Imprinting Control Region Mice Research Articles

Stimulation of Angiotensin II Receptor Subtype 2 Reduces Preeclampsia-like Symptoms in a Mouse Model of Preeclampsia.

Angiotensin II (AngII) receptor subtype 1 (AT1R) is involved in the pathogenesis of preeclampsia (PE). Angiotensin II receptor subtype 2 (AT2R) can antagonize the effects of AT1R, but its effects during pregnancy are not known. We investigated the effect of AT2R on the pathogenesis of PE using a mouse model and recently developed AT2R agonist (compound 21 [C21]). Blastocysts collected from pregnant imprinting control region (ICR) mice were incubated with adenovirus containing the CD40L gene and transferred into the uterine horns of pseudo-pregnant ICR mice to express PE-like features. Osmotic pumps were placed subcutaneously on the dorsal side with C21 or saline. C21 reduced the plasma soluble fms-like tyrosine kinase 1 (sFlt-1) concentration, ameliorating hypertension. The splenic T and B cell profiles in model mice were analyzed by flow cytometry. The gated percentage of IFN-γ-positive Th cells was significantly increased and the percentage of plasma cells in B cells was significantly decreased; however, the percentages were not altered by C21. sFlt-1 and soluble endoglin concentrations in plasma were measured with an enzyme-linked immunosorbent assay, and sFlt-1 was reduced. C21 could become a candidate PE drug as it ameliorated the pathophysiology of PE as a result of decreased production of sFlt-1.

Involvement of the vagus nerve and hepatic gene expression in serum adiponectin concentrations in mice.

Several humoral factors, such as adiponectin and urate, have been suggested to affect metabolic syndromes. Previously, we reported a reduction in blood adiponectin concentrations after a high-fructose diet partially via the vagus nerve in rats. Although a lithogenic diet (LD), i.e., supplementation of a normal control diet (CT) with 0.6% cholesterol and 0.2% sodium cholate, reduced blood adiponectin concentrations, the involvement of the vagus nerve in this mechanism remains unclear. To estimate the involvement of the vagus nerve in the regulation of blood adiponectin concentrations using an LD, male imprinting control region mice that had been vagotomized (HVx) or only laparotomized (Sham) were administered a CT or an LD for 10weeks. Serum adiponectin concentrations in the Sham-LD, HVx-CT, and HVx-LD groups were reduced by half compared with the Sham-CT group. The hepatic mRNA levels of fibroblast growth factor 21 (Fgf21), which reportedly stimulates adiponectin secretion from white adipose tissue, were lower in the LD groups compared with the CT groups. HepG2 hepatoma cells showed that various bile acids reduced the mRNA expression of FGF21. Moreover, the LD increased serum urate concentrations and reduced hepatic expressions of the acyl-CoA oxidase 1 (Acox1) mRNA and glucokinase, suggesting insufficient regeneration of ATP from AMP. In conclusion, serum adiponectin concentration may be regulated via the vagus nerve in normal mice, whereas a reduction of hepatic Fgf21 mRNA by bile acids may also lower serum adiponectin levels. Moreover, the LD may promote hepatic AMP accumulation and subsequently increase the serum urate concentration in mice.

Solid-phase synthesis and pathological evaluation of pyroglutamate amyloid-β3-42 peptide

Pyroglutamate amyloid-β3-42 (AβpE3-42) is an N-terminally truncated and pyroglutamate-modified Aβ peptide retaining highly hydrophobic, amyloidogenic, and neurotoxic properties. In Alzheimer’s disease (AD) patients, AβpE3-42 peptides accumulate into oligomers and induce cellular toxicity and synaptic dysfunction. AβpE3-42 aggregates further seed the formation of amyloid plaques, which are the pathological hallmarks of AD. Given that AβpE3-42 peptides play critical roles in the development of neurodegeneration, a reliable and reproducible synthetic access to these peptides may support pathological and medicinal studies of AD. Here, we synthesized AβpE3-42 peptides through the microwave-assisted solid-phase peptide synthesis (SPPS). Utilizing thioflavin T fluorescence assay and dot blotting analysis with anti-amyloid oligomer antibody, the amyloidogenic activity of synthesized AβpE3-42 peptides was confirmed. We further observed the cytotoxicity of AβpE3-42 aggregates in cell viability test. To examine the cognitive deficits induced by synthetic AβpE3-42 peptides, AβpE3-42 oligomers were intracerebroventricularly injected into imprinting control region mice and Y-maze and Morris water maze tests were performed. We found that AβpE3-42 aggregates altered the expression level of postsynaptic density protein 95 in cortical lysates. Collectively, we produced AβpE3-42 peptides in the microwave-assisted SPPS and evaluated the amyloidogenic and pathological function of the synthesized peptides.

Screening of Integrin Heterodimers Expressed Functionally on the Undifferentiated Spermatogonial Stem Cells in the Outbred ICR Mice.

Background and ObjectivesOutbred mice are widely used in toxicology, pharmacology, and fundamental biomedical research. However, there have been no reports of in vitro culture systems for spermatogonial stem cells (SSCs) derived from these mice.MethodsAs a step towards constructing a non-cellular niche supporting the in vitro maintenance of outbred mouse SSC self-renewal, we systematically investigated the types of integrin heterodimers that are expressed transcriptionally, translationally, and functionally in SSCs derived from Imprinting Control Region (ICR) mice.ResultsAmong the genes encoding 25 integrin subunits, integrin α1, α5, α6, α9, αV, and αE, and integrin β1 and β5 had significantly higher transcriptional levels than the other subunits. Furthermore, at the translational level, integrin α5, α6, α9, αV, αE, and β1 were localized on the surface of SSCs, but integrin α1 and β5 not. Moreover, significantly stronger translational expression than integrin α9 and αE was observed in integrin α5, α6, αV, and β1. SSCs showed significantly increased adhesion to fibronectin, laminin, tenascin C and vitronectin, and functional blocking of integrin α5β1, α6β1, α9β1 or αVβ1 significantly inhibited adhesion to these molecules.ConclusionsWe confirmed that integrin α5β1, α6β1, α9β1 and αVβ1 actively function on the surface of undifferentiated SSCs derived from outbred ICR mice.

Using a Dielectrophoretic Microfluidic Biochip Enhanced Fertilization of Mouse Embryo in Vitro.

Droplet microfluidics has appealed to many interests for its capability to epitomize cells in a microscale environment and it is also a forceful technique for high-throughput single-cell epitomization. A dielectrophoretic microfluidic system imitates the oviduct of mammals with a microchannel to achieve fertilization in vitro (IVF) of an imprinting control-region (ICR) mouse. We applied a microfluidic chip and a positive dielectrophoretic (p-DEP) force to capture and to screen the sperm for the purpose of manipulating the oocyte. The p-DEP responses of the oocyte and sperm were exhibited under applied bias conditions (waveform AC 10 Vpp, 1 MHz) for trapping 1 min. The insemination concentration of sperm nearby the oocyte was increased to enhance the probability of natural fertilization through the p-DEP force trapping. A simulation tool (CFDRC-ACE+) was used to simulate and to analyze the distribution of the electric field. The DEP microfluidic devices were fabricated using poly (dimethylsiloxane) (PDMS) and ITO (indium tin oxide)-glass with electrodes. We discuss the requirement of sperm in a DEP microfluidic chip at varied concentrations to enhance the future rate of fertilization in vitro for an oligozoospermia patient. The result indicates that the rate of fertility in our device is 17.2 ± 7.5% (n = 30) at about 3000 sperms, compatible with traditional droplet-based IVF, which is 14.2 ± 7.5% (n = 28).

Efficient production and transmission of CRISPR/Cas9-mediated mutant alleles at the IG-DMR via generation of mosaic mice using a modified 2CC method

Generation of mutant imprinting control region (ICR) mice using genome editing is an important approach for elucidating ICR functions. IG-DMR is an ICR in the Dlk1-Dio3 imprinted domain that contains functional regions—in both parental alleles—that are essential for embryonic development. One drawback of this approach is that embryonic lethality can occur from aberrant expression of the imprinted genes if IG-DMR gets mutated in either the paternal or maternal allele. To overcome this problem, we generated mosaic mice that contained cells with modified IG-DMR alleles and wild-type cells using the 2CC method that allowed for microinjection of the CRISPR/Cas9 constructs into a blastomere of 2-cell embryos. This method improved the birth rate of the founder pups relative to that obtained using the standard protocol. We also successfully produced mosaic mice in which the tandem repeat array sequence in the IG-DMR had been replaced by homology directed repair. Additionally, paternal transmission of the replaced allele caused aberrant expression of the imprinted genes due to hypomethylation of the IG-DMR, indicating that the replaced allele recapitulated our deletion model. Our results indicate that this method is useful for the generation of mutant mice in which a genomic locus essential for normal development has been genetically edited.

Abstract TP137: Suppression of Microrna Let-7a Expression Promotes Neurogenesis in Arginine Decarboxylase-neural Stem Cells After Ischemia

Introduction: Neural stem cells (NSCs) have been studied as promising novel treatments for central nervous system (CNS) disorders due to the potential of differentiation into neuron. To increase the therapeutic efficiency of NSCs, we have focused on microRNA let-7a ( let-7a ), known as the regulator of diverse cellular mechanisms and agmatine, an endogenous primary amine synthesized from decarboxylation of L-arginine catalyzed by arginine decarboxylase (ADC) and a neurotransmitter reported to affect neuroprotection in CNS. The purpose of this study is to investigate the role of arginine metabolic enzyme which regulated let-7a in neural stem cell differentiation. Methods: Cortical NSCs were isolated from pregnant imprinting control region mice and used for experiments after 2-3 passages. After let-7a mimic and agmatine treatment, we performed by RT-PCR, western blot and immunocytochemistry. To confirm the effects of arginine metabolic enzyme and let-7a in vivo , mice were subjected to distal middle cerebral artery occlusion (dMCAO). Following dMCAO, mice were treated agmatine by intraperitoneal injection, let-7a mimic by osmotic pump and human ADC gene delivery into the NSCs using retroviral vector ( vh ADC)-NSCs via intracranial injection at the corpus callosum. Results: In vitro study suggested that high levels of let-7a promoted the expression of TLX and c-Myc, as well as repressed DCX and ERK expression. In addition, agmatine attenuated the expression of TLX and increased the expression of ERK by negatively regulating let-7a . Also, after dMCAO, we found that vh ADC-NSC and let-7a co-treatment group reduced the infarct volume, increased the expression of DCX, Neurogenin2, Olig2, and attenuated the expression of GFAP. Conclusion: In the present study, we found that agmatine can enhance NSC differentiation into neurons by modulating expression of let-7a . In vivo study showed that vh ADC-NSC and let-7a co-treatment promoted neurogenesis and inhibited glial differentiation in the treatment of neural stem cell transplantation in ischemic condition. Therefore, our study suggests that the therapeutic efficiency of the vh ADC-NSC on neural stem cell differentiation through suppression of let-7a of the CNS diseases.

Progressive hearing loss in vitamin A-deficient mice which may be protected by the activation of cochlear melanocyte

Vitamin A deficiency (VAD) produces various pathologic phenotypes in humans and animals. However, evidence regarding the effect of VAD on hearing function has been inconsistent. In this study, we evaluated the effect of VAD on hearing function in two mouse models of VAD. Hearing ability was evaluated on the basis of auditory brainstem response from 3 to 20 weeks after birth in C57BL/6 (pigmented) and imprinting control region (albino) mice. The two mice strains were divided into the VAD (purified vitamin A-free diet from 7 days after pregnancy) and control (normal diet) groups. Albino VAD mice exhibited hearing loss after 6 weeks and became deaf at 18 weeks. Histological findings revealed degenerative changes in outer hair cells and neuronal loss in the spiral ganglion in albino VAD mice. In contrast, pigmented VAD mice, except those with middle-ear infection, showed no significant hearing loss. Interestingly, pigmented VAD mice exhibited melanocyte activation in the stria vascularis and upregulation of tyrosinase. Recovery of hearing after noise exposure was poorer in pigmented VAD mice than in control mice. In conclusion, complete VAD might be related to age-related or noise-induced hearing loss in mice, protection against which might involve melanocyte activation.

Astragalus root induces ovarian β‑oxidation and suppresses estrogen‑dependent uterine proliferation.

Continuous estrogen stimulation in the uterus has been known to cause excess proliferation of the functional layer of endometrium, resulting in endometrial hyperplasia and leading to infertility. Estrogens can modulate other nuclear receptor signaling pathways, such as peroxisome proliferator‑activated receptors (PPARs). Astragalus root (AsR) has exhibited strong PPARα agonistic activity. Female Imprinting Control Region mice were fed a powder diet that included 5%AsR hot water extract or 0.1% bezafibrate as a positive control for 56days to investigate AsR effects on the reproductive tract, ovary and uterus. AsR resulted in upregulation of the expression of uterine and ovarian PPARα mRNA by 2.5‑fold, and 1.5‑fold, respectively, compared with controls. AsR significantly increased ovarian expression levels of mitochondrial 2,4‑dienoyl‑CoA reductase (mDECR), an auxiliary enzyme involved in β‑oxidation. AsR‑fed mice also exhibited a significant increase in blood estradiol levels and tended to have higher ovary weight. AsR resulted in significantly decreased uterine weight and mDECR expression levels. It has been reported that a PPARα agonist suppresses the development of estrogen‑dependent endometrial hyperplasia. These findings raise the possibility that AsR suppresses estrogen‑dependent endometrial hyperplasia and ovarian dysfunction leading to infertility.

Establishment of a radiotelemetric recording technique in mice to investigate gastric slow waves: Modulatory role of putative neurotransmitter systems

What is the central question of this study? Gastric slow waves originating from the interstitial cells of Cajal-smooth muscle syncytium are usually studied in culture or in tissue segments, but nobody has described recordings of slow waves from awake, freely moving mice. Can radiotelemetry be used to record slow waves, and do they respond predictably to drug treatment? What is the main finding and its importance? Radiotelemetry can be used to record slow waves from awake, freely moving mice, permitting an examination of drug actions in vivo, which is crucial to drug discovery projects for characterizing the effects of drugs and metabolites on gastrointestinal function. The mouse is the most commonly used species in preclinical research, and isolated tissues are used to study slow waves from the interstitial cells of Cajal-smooth muscle syncytium of the gastrointestinal tract. The aim of this study was to establish a radiotelemetric technique in awake mice to record gastric myoelectric activity from the antrum to gain insight into the effects of endogenous modulatory systems on slow waves. Under general anaesthesia, two biopotential wires from a telemetry transmitter were sutured into the antrum of male ICR (imprinting control region) mice. The animals were allowed 1week to recover from surgery before the i.p. administration of drugs to stimulate or inhibit slow waves. The basal dominant frequency of slow waves was 6.96±0.43c.p.m., and the percentages of power in the bradygastric, normogastric and tachygastric ranges were 6.89±0.98, 37.32±1.72 and 34.38±0.77%, respectively (n=74). Nicotine at 1mgkg-1 increased normogastric power, but at 3mgkg-1 it increased bradygastric power (P<0.05). Metoclopramide at 10mgkg-1 increased normogastric power; sodium nitroprusside at 10mgkg-1 had latent effects on tachygastric power (P<0.05); and l-NAME at 10mgkg-1 had no effect (P>0.05). Nicotine and bethanechol also caused varying degrees of hypothermia (>1°C reductions; P<0.05). In conclusion, radiotelemetry can be used to record slow waves from awake, freely moving mice. In light of our findings, we recommend that studies assessing slow waves should also assess body temperature simultaneously.

Triphlorethol A, a Dietary Polyphenol from Seaweed, Decreases Sleep Latency and Increases Non-Rapid Eye Movement Sleep in Mice.

In our previous studies, we have demonstrated that marine polyphenol phlorotannins promote sleep through the benzodiazepine site of the gamma-aminobutyric acid type A (GABAA) receptors. In this follow-up study, the sleep-promoting effects of triphlorethol A, one of the major phlorotannin constituents, were investigated. The effect of triphlorethol A on sleep-wake architecture and profiles was evaluated based on electroencephalogram and electromyogram data from C57BL/6N mice and compared with the well-known hypnotic drug zolpidem. Oral administration of triphlorethol A (5, 10, 25, and 50 mg/kg) dose-dependently decreased sleep latency and increased sleep duration during pentobarbital-induced sleep in imprinting control region mice. Triphlorethol A (50 mg/kg) significantly decreased sleep latency and increased the amount of non-rapid eye movement sleep (NREMS) in C57BL/6N mice, without affecting rapid eye movement sleep (REMS). There was no significant difference between the effects of triphlorethol A at 50 mg/kg and zolpidem at 10 mg/kg. Triphlorethol A had no effect on delta activity (0.5–4 Hz) of NREMS, whereas zolpidem significantly decreased it. These results not only support the sleep-promoting effects of marine polyphenol phlorotannins, but also suggest that the marine polyphenol compound triphlorethol A is a promising structure for developing novel sedative hypnotics.

Selection of reference genes for expression analysis in mouse models of acute alcoholic liver injury.

Investigations of hepatic gene expression are crucial for determining the molecular factors involved in acute alcoholic liver injury. The results of liver molecular investigations may reveal etiologically important genomic alterations. Therefore, it is necessary to normalize gene expression data to identify stable genes, which may be used as a reference under different experimental conditions. The aim of the present study was to apply reverse transcription‑quantitative polymerase chain reaction analysis and use analysis software to investigate the expression stability of candidate reference genes in hepatic tissues from mice with acute alcoholic liver injury. The acute alcoholic liver injury models were established by the intragastric administration of alcohol (5mg/kg) in Imprinting Control Region mice. Total RNA was isolated from the mouse livers, following which the expression levels of seven reference genes, β-actin, glyceraldehyde 3-phosphate dehydrogenase (Gadph), glucuronidase β, hypoxanthine phosphoribosyltransferase 1 (Hprt1), 18S ribosomal RNA, TATA binding protein and β‑2 microglobulin, were examined, and gene expression stability was assessed using the geNorm, NormFinder and BestKeeper tools. The geNorm analysis revealed that the gene with the lowest variability was Hprt1. Hprt1 and Gapdh were validated as the optimal reference gene pair in all samples from all groups. The NormFinder and BestKeeper results showed that Hprt1 was the most stable gene in all samples. Alcohol induces endoplasmic reticulum (ER) stress, causing changes in the expression levels of ER stress‑associated genes. The stability of Hprt1 was verified by the expression analysis of ER stress‑associated genes, and gene expression levels in the ethanol groups were upregulated, with a significant difference in expression, compared with those in the control group. Therefore, Hprt1 was selected as the most stable gene, and Hprt1 and Gapdh were determined to be the optimum gene pair in mouse models of acute alcoholic liver injury. The reliability of the Hprt1 gene was confirmed by expression analysis of ER stress‑associated genes.

Icariin promotes osteoblastic differentiation in OVX mice via MAPK signaling pathway revealed by profiling

Objective: Icariin (ICA), an extract from epimedium, has been reported to be effective in promoting bone formation. The objective of the study is to search for the molecular targets of ICA in bone mesenchymal stem cells (bMSCs) from the mice with ovariectomy (OVX)-induced osteoporosis. Methods: Six-month-old Imprinting Control Region (ICR) mice that underwent OVX were treated with ICA. After three months, bone mass was evaluated by microcomputed tomography, morphometry and immunohistological detection. bMSCs were isolated from the femur and tibia to observe the self-renewal and differentiation capacities using colony-forming unit fibroblastic (CFU-F), colony-forming unit adipocyte (CFU-Adipo) and alkaline phosphatase (ALP) staining. In addition, microarray of bMSCs ex vivo was measured two weeks after ICA treatment and analyzed by heatmap and pathway analysis. The signaling pathway was further explored by western blot assay and inhibitors of p38 and ERK: SB203508 and PD98059. Results: [Formula: see text]CT displayed a decrease in bone mass for three months after OVX. ICA treatment increased the trabecular thickness (Tb.Th), osteoblast number while decreased osteoclast number, elevating osteocalcin (OC) protein levels in vivo and facilitating the self-renewal and osteoblastic differentiation of bMSCs ex vivo. Microarray data indicated ICA rescued several gene expressions that were dysregulated by OVX. Pathway analysis revealed that the core genes acted by ICA were highly involved in MAPK signaling pathway. Further study demonstrated ICA suppressed ERK while stimulated p38 phosphorylation to promote osteoblastic differentiation in vitro. Conclusion: ICA promotes osteoblastic differentiation of bMSCs in OVX mice. MAPK signaling pathway might be involved in the process.

Improvement of Diet-induced Obesity by Ingestion of Mushroom Chitosan Prepared from Flammulina velutipes.

The anti-obesity effects of mushroom chitosan prepared from Flammulina velutipes were investigated using an animal model with diet-induced obesity. In this study, 5-week-old imprinting control region (ICR) mice were divided into six groups of 10 mice each and fed different diets based on the MF powdered diet (standard diet) for 6 weeks: standard diet control group, high-fat diet control group (induced dietary obesity) consisting of the standard diet and 20% lard, and mushroom chitosan groups consisting of the high-fat diet with mushroom chitosan added at 100, 500, 1,000, and 2,000 mg/kg body weight. On the final day of the experiment, mean body weight was 39.1 g in the high-fat control group and 36.3 g in the 2,000 mg/kg mushroom chitosan group, compared to 35.8 g in the standard diet control group. In the mushroom chitosan groups, a dose-dependent suppression of weight gain and marked improvements in serum triglycerides, total cholesterol, LDL-cholesterol, and HDL-cholesterol were found. The mushroom chitosan groups showed fewer and smaller fat deposits in liver cells than the high-fat diet control group, and liver weight was significantly reduced. Glutamic oxaloacetic transaminase (GOT) and glutamate pyruvic transaminase (GPT), which are indices of the hepatic function, all showed dose-dependent improvement with mushroom chitosan administration. These results suggested that mushroom chitosan acts to suppress enlargement of the liver from fat deposition resulting from a high-fat diet and to restore hepatic function. The lipid content of feces showed a marked increase correlated with the mushroom chitosan dose. These findings suggest the potential use of mushroom chitosan as a functional food ingredient that contributes to the prevention or improvement of dietary obesity by inhibiting digestion and absorption of fats in the digestive tract and simultaneously promotes lipolysis in adipocytes.

Modulation of Neurogenesis through the Promotion of Energy Production Activity Is behind the Antidepressant-Like Effect of Colonial Green Alga, Botryococcus braunii.

Algae have been recognized as important resources providing functional components due to their capacity to exert beneficial effects on health. Therefore, there is increasing interest in investigating the biological activity of algae. In this study, we evaluated the antidepressant-like effect of the administration of 100 mg/kg/day of the ethanol extract of colonial green alga Botryococcus braunii (EEB) for 14 consecutive days in the forced swimming test (FST)-induced depression in imprinting control region (ICR) mice. Imipramine, a commercial antidepressant drug, was used as a positive control. In addition, we investigated the molecular mechanisms underlying the effect of EEB by measuring ATP production and by assessing any change in gene expression at the end of the treatment using real-time polymerase chain reaction (PCR) and microarray assays. We showed that the immobility time in the water-administered control (FST stress) group gradually increased from day 1 to day 14. However, treatment with EEB caused a significant decrease of immobility time in the FST compared with that in the FST stress group. Microarray and real-time PCR results revealed that EEB treatment induced variation in the expression of several genes associated with neurogenesis, energy metabolism, and dopamine synthesis. Interestingly, we revealed that only EEB treatment enhanced the promotion of energy production, while treatment with imipramine was ineffective. Our study provides the first evidence that B. braunii enhances energy production, which may contribute to the modulation of neurogenesis and to the enhancement of dopaminergic function, in turn potentially underlying the antistress- and antidepressant-like effects that we observed.

Quercetin alleviates generalized hyperalgesia in mice with induced adenomyosis.

Adenomyosis is a common gynecologic disorder characterized by the presence of endometrial glands and stroma within the myometrium. The present study investigated the effect of quercetin in neonatal Imprinting Control Region mice with tamoxifen-induced adenomyosis. The body weight and hotplate response latency of all mice was examined at 4, 8, 12 and 16 weeks after birth. The mice dosed with tamoxifen were divided into four groups: high- or low-quercetin group, valproic acid (VPA) group and untreated group. The group of mice that were neonatally administrated with the solvent only (no tamoxifen), received no treatment and served as a blank control group. After 3 weeks of drug treatment, the potential ability of quercetin to improve the generalized hyperalgesia in mice with induced adenomyosis was evaluated by determining the body weight, pain modulation, examining the myometrial infiltration by histology examination of the uterus and detecting the expression of transient receptor potential cation channel subfamily V member 1 (Trpv-1), phospho (p)-p38 mitogen activated protein kinase p-extracellular signal-regulated kinase (p-ERK) in DRG neurons via immunohistochemistry. The results demonstrated that treatment with quercetin improved the generalized hyperalgesia by extending the hotplate response latency, reduced myometrial infiltration and decreased the expression levels Trpv-1, p-p38 and p-ERK in dorsal root ganglion neurons. The results indicated that quercetin decreases the incidence of hyperalgesia in mice with tamoxifen-induced adenomyosis, and the potential mechanism is through reduced central sensitization, which may be a promising treatment for adenomyosis.

Maternal exposure to nanosized titanium dioxide suppresses embryonic development in mice.

Although nanoscale titanium dioxide (nano-TiO2) has been extensively used in industrial food applications and daily products for pregnant women, infants, and children, its potential toxicity on fetal development has been rarely studied. The main objective of this investigation was to establish the effects of maternal exposure of nano-TiO2 on developing embryos. Female imprinting control region mice were orally administered nano-TiO2 from gestational day 0 to 17. Our findings showed that Ti concentrations in maternal serum, placenta, and fetus were increased in nano-TiO2-exposed mice when compared to controls, which resulted in reductions in the contents of calcium and zinc in maternal serum, placenta, and fetus, maternal weight gain, placental weight, fetal weight, number of live fetuses, and fetal crown–rump length as well as cauda length, and caused an increase in the number of both dead fetuses and resorptions. Furthermore, maternal nano-TiO2 exposure inhibited development of the fetal skeleton, suggesting a significant absence of cartilage, reduced or absent ossification, and an increase in the number of fetuses with dysplasia, including exencephaly, spina bifida, coiled tail, scoliosis, rib absence, and sternum absence. These findings indicated that nano-TiO2 can cross the blood–fetal barrier and placental barrier, thereby delaying the development of fetal mice and inducing skeletal malformation. These factors may be associated with reductions in both calcium and zinc in maternal serum and the fetus, and both the placenta and embryos may be major targets of developmental toxicity following maternal exposure to nano-TiO2 during the prenatal period. Therefore, the application of nano-TiO2 should be carried out with caution.

Retinoic acid ameliorates photoaged skin through RAR‑mediated pathway in mice.

Retinoic acid (RA), the bioactive metabolite of vitamin A, has demonstrated efficacy in the treatment of photoaged skin; however, the mechanism of action of RA remains unclear. The aim of the present study was to examine whether the therapeutic effects of RA on photoaged skin are mediated by retinoic acid receptor (RAR) and/or retinoid X receptor (RXR) in mice, and to investigate the underlying mechanism. Photoaged skin in Imprinting Control Region mice was induced by repeated exposure to ultraviolet (UV) irradiation. Mice were randomly divided into nine groups: Normal; UV control; all‑trans retinoic acid (ATRA); ATRA+RAR antagonist; ATRA+RXR antagonist; RAR agonist; RAR agonist+RAR antagonist; RXR agonist; and RXR agonist+RXR antagonist. Masson's trichrome staining was used to examine skin collagen fibers. Hydroxyproline assays were used to determine collagen content. The protein expression of matrix metalloproteinase (MMP)‑3, MMP‑13, type I procollagen, c‑Jun and c‑Fos was detected using western blot analysis. The results demonstrated that ATRA and RAR agonist ameliorated the UV‑induced damage to skin collagen fibers, and increased the collagen content in photoaged skin through RAR. Furthermore, ATRA and RAR agonist stimulated type I procollagen protein expression, and inhibited MMP‑3, MMP‑13 and c‑Jun protein expression through RAR in photoaged skin. However, ATRA and RAR agonist exhibited no significant effect on the protein expression of c‑Fos in photoaged skin. These findings suggest that RA ameliorates photoaged skin through a RAR‑mediated signaling pathway in mice.

Anticholangiocarcinoma activity and toxicity of the Kaempferia galanga Linn. Rhizome ethanolic extract

BackgroundCholangiocarcinoma (CCA) is an important public health problem in several tropical and subtropical parts of the world particularly Thailand. Chemotherapy of CCA is largely ineffective and discovery and development of effective alternative drugs is urgently needed. The objective of the study was to confirm the anti-CCA potential as well as toxicity of the crude extract of Kaempferia galangal Linn. (rhizome) both in vitro and in animal models.MethodsThe ethanolic extract of K. galanga Linn. rhizome, ethyl-p-methoxycinnamate (EPMC) and 5-fluorouracil (5-FU) were evaluated for their cytotoxic activities against CCA cell line (CL-6) using MTT cell proliferation assay. Acute and subacute toxicity of the extract were evaluated in ICR (Imprinting Control Region) mice according to the OECD (International Organization for Economic Co-operation and Development) Guideline. Anti-CCA activity was evaluated in CCA- xenografted nude mice.ResultsResults of cytotoxicity test showed moderate activity of the extract and EPMC with median (95% confidence interval: 95% CI) 50% inhibitory concentration (IC50) of 64.2 (57.76–72.11) and 49.19 (48.16–52.29) μg/ml, respectively. The IC50 of 5-FU was 107.1 (103.53–109.64) μg/ml. The selectivity index (SI) values for the extract, EPMC and 5-FU against human normal cell line (OUMS) and cancer cell line (CL-6) were 2.2, 2.09 and 1.31, respectively. Toxicity testing revealed no overt toxic effect up to the maximum single oral dose of 5000 mg/kg body weight and up to daily dose of 1000 mg/kg body weight for 30 days. The extract at the maximum tolerated dose level of 1000 mg/kg body weight for 30 days exhibited promising anti-CCA activity in CL6-xenografted nude mice as determined by inhibitory activity on tumor growth (58.41%) and lung metastasis (33.3%), as well as prolongation of survival time (62 days).ConclusionThe K. galangal Linn. rhizome extract and its bioactive compound EPMC exhibited moderate cytotoxic activity against human CCA tumor (CL-6) cell line. Results of toxicity testing suggest that the extract was well tolerated up to the maximum single oral dose of 5000 mg/kg body weight and daily dose of 1000 mg/kg body weight for 30 days. The extract exhibited promising anti-CCA activity in CL6-xenografed nude mice as determined by significant inhibitory activity on tumor growth and lung metastasis, as well as prolongation of survival time.

Ethanol Extract of Pinus koraiensis Leaf Ameliorates Alcoholic Fatty Liver via the Activation of LKB1-AMPK Signaling In Vitro and In Vivo.

Although Pinus koraiensis leaf (PKL) was reported for its anti-diabetes, anti-obesity and anticancer effects as a folk remedy, the inhibitory effect of PKL on alcoholic fatty liver has never been elucidated yet. This study investigated the molecular mechanisms of PKL on alcoholic fatty liver in HepG2 cells, Sprague Dawley (SD) rats and Imprinting Control Region (ICR) mice. Pinus koraiensis leaf increased phosphorylation of liver kinase B1 (LKB1)/AMP-activated protein kinase signaling, low-density lipoprotein receptor and decreased fatty acid biosynthesis-related proteins such as sterol regulatory element-binding protein 1c, fatty acid synthase, 3-hydroxy-3-methylglutaryl-CoA reductase in HepG2 cells. In SD rats with 25% alcohol-induced fatty liver, PKL suppressed the levels of aspartate aminotransferase and triglyceride and also enhanced the activities of antioxidant enzymes including superoxide dismutase, glutathione peroxidase and glutathione s-transferase compared with untreated control. Furthermore, PKL increased serum alcohol dehydrogenase and serum aldehyde dehydrogenase, but decreased serum alcohol concentration in ICR mice after alcohol administration. Consistently, histochemical analysis revealed that PKL attenuated alcohol-induced fatty liver in SD rats. Overall, these findings suggest that PKL ameliorates alcohol-induced fatty liver via activation of LKB1-AMP-activated protein kinase and modulation of proteins related to lipogenesis synthesis, cholesterol synthesis and fatty acid oxidation. Copyright © 2017 John Wiley & Sons, Ltd.