小鼠C2C12肌管细胞,分别转染炎性反应相关蛋白的siRNA,利用上述实验方法检测细胞炎性分子和炎性反应相关蛋白的表达水平.结果 SD后骨骼肌组织中IL-1β表达水平升高,炎性反应相关蛋白XBP1和IRE1α表达水平显著增强.体外培养的C2C12肌管细胞中转染XBP1 siRNA和IRE1αsiRNA后,可显著抑制IL-1β的转录诱导表达.结论 SD通过诱导骨骼肌组织IRE1α/XBP1内质网应激反应途径异常活化进而介导炎性因子IL-1β表达水平升高.

Sleep loss leads to a spectrum of mood disorders such as anxiety disorders, bipolar disorder and depression in many individuals. However, the underlying mechanisms are largely unknown. In this study, sleep-disturbed animals were tested for anxiety and depressive behaviors. We then studied the effects of SD on hypothalamic-pituitary-adrenal (HPA) axis function by measuring serum and CSF levels of corticosterone (CORT), and at the end of the experiment, brains were collected to measure the circadian oscillations of clock genes expression in the hypothalamus, glial cell activation and inflammatory cytokine alterations. Our results indicated that SD for 3 days resulted in anxiety- and depressive-like behaviors. SD exaggerated cortisol response to HPA axis, significantly altered the circadian oscillations of clock genes, decreased the expression of tight junction protein ZO-1 and Claudin 5 and increased the number of GFAP-positive cells and Iba-1-positive cells and caused subsequent elevation of pro-inflammatory cytokines IL-6, IL-1β and TNFα. These findings demonstrated that SD for 3 days induced anxiety- and depression-like behaviors in rats in company with altering the circadian oscillations of clock genes and inducing neuroinflammation, indicating the underlying mechanism of sleep loss induced neuronal dysfunction.

Background: Sleep loss leads to a spectrum of mood disorders such as anxiety, cognitive dysfunction and motor coordination impairment in many individuals. However, the underlying mechanisms are largely unknown. Methods: In this study, we examined the effects of sleep deprivation (SD) on depression and the mechanism by subjecting rats to a slowly rotating platform for 3 days to mimic the process of sleep loss. Sleep-deprived animals were tested behaviorally for anxiety- and depressive-like behaviors. We further studied the effects of SD on hypothalamic-pituitary-adrenal (HPA) axis activity, and at the end of the experiment, brains were collected to measure the circadian clock genes expression in the hypothalamus, glial cell activation and inflammatory cytokine alterations. Results: Our results indicated that SD for 3 days resulted in anxiety- and depressive-like behaviors. SD exaggerated cortisol response to HPA axis, significantly altered the mRNA profile of circadian clock genes, and induced neuroinflammation by increasing the expression of glial cell markers, including the microglial marker ionized calcium-binding adapter molecule 1 (Iba1) and the astroglial marker glial fibrillary acidic protein (GFAP). The expression of M1 and M2 microglial markers (Arg-1 and CD206, respectively) and pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) were increased in the brain. Conclusion: These results indicated that SD for 3 days induced anxiety- and depression-like behaviors in rats by impairing the regulation of circadian clock genes and inducing neuroinflammation, ultimately resulting in brain injury.

Disturbed sleep is closely associated with an increased risk of metabolic diseases. However, the underlying mechanisms of circadian clock genes linking sleep and lipid profile abnormalities have not been fully elucidated. This study aimed to explore the important role of the circadian clock in regulating impaired cholesterol metabolism at an early stage of sleep deprivation (SD). Sleep disturbance was conducted using an SD instrument. Our results showed that SD increased the serum cholesterol levels. Concentrations of serum leptin and resistin were much lower after SD, but other metabolic hormone concentrations (adiponectin, glucagon, insulin, thyroxine, norepinephrine, and epinephrine) were unchanged before and after SD. Warning signs of cardiovascular diseases [decreased high density lipoprotein (HDL)-cholesterol and increased corticosterone and 8-hydroxyguanosine levels] and hepatic cholestasis (elevated total bile acids and bilirubin levels) were observed after SD. Cholesterol accumulation was also observed in the liver after SD. The expression levels of HMGCR, the critical enzyme for cholesterol synthesis, remained unchanged in the liver. However, the expression levels of liver CYP7A1, the enzyme responsible for the conversion of cholesterol into bile acids, significantly reduced after SD. Furthermore, expression of NR1D1, a circadian oscillator and transcriptional regulator of CYP7A1, strikingly decreased after SD. Moreover, NR1D1 deficiency decreased liver CYP7A1 levels, and SD could exacerbate the reduction of CYP7A1 expression in NR1D1−/− mouse livers. Additionally, NR1D1 deficiency could further increase serum cholesterol levels under SD. These results suggest that sleep disturbance can induce increased serum cholesterol levels and liver cholesterol accumulation by NR1D1 mediated CYP7A1 inhibition.

Health consequences of sleep loss or insufficient sleep have been proved to be associated with increased inflammation, which is considered as a risk factor for various diseases, including cardiovascular, metabolic, neurodegenerative diseases and cancer. However, the inflammation occurred in targeted or injured tissues after sleep loss has not been fully revealed. High-mobility group box 1 (HMGB1) plays a critical role in triggering and sustaining inflammatory responses by inducing cytokine releasing and recruiting leucocytes. The aim of this study is to address whether sleep deprivation (SD) could induce hepatic HMGB1 upregulation and the underlying mechanism involved. Samples of rat liver, blood and urine were collected after 48 h of SD. The expression levels of HMGB1, REV-ERBα and miR-122 were determined by ELISA, PCR and western blot assays. Binding sites of miR-122 within HMGB1-3’UTR were identified by luciferase reporter assay. SD induced increasing of HMGB1 expression in the rat liver, serum and urine. Interestingly, liver HMGB1 upregulation was only observed at the protein levels, while HMGB1 mRNA levels remained stable under the same conditions. Then, two miR-122 binding sites were identified within the 3’-UTR region of HMGB1. And the liver miR-122 expression levels were significantly decreased after SD. Furthermore, HMGB1 protein expression levels dramatically decreased in miR-122 mimic-transfected cells and significantly increased in miR-122 inhibitors-transfected cells compared with those in the control cells. Besides, SD inhibited the expression of central clock gene, REV-ERBα, in the liver. Knockdown of REV-ERBα expression significantly decreased miR-122 expression and increased HMGB1 expression. Down regulation of liver REV-ERBα and miR-122 is involved in local HMGB1 upregulation induced by SD. In addition, HMGB1 might function as potential biomarker for indicating the hepatic inflammatory responses induced by SD. Military Sciences Foundation of China (No. AWS17J014, BWS17J025), National Natural Sciences Foundation of China (81700759).

Our previous studies revealed that GADD45α is a liable protein, which undergoes MDM2-dependent constitutive ubiquitination and degradation in resting HepG2 hepatoma cells. Arsenite exposure induces ribosomal stress responses mediated by the ribosomal protein S7, which can block MDM2 activity and result in GADD45α accumulation and cell apoptosis. In the present study, we found that one of the catalytic subunits of IκB kinase (IKK), IKKβ, exerted a novel IKKα- and NF-κB-independent function in stabilizing MDM2 and therefore contributed to ubiquitination-dependent degradation of GADD45α in resting HepG2 cells. Arsenite stimulation induced transactivation of p53, which formed a complex with its downstream target, Ets-1, and then synergistically repressed IKKβ transcription, reduced MDM2 stability, and ultimately removed the inhibitory effect of MDM2 on GADD45α induction. In addition, DAPK1 functioned as an upstream protein kinase triggering p53/Ets-1-dependent IKKβ and MDM2 reduction and GADD45α accumulation, thus promoting apoptosis in HepG2 cells. Subsequent studies further revealed that the activation of the DAPK1/p53/Ets-1/IKKβ/MDM2/GADD45α cascade was a common signaling event in mediating apoptosis of diverse cancer cells induced by arsenite and other tumor therapeutic agents. Therefore, we conclude that data in the current study have revealed a novel role for IKKβ in negatively regulating GADD45α protein stability and the contribution of p53-dependent IKKβ reduction to mediating cancer cell apoptosis.