Epigenome-wide DNA methylation profiling of preeclamptic placenta according to severe features.

BACKGROUND: Preeclampsia (PE) is an obstetric disorder with significant morbidities for both the mother and fetus possibly caused by a failure of the placental trophoblast invasion. However, its pathophysiology largely remains unclear. Here, we performed DNA methylation profiling to determine whether differential patterns of DNA methylation correlate with PE and severe features of PE. MATERIALS AND METHODS: We extracted DNA from placental tissues of 13 normal, five PE, and eight PE pregnant women with severe features. Genome-wide DNA methylation analysis was performed using the Illumina HumanMethylation 850K BeadChip. New functional annotations of differentially methylated CpGs (DMCs) in PE were predicted using bioinformatics tools. RESULTS: Significant differences were evident for 398 DMCs, including 243 DMCs in PE and 155 DMCs in PE with severe features, compared with normal placental tissues. Of these, 12 hypermethylated DMCs and three hypomethylated DMCs were observed in both PE groups, thus were independent from severe features. Three hundred seventy-nine DMCs were identified by the presence or absence of severe features. Two hundred genes containing these DMCs were associated with developmental processes and cell morphogenesis. These genes were significantly associated with various PE complications such as disease susceptibility, viral infections, immune system diseases, endocrine disturbance, seizures, hematologic diseases, and thyroid diseases. CONCLUSIONS: This is the first study to investigate the genome-scale DNA methylation profiles of PE placentas according to severe features. The epigenetic variation in the placentas probably resulted in altered developmental processes and immune dysregulation, contributing to PE. This study provides basic information to refine the clinical and pathological mechanisms of the severe features in placenta-mediated PE.

Epigenome-wide base-resolution profiling of DNA methylation in chorionic villi of fetuses with Down syndrome by methyl-capture sequencing.

BACKGROUND: Epigenetic mechanisms provide an interface between environmental factors and the genome and are influential in various diseases. These mechanisms, including DNA methylation, influence the regulation of development, differentiation, and establishment of cellular identity. Here, we performed high-throughput methylome profiling to determine whether differential patterns of DNA methylation correlate with Down syndrome (DS). MATERIALS AND METHODS: We extracted DNA from the chorionic villi cells of five normal and five DS fetuses at the early developmental stage (12-13 weeks of gestation). Methyl-capture sequencing (MC-Seq) was used to investigate the methylation levels of CpG sites distributed across the whole genome to identify differentially methylated CpG sites (DMCs) and regions (DMRs) in DS. New functional annotations of DMR genes using bioinformatics tools were predicted. RESULTS: DNA hypermethylation was observed in DS fetal chorionic villi cells. Significant differences were evident for 4,439 DMCs, including hypermethylation (n = 4,261) and hypomethylation (n = 178). Among them, 140 hypermethylated DMRs and only 1 hypomethylated DMR were located on 121 genes and 1 gene, respectively. One hundred twenty-two genes, including 141 DMRs, were associated with heart morphogenesis and development of the ear, thyroid gland, and nervous systems. The genes were significantly associated with DS and various diseases, including hepatopulmonary syndrome, conductive hearing loss, holoprosencephaly, heart diseases, glaucoma, and musculoskeletal abnormalities. CONCLUSIONS: This is the first study to compare the whole-epigenome DNA methylation pattern of the chorionic villi cells from normal and DS fetuses at the early developmental-stage using MC-seq. Overall, our results indicate that the chorionic villi cells of DS fetuses are hypermethylated in all autosomes and suggested that altered DNA methylation may be a recurrent and functionally relevant downstream response to DS in human cells. This study provides basic information for future research focused on the pathophysiology of the DS and its potential effects, as well as the role DNA methylation plays in the early developmental stage of DS fetuses.

Involvement of α(2)-adrenergic receptor in the regulation of the blood glucose level induced by immobilization stress.

The blood glucose profiles were characterized after mice were forced into immobilization stress with various exposure durations. The blood glucose level was significantly enhanced by immobilization stress for 30 min or 1 h, respectively. On the other hand, the blood glucose level was not affected in the groups which were forced into immobilization stress for 2 or 4 h. We further examined the effect of yohimbine (an α2-adrenergic receptor antagonist) administered systemically or centrally in the immobilization stress model. Mice were pretreated intraperitoneally (i.p.; from 0.5 to 5 mg/kg), intracerebroventricularly (i.c.v.; from 1 to 10 µg/5 µl), or intrathecally (i.t.; from 1 to 10 µg/5 µl) with yohimbine for 10 min and then, forced into immobilization stress for 30 min. The blood glucose level was measured right after immobilization stress. We found that up-regulation of the blood glucose level induced by immobilization stress was abolished by i.p. pretreatment with yohimbine. And the immobilization stress-induced blood glucose level was not inhibited by i.c.v. or i.t. pretreatment with yohimbine at a lower dose (1 µg/5 µl). However, immobilization stress-induced blood glucose level was significantly inhibited by i.c.v. or i.t. pretreatment with yohimbine at higher doses (5 and 10 µg/5 µl). In addition, the i.p. (5 mg/kg), i.c.v. (10 µg/5 µl), or i.t. (10 µg/5 µl) pretreatment with yohimbine reduced hypothalamic glucose transporter 4 expression. The involvement of α2-adrenergic receptor in regulation of immobilization stress- induced blood glucose level was further confirmed by the i.p, i.c.v, or i.t pretreatment with idazoxan, another specific α2-adrenergic receptor antagonist. Finally, i.p., i.c.v., or i.t. pretreatment with yohimbine attenuated the blood glucose level in D-glucose-fed model. We suggest that α2-adrenergic receptors located at the peripheral, the brain and the spinal cord play important roles in the up-regulation of the blood glucose level in immobilization stress.

Interleukin-1ß (IL-1ß) increases pain behavior and the blood glucose level: possible involvement of glucocorticoid system.

The possible involvement of glucocorticoid system in interleukin-1ß (IL-1ß)-induced nociception and the blood glucose level was studied in ICR mice. In the first experiment, mice were treated intrathecally (i.t.) with IL-1ß (100 pg). Corticotrophin releasing hormone (CRH) mRNA (hypothalamus) and c-Fos mRNA (pituitary gland, spinal cord, and the adrenal gland) levels were measured at 30, 60 and 120 min after IL-1ß administration. We found that i.t. injection with IL-1ß increased CRH mRNA level in the hypothalamus. The IL-1ß administered i.t. elevated c-Fos mRNA levels in the spinal cord, pituitary and adrenal glands. Furthermore, i.t. administration of IL-1ß significantly increased the plasma corticosterone level up to 60 min. In addition, the adrenalectomy caused the reductions of the blood glucose level and pain behavior induced by IL-1ß injected i.t. in normal and D-glucose-fed groups. Furthermore, intraperitoneal (i.p.) pretreatment with RU486 (100mg/kg) attenuated the blood glucose level and pain behavior induced by IL-1ß administered i.t. in normal and D-glucose-fed groups. Our results suggest that IL-1ß administered i.t. increases the blood glucose level and pain behavior via an activation of the glucocorticoid system.

Antinociceptive profiles and mechanisms of orally administered coumarin in mice.

In the present study, the antinociceptive profiles of coumarin were examined in ICR mice. Coumarin administered orally (from 1 to 10 mg/kg) showed an antinociceptive effect in a dose-dependent manner as measured in the acetic acid-induced writhing test. Duration of antinociceptive action of coumarin maintained at least for 60 min. But, the cumulative response time of nociceptive behaviors induced by a subcutaneous (s.c.) formalin injection, intrathecal (i.t.) substance P (0.7 µg) or glutamate (20 µg) injection was not affected by coumarin. In addition, intracerebroventricular (i.c.v.) or intrathecal (i.t.) administration with coumarin (10-40 µg) attenuated acetic acid-induced writhing response in a dose dependent manner. Intraperitoneal (i.p.) pretreatment with naloxone (an opioid receptor antagonist) attenuated antinociceptive effect induced by coumarin in the writhing test. Furthermore, i.c.v. or i.t. pretreatment with naloxone (5 µg) reversed the decreased acetic acid-induced writhing response. However, methysergide (a 5-HT serotonergic receptor antagonist) or yohimbine (an α2-adrenergic receptor antagonist) did not affect antinociception induced by coumarin in the writhing test. Our results suggest that coumarin exerts a selective antinociceptive property in the acetic acid-induced visceral-derived pain model. Furthermore, the antinociceptive effect of coumarin may be mediated by activation of central opioid receptors, but not serotonergic and adrenergic receptors.

Effect of cholera toxin administered supraspinally or spinally on the blood glucose level in pain and d-glucose fed animal models.

In the present study, the effect of intrathecal (i.t.) or intracerebroventricular (i.c.v.) administration with cholera toxin (CTX) on the blood glucose level was examined in ICR mice. The i.t. treatment with CTX alone for 24 h dose-dependently increased the blood glucose level. However, i.c.v. treatment with CTX for 24 h did not affect the blood glucose level. When mice were orally fed with D-glucose (2 g/kg), the blood glucose level reached to a maximum level at 30 min and almost returned to the control level at 120 min after D-glucose feeding. I.c.v. pretreatment with CTX increased the blood glucose level in a potentiative manner, whereas i.t. pretreatment with CTX increased the blood glucose level in an additive manner in a D-glucose fed group. In addition, the blood glucose level was increased in formalin-induced pain animal model. I.c.v. pretreatment with CTX enhanced the blood glucose level in a potentiative manner in formalin-induced pain animal model. On the other hand, i.t. pretreatment with CTX increased the blood glucose level in an additive manner in formalin-induced pain animal model. Our results suggest that CTX administered supraspinally or spinally differentially modulates the regulation of the blood glucose level in D-glucose fed model as well as in formalin-induced pain model.

Effect of GABA receptor agonists or antagonists injected spinally on the blood glucose level in mice.

The possible roles of gamma-amino butyric acid (GABA) receptors located in the spinal cord for the regulation of the blood glucose level were studied in ICR mice. We found in the present study that intrathecal (i.t.) injection with baclofen (a GABAB receptor agonist; 1-10 µg/5 µl) or bicuculline (a GABAA receptor antagonist; 1-10 µg/5 µl) caused an elevation of the blood glucose level in a dose-dependent manner. The hyperglycemic effect induced by baclofen was more pronounced than that induced by bicuculline. However, muscimol (a GABAA receptor agonist; 1-5 µg/5 µl) or phaclofen (a GABAB receptor antagonist; 5-10 µg/5 µl) administered i.t. did not affect the blood glucose level. Baclofen-induced elevation of the blood glucose was dose-dependently attenuated by phaclofen. Furthermore, i.t. pretreatment with pertussis toxin (PTX; 0.05 or 0.1 µg/5 µl) for 6 days dose-dependently reduced the hyperglycemic effect induced by baclofen. Our results suggest that GABAB receptors located in the spinal cord play important roles for the elevation of the blood glucose level. Spinally located PTX-sensitive G-proteins appear to be involved in hyperglycemic effect induced by baclofen. Furthermore, inactivation of GABAA receptors located in the spinal cord appears to be responsible for tonic up-regulation of the blood glucose level.

Mechanisms involved in the antinociceptive effects of orally administered oleanolic acid in the mouse.

The antinociceptive effects of oleanolic acid were examined in ICR mice. Oleanolic acid administered orally (1, 5 and 10 mg/kg) showed an antinociceptive effect in a dose-dependent manner as measured in the acetic acid-induced writhing test. In the time- course study, duration of antinociceptive action of oleanolic acid maintained at least for 60 min. In addition, the cumulative nociceptive response time for intraplantar formalin injection (2nd phase), intrathecal injection of substance P (0.7 µg) or glutamate (20 µg) was diminished by oleanolic acid. Intraperitoneal (i.p.) pretreatment with naloxone (opioid receptor antagonist) or methysergide (5-HT serotonergic receptor antagonist) attenuated antinociceptive effect induced by oleanolic acid in the writhing test. However, yohimbine (adrenergic receptor antagonist) did not affect antinociception induced by oleanolic acid. The results indicate that oleanolic acid shows an antinociceptive property in various pain models such as writhing, formalin, substance P and glutamate pain tests. Furthermore, this antinociceptive effect of oleanolic acid may be mediated by opioidergic and serotonergic receptors, but not adrenergic receptors.

Repaglinide, but not nateglinide administered supraspinally and spinally exerts an anti-diabetic action in d-glucose fed and streptozotocin-treated mouse models.

We have recently demonstrated that some anti-diabetic drugs such as biguanide and thizolidinediones administered centrally modulate the blood glucose level, suggesting that orally administered anti-diabetic drugs may modulate the blood glucose level by acting on central nervous system. The present study was designed to explore the possible action of another class of anti-diabetic drugs, glinidies, administered centrally on the blood glucose level in ICR mice. Mice were administered intracerebroventricularly (i.c.v.) or intrathecally (i.t.) with 5 to 30 µg of repaglinide or nateglinide in D-glucose-fed and streptozotocin (STZ)-treated models. We found that i.c.v. or i.t. injection with repaglinide dose-dependently attenuated the blood glucose level in D-glucose-fed model, whereas i.c.v. or i.t. injection with nateglinide showed no modulatory action on the blood glucose level in D-glucose-fed model. Furthermore, the effect of repaglinide administered i.c.v. or i.t. on the blood glucose level in STZ-treated model was studied. We found that repaglinide administered i.c.v. slightly enhanced the blood glucose level in STZ-treated model. On the other hand, i.t. injection with repaglinide attenuated the blood glucose level in STZ-treated model. The plasma insulin level was enhanced by repaglinide in D-glucose-fed model, but repaglinide did not affect the plasma insulin level in STZ-treated model. In addition, nateglinide did not alter the plasma insulin level in both D-glucose-fed and STZ-treated models. These results suggest that the anti-diabetic action of repaglinide appears to be, at least, mediated via the brain and the spinal cord as revealed in both D-glucose fed and STZ-treated models.

Central anti-diabetic action of biguanide and thizolidinediones in D-glucose fed and streptozotocin-treated mouse models.

BACKGROUND: In the present study, the possible anti-diabetic action of biguanide and thiazolidinediones administered supraspinally or spinally was studied in ICR mice. METHODS: Mice were intracerebroventricular (i.c.v.) or intrathecal (i.t.) treated with 20 or 30 µg metformin, pioglitazone and rosiglitazone in d-glucose fed and streptozotocin-treated models, and blood glucose levels was measured at 30, 60 and 120 min after i.c.v. or i.t. administration. RESULTS: We found that i.c.v. injection with metformin or rosiglitazone slightly attenuated the blood glucose level in d-glucose fed model, whereas pioglitazone showed no effect on the blood glucose level in d-glucose fed model. The i.t. administration with metformin, pioglitazone or rosiglitazone did not alter the blood glucose level in d-glucose fed model. We also assessed the possible roles of biguanide and thiazolidinedione in the regulation of the blood glucose level in streptozotocin-treated model. We found in the present study that i.c.v. or i.t. administration with metformin caused a pronounced attenuation of the blood glucose level in streptozotocin-treated model. However, rosiglitazone administered i.c.v. did not affect the blood glucose level in streptozotocin-treated model. CONCLUSIONS: Our results suggest that the anti-diabetic actions of metformin and rosiglitazone appear to be mediated via the brain regions as revealed in d-glucose fed animal model. Furthermore, metformin administered supraspinally or spinally may be effective for treating type I diabetes mellitus as revealed in streptozotocin-treated mouse model.

Hop extract produces antinociception by acting on opioid system in mice.

In the present study, the antinociceptive profiles of hop extract were characterized in ICR mice. Hop extract administered orally (from 25 to 100 mg/kg) showed an antinociceptive effect in a dose-dependent manner as measured in the acetic acid-induced writhing test. Antinociceptive action of hop extract was maintained at least for 60 min. Moreover, cumulative response time of nociceptive behaviors induced with intraplantar formalin injection was reduced by hop extract treatment during the 2nd phases. Furthermore, the cumulative nociceptive response time for intrathecal injection of substance P (0.7 µg) or glutamate (20 µg) was diminished by hop extract. Intraperitoneal pretreatment with naloxone (an opioid receptor antagonist) attenuated antinociceptive effect induced by hop extract in the writhing test. However, methysergide (a 5-HT serotonergic receptor antagonist) or yohimbine (an α(2)-adrenergic receptor antagonist) did not affect antinociception induced by hop extract in the writhing test. Our results suggest that hop extract shows an antinociceptive property in various pain models. Furthermore, the antinociceptive effect of hop extract may be mediated by opioidergic receptors, but not serotonergic and α(2)-adrenergic receptors.

Effect of Agrimonia pilosa Ledeb Extract on the Antinociception and Mechanisms in Mouse.

In the present study, the antinociceptive profiles of Agrimonia pilosa Ledeb extract were examined in ICR mice. Agrimonia pilosa Ledeb extract administered orally (200 mg/kg) showed an antinociceptive effect as measured by the tail-flick and hot-plate tests. In addition, Agrimonia pilosa Ledeb extract attenuated the writhing numbers in the acetic acid-induced writhing test. Furthermore, the cumulative nociceptive response time for intrathecal (i.t.) injection of substance P (0.7 µg) was diminished by Agrimonia pilosa Ledeb extract. Intraperitoneal (i.p.) pretreatment with yohimbine (α(2)-adrenergic receptor antagonist) attenuated antinociceptive effect induced by Agrimonia pilosa Ledeb extract in the writhing test. However, naloxone (opioid receptor antagonist) or methysergide (5-HT serotonergic receptor antagonist) did not affect antinociception induced by Agrimonia pilosa Ledeb extract in the writhing test. Our results suggest that Agrimonia pilosa Ledeb extract shows an antinociceptive property in various pain models. Furthermore, this antinociceptive effect of Agrimonia pilosa Ledeb extract may be mediated by α(2)-adrenergic receptor, but not opioidergic and serotonergic receptors.

Interleukin-1ß (IL-1ß) increases pain behavior and the blood glucose level: possible involvement of sympathetic nervous system.

The relationship between interleukin-1ß (IL-1ß)-induced nociception and the blood glucose level was studied in ICR mice. We found in the present study that intrathecal (i.t.) injection of IL-1ß increased pain behavior. In addition, i.t. IL-1ß injection caused an elevation of the blood glucose level. The time-course study showed that maximal blood glucose level was observed 30 and 60 min after i.t. IL-1ß administration. Furthermore, i.t. injection of IL-1ß enhanced the blood glucose level when mice were orally fed with d-glucose. The i.t. administration of IL-1ß antagonist (AF12198) inhibited the hyperglycemia and pain behaviors induced by IL-1ß. We found in the present study that adrenal tyrosine hydroxylase (TH) mRNA level was also increased by i.t. IL-1ß injection. Furthermore, intraperitoneal (i.p.) pretreatment with phentolamine (an α(1)-adrenergic blocker) or yohimbine (an α(2)-adrenergic blocker) significantly attenuated the blood glucose level and pain behavior induced by IL-1ß administered i.t. However, the blood glucose level and pain behavior were not affected by butoxamine (a ß(2)-adrenergic blocker), whereas metoprolol (a ß(2)-adrenergic blocker) enhanced IL-1ß-induced blood glucose level and pain behavior in mice fed with d-glucose. However, its effect was not statistically significant. Our results suggest that IL-1ß administered i.t. increases the blood glucose level via an activation of α adrenergic nervous system.

The analgesic effects and mechanisms of orally administered eugenol.

In the present study, the antinociceptive profiles of eugenol were examined in ICR mice. Eugenol administered orally (from 1 to 10 mg/kg) showed an antinociceptive effect in a dose-dependent manner as measured in the acetic acid-induced writhing test. Duration of antinociceptive action of eugenol maintained at least for 30 min. Moreover, the cumulative response time of nociceptive behaviors induced by an intraplantar formalin injection was reduced by eugenol treatment during the 2(nd) phases. Furthermore, the cumulative nociceptive response time for intrathecal injection of substance P (0.7 µg) or glutamate (20 µg) was diminished by eugenol. Intraperitoneal pretreatment with yohimbine (α2-adrenergic receptor antagonist) or naloxone (opioid receptor antagonist) attenuated antinociceptive effect induced by eugenol in the writhing test. However, methysergide (5-HT serotonergic receptor antagonist) did not affect antinociception induced by eugenol in the writhing test. Our results suggest that eugenol shows an antinociceptive property in various pain models. Furthermore, this antinociceptive effect of eugenol may be mediated by α2-adrenergic and opioidergic receptors, but not serotonergic receptor.

The regulation of blood glucose level in physical and emotional stress models: possible involvement of adrenergic and glucocorticoid systems.

This study was done to determine the effect of stress on blood glucose regulation in ICR mice. The stress was induced by the electrical foot shock-witness model. Blood glucose level was found to be increased in the electrical foot shock-induced physical stress group. Furthermore, the blood glucose levels were also elevated in the emotional stress group in both physical and emotional stress groups. The blood glucose level reached maximum 30 min after stress stimulation and returned to normal level 2 h after stress stimulation in both physical and emotional stress groups. Subsequently, we observed that intraperitoneal injection of phentolamine (an α1-adrenergic receptor antagonist), yohimbine (an α2-adrenergic receptor antagonist) or RU486 (a glucocorticoid receptor blocker) significantly inhibited blood glucose level induced by both physical and emotional stress. The results of our study suggest that physical and emotional stress increases blood glucose level via activation of adrenergic and glucocorticoid system.

Characterization of blood glucose level regulation in mouse opioid withdrawal models.

The regulation of blood glucose level in intracerebroventricular (i.c.v.) administration with opioid alone or opioid withdrawal model was studied in ICR mice. In the first group, we found that i.c.v. administered morphine or beta-endorphin alone causes an elevation of blood glucose level. Blood glucose level induced by i.c.v. morphine or beta-endorphin began to increase within 30min and reached maximal level at 1h, decreasing to the basal level after 2h. In another group, we observed that intraperitoneal (i.p.) injection with naloxone (10mg/kg) post-treated 3h after either a single i.c.v. injection with morphine or beta-endorphin did not affect the increased blood glucose level in either group. In the next study, we observed that multiple (1 time/day for 3 days) i.c.v. injection with morphine alone significantly increased the blood glucose level. However, i.p. injection with naloxone post-treated 3h after the last i.c.v. injection with morphine caused a decrease of blood glucose level. We found that multiple (1 time/day for 3 days) i.c.v. injections with beta-endorphin did not affect the blood glucose level. Furthermore, i.p. injection with naloxone did not affect the blood glucose level in the mice injected with multiply beta-endorphin. Our results suggest that both morphine and beta-endorphin administered i.c.v. acutely increases the blood glucose level. However, blood glucose levels in the groups of multiply administered morphine alone, beta-endorphin alone, and naloxone-treated withdrawal model in multiply injected morphine and beta-endorphin appear to be differentially regulated.