Per2 mRNA Expression Research Articles

Microcurrent stimulation activates the circadian machinery in mice

The circadian rhythm, which regulates various body functions, is transcriptionally controlled by a series of clock gene clusters. The clock genes are related to the pathology of various kinds of diseases, which in turn, is related to aging. Aging in humans is a worldwide problem; it induces sleep disorders and disruption of the circadian rhythm. It also decreases ocular vision and appetite and weakens the synchronization of clock genes by light and food. Therefore, a simple method for the synchronization of clock genes in the body is required. In this study, the influence of microcurrent stimulation (MCS) on the circadian machinery in wild-type (WT) and Clock mutant (Clk/Clk) mice was investigated. MCS induced Per1 mRNA expression in cultured mouse astrocytes; cAMP response element (CRE) in the Per1 mouse promoter was found to be important for the induction of Per1 mRNA. In addition, MCS increased the Per1 mRNA levels in mouse livers and caused the phase advance of the Per1 expression rhythm. The protein expression rhythm of phosphor-cAMP response element-binding protein (pCREB) was altered and the phase of expression of pCREB protein advanced. Finally, the influence of MCS on the locomotor activity rhythm in WT and Clk/Clk mice was investigated. MCS caused the phase advance of the locomotor activity rhythm in WT and Clk/Clk mice. The results of this study indicate that MCS activated the clock machinery in mice; MCS may thus improve the quality of new treatment modalities in the future.

<p>Chronic chemotherapy with paclitaxel nanoparticles induced apoptosis in lung cancer in vitro and in vivo</p>

AimPaclitaxel (PTX) is an effective antitumor drug. Previous research demonstrated that paclitaxel nanoparticles (PTX-NPs) exhibited the greatest antitumor effect at 15 hours after light onset (15 HALO), but the mechanism in chronic chemotherapy is still unknown. In our study, we investigated whether PTX-NPs regulated Period2 (Per2) during chronic chemotherapy to induce apoptosis in vivo and in vitro.MethodsTo improve the antitumor effect and reduce organ damage induced by PTX treatment, PTX-NPs were prepared using a film dispersion method. Then, A549 cells were treated with PTX-NPs at 0, 5, 10, 15, and 20 HALO. An annexin/PI V-FITC apoptosis kit was measured for apoptosis, and PI was analyzed for cell cycle. The relative mechanism was detected by RT-PCR and Western blotting. Tumor volume and weight were measured to evaluate the antitumor effect of the PTX-NPs, and H&E staining was performed to assess organ damage.ResultsCell cycle analysis demonstrated that PTX-NPs blocked cell cycle in G2 phase and that the ratio of cell death was significantly increased in A549 cells, while the ratios of cells in G2 phase and of apoptotic cells were highest at 15 HALO. Evaluation of in vivo antitumor activity revealed that PTX-NPs inhibited tumor growth and decreased tumor weight at 15 HALO. RT-PCR and Western blotting demonstrated that PTX-NPs upregulated Per2 mRNA and protein expression, and the highest Per2 expression was observed at 15 HALO in vivo and in vitro. Meanwhile, Bax mRNA and protein expression was upregulated, while Bcl-2 mRNA and protein expression was downregulated after PTX-NPs treatment in vivo. Moreover, H&E staining revealed that PTX-NPs reduced liver damage at 15 HALO.ConclusionPTX-NPs exhibited the most effective antitumor activity and reduced liver damage at 15 HALO through upregulation of Per2 expression to induce apoptosis in vivo and in vitro.

Smad3 Suppresses Epithelial Cell Migration and Proliferation via the Clock Gene Dec1, Which Negatively Regulates the Expression of Clock Genes Dec2 and Per1

Smad3 has circadian expression; however, whether Smad3 affects the expression of clock genes is poorly understood. Here, we investigated the regulatory mechanisms between Smad3 and the clock genes Dec1, Dec2, and Per1. In Smad3 knockout mice, the amplitude of locomotor activity was decreased, and Dec1 expression was decreased in the suprachiasmatic nucleus, liver, kidney, and tongue compared with control mice. Conversely, Dec2 and Per1 expression was increased compared with that of control mice. In Smad3 knockout mice, immunohistochemical staining revealed that Dec1 expression decreased, whereas Dec2 and Per1 expression increased in the endothelial cells of the kidney and liver. In NIH3T3 cells, Smad3 overexpression increased Dec1 expression, but decreased Dec2 and Per1 expression. In a wound-healing experiment that used Smad3 knockout mice, Dec1 expression decreased in the basal cells of squamous epithelium, promoting wound healing of the mucosa. Finally, the migration and proliferation of Smad3 knockdown squamous carcinoma cells was suppressed by Dec1 overexpression but was promoted by Dec2 overexpression. Dec1 overexpression decreased E-cadherin and proliferating cell nuclear antigen expression, whereas these expression levels were increased by Dec2 overexpression. These results suggest Smad3 is relevant to circadian rhythm and regulates cell migration and proliferation through Dec1, Dec2, and Per1 expression.

Temporal dynamics of cortisol-associated changes in mRNA expression of glucocorticoid responsive genes FKBP5, GILZ, SDPR, PER1, PER2 and PER3 in healthy humans

Secretion of the stress hormone cortisol follows a circadian rhythm and is stimulated following stress exposure. Cortisol regulates the transcription of several genes, primarily through activation of the glucocorticoid receptor (GR). Previously, we showed an upregulation of PERIOD genes PER1 and PER3 after pharmacological/glucocorticoid challenge in vivo and in vitro. The current study aims to investigate the temporal association between unstimulated, diurnal cortisol secretion and the expression of selected GR-target genes (PER1, PER2, PER3, FKBP5, GILZ and SDPR) in vivo to determine the timing of the most pronounced coupling between cortisol and mRNA expression.Unstimulated plasma and saliva cortisol concentrations and gene expression levels in whole blood were measured every 15 min from early morning until 16:00 h in 18 healthy men. Time-lagged correlations of cortisol concentrations with mRNA expression levels were assessed allowing lags between -240 and + 240 min.Strong positive correlations at non-zero lags between cortisol levels and the expression of FKBP5 (plasma: r = 0.74 (CI = 0.65-0.81), p < 0.001, lag + 90 min; saliva: r = 0.71 (CI = 0.61-0.78), p < 0.001, lag + 75 min), and GILZ (plasma: r = 0.59 (CI = 0.46-0.69), p < 0.001, lag + 30 min; saliva r = 0.53 (CI = 0.41-0.63), p < 0.001, lag +15 min) were observed. Expressions of PERIOD genes and SDPR correlated only weakly with cortisol (all |r| < 0.25).Our findings demonstrate strong correlations between cortisol secretion and gene expression in humans under unstimulated conditions. The observed time-lags can guide future research aiming to characterize glucocorticoid-dependent gene expression in clinical samples with stress-related disorders.

Bisphenol A Alters Bmal1, Per2, and Rev-Erba mRNA and Requires Bmal1 to Increase Neuropeptide Y Expression in Hypothalamic Neurons.

Bisphenol A (BPA), a ubiquitous environmental endocrine disruptor, is considered an obesogen. However, its role in the hypothalamic control of energy balance remains largely unexplored. Because disruption of the circadian clock is tightly associated with metabolic consequences, we explored how BPA affects the components of the molecular circadian clock in the feeding-related neurons of the hypothalamus. In immortalized POMC and NPY/AgRP-expressing hypothalamic cell lines and primary culture, we describe how BPA significantly alters mRNA expression of circadian clock genes Bmal1,Per2, and Rev-Erbα. Furthermore, we use newly generated Bmal1-knockout (KO) hypothalamic cell lines to link the BPA-induced neuropeptide dysregulation to the molecular clock. Specifically, BPA increased Npy, Agrp, and Pomc mRNA expression in wild type hypothalamic cells, whereas the increase in Npy, but not Agrp or Pomc, was abolished in cell lines lacking BMAL1. In line with this increase, BPA led to increased BMAL1 binding to the Npy promotor, potentially increasing Npy transcription. In conclusion, we show that BPA-mediated dysregulation of the circadian molecular clock is linked to the deleterious effects of BPA on neuropeptide expression. Furthermore, we describe hypothalamic Bmal1-KO cell lines to study the role of BMAL1 in hypothalamic responses to metabolic, hormonal, and environmental factors.

Implicit time-place conditioning alters Per2 mRNA expression selectively in striatum without shifting its circadian clocks

Animals create implicit memories of the time of day that significant events occur then anticipate the recurrence of those conditions at the same time on subsequent days. We tested the hypothesis that implicit time memory for daily encounters relies on the setting of the canonical circadian clockwork in brain areas involved in the formation or expression of context memories. We conditioned mice to avoid locations paired with a mild foot shock at one of two Zeitgeber times set 8 hours apart. Place avoidance was exhibited only when testing time matched the prior training time. The suprachiasmatic nucleus, dorsal striatum, nucleus accumbens, cingulate cortex, hippocampal complex, and amygdala were assessed for clock gene expression. Baseline phase dependent differences in clock gene expression were found in most tissues. Evidence for conditioned resetting of a molecular circadian oscillation was found only in the striatum (dorsal striatum and nucleus accumbens shell), and specifically for Per2 expression. There was no evidence of glucocorticoid stress response in any tissue. The results are consistent with a model where temporal conditioning promotes a selective Per2 response in dopamine-targeted brain regions responsible for sensorimotor integration, without resetting the entire circadian clockwork.

Peripheral Neuroimmunoendocrine Interactions: Contribution of TNFRp55 to the Circadian Synchronization of Progesterone and Cytokine Production in Joints of Mice in Late Pregnancy

Objective: Circadian rhythms are generated by the suprachiasmatic nucleus of the hypothalamus and involve rhythmic expression of clock genes and proteins. This rhythmicity is transferred to peripheral tissues by neural and hormonal signals. Late pregnancy is considered a state of inflammation which impacts on peripheral tissues such as joints. Tumor necrosis factor (TNF) mediates inflammatory and circadian responses through its p55 receptor (TNFRp55). Neuroimmunoendocrine interactions in joints have not been studied completely. The purpose of this study was to analyze these interactions, investigating the circadian rhythms of progesterone (Pg) and pro- and anti-inflammatory cytokines in the joints at the end of pregnancy (gestational day 18). Moreover, the impact of TNFRp55 deficiency on these temporal oscillations was explored. Methods: Wild-type and TNFRp55-deficient (KO) C57BL/6 mice were kept under constant darkness in order to study their endogenous circadian rhythms. The expression of the clock genes Bmal1 and Per1 at circadian time 7 was studied by reverse transcription polymerase chain reaction in the ankle joints of nonpregnant and pregnant (gestational day 18) mice. In late pregnancy, Pg and the cytokines interleukin 17 (IL-17), IL-6, and IL-10 were measured in the joints throughout a 24-h period by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. Results: A significant increase in Bmal1 and Per1 mRNA expression was detected in the joints of pregnant KO mice. Furthermore, KO mice displayed a desynchronization of articular Pg and cytokine production. Conclusions: Our results show that TNF, via TNFRp55 signaling, modulates articular Pg and cytokine circadian rhythms in late pregnancy. These findings suggest a temporal neuroimmunoendocrine association in peripheral tissues in late pregnancy.

Maternal supplementation with calcium varying with feeding time daily during late pregnancy affects lipid metabolism and transport of placenta in pigs

To investigate effects of Ca level varying with feeding time daily in sows during late pregnancy on placental lipid metabolism and transport in pigs, sixty pregnant sows were assigned to 3 groups: the CON group was fed low-Ca diet with 11.25 g CaCO3 at 0600 h and 1500 h, H-L group was fed low-Ca diet with 22.5 g CaCO3 at 0600 h and low-Ca diet at 1500 h, and L-H group was fed low-Ca diet at 0600 h and low-Ca diet with 22.5 g CaCO3 at 1500 h, respectively. Serum from sows and umbilical cord and placenta were collected during delivery. Results showed that, compared with the CON group, H-L feeding significantly increased maternal serum total triglyceride (TG) and umbilical serum high-density lipoprotein (HDL) (P < 0.05). The results showed that long chain fatty acid (FA) contents in placenta were significantly increased in H-L and L-H groups (P < 0.05). Experiments on genes involved in glycolipid metabolism showed that H-L or L-H feeding inhibited mRNA expression of GLUT3, GLUT4, FAS, FABP1, FABPpm, FAT/CD36, while activated the mRNA expression of FASD1, FASD2 and SCD in placenta (P < 0.05). In addition, experiments on genes involved in biological clock showed that L-H feeding sequence activated the mRNA expression of per1 and clock, while H-L and L-H feeding sequence inhibited mRNA expression of per2 in placenta (P < 0.05). It is concluded that maternal supplementation with Ca varying with feeding time daily during late pregnancy affects placental lipid metabolism and transport in pigs by regulating the mRNA expression related to lipid metabolism and the circadian clock.

Short-term propofol anaesthesia down-regulates clock genes expression in the master clock

ABSTRACTBackground: Propofol anesthesia triggers phase-advances of circadian rhythms controlled by the suprachiasmatic nuclei (SCN), the master clock. Besides, inhalational anesthesia has been associated with a subsequent reduction of Per2 mRNA levels in the whole brain of rodents. The acute effects of propofol anesthesia per se on the SCN molecular clockwork remain unclear. Here we aim to study the expression of Per1 and Per2 clock genes in the SCN of rats exposed to constant darkness after a single dose of propofol. Methods: Thirty 2-months old rats were randomly divided into 2 groups receiving a single dose of either 120 mg/kg propofol 1% (n=15), or intralipid® 10% (n=15) in late day (projected circadian time (CT) 10, i.e., 10h after the expected time of lights on). Thereafter, rat brains were sampled in darkness 1h, 2h or 3h after the treatment (projected CT11, CT12 or CT13). Expression of Per1 and Per2 mRNA was analyzed by in situ hybridization in SCN coronal sections. Results: Per1 expression was affected by time and treatment. Per1 expression in the SCN after propofol treatment decreased at CT11 and CT12 when compared to the vehicle group. For Per2 expression, we observed only a treatment effect. Observed in dark conditions without hypothermia or/and concomitant surgery, such down-regulation of clock genes Per is only correlated to propofol treatment. This may explain “jet-lag-like” symptoms described by patients after anesthesia. Conclusion: We show here for the first time that short-term propofol anesthesia leads to a transient down-regulation of Per1 and Per2 expression in the SCN.

Electroconvulsive Seizure Alters the Expression and Daily Oscillation of Circadian Genes in the Rat Frontal Cortex.

ObjectiveElectroconvulsive therapy (ECT) is the most effective treatment for mood disorders. Accumulating evidence has suggested the important role of circadian genes in mood disorders. However, the effects of ECT on circadian genes have not been systemically investigated. MethodsWe examined the expression and daily oscillation of major circadian genes in the rat frontal cortex after electroconvulsive seizure (ECS). ResultsFirstly, mRNA and protein level were investigated at 24 hr after single ECS (E1X) and repeated ECS treatements for 10 days (E10X), which showed more remarkable changes after E10X than E1X. mRNA expression of Rorα, Bmal1, Clock, Per1, and Cry1 was decreased, while Rev-erbα expression was increased at 24 hr after E10X compared to sham. The proteins showed similar pattern of changes. Next, the effects on oscillation and rhythm properties (mesor, amplitude, and acrophase) were examined, which also showed more prominent changes after E10X than E1X. After E10X, mesor of Rorα, Bmal1, and Cry1 was reduced, and that of Rev-erbα was increased. Five genes, Rev-erbα, Bmal1, Per1, Per2, and Cry2, showed earlier acrophase after E10X. Conclusion The findings suggest that repeated ECS induces reduced expression and phase advance of major circadian genes in the in vivo rat frontal cortex.

Histone deacetylase inhibitors induce the expression of tumor suppressor genes Per1 and Per2 in human gastric cancer cells.

Period circadian regulator (Per)1 and Per2 genes are involved in the molecular mechanism of the circadian clock, and exhibit tumor suppressor properties. Several studies have reported a decreased expression of Per1, Per2 and Per3 genes in different types of cancer and cancer cell lines. Promoter methylation downregulates Per1, Per2 or Per3 expression in myeloid leukemia, breast, lung, and other cancer cells; whereas histone deacetylase inhibitors (HDACi) upregulate Per1 or Per3 expression in certain cancer cell lines. However, the transcriptional regulation of Per1 and Per2 in cancer cells by chromatin modifications is not fully understood. The present study aimed to determine whether HDACi regulate Per1 and Per2 expression in gastric cancer cell lines, and to investigate changes in chromatin modifications in response to HDACi. Treatment of KATO III and NCI-N87 human gastric cancer cells with sodium butyrate (NaB) or Trichostatin A (TSA) induced Per1 and Per2 mRNA expression in a dose-dependent manner. Chromatin immunoprecipitaion assays revealed that NaB and TSA decreased lysine 9 trimethylation on histone H3 (H3K9me3) at the Per1 promoter. TSA, but not NaB increased H3K9 acetylation at the Per2 promoter. It was also observed that binding of Sp1 and Sp3 to the Per1 promoter decreased following NaB treatment, whereas Sp1 binding increased at the Per2 promoter of NaB- and TSA-treated cells. In addition, Per1 promoter is not methylated in KATO III cells, while Per2 promoter was methylated, although NaB, TSA, and 5-Azacytidine do not change the methylated CpGs analyzed. In conclusion, HDACi induce Per1 and Per2 expression, in part, through mechanisms involving chromatin remodeling at the proximal promoter of these genes; however, other indirect mechanisms triggered by these HDACi cannot be ruled out. These findings reveal a previously unappreciated regulatory pathway between silencing of Per1 gene by H3K9me3 and upregulation of Per2 by HDACi in cancer cells.

A Free-Choice High-Fat High-Sugar Diet Alters Day-Night Per2 Gene Expression in Reward-Related Brain Areas in Rats.

Under normal light–dark conditions, nocturnal rodents consume most of their food during the dark period. Diets high in fat and sugar, however, may affect the day–night feeding rhythm resulting in a higher light phase intake. In vitro and in vivo studies showed that nutrients affect clock-gene expression. We therefore hypothesized that overconsuming fat and sugar alters clock-gene expression in brain structures important for feeding behavior. We determined the effects of a free-choice high-fat high-sugar (fcHFHS) diet on clock-gene expression in rat brain areas related to feeding and reward and compared them with chow-fed rats. Consuming a fcHFHS diet for 6 weeks disrupted day–night differences in Per2 mRNA expression in the nucleus accumbens (NAc) and lateral hypothalamus but not in the suprachiasmatic nucleus, habenula, and ventral tegmental area. Furthermore, short-term sugar drinking, but not fat feeding, upregulates Per2 mRNA expression in the NAc. The disruptions in day–night differences in NAc Per2 gene expression were not accompanied by altered day–night differences in the mRNA expression of peptides related to food intake. We conclude that the fcHFHS diet and acute sugar drinking affect Per2 gene expression in areas involved in food reward; however, this is not sufficient to alter the day–night pattern of food intake.

Abnormal circadian locomotor rhythms and Per gene expression in six-month-old triple transgenic mice model of Alzheimer’s disease

Circadian rhythm disturbance (CRD) is one of the iconic manifestations in Alzheimer’s disease (AD), a disease tightly associated with age, but the characteristics and gender difference of CRD occurred in AD have not been well demonstrated. Using 6-month-old triple transgenic AD mouse model (3xTg-AD) without obvious brain pathological changes, we demonstrated the gender difference of CRD at this age. We further showed abnormal Per gene expression in the central clock suprachiasmatic nucleus (SCN) of the 3xTg-AD mice. Specifically, compared with the wide type (WT) mice, the 3xTg-AD mice showed disrupted circadian locomotor rhythms both at LD (light-dark 12 h:12 h) and DD (constant dark) conditions, such as increased activities in the resting phase, decreased and scattered activities in the active phase, decreased overall activity intensities, amplitude, robustness, and increased intradaily variability. We further observed that 3xTg-AD female mice showed obviously less CRD compared with the 3xTg-AD male mice, and female mice of both WT and 3xTg-AD were more active in locomotor activity. Accordingly, 3xTg-AD mice showed a phase delay in the expression of Per1 and Per2 mRNA in the SCN, with the levels of Per1 and Per2 mRNA were significantly lower than that of WT mice at specific time points. We conclude that 3xTg-AD mice exhibit behavioral CRD at the age of six months with male gender preference, and these phenomena are at least partly associated with the alteration of Per1 and Per2 transcription patterns in the SCN.

Modeling the interactions of sense and antisense Period transcripts in the mammalian circadian clock network.

In recent years, it has become increasingly apparent that antisense transcription plays an important role in the regulation of gene expression. The circadian clock is no exception: an antisense transcript of the mammalian core-clock gene PERIOD2 (PER2), which we shall refer to as Per2AS RNA, oscillates with a circadian period and a nearly 12 h phase shift from the peak expression of Per2 mRNA. In this paper, we ask whether Per2AS plays a regulatory role in the mammalian circadian clock by studying in silico the potential effects of interactions between Per2 and Per2AS RNAs on circadian rhythms. Based on the antiphasic expression pattern, we consider two hypotheses about how Per2 and Per2AS mutually interfere with each other's expression. In our pre-transcriptional model, the transcription of Per2AS RNA from the non-coding strand represses the transcription of Per2 mRNA from the coding strand and vice versa. In our post-transcriptional model, Per2 and Per2AS transcripts form a double-stranded RNA duplex, which is rapidly degraded. To study these two possible mechanisms, we have added terms describing our alternative hypotheses to a published mathematical model of the molecular regulatory network of the mammalian circadian clock. Our pre-transcriptional model predicts that transcriptional interference between Per2 and Per2AS can generate alternative modes of circadian oscillations, which we characterize in terms of the amplitude and phase of oscillation of core clock genes. In our post-transcriptional model, Per2/Per2AS duplex formation dampens the circadian rhythm. In a model that combines pre- and post-transcriptional controls, the period, amplitude and phase of circadian proteins exhibit non-monotonic dependencies on the rate of expression of Per2AS. All three models provide potential explanations of the observed antiphasic, circadian oscillations of Per2 and Per2AS RNAs. They make discordant predictions that can be tested experimentally in order to distinguish among these alternative hypotheses.

Mice Lacking EGR1 Have Impaired Clock Gene (BMAL1) Oscillation, Locomotor Activity, and Body Temperature.

Early growth response transcription factor 1 (EGR1) is expressed in the suprachiasmatic nucleus (SCN) after light stimulation. We used EGR1-deficient mice to address the role of EGR1 in the clock function and light-induced resetting of the clock. The diurnal rhythms of expression of the clock genes BMAL1 and PER1 in the SCN were evaluated by semi-quantitative in situ hybridization. We found no difference in the expression of PER1 mRNA between wildtype and EGR1-deficient mice; however, the daily rhythm of BMAL1 mRNA was completely abolished in the EGR1-deficient mice. In addition, we evaluated the circadian running wheel activity, telemetric locomotor activity, and core body temperature of the mice. Loss of EGR1 neither altered light-induced phase shifts at subjective night nor affected negative masking. Overall, circadian light entrainment was found in EGR1-deficient mice but they displayed a reduced locomotor activity and an altered temperature regulation compared to wild type mice. When placed in running wheels, a subpopulation of EGR1-deficient mice displayed a more disrupted activity rhythm with no measurable endogenous period length (tau). In conclusion, the present study provides the first evidence that the circadian clock in the SCN is disturbed in mice deficient of EGR1.

Dynamic feeding low and high methionine diets affect the diurnal rhythm of amino acid transporters and clock related genes in jejunum of laying hens

ABSTRACTThis study was conducted to investigate effects of dynamic feeding low and high methionine (Met) diets on circadian clock gene expression in jejunum of laying hens. A total of 180 laying hens (Brown Hy-line, 41 weeks old) were allocated into 3 groups with 6 replicates each. The CON group received diet containing 0.30% Met, LH group received low Met diet (0.27% Met) at 07:30 h and high Met diet (0.33% Met) at 15:30 h, while HL group received high Met diet at 07:30 h and low Met diet at 15:30 h. After 10 weeks, jejunum samples were collected at 4 h intervals in a daily cycle initial starting at 07:30 h before feeding. Results showed that dynamic feeding of Met significantly affected the wave of mRNA expression of B0AT1, LAT1, SNAT2, CAT-2, and y+LAT2 by feeding regime during a day (P<0.05). Dynamic feeding of Met also influenced the fluctuation of relative mRNA expression of Clock, Bmal1, Cry1, Cry2, Per2, and Per3 significantly in hens during a day (P<0.05). These results demonstrated that dynamic feeding different Met level diets of hens affected the rhythmic fluctuation of amino acid transporters and may affected intestinal function by influencing the clock-related genes further.

Passionflower Extract Induces High-amplitude Rhythms without Phase Shifts in the Expression of Several Circadian Clock Genes in Vitro and in Vivo

Circadian rhythms play key roles in the regulation of physiological and behavioral systems including wake-sleep cycles. We evaluated the effects of passionflower (aerial parts of Passiflora incarnata Linnaeus) extract (PFE) on circadian rhythms using NIH3T3 cells and mice. PFE (100 μg/mL) induced high-amplitude rhythms in the expression of period circadian protein (Per) 2, cryptochrome (Cry) 1, superoxide dismutase (SOD) 1, and glutathione peroxidase (GPx) in vitro from 12 h after a treatment with serum-rich medium. Isovitexin 2"-O-glucoside, isoschaftoside, and homoorientin, which were purified from PFE, also significantly enhanced Per2 mRNA expression at 20 h. An oral treatment with PFE (100 mg/kg/day) at zeitgeber time (ZT) 0 h for 15 days improved sleep latencies and sleeping times in the pentobarbital-induced sleep test in mice, similar to muscimol (0.2 mg/kg, i.p.). PFE induced high-amplitude rhythms without obvious phase shifts in serum corticosterone levels and the expression of Per1, Per2, and Cry1 in the liver as well as NIH3T3 cells. However, in the cerebrum, PFE enhanced the circadian expression of brain-muscle ARNT-like protein (Bmal) 1, circadian locomotor output cycles kaput (Clock), and Per1. Regarding this difference, we suggest the involvement of several neurotransmitters that influence the circadian rhythm. Indeed, PFE significantly increased dopamine levels at ZT 18 h, and then affected the mRNA expression of the synthetic and metabolic enzymes such as monoamine oxidase (MAO), catechol-O-methyltransferase (COMT), and glutamic acid decarboxylase (GAD). The results obtained show that PFE positively modulates circadian rhythms by inducing high-amplitude rhythms in the expression of several circadian clock genes.

MC3T3-E1 세포에서 BMP2에 의한 조골세포의 분화에 일주기 유전자 Per1이 미치는 영향

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that play important roles in a variety of cellular functions. Among BMP family members, BMP2 efficiently promotes osteoblast differentiation through Smad-mediated runt-related transcription factor 2 (Runx2) expression. Several recent studies suggest that BMPs are associated with clock genes, in particular Bmal1. Bmal1 protein heterodimerizes with Clock protein and then induces period 1 (Per1) expression. However, the role of Per1 on osteoblast differentiation remains unclear. In this study, we investigated whether Per1 is involved in osteoblast differentiation. MC3T3-E1 cells were treated with BMP2 for induction of osteoblastic differentiation. Osteogenic maker gene and Per1 mRNA expression were measured using real-time PCR. Interestingly, BMP2 treatment induced Per1 mRNA expression in MC3T3-E1 cells. To further investigate the function of Per1 on osteoblast differentiation, MC3T3-E1 cells were transiently transfected with pCMV-Per1. Per1 overexpression increased Runx2 mRNA and protein levels. Also, mRNA expression and promoter activity of osteocalcin were upregulated by Per1 overexpression. To investigate the effect of interaction between Per1 and osteogenic condition, MC3T3-E1 cells were cultured in osteogenic medium containing ascorbic acid and β-glycerophosphate. Osteogenic medium-induced ALP staining level and mineralization were synergistically increased by overexpression of Per1. Taken together, these results demonstrate that Per1 is a positive regulator of osteoblast differentiation.

Postnatal Ontogeny of the Circadian Expression of the Adrenal Clock Genes and Corticosterone Rhythm in Male Rats.

The postnatal synchronization of the circadian variation of the adrenal clock genes in mammals remains unknown. We evaluated the postnatal ontogeny of daily variation of clock genes (Clock/Bmal1/Per1/Per2/Per3/Cry1/Cry2/Rorα/Rev-Erbα) and steroidogenesis-related genes (Star and Mc2r) in rat adrenals and its relationship with the emergence of plasma corticosterone rhythm using cosinor analysis. Plasma corticosterone circadian rhythm was detected from postnatal day (P)1, with morning acrophase, between zeitgeber time (ZT)0 and ZT2. From P14, there was a nocturnal acrophase of corticosterone at ZT20, which was associated with pups' eye opening. From P3 there was a circadian variation of the mRNA expression of Bmal1, Per2, Per3, and Cry1 genes with morning acrophase, whereas Rev-Erbα had nocturnal acrophase. From P14, Bmal1, Per2, Per3, and Cry1 acrophases advanced by approximately 10 hours, as compared with early neonatal days, becoming vespertine-nocturnal. In all postnatal ages, Per2 and Cry1 circadian profiles were synchronized in phase with the circadian rhythm of plasma corticosterone, whereas Bmal1 was in antiphase. An adult-like Star circadian rhythm profile was observed only from P21. In conclusion, our original data demonstrated a progressive postnatal maturation of the circadian variation of the adrenal clock genes in synchrony with the development of the corticosterone circadian rhythm in rats.

Effect of 1a, 25(OH)2D3 on circadian clock gene expression in cardiac myocytes

Objective To explore the effect of 1a, 25(OH)2D3 on circadian clock gene expressions in cardiac myocytes. Methods Cultured cardiac myocytes isolated from 7-day-old Sprague-Dawley(SD) rats were identified by immunofluorescence.The medium including 1a, 25(OH)2D3 (final concentrations were 0 nmol/L, 1 nmol/L, 10 nmol/L, 50 nmol/L and 100 nmol/L) were added to primary myocardial cells to culture for 2 h and then total RNA was extracted.Real-time polymerase chain reaction (RT-PCR) was applied to analyze myocardial cells circadian clock gene (Bmal1, Per2, Rev-erba) transcript levels to determine optimum concentration of 1a, 25(OH)2D3.Then, the primary myocardial cells cultured for 72 h were divided into 3 groups: the control group was of serum-free culture medium; serum shock group was of DMEM containing 50%volume fraction of horse serum cultured 2 h; 1a, 25(OH)2D3 treatment group receiving 1a, 25(OH)2D3 at optimal concentration cultured 2 h. The cells were collected at 7 time points (0 h, 4 h, 8 h, 12 h, 16 h, 20 h, 24 h) and then total RNA was extracted.RT-PCR was applied to analyze circadian clock gene (Bmal1, Per2, Rev-erba) transcript levels in the myocardial cells. Results In the presence of 50 nmol/L 1 a, 25(OH)2D3, the Bmal1 mRNA expression showed the highest level, but the Per2 and Rev-erba mRNA expression levels were minimum.Compared with the control group, both 1a, 25(OH)2D3 treatment group and serum shock group caused day-cycle rhythmic oscillation in circadian clock genes(Bmal1, Per2, Rev-erba) in the cardiac myocytes.And the expressions pattern of Bmal1 and Per2 genes were in the opposite phase.While Bmal1 gene expression appeared at peak at 12 h, Per2 gene expression appeared in a trough.Expression of Rev-erba gene trend began to rise at 8 h, and the highest expression level appeared at 12-16 h. Conclusions 1a, 25(OH)2D3 can affect Bmal1, Per2 and Rev-erba mRNA expressions of circadian clock genes in the cardiac myocytes. Key words: 1a, 25(OH)2D3; Cardiac myocytes; Circadian clock; Circadian clock gene