Female ICR Mice Research Articles

GnRH agonist improves pregnancy outcome in mice with induced adenomyosis by restoring endometrial receptivity.

PurposeAdenomyosis has a negative impact on female fertility. GnRH agonist treatment can improve pregnancy outcomes in women with adenomyosis. However, the impact of GnRH agonist upon endometrium receptivity of patients with adenomyosis remains unclear. In this study, endometrial receptivity and pregnancy outcome were investigated using a mouse model of adenomyosis.Materials and methodsAdenomyosis was induced in 12 female ICR mice, neonatally treated with tamoxifen, while another six female mice (control group) received solvent only. At 75 days, the induced adenomyosis group was randomly divided into two groups: an untreated group and a group treated with GnRH agonist (n = 6 each). Sixty days later, the mice were mated and pregnancy outcomes were observed and compared among the three groups (n = 6 each). In a parallel experiment using the same treatment regimes, uterus samples were collected on day 4 of pregnancy for immunohistochemistry, gene (quantitative polymerase chain reaction) and protein expression (Western blot), and scanning electron microscopy analyses.ResultsWe found that the average live litter size was reduced in the adenomyosis compared with control group (8 ± 0.56 versus 13 ± 0.71; P = 0.03). However, the litter size was significantly increased in the treated with GnRH agonist group compared with the untreated group (12 ± 0.35 versus 8 ± 0.56; P = 0.04). The uterine expression levels of Hoxa10, Hoxa11, Lif and integrin b3 mRNA and protein were decreased in the adenomyosis group, and were significantly increased after GnRH agonist treatment. Additionally, pinopodes were reduced in number and poorly developed in mice with induced adenomyosis. However, pinopodes were abundant and well-developed in the GnRH agonist treatment group.ConclusionAdenomyosis may have an adverse impact on endometrial receptivity and reduce pregnancy outcomes in mice. However, GnRH agonist may improve the pregnancy outcome by partially restoring endometrial receptivity.

P-158 - SOD1 haploinsufficiency exerts deleterious effects on the testis via activation of the autoimmune response in NZW mice

The New Zealand black (NZB) × New Zealand white (NZW) F1 hybrid mouse spontaneously develops an SLE-like autoimmune condition. ROS originated from the NZB background have a functional role in triggering the fatal responses in the F1 mice, whereas NZW mice show no obvious abnormal phenotype. In the current study, we evaluated the impact of ROS, which is elevated by SOD1 deficiency, on the latent abnormality in the immune system of the NZW mice. We analyzed SOD1+/−/NZW male mice from viewpoints of fecundity, spermatogenesis, and immune responses. Breeding SOD1+/−/NZW male mice with SOD1+/+/NZW or SOD1+/+/ICR female mice also did not give birth any SOD1+/− mice. SOD1+/−/NZW male mice showed significant reduction in the testis weight and sperm count and significant increase in the number of morphologically abnormal seminiferous tubules in testes. We also found that the plasma IgG autoantibodies reactive to testicular proteins were elevated in the SOD1+/−/NZW mice. Oxidative stress marker in the testis was increased in the SOD1+/−/NZW mice. These results collectively provide notion that the oxidative stress caused by SOD1 haploinsufficiency exerts deleterious effects on the testis via activation of the autoimmune response in SOD1+/−/NZW male mice.

Multigenerational effects of maternal cigarette smoke exposure during pregnancy on sperm counts of F1 and F2 male offspring

Animal models and human studies showed that in utero cigarette smoke exposure decreases sperm counts of offspring. This study used a mouse model to investigate the effects of maternal exposure to cigarette smoke on reproductive systems in F1 and F2 male offspring. Female ICR mice were exposed either to clean air or to cigarette smoke during pregnancy at the post-implantation stage. Epididymal sperm counts were decreased in a cigarette smoke dose–dependent manner in F1 (by 40–60%) and F2 males (by 23–40%) at postnatal day 56. In F1, the seminiferous epithelium heights were lower in the cigarette smoke–exposed groups than in the control group, and these effects were sustained in F2 males. Results suggest that maternal cigarette smoke exposure during pregnancy can have a multigenerational adverse effect on sperm counts in male offspring, which is mediated through in utero exposure of fetal germ cells to cigarette smoke.

Influence of pretreatment of insulin on phosphorylation of extracellular receptor kinase by gonadotropin releasing hormone in cultured mouse granulosa gells

Background: To investigate the influence of pretreatment of insulin on the phosphorylation of extracellular receptor kinase (ERK) by gonadotropin releasing hormone (GnRH) in cultured mouse granulosa cells. Materials and Methods: The granulosa cells from the mouse were collected simultaneously from the ovaries of gonadotropin-primed ICR female mice. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the expression of the receptors of GnRH and insulin. The granulosa cells were cultured with ten nM of insulin, ten nM of GnRH, and ten nM of insulin pretreatment before ten nM of GnRH. Western blotting was used for analysis of phosphorylation of ERK. Student t-test and Dunnett’s comparison test were used for statistical analysis and statistical significance was defined as p Results: The authors confirmed the presence of receptors of GnRH and insulin in the mouse granulosa cells by RTPCR. Both insulin and GnRH could activate ERK phosphorylation. Pretreatment of insulin for 30 minutes before GnRH treatment inhibited ERK phosphorylation by GnRH. Conclusion: Insulin might have a negative effect of GnRH regulation of mouse ovarian physiology by inhibition of GnRH activation of ERK. Based on these study results, the authors demonstrated the role of insulin as a delicate modulator of reproductive function.

UTX Affects Neural Stem Cell Proliferation and Differentiation through PTEN Signaling.

SummaryNeural stem cell (NSC) proliferation and differentiation in the developing brain is a complex process precisely regulated by intrinsic and extrinsic signals. Although epigenetic modification has been reportedly involved in the regulation of the cerebral cortex, whether UTX, an H3K27me3 demethylase, regulates the development of cerebral cortex during the embryonic period is unclear. In this study, we demonstrate that Utx deficiency by knockdown and conditional knockout increases NSC proliferation and decreases terminal mitosis and neuronal differentiation. Furthermore, we find that impairment of cortical development caused by lack of Utx is less significant in males than in females. In addition, UTX demethylates H3K27me3 at the Pten promoter and promotes Pten expression. P-AKT and P-mTOR levels are increased in the Utx conditional knockout cortices. Finally, Utx or Pten overexpression can rescue the impairment of brain development caused by Utx loss. These findings may provide important clues toward deciphering brain diseases.

Syringin prevents bone loss in ovariectomized mice via TRAF6 mediated inhibition of NF-κB and stimulation of PI3K/AKT

BackgroundSyringin, also called eleutheroside B, is a main bioactive phenolic glycoside in Acanthopanax senticosus (Rupr. et Maxim.) Harms. Based on the “kidney dominates bone” theory of TCM, A. senticosus can strengthen bone and Syringin may be one of the responsibilities. PurposeThe objectives of this study were to estimate the osteoporotic activity of Syringin and reveal the possible molecular mechanisms in vivo. MethodsSixty female ICR mice were randomly assigned into sham operated group (SHAM, treated with vehicle) and five ovariectomized subgroups (n = 10 each), treated with vehicle as OVX group, estradiol valerate (EV, 1 mg/kg/day) as positive group, and Syringin (10, 20 and 40 mg/kg/day) as low, moderate and high dosage groups. The therapeutic effect of Syringin against osteoporosis was systematically analyzed by determining the bone mineral density (BMD), bone biomechanical properties, bone microarchitecture and serum biochemical parameters, and the molecular mechanism was also evaluated. ResultsAfter three months of orally administrated intervention, Syringin (10, 20 and 40 mg/kg/day) significantly improved the BMD, bone maximum load and trabecular bone microarchitecture in ovariectomized mice, evidenced by the increased bone mineral content, tissue mineral content, tissue mineral density, trabecular thickness and trabecular number, as well as the decreased trabecular separation in OVX mice. Meanwhile, the activities of tartrate-resistant acid phosphatase, deoxypyridinoline and cathepsin K in OVX mice were also inhibited by Syringin, while the increased body weight and decreased uterus weight seemed not influenced by Syringin administration. Concerning the underlying molecular mechanisms, Syringin significantly downregulated the expression of tumor-necrosis factor receptor-associated factor 6 (TRAF6), nuclear factor kappa B (NF-κB) and receptor activator of nuclear factor kappa B ligand (RANKL) proteins levels, upregulated the expression of osteoprotegerin (OPG), phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) levels, suggesting that Syringin prevented bone lost by TRAF6-mediated inhibition of NF-κB and stimulation of PI3K/AKT, and subsequently increasing the OPG/RANKL ratio and inhibiting the osteoclastogenesis, finally promoting bone formation. ConclusionsAll of the data implied Syringin possessed the potent anti-osteoporosis activity on ovariectomized mice, and the underlying molecular mechanism may be related to the NF-κB and PI3K/AKT signaling pathways.

Effect of two kinds of fermented food on blood glucose and lipids in induced diabetic mice

To investigate the effect of two kinds of fermented food, noni juice and natto, on blood glucose and lipids in induced diabetic mice. Female (ICR) mice were induced into diabetes by an injection of alloxan (55 mg/kg, i. v.). After 72 hours, those mice whose fasting blood glucose levels were over 12.00 mmol/L and urine sugar was strongly positive (+ + +) were regarded as diabetical model and were randomly divided into three groups (n=10):diabetical model (DM) group, noni juice (NJ) group and natto (NT) group. Another ten normal female ICR mice were taken as normal control (NC) group. The mice in NJ and NT groups were gavaged with noni juice (25.0 ml/kg) and natto (0.6 g/kg) respectively. The other two groups were given normal saline (25.0 ml/kg). Continuous gavage administration was given for 30 days, the water-drinking volume and food-intake were recorded. After 1.5 h of the last administration, the glucose tolerance of mice was measured. Finally, the changes in glycated serum protein(GSP), insulin(Ins) and blood lipids of blood samples of mice, taken from the femoral artery, were determined. Compared to the NC group, the water-drinking amount and food-intake, GSP and total cholesterol (TC), triglyceride(TG) and low density lipoprotein (LDL) in DM group were increased significantly (P<0.01), while glucose tolerance, Ins and high density lipoprotein (HDL) were decreased significantly (P<0.01). However, when it came to DM group, NJ and NT could significantly (P<0.01 or P<0.05) reduce, GSP, TG and LDL, meanwhile improve glucose tolerance, Ins and HDL (P<0. 01). Both noni juice and natto could reduce the blood glucose levels in induced diabetical mice and improve blood lipids, which suggested that they may have certain application value in prevention and treatment of diabetic mellitus.

Ingenol-3-Angelate Suppresses Growth of Melanoma Cells and Skin Tumor Development by Downregulation of NF-κB-Cox2 Signaling.

BackgroundA recent focus in skin cancer prevention intervenes though modulating molecular links between inflammation and cell growth signaling, such as NF-κB. This study elucidates the effect of a non-tumor promoting phorbol ester, ingenol-3-angelate (I3A), on the growth of human melanoma cells and on the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin inflammation and 7,12-Dimethylbenz(a)anthracene (DMBA)-induced skin carcinoma in mice.Material/MethodsCell viability was assessed by MTT assay, cell proliferation by clonogenic assay, apoptosis and cell cycle arrest was analyzed by flow cytometry, protein expression was studied by IHC and Western blotting, and gene expression by qPCR.ResultsI3A suppressed the survival and proliferation of human melanoma cells with estimated IC50 values around 38 and 46 μM for A2058 and HT144 cell, respectively. I3A activated the protein levels of PKCδ and PKCɛ, which induced apoptosis by activating caspase-9 and caspace-3 followed by lowering of mitochondrial membrane potential and enhancing DNA fragmentation. I3A induced G1 phase cell cycle arrest as well as G2/M phase arrest in both cell lines. I3A inhibited the levels of NF-κB p65 protein as well as phosphorylation of p65 and its nuclear translocation. I3A suppressed the gene expression of NF-κB, COX-2 and iNOS. I3A inhibited TPA-induced inflammation and epidermal hyperplasia in female ICR mice by downregulating NF-κB and iNOS. I3A suppressed the growth of skin tumor in DMBA-induced mice in dose-dependent manner.ConclusionsThe mechanism of I3A induces apoptosis in human melanoma cells and suppresses skin inflammation and carcinoma via downregulation of NF-κB-iNOS-COX-2 signaling.

Dienogest suppresses the activation of primordial follicles and preserves the primordial follicle stockpile for fertility in mice.

The aim of the present study was to characterize the effect of long-term usage of dienogest, a fourth-generation progestin that possesses progestogen and anti-androgen activities, on the stockpile of oocytes and fertility after administration. Female ICR mice (100 days old) were divided into a dienogest group and a control group. The mice received 16 consecutive subcutaneous injections of 5 mg dienogest dissolved in corn oil or corn oil as a vehicle control every 4 days. The mice treated with dienogest had more total offspring and larger litter sizes after the final administration than the mice treated with the vehicle control. Greater numbers of primordial follicles were detected at both 4 and 80 days after the final administration. No significant differences were found in serum anti-Müllerian hormone concentrations at 4 and 80 days after the final dienogest administration. The ratio of primary to primordial follicles was decreased in 3-day-old newborn ovaries cultured for 4 days with dienogest (10-7, 10-6 and 10-5 mol/l) compared with ovaries cultured without dienogest. The results of the present study indicate that dienogest suppresses the activation of primordial follicles during its administration and preserves the primordial follicle stockpile and subsequent fertility in mice.

Subchronic Oral Dose Toxicity Study of Enterococcus Faecalis 2001 (EF 2001) in Mice

As a part of general toxicity studies of Enterococcus Faecalis 2001 (EF 2001) prepared using heat-treatment bacillus mort body EF 2001 in mice, this study examined the toxicity of EF 2001 in single and repeated administrations following the previous report in order to apply this product to preventive medicine. The safety of oral ingestion of EF 2001 was examined in 6-week-old male and female ICR mice with 1,000 mg/kg, 3,000 mg/kg and 5,000 mg/kg body weight/day administrated by gavage of the maximum acceptable dose of EF 2001. The study was conducted using distilled water as a control following the methods for general toxicity studies described in the “Guidelines for Non-clinical Studies of Pharmaceutical Products 2002”. As a control, 1) observation of general conditions, 2) measurement of body weight, 3) determination of food consumption, 4) determination of water consumption, 5) blood test and urinalysis and 6) pathological examination were performed for the administration of EF 2001. Mice received EF 2001 for 13 weeks and results were compared with those of the control group that received distilled water. The results of the above examinations revealed no significant differences between control and EF 2001 groups for both males and females. Thus, no notable toxicity was confirmed with single and repeated oral administrations of EF 2001. Oral administration in the above doses did not result in abnormal symptoms or death during the observation period. No abnormalities in blood cell count or organ weights were seen. Without any evidence of toxicity to cells and organs, EF 2001 is speculated to not adversely affect living organisms. The 50% lethal dose of EF 2001 with oral administration in mice is estimated to be greater than 5,000 mg/kg body weight/day for both male and female mice. Therefore, LD50 value for animals was 5,000 mg/kg or more.

Effects of perinatal fluoride exposure on the expressions of miR-124 and miR-132 in hippocampus of mouse pups

To investigate the effects of perinatal fluoride exposure on learning and memory ability of mouse offspring, ICR female mice were received different doses of sodium fluoride (0, 25, 50, 100 mg/L NaF) from pregnant day 7 to lactational day 21. Pups were exposed to fluoride through the cord blood and breast milk. Open field test showed that compared to the control group, perinatal fluoride exposure significantly decreased the number of entries into the center zone in 100 mg/L NaF group. In the eight-arm maze test, the number of working memory errors, reference memory errors, and the total arm entries were significantly increased in fluoride treatment groups, compared to the control group. Additionally, 100 mg/L NaF significantly elevated the expression levels of miR-124, miR-132, and DiGeorge syndrome chromosomal region 8 (DGCR8) in hippocampus of mouse pups at postnatal day (PND) 21. Contrarily, methyl CpG binding protein 2 (MeCP2) were dramatically reduced in 50 and 100 mg/L NaF groups, while cAMP-response element binding protein (CREB) mRNA level was significantly decreased in all fluoride groups. These findings suggested that the impairment of learning and memory in mouse offspring induced by perinatal fluoride exposure may partly result from the enhanced miR-124 and miR-132 and the alterations of their target genes.

Maternal Exposure to Nano Titanium Dioxide Induces Neurotoxic Effects in Offspring Mice

Although nano-TiO2 has been extensively used in food industrial application, and in daily products for infants and children, its potential neurotoxicity presents a significant concern. As previous studies have demonstrated the neurological effects of nano-TiO2 in mice, the main objective of the current investigation was to establish the effects of prenatal exposure of nano-TiO2 on developing embryos and offspring. ICR female mice were orally administered (1, 2, and 3 mg/kg body weight) nano-TiO2 from prenatal day 2 to postnatal day 21. Morphological changes, learning and memory, and potential target molecules of neurodevelopmental toxicity in offspring mice of nano-TiO2-exposed mothers were examined. The results showed that nano-TiO2 could translocate from prenatally exposed maternal to offspring hippocampi, resulting in hippocampal damage including: a 29.07%-61.4% reduction in spatial recognition; a 22.55% -61.34% reduction in total dendritic length; a 53.57%-71.42% reduction in dendritic branch number; and a 27.72%-75.74% reduction in spine density in offspring CA1 pyramidal cells. Furthermore, expression of several proteins involved in dendritic development was decreased including: a 47.62%-73.81% in microtubule-associated proteins; a 14.58%-54.17% in mitotic kinesin-like protein 1; a 1.14%-31.82% in collapsin response mediator protein 3; a 65.17%-79.05% in neuregulin tyrosine kinase receptor 4; a 1.13%-76.92% in kinesin superfamily protein 17; and a 25.73%-61.4% in post synaptic density protein -95 in offspring hippocampi. Hippocampal neurons may present a major target of neurotoxicity in offspring following maternal exposure to nano-TiO2 during the prenatal period. Therefore, food application of nano-TiO2 should be performed with caution.

Protocatechuic Acid Attenuates Trabecular Bone Loss in Ovariectomized Mice.

Primary osteoporosis is a disease related to excessive bone resorption due to estrogen insufficiency that occurs postmenopause. Protocatechuic acid (PCA), or 3,4-dihydroxybenzoic acid, is a common compound present in numerous plants. Although numerous biological activities of PCA have been identified, its antiosteoporotic function has not been well established. In this study, the antiosteoporotic activity of PCA supplementation was determined in ovariectomized (OVX) female ICR mice at 12 weeks after OVX. The biomechanical properties of a bone were evaluated by microcomputed tomography. The signaling molecules associated with osteoclast differentiation were determined in bone marrow cells through immunoblot or RT-PCR. Oral supplementation with PCA (20 mg/kg/day) significantly ameliorated the OVX-mediated stimulation of osteoclast activity based on decreases in serum levels of receptor activator of nuclear factor κB ligand (RANKL), osteocalcin, and bone alkaline phosphatase and increase in serum osteoprotegerin (each group, n = 6; p < 0.05). In addition, the OVX-induced decreases in mRNA expression levels of cathepsin K, calcitonin receptor, nuclear factor of activated T cell cytoplasmic 1 (NFATc1), and tumor necrosis factor (TNF) receptor-associated factor-6 (TRAF6) in bone marrow cells were significantly attenuated (each group, n = 6; p < 0.05). Finally, the loss of trabecular bone and changes in biomechanical properties of a bone were significantly improved by supplementation with 20 mg/kg PCA (each group, n = 6; p < 0.05). Collectively, our results show that PCA supplement suppressed trabecular bone loss in OVX mice and therefore might be an effective alternative approach for preventing the progression of postmenopausal osteoporosis.

菇类菌丝复合性饮品之基因毒性及急性毒性试验探讨

以樟芝(Antrodiacinnamomea)菌丝体发酵液为主另外添加各1% (v/v)舞茸(Grifolafrondosa)子实体浓缩萃取液、巴西蘑菇(Agaricusblazei)菌丝体40倍浓缩发酵滤液、桑黄(Phellinus linteus)子实体浓缩萃取液及云芝(Coriolus versicolor)菌丝体40倍浓缩发酵滤液再经调整糖酸比而成。为了解其安全性，乃以三项基因毒及小鼠口服急性毒性试验来评估。结果显示，在五株沙门氏杆菌回复突变测试中，不论在有无S9代谢活化的情况下，每个测试剂量组之回复突变菌落数均未达阴性对照组的两倍以上，显示其不具致突变之性质。体外哺乳类细胞染色体变异测试之结果经卜瓦松分布分析后显示，在短时间处理以及长时间处理各组中，皆未观察到具染色体变异之细胞数，与阴性对照组有显著差异。故对哺乳类细胞CHO-K1不具有致染色体变异之基因毒性。生体内哺乳类动物细胞微核试验，亦未见具诱发小鼠周边血球细胞产生微核的能力，亦不影响其造血机能。口服急性毒性试验结果显示，对ICR雄鼠及雌鼠之口服单一极限急性毒性剂量10 ml/kg bw下，并未发现死亡及临床异常症状。 Concentrated aqueous extract from Grifolafrondosa fruit body (1% v/v), Agaricusblazai mycelia (40X), Phellinus linteus fruit body (50X) and Coriolus versicolor mycelia (40X) was added to the mycelium fermentation broth of Antrodia cinnamomea to produce a mushroom mycelia complex drink after the adjustment of the sweet and sour ratio. To better understand the safety of this drink, a battery of three genotoxicity tests and an acute toxicity test in mice were carried out in this study. Results showed that this complex drink did not increase the number of revertant colonies in any of the five test strains when compared with the negative control plates, regardless of the metabolic activation, in contrast to twice or more differences for five positive controls, indicating that the drink present no mutagenic activity. In the chromosome aberration test, neither short-term nor continuous treatment induced higher frequency of aberrations that were significantly different from negative controls. This result demonstrated that the mushroom mycelia complex drink was not genotoxic to CHO-K1 cells. In the mammalian in vivo micronucleus test, this drink did not increase the frequency of micronucleated erythrocytes in peripheral blood and had no significant effects on hematopoietic parameters. In the acute toxicity test, a single administration of the drink at dose of 10 ml/kg bw did not cause any morbidity or mortality in both male and female ICR mice.

Intravenous human endothelial progenitor cell administration into aged mice enhances embryo development and oocyte quality by reducing inflammation, endoplasmic reticulum stress and apoptosis.

Stem cell therapy has been proposed to restore the function and structure of injured tissues. In the present study, we investigated the ability of human endothelial progenitor cells (hEPCs) to attenuate ovarian aging and dysfunction. Female ICR mice aged 4 and 6 months were injected with cultured hEPCs. Cultured hEPCs were injected intravenously twice with 5 × 104 cells with a 4 day interval. After pregnant mare serum gonadotropin and human chorionic gonadotropin stimulation, oocytes and ovaries of aged mice were collected, cumulus-free oocytes were activated by SrCl2 and gene expression levels related to inflammation, apoptosis, follicle development and endoplasmic reticulum (ER) stress in ovaries were compared. Administration of hEPCs attenuated the level of inflammatory cytokines and adverse apoptotic factor, as well as reducing ER stress in the ovaries. Increased cleavage and blastocyst formation rates and cell numbers in blastocysts from hEPCs-treated aged mice vs. same aged control mice demonstrated a protective function of hEPCs against reproductive aging. Based on these data, we suggest that treatment with hEPCs attenuates reproductive aging and dysfunction potentially via regulation of inflammation, apoptosis and ER stress.

Effect of isoflavone-enriched whole soy milk powder supplementation on bone metabolism in ovariectomized mice.

BACKGROUND/OBJECTIVEThere is intense interest in soy isoflavone as a hormone replacement therapy for the prevention of postmenopausal osteoporosis. A new kind of isoflavone-enriched whole soy milk powder (I-WSM) containing more isoflavones than conventional whole soy milk powder was recently developed. The aim of this study was to investigate the effects of I-WSM on bone metabolism in ovariectomized mice.MATERIALS/METHODSSixty female ICR mice individually underwent ovariectomy (OVX) or a sham operation, and were randomized into six groups of 10 animals each as follows: Sham, OVX, OVX with 2% I-WSM diet, OVX with 10% I-WSM diet, OVX with 20% I-WSM diet, and OVX with 20% WSM diet. After an 8-week treatment period, bone mineral density (BMD), calcium, alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP) 5b, osteocalcin (OC), procollagen 1 N-terminal propeptide (P1NP), and osteoprotegenin (OPG) were analyzed.RESULTSBMD was significantly lower in the OVX group compared to the Sham group but was significantly higher in OVX + 10% I-WSM and OVX + 20% I-WSM groups compared to the OVX group (P < 0.05). Serum calcium concentration significantly increased in the OVX + 10% and 20% I-WSM groups. Serum ALP levels were significantly lower in the OVX + 10% and 20% I-WSM groups compared to the other experimental groups (P < 0.05). OC was significantly reduced in the OVX group compared to the Sham group (P < 0.05), but a dose-dependent increase was observed in the OVX groups supplemented with I-WSM. P1NP and OPG levels were significantly reduced, while TRAP 5b level was significantly elevated in the OVX group compared with the Sham group, which was not affected by I-WSM (P < 0.05).CONCLUSIONSThis study suggests that I-WSM supplementation in OVX mice has the effect of preventing BMD reduction and promoting bone formation. Therefore, I-WSM can be used as an effective alternative to postmenopausal osteoporosis prevention.

Drug Compatibility and Distribution Assessments of a Bupivacaine-Polymer Formulation Measured Following Site-Directed Injection in Mice

Background: A long-acting local anaesthetic agent would enable superior post-operative analgesia. Our slow-release bupivacaine-polymer formulation comprising 15% bupivacaine loaded into a poly (DL-lactic acid co castor oil 3:7), DL-LA:CO was effective for up to 96 hours. In this study we determined in vivo drug levels of the formulation from plasma and tissue assay and we tested the feasibility of drug level assessments from an exudate sample collected in a novel device near the injection-site. Methods: Prior to formulation injection, a titanium cylinder dual-open-ended chamber was implanted in the dorso-lumber region of female ICR mice, to be used as a biological compartment. Following a healing period, the bupivacaine-polymer formulation was injected close to the sciatic nerve. Formulation assay samples were collected over one-week post-injection; exudate was drawn from the chamber in the anaesthetised animal; and blood and tissue samples from the sacrificed animal. Bupivacaine distribution in the exudate and plasma samples was determined by liquid chromatography–mass spectrometry developed for these experiments, and in tissue samples by high -performance liquid chromatography. Results: Bupivacaine was detected during the first 24h following formulation injection in all three samples (exudate, plasma and tissue), and decreased to almost undetectable levels by day 7 post-injection. Conclusion: Bupivacaine drug assay measured from an exudate sample may be applicable in addition or as a replacement for standard plasma and tissue assay in studies of drug pharmacokinetic distribution. The high sensitivity and simplicity of the metallic chamber implant to detect drug concentration and exudate analysis was confirmed in sever hepatic impairment.

Acute toxicity test of triple fermented barley extracts (fbe) in mice after oral administration

The objective of this study was to obtain information on acute (single) oral dose toxicity of triple fermented (using saccharification, Saccharomyces cerevisiae, and Weissella cibaria) barley extracts (Hordeum vulgare L., FBe) in female and male ICR mice, to facilitate the development of natural products as medicinal ingredients and functional food. To investigate toxicity and identify target organs, FBe was orally administered once to female and male ICR mice at doses of 2000, 1000, 500, and 0 mg/kg (of body weight) in a volume of 20 mL/kg, dissolved in distilled water. The mortality, change in body weight, and clinical signs were monitored for 14 days following treatment. Additionally, gross observations of the organs, changes in organ weights, and histopathological measurements of principle organs were compared between treatment and control groups of the same sex. The results showed that a single oral dose of FBe was not associated with mortality in any of the treatment groups over the 14-day experimental period. No FBe treatment-related changes in body and organ weights, clinical signs, or necropsy and histopathological findings were detected in this experiment. The results obtained in this study suggest that FBe is non-toxic in mice and is therefore likely to be safe for clinical use. The LD50 (lethal dose in 50% of animals) and approximate LD in mice following a single oral dose of FBe were determined to be greater than 2000 mg/kg, the recommended dose limit for both female and male rodents. In addition, no specific organ targets or clinical signs were detected.

Dangguijakyak-san ameliorates memory deficits in ovariectomized mice by upregulating hippocampal estrogen synthesis

BackgroundDangguijakyak-san (DJS) is an herbal formulation that has been clinically applicable for treating postmenopausal symptoms and neurological disorders. It is reported that hippocampal estrogen attenuates memory impairment via neuroprotection and synaptogenesis. However, the effect of DJS on hippocampal estrogen synthesis remains unknown. In this study, we explored the effect of DJS and its neuroprotective mechanism against memory impairment in ovariectomized (OVX) mice, with respect to hippocampal estrogen stimulation.MethodsCell cultures were prepared from the hippocampi of 18-day-old embryos from timed pregnant Sprague–Dawley rats. The hippocampi were dissected, collected, dissociated, and plated in 60-mm dishes. The cells were treated with DJS for 48 h and the supernatant was collected to determine estrogen levels. Female ICR mice (8-weeks-old) were housed for 1 week and ovariectomy was performed to remove the influence of ovary-synthesized estrogens. Following a 2-week post-surgical recovery period, the mice were administrated with DJS (50 and 100 mg/kg/day, p.o.) or 17β-estradiol (200 μg/kg/day, i.p.) once daily for 21 days. Hippocampal and serum estrogen levels were determined using enzyme-linked immunosorbent assay kit. Memory behavioral tests, western blot, and immunohistochemical analyses were performed to evaluate the neuroprotective effects of DJS in this model.ResultsDJS treatment promoted estrogen synthesis in primary hippocampal cells and the hippocampus of OVX mice, resulting in the amelioration of OVX-induced memory impairment. Hippocampal estrogen stimulated by DJS treatment contributed to the activation of cAMP response element-binding protein and synaptic protein in OVX mice.ConclusionDJS may attenuate memory deficits in postmenopausal women via hippocampal estrogen synthesis.

Effect of high-fat diet on secreted milk transcriptome in midlactation mice.

High-fat diet (HFD) during lactation alters milk composition and is associated with development of metabolic diseases in the offspring. We hypothesized that HFD affects milk microRNA (miRNA) and mRNA content, which potentially impact offspring development. Our objective was to determine the effect of maternal HFD on secreted milk transcriptome. To meet this objective, 4 wk old female ICR mice were divided into two treatments: control diet containing 10% kcal fat and HFD containing 60% kcal fat. After 4 wk on CD or HFD, mice were bred while continuously fed the same diets. On postnatal day 2 (P2), litters were normalized to 10 pups, and half the pups in each litter were cross-fostered between treatments. Milk was collected from dams on P10 and P12. Total RNA was isolated from milk fat fraction of P10 samples and used for mRNA-Seq and small RNA-Seq. P12 milk was used to determine macronutrient composition. After 4 wk of prepregnancy feeding HFD mice weighed significantly more than did the control mice. Lactose and fat concentration were significantly ( P < 0.05) higher in milk of HFD dams. Pup weight was significantly greater ( P < 0.05) in groups suckled by HFD vs. control dams. There were 25 miRNA and over 1,500 mRNA differentially expressed (DE) in milk of HFD vs. control dams. DE mRNA and target genes of DE miRNA enriched categories that were primarily related to multicellular organismal development. Maternal HFD impacts mRNA and miRNA content of milk, if bioactive nucleic acids are absorbed by neonate differences may affect development.