Circadian misalignment impairs oligodendrocyte myelination via Bmal1 overexpression leading to anxiety and depression-like behaviors.

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

Interaction of Bmal1 and eIF2α/ATF4 pathway was involved in Shuxie compound alleviation of circadian rhythm disturbance-induced hepatic endoplasmic reticulum stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Shuxie Compound (SX) combines the composition and efficacy of Suanzaoren decoction and Huanglian Wendan decoction. It can soothe the liver, regulate the qi, nourish the blood and calm the mind. It is used in the clinical treatment of sleep disorder with liver stagnation. Modern studies have proved that circadian rhythm disorder (CRD) can cause sleep deprivation and liver damage, which can be effectively ameliorated by traditional Chinese medicine to soothe the liver stagnation. However, the mechanism of SX is unclear. AIM OF THE STUDY: This study was designed to demonstrate the impact of SX on CRD in vivo, and confirm the molecular mechanisms of SX in vitro. MATERIALS AND METHODS: The quality of SX and drug-containing serum was controlled by UPLC-Q-TOF/MS, which were used in vivo and in vitro experiments, respectively. In vivo, a light deprivation mouse model was used. In vitro, a stable knockdown Bmal1 cell line was used to explore SX mechanism. RESULTS: Low-dose SX (SXL) could restore (1) circadian activity pattern, (2) 24-h basal metabolic pattern, (3) liver injury, and (4) Endoplasmic reticulum (ER) stress in CRD mice. CRD decreased the liver Bmal1 protein at ZT15, which was reversed by SXL treatment. Besides, SXL decreased the mRNA expression of Grp78/ATF4/Chop and the protein expression of ATF4/Chop at ZT11. In vitro experiments, SX reduced the protein expression of thapsigargin (tg)-induced p-eIF2α/ATF4 pathway and increase the viability of AML12 cells by increasing the expression of Bmal1 protein. CONCLUSIONS: SXL relieved CRD-induced ER stress and improve cell viability by up-regulating the expression of Bmal1 protein in the liver and then inhibiting the protein expression of p-eIF2α/ATF4.

The Circadian System Is Essential for the Crosstalk of VEGF-Notch-mediated Endothelial Angiogenesis in Ischemic Stroke.

Ischemic stroke is a major public health problem worldwide. Although the circadian clock is involved in the process of ischemic stroke, the exact mechanism of the circadian clock in regulating angiogenesis after cerebral infarction remains unclear. In the present study, we determined that environmental circadian disruption (ECD) increased the stroke severity and impaired angiogenesis in the rat middle cerebral artery occlusion model, by measuring the infarct volume, neurological tests, and angiogenesis-related protein. We further report that Bmal1 plays an irreplaceable role in angiogenesis. Overexpression of Bmal1 promoted tube-forming, migration, and wound healing, and upregulated the vascular endothelial growth factor (VEGF) and Notch pathway protein levels. This promoting effect was reversed by the Notch pathway inhibitor DAPT, according to the results of angiogenesis capacity and VEGF pathway protein level. In conclusion, our study reveals the intervention of ECD in angiogenesis in ischemic stroke and further identifies the exact mechanism by which Bmal1 regulates angiogenesis through the VEGF-Notch1 pathway.

Melatonin upregulates BMAL1 to attenuate chronic sleep deprivation-related cognitive impairment by alleviating oxidative stress.

PURPOSE: To investigate the mechanism underlying the regulatory effect of melatonin on chronic sleep deprivation-related cognitive impairment. METHODS: Chronic sleep deprivation (CSD) model was established using the MMPM method. After the model was established, melatonin receptor agonist and inhibitor were given, respectively. Water maze was conducted to record the escape latency and the duration of crossing the platform of space exploration. The concentration of TNF-α, IL-6, MDA, and SOD was measured by ELISA. Immunofluorescence was used to determine the expression level of CD86 and CD206, while the mRNA expression of Bax, Bcl-2, P65, IκB, and BMAL1 was detected by qPCR. Western blotting assay was utilized to determine the protein expression of Bax, Bcl-2, P65, p-P65, IκB, p-I κB, and BMAL1. RESULTS: Compared with the control, the escape latency was greatly increased on the second and third day, accompanied by the increased expression of TNF-α, IL-6, MDA, and SOD in serum. Furthermore, dramatically upregulated Bax, Bcl-2, P65, IκB, and CD86 were observed in the model group, accompanied by the declined expression level of BMAL1 and CD206. Compared with the model group, the escape latency was declined, the concentration of TNF-α, IL-6, MDA, and SOD was decreased, the expression level of Bax, Bcl-2, P65, IκB, and CD86 was declined, and the level of BMAL1 and CD206 was promoted by the treatment of the melatonin agonist, while the opposite results were observed under the treatment of the melatonin inhibitor. CONCLUSION: Melatonin upregulates BMAL1 to attenuate chronic sleep deprivation-related cognitive impairment by alleviating oxidative stress.

The circadian clock gene ARNTL overexpression suppresses oral cancer progression by inducing apoptosis via activating autophagy.

The study aimed to explore tumor suppressor mechanism of ARNTL from the perspective of autophagy in oral cancer. Human oral squamous carcinoma HN6 cells stably overexpressing ARNTL were established, cell viability and apoptosis were detected by CCK-8 and TUNEL assays, and intracellular autophagosomes were observed under electron microscopy. Western Blot detected expressions of Beclin1, LC3 II/I, ATG-12, P62, BAX and BCL-2. Bafilomycin A1 was used to detect autophagic flux, and Western Blot was used to detect changes of LC3II and P62 proteins. Autophinib was added to cells with ARNTL overexpression for recovery experiments, and cell proliferation and apoptosis were detected by flow cytometry. In vivo tumorigenesis experiment was used to evaluate the in vivo anti-tumor efficacy of ARNTL, and Western blot simultaneously detected ARNTL, LC3 II/I, Beclin1, P62 and ATG-12 expressions. ARNTL overexpression promoted apoptosis and autophagy and inhibited cell viability. In ARNTL-overexpressing cells, expressions of Beclin1, LC3 II/I, and BAX were significantly up-regulated, while P62 and BCL-2 expressions were decreased, and ATG-12 expression wasn't significantly changed. When the autophagy inhibitor Autophinib was used, expressions of elevated BAX and decreased BCL-2 were reversed effectively, as were decreased cell proliferation index and increased apoptosis index. An in vivo tumorigenesis assay also showed ARNTL overexpression inhibited tumor growth, and autophagy-related protein expressions were consistent with the in vitro data. The research demonstrated for the first time that ARNTL induced apoptosis and inhibited cell proliferation dependent on autophagy in oral cancer, which provides theoretical basis for potential therapeutic targets.

Hepatitis B and circadian rhythm of the liver.

The circadian rhythm in humans is determined by the central clock located in the hypothalamus's suprachiasmatic nucleus, and it synchronizes the peripheral clocks in other tissues. Circadian clock genes and clock-controlled genes exist in almost all cell types. They have an essential role in many physiological processes, including lipid metabolism in the liver, regulation of the immune system, and the severity of infections. In addition, circadian rhythm genes can stimulate the immune response of host cells to virus infection. Hepatitis B virus (HBV) infection is the leading cause of liver disease and liver cancer globally. HBV infection depends on the host cell, and hepatocyte circadian rhythm genes are associated with HBV replication, survival, and spread. The core circadian rhythm proteins, REV-ERB and brain and muscle ARNTL-like protein 1, have a crucial role in HBV replication in hepatocytes. In addition to influencing the virus's life cycle, the circadian rhythm also affects the pharmacokinetics and efficacy of antiviral vaccines. Therefore, it is vital to apply antiviral therapy at the appropriate time of day to reduce toxicity and improve the effectiveness of antiviral treatment. For these reasons, understanding the role of the circadian rhythm in the regulation of HBV infection and host responses to the virus provides us with a new perspective of the interplay of the circadian rhythm and anti-HBV therapy. Therefore, this review emphasizes the importance of the circadian rhythm in HBV infection and the optimization of antiviral treatment based on the circadian rhythm-dependent immune response.

Resveratrol Induces the Fasting State and Alters Circadian Metabolism in Hepatocytes.

Resveratrol is a nutritional substance that has both metabolic and circadian effects. While some studies indicate a correlation between resveratrol and reduced gluconeogenesis, others propose the opposite. Our aim was to study the metabolic effect of resveratrol around the circadian clock in order to determine more accurately the hepatic signaling pathways involved. AML-12 hepatocytes were treated with resveratrol and clock and metabolic markers were measured around the clock. Resveratrol-treated AML-12 hepatocytes showed reduced ratio of the following key metabolic factors: phosphorylated PP2A to total PP2A (pPP2A/PP2A), pAKT/AKT, pFOXO1/FOXO1 and pAMPK/AMPK, indicating inhibition of AKT and AMPK, but activation of PP2A and FOXO1. In addition, the levels of phosphorylated mTOR were low after resveratrol treatment. The levels of the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) were significantly higher after resveratrol treatment. In accordance with the reduced mTOR activity, the ratio of pBMAL1/BMAL1, the clock transcription factor, also decreased. Bmal1 mRNA oscillated robustly in AML-12 hepatocytes, but resveratrol treatment led to a phase advance and a decrease in its amplitude, similarly to the effect on Srebp1c and Pgc1α mRNA. After resveratrol treatment, daily mRNA levels of Bmal1, Sirt1 and Srebp1c were significantly higher. Resveratrol changes the circadian expression of metabolic and clock genes activating the fasting state and inducing the PP2A-FOXO1-PEPCK pathway.

L-Theanine inhibits melanoma cell growth and migration via regulating expression of the clock gene BMAL1.

PURPOSE: L-Theanine is a unique non-protein amino acid found in green tea, which has been identified as a safe dietary supplement. It has been reported that L-theanine exerts various biological activities. In this study, we explored the anti-cancer effects of L-theanine on melanoma cells. METHODS: A375, B16-F10, and PIG1 cell lines were used in the present study. EdU labeling, TUNEL and Annexin V/PI staining, wound-healing, and transwell migration assay were performed to detect the effects of L-theanine on melanoma cell proliferation, apoptosis, and migration. Brain and muscle Arnt-like protein 1 (BMAL1) was knocked down in melanoma cells to evaluate if L-theanine plays the anti-cancer role through regulating circadian rhythm of melanoma cells. The western blot, qRT-PCR, and dual luciferase assay were performed to explore the mechanism involved in the effects of L-theanine on melanoma cells. RESULTS: L-Theanine apparently reduced the viability of melanoma cells. Further experiments showed that L-theanine attenuated the proliferation and migration, and promoted apoptosis of melanoma cells. L-Theanine significantly enhanced the expression of BMAL1, a clock gene in melanoma cells. Down-regulation of BMAL1 suppressed the anti-cancer effects of L-theanine on melanoma cells. Further experiments indicated that the p53 transcriptional activity raised by L-theanine was dependent on BMAL1 expression in melanoma cells. CONCLUSION: L-Theanine exerts the anti-cancer effect on melanoma cells through attenuating the proliferation and migration, and promoting apoptosis of them, which is dependent on the regulation of the clock gene Bmal1 in melanoma cells.