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.

[Xiongcan Yishen Formula up-regulates the clock gene expressions in the testis tissue of the rats with late-onset hypogonadism].

OBJECTIVE: To investigate the effect of Xiongcan Yishen Formula (XYF) on the expressions of the clock genes in the testis tissue of the rats with late-onset hypogonadism (LOH). METHODS: Forty-eight 8-week-old male SD rats were randomly divided into 6 groups, normal control, model control, testosterone propionate (TP), and low-, medium- and high-dose XYF. The LOH model was made in the later 5 groups of rats by intraperitoneal injection of D-galactose at 480 mg/kg/d for 56 successive days, while the normal controls were injected with the same volume of normal saline. After modeling, the rats in the low-, medium- and high-dose XYF groups were treated intragastrically with XYF at 10.4, 20.8 and 41.6 g/kg/d, bid, respectively, those in the normal and model control groups with the same volume of distilled water, and those in the TP group intramuscularly with TP at 5.21 mg/kg/d, qd alt, all for 28 days. After treatment, the supernatant was obtained for measurement of the serum T level by ELISA, and the testis tissue collected for determination of the mRNA and protein expressions of BMAL1, NR1D1, PER2, CRY1, StAR and CYP11A1 by RT-qPCR and Western blot. RESULTS: Compared with the normal controls, the rats in the LOH model control group showed significantly decreased serum T and mRNA and protein expressions of BMAL1, NR1D1, PER2, CRY1, StAR and CYP11A1 (P < 0.05). In comparison with the findings in the model controls, the T level was remarkably increased in the TP and XYF groups (P < 0.05), the expressions of StAR mRNA and CYP11A1 mRNA and protein markedly up-regulated in the high-dose XYF group (P < 0.05), and so was the expression of the StAR protein in the XYF and TP groups (P < 0.05), those of BMAL1 and NR1D1 proteins and PER2 mRNA and protein in the high-dose XYF group (P < 0.05), those of BMAL1 mRNA and CRY1 protein in the medium- and high-dose XYF groups (P < 0.05), that of NR1D1 mRNA in the XYF and TP groups (P < 0.05), and that of CRY1 mRNA in the medium- and high-dose XYF and TP groups (P < 0.05). CONCLUSION: Xiongcan Yishen Formula could up-regulate the expressions of the clock genes in the testis tissue of the LOH rats and increase the serum T level as well, which may underlie the mechanisms of Xiongcan Yishen Formula acting on LOH.

Protective effects of brain and muscle ARNT-like gene 1 on oxidized low-density lipoprotein-induced human brain microvascular endothelial cell injury by alleviating ferroptosis.

Ferroptosis plays an important role in atherosclerotic cerebrovascular diseases. The brain and muscle ARNT-like gene 1 (BMAL1) is an important mediator in the progression of cerebrovascular diseases. However, whether BMAL1 regulates ferroptosis in atherosclerotic cerebrovascular diseases remains obscure. Here, human brain microvascular endothelial cells (HBMECs) were exposed to oxidized low-density lipoprotein (ox-LDL) to imitate cerebrovascular atherosclerosis. It was found that ox-LDL treatment induced ferroptosis events and reduced BMAL1 expression in HBMECs, which could be reversed by ferroptosis inhibitor ferrostatin-1. Furthermore, BMAL1 overexpression markedly mitigated ox-LDL-induced ferroptosis events and cell damage. Moreover, BMAL1 overexpression significantly promoted nuclear factor erythroid 2-related factor 2 (Nrf2) expression in HBMECs under ox-LDL conditions. And, Nrf2 silencing attenuated the protective effects of BMAL1 on ox-LDL-stimulated HBMEC damage and ferroptosis. Altogether, our findings delineate the cerebrovascular protective role of BMAL1/Nrf2 by antagonizing ferroptosis in response to ox-LDL stimulation and provide novel perspectives for therapeutic strategies for atherosclerotic cerebrovascular diseases.

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.

BMAL1 regulates osteoblast differentiation through mTOR/GSK3ß/ß-catenin pathway.

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 GSK3ß/ß-catenin signaling with decreased ß-catenin expression and GSK-3ß phosphorylation at serine 9, while GSK3ß 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 ß-catenin expression and GSK-3ß phosphorylation. Collectively, overall data indicated that circadian gene Bmal1 regulated osteoblast differentiation and inflammatory response in an mTOR/GSK3ß/ß-catenin-dependent manner, and thereby may contribute to the mineralization process and bone modeling/remodeling.

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 System Is Essential for the Crosstalk of VEGF-Notch-mediated Endothelial Angiogenesis in Ischemic Stroke / 神经科学通报·英文版

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.

Seasonal and temporal variation in the placenta during melatonin supplementation in a bovine compromised pregnancy model.

Compromised pregnancies result in a poorly functioning placenta restricting the amount of oxygen and nutrient supply to the fetus resulting in intrauterine growth restriction (IUGR). Supplementing dietary melatonin during a compromised pregnancy increased uteroplacental blood flow and prevented IUGR in a seasonal-dependent manner. The objectives were to evaluate seasonal melatonin-mediated changes in temporal alterations of the bovine placental vascularity and transcript abundance of clock genes, angiogenic factors, and nutrient sensing genes in 54 underfed pregnant Brangus heifers (Fall, n = 29; Summer, n = 25). At day 160 of gestation, heifers were assigned to treatments consisting of adequately fed (ADQ-CON; 100% NRC; n = 13), nutrient restricted (RES-CON; 60% NRC; n = 13), and ADQ or RES supplemented with 20 mg/d of melatonin (ADQ-MEL, n = 13; RES-MEL, n = 15). The animals were fed daily at 0900 hours until day 240 where Cesarean sections were performed in the morning (0500 hours) or afternoon (1300 hours) for placentome collections. In both seasons, we observed a temporal alteration of the core clock genes in the cotyledonary tissue in a season-dependent manner. In the fall, ARNTL, CLOCK, NR1D1, and RORA transcript abundance were decreased (P ≤ 0.05) in the afternoon compared to the morning; whereas in the summer, ARNTL, PER2, and RORA expression were increased (P ≤ 0.05) in the afternoon. Interestingly, in both seasons, there was a concomitant temporal increase (P ≤ 0.05) of cotyledonary blood vessel perfusion and caruncular melatonin receptor 1A transcript abundance. Melatonin supplementation did not alter the melatonin receptor 1A transcript abundance (P > 0.05), however, in the summer, melatonin supplementation increased cotyledonary VEGFA, CRY1, and RORA (P ≤ 0.05) transcript abundance. In addition, during the summer the placentomes from underfed dams had increased average capillary size and HIF1α transcript abundance compared to those adequately fed (P ≤ 0.05). In conclusion, these data indicate increased cotyledonary blood vessel size and blood distribution after feeding to better facilitate nutrient transport. Interestingly, the maternal nutritional plane appears to play a crucial role in regulating the bovine placental circadian clock. Based on these findings, the regulation of angiogenic factors and clock genes in the bovine placenta appears to be an underlying mechanism of the therapeutic effect of dietary melatonin supplementation in the summer.

Maternal nutrient restriction during the last trimester of pregnancy impairs the fetal development, increases morbidity and mortality, and reduces its performance in adult life. Animals with compromised pregnancies exhibit a reduction in uterine blood flow thereby limiting the nutrients available for the fetus to grow and develop. Melatonin, a hormone that many people use as a sleep aid, could be a solution as a potential therapeutic in cattle since it has antioxidant properties and has been shown to regulate blood flow and rescue fetal weight during compromised pregnancies. In the current study, we examined the changes in placental vascularity and gene expression when supplementing underfed dams with dietary melatonin during late gestation in a group of fall-calving and spring-calving heifers. Contrary to our hypothesis melatonin did not control the placental circadian clock gene network, while maternal nutrient restriction disrupted the gene expression in the placenta. Furthermore, this study found that gene expression in the placenta is seasonally dependent.

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.

Anti-influenza virus activity of the REV-ERBα agonist SR9009 and related analogues.

REV-ERBα is a member of the nuclear receptor superfamily of transcription factors that aids in the regulation of many diseases. However, the prospect of using REV-ERBα for anti-influenza virus treatment remains poorly described, and there is an urgent need to develop effective anti-influenza agents due to the emergence of drug-resistant influenza viruses. In this study, eight SR9009 analogues were designed, synthesized, and evaluated for their biological activities against multiple influenza virus strains (H1N1, H3N2, adamantane- and oseltamivir-resistant H1N1 and influenza B virus), using ribavirin as the positive control. SR9009 and its analogues showed low micromolar or submicromolar EC50 values and exhibited modestly improved antiviral potency compared to that of ribavirin. In particular, compound 5a possessed the most potent inhibitory activity (EC50 = 0.471, 0.644, 1.644, 0.712 and 0.661 µM for A/PR/8/34, A/WSN/33, A/Wisconsin/67/2005, B/Yamagata/16/88 and Hebei/SWL1/2006, respectively). Cotransfection assays showed that all synthesized derivatives efficaciously suppressed transcription driven by the Bmal1 promoter. Mechanistic study results indicated that 5a efficiently inhibited IAV replication and interfered with the ealry stage of influenza virus life cycle. In addition, we found that 5a upregulated the key antiviral interferon-stimulated genes MxA, OAS2 and CH25H. Further in-depth transcriptome analysis revealed a series of upregulated genes that may contribute to the antiviral activities of 5a. These findings may provide an important direction for the development of new host-targeted broad-spectrum antiviral agents.

Efficacy of Kushen decoction on high-fat-diet-induced hyperlipidemia in rats.

OBJECTIVE: To investigate the efficacy and underlying mechanisms of action of Kushen decoction on high-fat-diet-induced hyperlipidemia in rats using RNA-seq technology. METHODS: The efficacy of a Kushen decoction, at a concentration of 1 mL/g of crude medicine prepared according to the method commonly used in clinical practice, was investigated on 24 specific pathogen-free male Sprague-Dawley rats. Liver tissues were compared using RNA-Seq technology. The differentially expressed genes were further investigated by real-time fluorescent quantitative polymerase chain reaction (qPCR and Western blot (WB). RESULTS: Serum triglycerides (TG), liver low-density lipoprotein cholesterol (LDL-C), body weight, body length, and Lee's index were significantly increased in the untreated hyperlipidemia-induced group (model) compared with the control group, whereas liver high-density lipoprotein cholesterol (HDL-C) was significantly decreased. Serum TG, liver LDL-C, bodyweight, and Lee's index were decreased in the high-dose Kushen decoction group (HDKS) compared with the model group, whereas liver HDL-C was significantly increased. Similarly, liver TG tended to decline in the HDKS group. Comparison of the gene expression profiles in the livers from different groups indicated that the Kushen decoction significantly affected metabolic pathways, PPAR signalling pathway, and circadian rhythm ( ≤ 0.05), with the genes ARNTL, PER3, and CLOCK being differentially expressed. qPCR and WB analysis confirmed the differential expression of the genes discovered by transcriptomics analysis. CONCLUSION: The Kushen decoction may achieve a lipid-lowering effect on hyperlipidemic rats by regulating metabolic pathways and the circadian rhythm pathway and in particular, their related genes ARNTL, PER3, and CLOCK.

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.

The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication.

The circadian clock regulates immune responses to microbes and affects pathogen replication, but the underlying molecular mechanisms are not well understood. Here we demonstrate that the circadian components BMAL1 and REV-ERBα influence several steps in the hepatitis C virus (HCV) life cycle, including particle entry into hepatocytes and RNA genome replication. Genetic knock out of Bmal1 and over-expression or activation of REV-ERB with synthetic agonists inhibits the replication of HCV and the related flaviruses dengue and Zika via perturbation of lipid signaling pathways. This study highlights a role for the circadian clock component REV-ERBα in regulating flavivirus replication.