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Background As a laboratory animal resource, the ICR mouse is commonly used in a variety of research fields. However, information on differences in exercise-related characteristics in ICR mice derived from different lineages and the underlying mechanisms remains to be elucidated. In this study, we investigated the intrinsic exercise capacity and a magnitude of response to acute exercise, and sought to identify mechanisms contributing to difference in Korl:ICR (a novel ICR lineage recently established by the National Institute of Food and Drug Safety Evaluation, Korea) and two commercialized ICR lineages derived from different origins (viz., A:ICR mouse from Orient Bio Com, the United States, and B:ICR mouse from Japan SLC Inc., Japan). Results Results showed that despite no significant difference in body weight and weight-proportioned tissue mass of heart and skeletal muscles among groups, the relatively low intrinsic exercise capacity and exaggerated response to acute exercise were identified in B:ICR comparted with Korl:ICR and A:ICR, as reflected by total work and lactate threshold (LT). Also, the mitochondrial efficiency expressed as the complex 1 and complex 1 + 2 respiratory control ratio (RCR) values for cardiac mitochondrial O2 consumption in B:ICR was significantly lower than that in Korl:ICR with higher level of state 2 respiration by glutamate/malate and UCP3 expression in cardiac muscle. Conclusions Taken together, these results indicate that the intrinsic exercise capacity of ICR mouse varies according to lineages, suggesting the role of cardiac mitochondrial coupling efficiency as a possible mechanism that might contribute to differences in the intrinsic exercise capacity and magnitude of response to exercise.

BackgroundThe oral administration of polystyrene-microplastics (PS-MPs) causes chronic constipation of ICR mice, but there are no reports on their effects on the inflammatory response in the colon. To determine if the oral administration of MPs causes inflammation in the colon, the changes in the apoptosis-associated speck like protein containing a caspase recruitment domain (ASC)-inflammasome pathway, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway, and inflammatory cytokine expression were evaluated in the mid colon of ICR mice treated with 0.5 μm size PS-MPs for two weeks.ResultsThe thicknesses of the mucosa, muscle, flat luminal surface, and crypt layer were decreased significantly (p ConclusionsTherefore, the present study suggests that PS-MPs can be a novel cause of an inflammatory response in the mid colon of ICR mice.

Significant phenotypes for constipation were detected in complement 3 (C3) knockout (KO) mice, although no research has been conducted on an association with alteration of gut microbiota. To investigate the effects of dysbiosis on fecal microbiota from C3 KO mice with constipation, the composition of fecal microbiota was characterized in mid-colons of 16-week-old C3 KO mice, and their function for defecation delay development was examined after fecal microbiota transplantation (FMT) of C3 KO mice. Some significant alterations in constipation phenotypes, including stool parameters and histopathological structure, were detected in 16-week-old C3 KO mice compared to those of wild-type (WT) mice. Fecal microbiota of C3 KO mice exhibited decreases in Anaerocolumna, Caecibacterium, Christensenella, Kineothrix, and Oscillibacter populations and increases in Prevotellamassilia, Reuthenibacterium, Prevotella, Eubacterium, Culturomica, Bacteroides, and Muribaculum populations. In FMT study, key stool parameters, including weight and water content, were remarkably declined in a transplanted KO (KFMT) group of antibiotics-induced depletion of microbiota (AiDM)-WT and AiDM-KO mice, and a similar change was observed in fecal morphology. However, intestine length decreased in only the KFMT group of AiDM-WT mice compared with that of AiDM-KO mice. The mucosal layer and muscle thickness were commonly decreased in the KFMT group of AiDM-WT and AiDM-KO mice, and significant alterations in the crypt structure of Lieberkuhn and molecular regulators, including AQP8, C-kit, and 5-HT, were observed in the same group. Taken together, results of the present study indicate that dysbiosis of fecal microbiota from C3 KO mice with constipation phenotypes has a key role in the induction and regulation of defecation delay.

The therapeutic effects of mulberry (Morus alba) leaves on lipid metabolism, including lipogenesis, lipolysis and hyperlipidemia are widely known, although their fermented products are yet to be applied. To investigate the therapeutic effects of a novel extract of mulberry leaves fermented with Cordyceps militaris (EMfC) on lipid metabolism, the lipid profile of serum, lipid accumulation, lipolytic activity and lipogenesis regulation were measured in high fat diet (HFD)-induced obese C57BL/6 mice treated with EMfC for 12 weeks. Briefly, the concentrations of low-density lipoprotein, triglyceride, total cholesterol and glucose significantly decreased in the serum of the HFD+EMfC treated group when compared with the HFD+Vehicle treated group, while the levels of high-density lipoprotein increased in the HFD+EMfC group. The amount of abdominal fat and the size of adipocytes were significantly lower in the HFD+EMfC treated group when compared with the HFD+Vehicle treated group. The weight and number of lipid droplets of liver tissue exhibited a similar decrease. Furthermore, the mRNA levels of peroxisome proliferator-activated receptor-γ for adipogenesis as well as adipocyte protein 2 and Fas cell surface death receptor for lipogenesis reduced following EMfC treatment for 12 weeks. Phosphorylation of perilipin and hormone-sensitive lipase, and in the adipose triglyceride lipase expression showed a significant increase in the HFD+EMfC treated group. These results indicated that EMfC may prevent fat accumulation in the HFD-induced obese C57BL/6 mice through the inhibition of lipogenesis and by stimulating lipolysis. Thus, the results provide evidence for the potential use of EMfC as an anti-obesity complex in the treatment of obesity.

Cognitive impairment responses are important research topics in the study of degenerative brain diseases as well as in understanding of human mental activities. To compare response to scopolamine (SPL)-induced cognitive impairment, we measured altered parameters for learning and memory ability, inflammatory response, oxidative stress, cholinergic dysfunction and neuronal cell damages, in Korl:ICR stock and two commercial breeder stocks (A:ICR and B:ICR) after relevant SPL exposure. In the water maze test, Korl:ICR showed no significant difference in SPL-induced learning and memory impairment compared to the two different ICRs, although escape latency was increased after SPL exposure. Although behavioral assessment using the manual avoidance test revealed reduced latency in all ICR mice after SPL treatment as compared to Vehicle, no differences were observed between the three ICR stocks. To determine cholinergic dysfunction induction by SPL exposure, activity of acetylcholinesterase (AChE) assessed in the three ICR stocks revealed no difference of acetylcholinesterase activity. Furthermore, low levels of superoxide dismutase (SOD) activity and high levels of inflammatory cytokines in SPL-treated group were maintained in all three ICR stocks, although some variations were observed between the SPLtreated groups. Neuronal cell damages induced by SPL showed similar response in all three ICR stocks, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, Nissl staining analysis and expression analyses of apoptosis-related proteins. Thus, the results of this study provide strong evidence that Korl:ICR is similar to the other two ICR. Stocks in response to learning and memory capacity.

Indirect evidence has determined the possibility that microplastics (MP) induce constipation, although direct scientific proof for constipation induction in animals remains unclear. To investigate whether oral administration of polystyrene (PS)-MP causes constipation, an alteration in the constipation parameters and mechanisms was analyzed in ICR mice, treated with 0.5 μm PS-MP for 2 weeks. Significant alterations in water consumption, stool weight, stool water contents, and stool morphology were detected in MP treated ICR mice, as compared to Vehicle treated group. Also, the gastrointestinal (GI) motility and intestinal length were decreased, while the histopathological structure and cytological structure of the mid colon were remarkably altered in treated mice. Mice exposed to MP also showed a significant decrease in the GI hormone concentration, muscarinic acetylcholine receptors (mAChRs) expression, and their downstream signaling pathway. Subsequent to MP treatment, concentrations of chloride ion and expressions of its channel (CFTR and CIC-2) were decreased, whereas expressions of aquaporin (AQP)3 and 8 for water transportation were downregulated by activation of the mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB signaling pathway. These results are the first to suggest that oral administration of PS-MP induces chronic constipation through the dysregulation of GI motility, mucin secretion, and chloride ion and water transportation in the mid colon.

BackgroundInflammatory bowel disease (IBD), including both Crohn’s disease and ulcerative colitis, are chronic human diseases that are challenging to cure and are often unable to be resolved. The inbred mouse strain C57BL/6 N has been used in investigations of IBD as an experimental animal model. The purpose of the current study was to compare the inflammatory responsiveness of C57BL/6NKorl mice, a sub-strain recently established by the National Institute of Food and Drug Safety Evaluation (NIFDS), with those of C57BL/6 N mice from two different sources using a dextran sulfate sodium (DSS)-induced colitis model.ResultsMale mice (8 weeks old) were administered DSS (0, 1, 2, or 3%) in drinking water for 7 days. DSS significantly decreased body weight and colon length and increased the colon weight-to-length ratio. Moreover, severe colitis-related clinical signs including diarrhea and rectal bleeding were observed beginning on day 4 in mice administered DSS at a concentration of 3%. DSS led to edema, epithelial layer disruption, inflammatory cell infiltration, and cytokine induction (tumor necrosis factor-α, interleukin-6, and interleukin-1β) in the colon tissues. However, no significant differences in DSS-promoted abnormal symptoms or their severity were found between the three sub-strains.ConclusionsThese results indicate that C57BL/6NKorl mice responded to DSS-induced colitis similar to the generally used C57BL6/N mice, thus this newly developed mouse sub-strain provides a useful animal model of IBD.

Alterations in complement component 3 (C3) expression has been reported to be linked to several bowel diseases including Crohn's disease, inflammatory bowel disease, and ulcerative colitis; however, the association with constipation has never been investigated. In this study, we aimed to investigate the correlation between C3 regulation and constipation development using a C3 deficiency model. To achieve these, alterations in stool excretion, transverse colon histological structure, and mucin secretion were analyzed in FVB/N-C3em1Hlee /Korl (C3 knockout, C3 KO) mice with the deletion of 11 nucleotides in exon 2 of the C3 gene. The stool excretion parameters, gastrointestinal transit, and intestine length were remarkably decreased in C3 KO mice compared with wild-type (WT) mice, although there was no specific change in feeding behavior. Furthermore, C3 KO mice showed a decrease in mucosal and muscle layer thickness, alterations in crypt structure, irregular distribution of goblet cells, and an increase of mucin droplets in the transverse colon. Mucin secretion was suppressed, and they accumulated in the crypts of C3 KO mice. In addition, the constipation phenotypes detected during C3 deficiency were confirmed in FVB/N mice treated with C3 convertase inhibitor (rosmarinic acid (RA)). Similar phenotypes were observed with respect to stool excretion parameters, gastrointestinal transit, intestine length, alterations in crypt structure, and mucin secretion in RA-treated FVB/N mice. Therefore, the results of the present study provide the first scientific evidence that C3 deficiency may play an important role in the development of constipation phenotypes in C3 KO mice.

(1) Background: We characterized a novel animal model with obesity-induced constipation because constipation is rarely known in genetically engineered mice (GEM), (2) Methods: The changes in the constipation parameters and mechanisms were analyzed in CRISPR-Cas9-mediated leptin (Lep) knockout (KO) mice from eight to 24 weeks, (3) Results: Significant constipation phenotypes were observed in the Lep KO mice since 16 weeks old. These mice showed a significant decrease in the gastrointestinal motility, mucosal layer thickness and ability for mucin secretion as well as the abnormal ultrastructure of Lieberk&uuml, hn crypts in the transverse colon. The density or function of the enteric neurons, intestinal Cajal cells (ICC), smooth muscle cells, and the concentration of gastrointestinal (GI) hormones for the GI motility were remarkably changed in Lep KO mice. The downstream signaling pathway of muscarinic acetylcholine receptors (mAChRs) were activated in Lep KO mice, while the expression of adipogenesis-regulating genes were alternatively reduced in the transverse colon of the same mice, (4) Conclusions: These results provide the first strong evidence that Lep KO mice can represent constipation successfully through dysregulation of the GI motility mediated by myenteric neurons, ICC, and smooth muscle cells in the transverse colon during an abnormal function of the lipid metabolism.

Background: Complement component 3 (C3) receptors play an important role as inflammatory mediators in the innate immune system, although their mechanisms were not well studied during constipation. Objective: The aim of this study is to investigate the regulatory role of C3 and its receptors' downstream signaling during constipation. Methods: Alterations in the C3, C3a receptor (C3aR), and C3b receptor (C3bR) expressions, PI3K/AKT pathway, RhoA/MLC pathway, MAP kinase pathway, and inflammatory cytokine expressions were measured in the mid colon of loperamide (Lop) treated SD rats. Results: Lop treatment successfully induced constipation phenotypes, including decreased stool parameters and histological structure alterations. The expression levels of C3 were significantly increased, whereas expressions of C3aR and C3bR were decreased during Lop-induced constipation. Moreover, significant upregulation was observed in the phosphorylation levels of PI3K, AKT, and GSK3β in mid colons of Lop treated SD rats. The expression of RhoA and phosphorylation of MLC were also enhanced in the Lop treated group. Furthermore, a similar pattern was detected in the MAP kinase pathway and inflammatory cytokine expressions. Subsequent to the Lop treatment, the phosphorylation of ERK and p38, as well as the mRNA levels of NF-κB, TNF-α, IL-6 and IL-1α were remarkably increased in the mid colon. Conclusion: These results indicate that Lop-induced constipation is tightly linked to the downregulation of C3aR and C3bR expressions, and upregulation of the C3 and C3Rs downstream signaling pathway, including PI3K/AKT, RhoA/MLC, and MAP kinase pathways as well as inflammatory cytokine expressions in the mid colon of SD rats.

The efficacy of &alpha, cubebenoate isolated from Schisandra chinensis has been previously studied in three disease areas, namely inflammation, sepsis, and allergy, and its role in other diseases is still being explored. To identify the novel function of &alpha, cubebenoate on lipid metabolism and related inflammatory response, alterations in fat accumulation, lipogenesis, lipolysis, and inflammasome activation were measured in 3T3-L1 preadipocytes and primary adipocytes treated with &alpha, cubebenoate. Lipid accumulation significantly decreased in MDI (3-isobutyl-1-methylxanthine, dexamethasone, and insulin)-stimulated 3T3-L1 adipocytes treated with &alpha, cubebenoate without any significant cytotoxicity. The mRNA levels of peroxisome proliferator-activated receptor (PPAR)&gamma, and CCAAT-enhancer binding protein (C/EBP) &alpha, for adipogenesis, as well as adipocyte fatty acid binding protein 2 (aP2) and fatty acid synthetase (FAS) for lipogenesis, were reduced after &alpha, cubebenoate treatment, while cell cycle arrest at G2/M stage was restored in the same group. &alpha, cubebenoate treatment induced glycerol release in primary adipocytes and enhanced expression of lipolytic proteins (HSL, perilipin, and ATGL) expression in MDI-stimulated 3T3-L1 adipocytes. Inflammasome activation and downstream cytokines expression were suppressed with &alpha, cubebenoate treatment, but the expression of insulin receptor signaling factors was remarkably increased by &alpha, cubebenoate treatment in MDI-stimulated 3T3-L1 adipocytes. These results indicate that &alpha, cubebenoate may play a novel role as lipogenesis inhibitor, lipolysis stimulator, and inflammasome suppressor in MDI-stimulated 3T3-L1 adipocytes. Our results provide the possibility that &alpha, cubebenoate can be considered as one of the candidates for obesity management.

Background Depression is a serious and common psychiatric disorder generally affecting more women than men. A woman’s risk of developing depression increases steadily with age, and higher incidence is associated with the onset of menopause. Here we evaluated the antidepressant properties of Asparagus cochinchinensis (AC) extract and investigated its underlying mechanisms in a rat menopausal depression model. Methods To model this menopausal depression, we induced a menopause-like state in rats via ovariectomy and exposed them to chronic unpredictable mild stress (CUMS) for 6 weeks, which promotes the development of depression-like symptoms. During the final 4 weeks of CUMS, rats were treated with either AC extract (1000 or 2000 mg/kg, PO), which has been reported to provide antidepressant effects, or with the tricyclic antidepressant imipramine (10 mg/kg, IP). Results We report that CUMS promotes depression-like behavior and significantly increases serum corticosterone and inflammatory cytokine levels in the serum of ovariectomized (OVX) rats. We also found that CUMS decreases the expression of brain-derived neurotrophic factor (BDNF) and its primary receptor, tropomyosin receptor kinase B (TrkB), in OVX rats, and treatment with AC extract rescues both BDNF and TrkB expression levels. Conclusion These results suggest that AC extract exerts antidepressant effects, possibly via modulation of the BDNF-TrkB pathway, in a rat model of menopausal depression.

The kainic acid-induced seizure mouse model is widely used in epilepsy research. In this study, we applied kainic acid to the subcutaneous injections of three different sources of DBA/2 mice to compare and evaluate the seizure response. The three mouse sources consisted of DBA/2Kor1 (Korea FDA source), DBA/2A (USA source), and DBA/2 (Japan source), and were purchased from different vendors. To compare the responses of DBA/2 mice to kainic acid injections, we examined the survival rate, seizure phenotype scoring, and behavioral changes. We also evaluated brain lesions using histopathological analysis. Following the administration of kainic acid, almost half of the cohort survived, and the seizure phenotype displayed a moderate level of sensitivity (2 ~ 4 out of 6). In the histopathologic analysis, there was no change in morphological features, and levels of glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba-1) increased in the kainic acid-treated groups. However, there was no difference in the neuronal nuclei (NeuN) expression level. All the data showed that the responses in the kainic acid-treated group were similar across the three strains. In conclusion, our results suggest that the three sources of DBA/2 mice (DBA/2Kor1, DBA/2A, and DBA/2B) have similar pathological responses to kainic acid-induced seizures.

Differences in responsiveness of BALB/c substrains have been investigated in various fields, including diabetes induction, corpus callosum deficiency, virus-induced demyelinating disease, aggressive behavior and osteonecrosis. However, induction efficacy of skin tumor remains untried. We therefore investigated the influence of BALB/c substrain backgrounds on the skin tumor induction efficacy in response to DMBA (7,12-Dimethylbenz[a]anthracene) and TPA (12-O-tetradecanoylphorbol-13-acetate) cotreatment. Alterations in the levels of tumor growth related factors, histopathological structure, and the expression to tumor related proteins were measured in three BALB/c substrains (BALB/cKorl, BALB/cA and BALB/cB) after exposure to DMBA (25 μg/kg) and three different doses of TPA (2, 4 and 8 μg/kg). The average number and induction efficacy of tumors in response to DMBA+TPA treatment were significantly greater in the BALB/cKorl substrain than in BALB/cA and BALB/cB. However, cotreatment with DMBA+TPA induced similar responses for body and organ weights of all three substrains. Few differences were detected in the serum analyzing factors, while similar responsiveness was observed for blood analyzing factors after DMBA+TPA treatment. Furthermore, the three BALB/c substrains exhibited similar patterns in their histopathological structure in DMBA+TPA-induced tumors. The expression levels of apoptotic proteins and tumor related proteins were constantly maintained in all three BALB/c substrains treated with DMBA+TPA. In addition, the responsiveness to cisplatin treatment was overall very similar in the three BALB/c substrains with DMBA+TPA-induced tumors. Taken together, these results indicate that genetic background of the three BALB/c substrains does not have a major effect on the DMBA+TPA-induced skin carcinogenesis and therapeutic responsiveness of cisplatin, except induction efficacy.

Nonalcoholic fatty liver disease (NAFLD) has become one of the most common medical problems. Inhibition of lipogenesis and promotion of lipolysis are two ways to prevent NAFLD. In this study, oleic acid-induced HepG2 cells are used as a NAFLD cell model to test whether s-petasin exerts inhibition of lipogenesis and promotion of the lipolysis effect. The results showed that s-petasin significantly inhibited the lipid level in oleic acid-induced HepG2 cells. Moreover, results showed that the triacylglycerol level was reduced by s-petasin in oleic acid-induced HepG2 cells. Western blot assay revealed that s-petasin stimulated phosphorylation of AMPKα and ACCα. The results also demonstrated that s-petasin can inhibit lipogenesis and enhance triacylglycerol turnover by down-regulation of FAS and SCD-1 and up-regulation of ATGL and HSL through the AMPK signaling-dependent regulation of transcriptional factors, FKHR and SREBP-1. This in vitro study indicates that s-petasin has potential as a candidate compound for NAFLD therapy.

A correlation between endoplasmic reticulum (ER) stress and laxative effects was first reported in a constipation model treated with an aqueous extract of Liriope platyphylla (AEtLP) roots. To investigate the correlation between the laxative effect of uridine (Urd) and ER stress response, alterations in the key parameters for ER stress were measured in loperamide (Lop) induced constipation Sprague Dawley (SD) rats treated with Urd. The efficacy of the laxative effect of Urd was notable on the symptoms of chronic constipation, including alteration of stool parameters and structure of the transverse colon, in Lop induced constipated SD rats. In the PERK/eIF2-ATF4 pathway of ER stress response, the levels of eukaryotic initiation factor 2 alpha (eIF2a) phosphorylation and DNA damage-inducible protein (GADD34) transcripts were significantly enhanced in the Lop+Vehicle treated group. However, the levels were restored in the Lop+Urd treated group, although few differences were detected in the decrease rate. Similar changes were observed for levels of inositol-requiring enzyme 1 beta (IRE1ß) phosphorylation and X-box binding protein 1 (XBP-1) transcript in the IRE1α/XBP pathway. Furthermore, the number of ER stress-induced apoptotic cells and Bax and Bcl-2 expression were recovered in the Lop+Urd treated group compared to the Lop+Vehicle treated group. The results of the present study therefore provide first evidence that the laxative effects of Urd may be tightly correlated with the recovery of ER stress response in constipation models.

This study was conducted to compare the multiple low-dose streptozotocin (MLDS)-induced diabetic patterns of Korl:ICR, A:ICR, and B:ICR mice obtained from three different sources. Six-week-old male ICR mice were obtained from three difference sources. Korl:ICR mice were kindly provided by the National Institute of Food and Drug Safety Evaluation (NIFDS). The other two groups of ICR mice were purchased from different vendors located in the United States (A:ICR) and Japan (B:ICR). All ICR mice that received MLDS exhibited overt diabetic symptoms throughout the study, and the incidence and development of diabetes mellitus were similar among the three ICR groups. The diabetic mice exhibited hyperglycemia, loss of body weight gain, decreased plasma insulin levels, impaired glucose tolerance, decreased number of insulin-positive cells, and decreased size of β-cells in the pancreas. The diabetes symptoms increased as the blood glucose level increased in the three ICR groups. In particular, the level of blood glucose in the STZ-treated group was higher in Korl:ICR and A:ICR mice than in B:ICR mice. The plasma insulin levels, glucose tolerance, blood chemistry, and morphological appearance of the pancreas were very similar in the ICR groups obtained from the three different sources. In conclusion, our results suggest that Korl:ICR, A:ICR, and B:ICR mice from different sources had similar overall responses to multiple low-dose STZ to induce diabetes mellitus.

This study investigated the laxative effects of phlorotannins (Pt) derived from Ecklonia cava (E. cave) on chronic constipation by evaluating alterations in stool parameters, gastrointestinal motility, histopathological structure, mucin secretion, gastrointestinal hormones, muscarinic cholinergic regulation, and fecal microbiota in SD rats with loperamide (Lop)-induced constipation subjected to Pt treatment. Stool-related parameters (including stool number, weight, and water contents), gastrointestinal motility, and length of intestine were significantly enhanced in the Lop+Pt-treated group as compared to the Lop+Vehicle-treated group. A similar recovery was detected in the histopathological and cytological structure of the mid-colon of Lop+Pt-treated rats, although the level of mucin secretion remained constant. Moreover, rats with Lop-induced constipation subjected to Pt treatment showed significant improvements in water channel expression, gastrointestinal hormone secretions, and expression of muscarinic acetylcholine receptors M2/M3 (mAChRs M2/M3) and their mediators of muscarinic cholinergic regulation. Furthermore, the Lop+Pt-treated group showed a significant recovery of Bifidobacteriaceae, Muribaculaceae, Clostridiaceae, and Eubacteriaceae families in fecal microbiota. Taken together, these results provide the first evidence that exposure of SD rats with Lop-induced constipation to Pt improves the constipation phenotype through the regulation of membrane water channel expression, GI hormones, the mAChR signaling pathway, and fecal microbiota.

Vitamin D3 is a fat-soluble secosteroid responsible for enhancing intestinal absorption of calcium, iron, and other materials. Vitamin D3 deficiency, therefore, can cause health problems such as metabolic diseases, and bone disorder. Female sex hormones including estrogen and progesterone are biosynthesized mainly in the granulosa cells of ovary. In this study, we isolated granulosa cells from porcine ovary and cultured for the experiments. In order to examine the effect of vitamin D3 on the ovarian granulosa cells, the mRNA and protein levels of genes were analyzed by real-time PCR and Western blot assay. The production of estrogen from the granulosa cells was also measured by the ELISA assay. Genes associated with follicle growth were not significantly altered by vitamin D3. However, it increases expression of genes involved in the estrogen-biosynthesis. Further, estrogen concentrations in porcine granulosa cell-cultured media increased in response to vitamin D3. These results showed that vitamin D3 is a powerful regulator of sex steroid hormone production in porcine granulosa cells, suggesting that vitamin D deficiency may result in inappropriate sexual development of industrial animals and eventually economic loss.

The aim of the study was to evaluate ultrasonographic findings, affected age, hematology, blood chemistry and clinical signs according to open or closed cervix in 102 bitches presented for treatment of pyometra. The prevalence of pyometra according to breed was observed in Maltese 22.5%, Yorkshire Terrier 13.7% and Shih Tzu 12.7%. The mean age of dogs was 9.6 ± 0.3 years, and open cervix pyometra was more prevalent than closed cervix pyometra. Clinical signs included anorexia, vaginal discharge, depression, polyuria/polydipsia, vomiting, and abdominal distension. The concentration of BUN and the activity of ALP in dogs with closed cervix pyometra were significantly higher than those in dogs with open cervix pyometra (p < 0.05). The white blood cell and neutrophils in dogs with closed cervix pyometra were significant higher than those in dogs with open cervix pyometra (p < 0.05). Ultrasonographic findings of the uterus with open or closed cervix pyometra showed variable patterns. The uterine wall was variable in appearance, from thick and irregular to smooth and thin. The uterine wall was thicker in open cervix pyometra than in closed cervix pyometra. The luminal cavity included smaller amount of anechoic fluid in open cervix pyometra than in closed cervix pyometra.

The laxative effects of aqueous extract of Liriope platyphylla (AEtLP) on loperamide (Lop)‑induced constipation have been reported; however, the key compounds and the mechanism underlying these effects remain unclear. Therefore, the laxative effects of five candidates derived from L. platyphylla: Diosgenin (DG), 5-hydroxymethylfurfural (5-HMF), adenosine (AD), hydroxypropyl cellulose (HPC) and uridine (UD) were investigated by examining the alteration of G protein α (Gα) expression, protein kinase C (PKC) phosphorylation and inositol triphosphate (IP3) concentration levels in the 5-hydroxytryptamine (5‑HT; serotonin) receptor signaling pathway. Primary rat intestine smooth muscle cells (pRISMCs), intestinal epithelial cells (IEC)‑18 and B35 cells were cotreated with Lop and the five compounds in order to screen the candidates. AEtLP, prucalopride (PCP) and bisacodyl (BS) served as positive controls. In pRISMCs, Gα expression levels were recovered in the majority of candidate‑treated groups, whereas PKC phosphorylation recovery was observed only in the DG, 5‑HMF and AD treatment groups. In IEC‑18 cells, the AD treatment group mimicked the effects of PCP on PKC phosphorylation levels, whereas the DG, 5‑HMF, HPC and UD treatment groups mimicked the effects of AEtLP and BS. In B35 cells, a greater upregulation of PKC phosphorylation levels were observed in the UD treatment group compared with the PCP and BS treatment groups, whereas DG, 5‑HMF and AD treatment reduced the PKC phosphorylation levels to a greater extent than AEtLP treatment. However, effects similar to AEtLP, PCP and BS on Gα expression levels were not detected in any treatment groups in IEC‑18 and B35 cells. Furthermore, the level of IP3 was enhanced only in pRISMCs, in which all five candidates were effective, while the greatest concentration was observed in the UD treatment group. In conclusion, the results of the present study suggest that UD may be considered the compound with the greatest laxative activity, which may regulate the 5‑HT receptor signaling pathway.

A dysfunction of endoplasmic reticulum (ER) stress response can result in various diseases, including cancer, inflammation, diabetes and neurodegenerative disorders. To investigate whether ER stress response can play an essential role in the induction and treatment of chronic constipation, alterations in the key parameters for ER stress were measured in loperamide (Lop) induced constipation Sprague Dawley (SD) rats treated with aqueous extracts of Liriope platyphylla (AEtLP), which has been shown to have a laxative effect. Symptoms of chronic constipation including alteration of stool parameters and the transverse colon’s structure were successfully induced by Lop treatment. Laxative effects such as enhancement of stools parameters, recovery of the mucosa thickness, increased muscle thickness and recovery of flat luminal surface were also observed in the Lop+AEtLP treated group. Furthermore, enhancement of eukaryotic initiation factor 2 alpha (eIF2α) phosphorylation and inositol-requiring enzyme 1 beta (IRE1β) expression, key indicators for ER stress, that were observed in the Lop+vehicle treated group were significantly recovered in the Lop+AEtLP treated group, although the phosphorylation level of c-Jun N-terminal protein kinase (JNK) remained constant. Moreover, alterations in the transcription level of the marker genes X-box binding protein 1 (XBP-1) and growth arrest and DNA damage-inducible protein (GADD34) were similar to those of eIF2α and IRE1β. However, their level was slightly or completely recovered after AEtLP treatment. Overall, this study provides the first evidence that ER stress response may be tightly correlated with chronic constipation induced by Lop treatment, as well as the laxative effects of AEtLP.

Non-alcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease worldwide. It is characterized by the accumulation of lipids without alcohol intake and often progresses to non-alcoholic steatohepatitis (NASH), liver fibrosis, and end-stage liver diseases such as cirrhosis or cancer. Although animal models have greatly contributed to the understanding of NAFLD, studies on the disease progression in humans are still limited. In this study, we used the recently reported high-fat L-methionine-defined and choline-deficient (HFMCD) diet to rapidly induce NASH and compared the responses to HFMCD in ICR mice from three different countries: Korea (supplied by the National Institute of Food and Drug Safety Evaluation), USA, and Japan during 6 weeks. Feeding HFMCD did not cause significant differences in weight gain in comparison with mice fed control diet. Relative weight of the liver increased gradually, while the relative weight of the kidneys remained unchanged. The parameters of liver injury (serum activities of alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase) increased rapidly from 1 week and remained elevated for as long as 6 weeks. Histopathological analysis showed that the accumulation of hepatic lipids induced by HFMCD was prominent at 1 week after diet supplementation and increased further at 6 weeks. Inflammatory markers were significantly increased in a time-dependent manner by HFMCD. The mRNA levels of TNF-α and IL-6 were elevated approximately 15-fold relative to control diet and that of IL-1β was increased more than 20-folds at 6 week after the onset of HFMCD intake. In addition, mRNA expression of fibrosis markers such as α-SMA, TGFβ1, and Col1a1 were also significantly increased at 6 week. In summary, the responses of Korl:ICR mice by intake of HFMCD diet were similar to those of ICR mice from other sources, which suggests that Korl:ICR mice is also a useful resource to study the pathogenesis of diet-induced NAFLD.

Asparagus cochinchinensis is a medical plant that has long been used to treat fever, cough, kidney disease, breast cancer, inflammatory disease and brain disease in northeast Asian countries. Although several studies have been conducted on the anti-neuroinflammatory effects of A. cochinchinensis, the correlation between these effects and nerve growth factor (NGF) has not yet been examined. In this study, we investigated the effects of an aqueous extract of A. cochinchinensis (AEAC) on the secretion and action mechanism of NGF in neuronal cells. The concentration of the NGF protein in the supernatant collected from cultured cells increased significantly in B35 cells treated with AEAC in comparison with the vehicle-treated group without any specific cytotoxicity. Furthermore, the mRNA expression of NGF showed a very similar pattern to its protein concentration. To examine the bioactivity of NGF secreted from B35 cells, undifferentiated PC12 cells were cultured in an AEAC-conditioned medium and neuritic outgrowth was observed. The dendrite length of PC12 cells in the AEAC-treated group was significantly higher than that in the vehicle-treated group. Moreover, the level of the downstream effectors p-TrkA and p-ERK of the high-affinity NGF receptor was significantly higher in the AEAC-treated group, while the expression of the downstream effectors of the low-affinity NGF receptor was significantly lower in the same group. These results suggest that AEAC may contribute to the regulation of NGF expression and secretion in neuronal cells; it is therefore an excellent candidate for further investigation as a therapeutic drug for neurodegenerative diseases.

Age-related changes are associated with susceptibility to infection, malignancy, autoimmunity, response to vaccination and wound healing. To investigate the relationship of several pathological phenotypes of allergic inflammationto age, alterations in theIL-4 derived luciferase signal and general phenotype biomarkers were measured in young (2-month-old) and old (12-month-old) IL-4/Luc/CNS-1 transgenic (Tg) mice with phthalic anhydride (PA)-induced allergic inflammationfor 2 weeks. There was no difference in the ear phenotypes and thickness between young and old mice, although these levels were higher in the PA-treated group thantheacetone-olive oil (AOO)-treated group. The luciferase signal was detected in the mesenteric lymph node (ML), thymus and pancreas of both young and old PA-treated mice, but showed a greater increasein old Tg mice (exceptin thethymus). Agreaterincrease inthe epidermal thickness and dermal thickness was measured in old PA-treated mice than young PA-treated mice, while total mast cell number remainedconstant in both groups. Furthermore, the concentration of IgE was greater in young PA-treated mice than in old PA-treated mice,as wasthe expression of VEGF and IL-6. Taken together, theresults of this study showed that an animal?s age is an important factor that must be considered when PA-induced allergic inflammation in IL-4/Luc/CNS-1 Tg mice areinvestigated to screen for allergens and therapeutic compounds.

Depression is a serious medical illness that is one of the most prevalent psychiatric disorders. Corticosterone (CORT) increases depression-like behavior, with some effects on anxiety-like behavior. 2-Phenethylamine (PEA) is a monoamine alkaloid that acts as a central nervous system stimulant in humans. Here, we show that PEA exerts antidepressant effects by modulating the Brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB)/cAMP response element binding protein (CREB) signaling pathway in CORT-induced depression. To investigate the potential effects of PEA on CORT-induced depression, we first treated CORT (50 &mu, M)-induced hippocampal neurons with 100 &mu, M PEA for 24 h. We found that treatment with CORT altered dendritic spine architecture, however, treatment with PEA rescued dendritic spine formation via regulation of BDNF/TrkB/CREB signaling. Next, we used a mouse model of CORT-induced depression. Mice were treated with CORT (20 mg/kg) for 21 days, followed by assessments of a battery of depression-like behaviors. During the final four days of CORT exposure, the mice were treated with PEA (50 mg/kg). We found that CORT injection promoted depression-like behavior and significantly decreased BDNF and TrkB expression in the hippocampus. However, treatment with PEA significantly ameliorated the behavioral and biochemical changes induced by CORT. Our findings reveal that PEA exerts antidepressant effects by modulating the BDNF/TrkB/CREB signaling pathway in a mouse model of CORT-induced depression.

The NAD(P)H:quinone oxidoreductase 1 (NQO1) gene encodes a cytosolic flavoenzyme that catalyzes the two-electron reduction of quinones to hydroquinones. A polymorphic form of NQO1 is associated with mood disorders such as schizophrenia. However, the role of NQO1 in dopaminergic system has not yet been elucidated. To determine the role of NQO1 in the dopaminergic system, we investigated pharmaco-behavioral effects of d-amphetamine using NQO1-deficienct mice. According to our comparative study involving NQO1+/+ and NQO1-/- mice, NQO1 deficiency increased d-amphetamine-induced psychomotor activity and psychological dependency compared to wild-type mice. Basal and d-amphetamine-induced dopamine levels were also enhanced by NQO1 deficiency. In NQO1-/- mice, neural activation induced by d-amphetamine was higher in dorsolateral striatum, but not in dorsomedial and ventral striata. Although protein level of CaMKIIα, which is a key player in amphetamine-induced dopamine efflux, was decreased in striata of NQO1-/- mice, phosphorylation of CaMKIIα was markedly enhanced in NQO1-/- mice compared to wild-type mice. Interestingly, experiments with pharmacological antagonist showed that D2 antagonist-induced suppression of locomotion required activation of NQO1. Moreover, the rewarding effect in response to D1 agonist was increased by NQO1 deficiency. These results suggest that striatal NQO1 is of considerable interest to understand the mechanism of dopaminergic regulation of psychiatric disorders.

Conflicting results for morusin activity during adipogenic differentiation are reported in 3T3-L1 adipocytes and cancer cells. To elucidate the influence of morusin on fat metabolism, their anti-obesity effects and molecular mechanism were investigated in 3T3-L1 cells and primary adipocytes. Morusin at a dose of less than 20 &micro, M does not induce any significant change in the viability of 3T3-L1 adipocytes. The accumulation of intracellular lipid droplets in 3T3-L1 adipocytes stimulated with 0.5 mM 3-isobutyl-1-methylxanthine, 1 &micro, M dexamethasone, 10 &micro, g/mL insulin in DMEM containing 10% FBS (MDI)-significantly reduces in a dose-dependent manner after morusin treatment. The phosphorylation level of members in the MAP kinase signaling pathway under the insulin receptor downstream also decrease significantly in the MDI + morusin-treated group compared to MDI + vehicle-treated group. Also, the expression of adipogenic transcription factors (PPAR&gamma, and C/EBP&alpha, ) and lipogenic proteins (aP2 and FAS) are significantly attenuated by exposure to the compound in MDI-stimulated 3T3-L1 adipocytes. Furthermore, the decrease in the G0/G1 arrest of cell cycle after culturing in MDI medium was dramatically recovered after co-culturing in MDI + 20 &micro, M morusin. Moreover, morusin treatment induces glycerol release in the primary adipocytes of SD rats and enhances lipolytic protein expression (HSL, ATGL, and perilipin) in differentiated 3T3-L1 adipocytes. Overall, the results of the present study provide strong evidence that morusin inhibits adipogenesis by regulating the insulin receptor signaling, cell cycle and adipogenic protein expression as well as stimulating lipolysis by enhancing glycerol release and lipolytic proteins expression.

BackgroundAlthough constipation has been researched in various neurological disorders, including Parkinson's disease (PD) and spinal cord injury (SCI), the pathological mechanism of this symptom has not been investigated in Alzheimer's disease (AD) associated with loss of nerve cells in the brain. This study was undertaken to gain scientific evidences for a molecular correlation between constipation and AD.MethodsTo understand the etiology, we measured alterations in various constipation parameters, muscarinic acetylcholine receptors (mAChRs) and endoplasmic reticulum (ER) stress response, in 11-month-old Tg2576 transgenic (Tg) mice showing AD-like phenotypes.ResultsA high accumulation of amyloid beta (Aβ) peptides, a key marker of AD pathology, were detected in the cortex and hippocampus of Tg mice. Furthermore, significant alterations were observed in various constipation parameters including stool weight, histological structure, cytological structure and mucin secretion in Tg2576 mice. Moreover, M2 and M3 expression and the downstream signaling pathways of mAChRs were decreased in the Tg group, as compared with non-Tg (NT) group. Furthermore, activation of ER stress proteins and alteration of ER structure were also detected in the same group.ConclusionsThe results of the present study provide strong novel evidence that the neuropathological constipation detected in Tg2576 mice is linked to dysregulation of the mAChR signaling pathways and ER stress response.

Alkylphenols such as 4-tert-octylphenol (OP), nonylphenol, and bisphenol A are classified as endocrine-disrupting chemicals (EDCs). Digestion and metabolism of food are controlled by many endocrine factors, including insulin, glucagon, and estrogen. These factors are differentially regulated during pregnancy. The alteration of nutritional intake and fat metabolism may affect the maintenance of pregnancy and supplementation of nutrients to the fetus, and therefore can cause severe metabolic diseases such as ketosis, marasmus and diabetes mellitus in pregnant individuals. In this study, we examined the effects of OP on fat metabolism in pregnant rats. Ethinyl estradiol (EE) was also administered as an estrogenic positive control. In our results, rats treated with OP showed significantly reduced body weights compared to the control group. In addition, histological analysis showed that the amount of fat deposited in adipocytes was reduced by OP treatment. To study the mechanism of action of OP in fat metabolism, we examined the expression levels of fat metabolism-associated genes in rat adipose tissue and liver by real-time PCR. OP and EE negatively regulated the expression of lipogenic enzymes, including FAS (fatty acid synthase), ACC-1 (acetyl-CoA carboxylase-1), and SCD-1 (stearoyl-CoA desaturase-1). The levels of lipogenic enzyme-associated transcription factors such as C/EBP-α (CAAT enhancer binding protein alpha) and SREBP-1c (sterol regulatory element binding protein-1c) were also reduced in both liver and adipose tissue. In summary, these findings suggest that OP has adverse effects on fat metabolism in pregnant rats and inhibits fat deposition via regulating lipogenic genes in the liver and adipose tissue. The altered fat metabolism by OP may affect the nutrition balance during pregnancy and can cause metabolism-related diseases.

The present study aimed to examine potential biomarkers for the diagnosis of endometriosis. A plasma-based proteomic approach, including 2-dimentional electrophoresis and mass spectrometry, was used. Samples were obtained from patients with (n=15) and without (n=15) endometriosis, or from mice with surgically induced endometriosis. Seven spots corresponding to six differentially expressed proteins were identified in the human plasma samples. However, only haptoglobin (Hp) was identified to be significantly decreased in the plasma levels of patients with endometriosis (P

We hypothesized that the administration of Cheonggukjang (CKJ) would exert positive effects on factors implicated with growth in Sprague-Dawley (SD) rats. To test this hypothesis, we measured specific aspects of bone and organ growth in male SD rats that were treated for 6 weeks with 3 concentrations of CKJ. Although the CKJ extract contained high concentrations of flavonoids and phenolic compounds, no significant differences in body length, organ weights, or femur weight were detected between the CKJ- and vehicle-treated groups. However, thicknesses of the epiphyseal growth plate in the proximal femoral epiphysis and the compact bone in the linea aspera were broadest in the femur of the 2 CKJ-treated groups when compared with the vehicle-treated groups. Furthermore, the levels of growth hormone (GH) and calcium ion were higher in the sera of the high-concentration CKJ-treated groups, whereas the expression level of GH receptor was higher in muscle tissue of all CKJ-treated groups and in the liver tissue of the high-concentration CKJ-treated group. In the GH receptor downstream signaling pathway, the phosphorylation levels of Akt and Erk were expressed differently between liver and muscle tissues upon CKJ treatment. However, the phosphorylation level of STAT5 was very similar to the expression level of the GH receptor in all CKJ-treated groups. These results indicate that CKJ extract may increase the thickness of the epiphyseal growth plate and the compact bone of the femur, elevate GH secretion, and stimulate regulation of the GH receptor downstream signaling pathway in the liver and muscle tissues of SD rats.

Amyloid β (Αβ) has been reported to be responsible for the functional and structural abnormalities of Alzheimer's disease (AD) through the induction of oxidative stress. The aim of this study was to determine whether or not treatment of transgenic (Tg) mice with green tea catechin (GTC), a radical scavenger, improves AD phenotypes. To test this, 7-month-old Tg mice were treated with a low (1 mg) or high (10 mg) dose of GTC for 6 months. Surprisingly, GTC-treated Tg mice exhibited significant decreases in behavioral impairment, Aβ-42 production, APP-C99/89 expression, γ-secretase component and Wnt protein levels, γ-secretase activity and MAPK activation. In contrast, the levels of APP-C83 protein and enzyme activities (α-secretase, neprilysin and Pin1) were elevated in the GTC-treated groups. Moreover, GTC-treated groups showed lower levels of total cholesterol and low-density lipoprotein cholesterol, whereas the level of high-density lipoprotein cholesterol increased. These results provide the first experimental evidence that GTC improves AD phenotypes, thereby suggesting that GTC can be used in the prevention of AD or treatment of AD patients.

Reserpine, an anti-hypertensive drug, is able to positively modulate several phenotypes associated with toxicity in a Caenorhabditis elegans model of Alzheimer`s disease (AD). We investigated into the therapeutic effects of reserpine on mammalian neurodegenerative disorders, and found that significant alteration of the key factors influencing AD was detected in Tg2576 mice after reserpine treatment for 30 days. The aggressive behavior of Tg2576 mice was significantly improved upon reserpine treatment, whereas their social contact was consistently maintained. Furthermore, the levels of -42 peptide in the hippocampus of the brain and blood serum were lower in the reserpine-treated group than in the vehicle-treated group. Among g-secretase components, the expression levels of PS-2, Pen-2, and APH-1 were slightly lower in reserpine-treated Tg2576 mice, although a significant change in nicastrin (NCT) expression was not detected. Furthermore, the serum level of nerve growth factor (NGF) increased in reserpine-treated Tg2576 mice compared with vehicle-treated mice. Among down-stream effectors of the NGF receptor TrkA signaling pathway, reserpine treatment induced elevation of TrkA phosphorylation and reduction of ERK phosphorylation. In addition, in the NGF receptor signaling pathway, the expression levels of and Bcl-2 were enhanced in reserpine-treated Tg2576 mice compared with vehicle-treated mice, whereas the expression level of RhoA declined. Overall, these results suggest that reserpine can help relieve AD pathogenesis in Tg2576 mice through downregulation of -42 deposition, alteration of -secretase components, and regulation of NGF metabolism.

In order to investigate the effects of a fermented soybean product (Chungkookjang, CKJ) on nerve growth factor (NGF) metabolism, NGF secretion ability and its related signaling pathway were analyzed in B35 neuronal cells and the Tg2576 mouse model of Alzheimer's disease (AD). In B35 cells, the concentration of NGF significantly increased upon treatment with Taegwang (TG)-CKJ and Shinhwa (SH)-CKJ extracts compared with vehicle. Further, a significant increase in PC12 cell length as well as the phsophorylation levels of TrkA and Akt, which are members of a high affinity NGF receptor signaling pathway, were observed after treatment with TG-CKJ and SH-CKJ conditional medium (CM). On the other hand, there was no difference in activation of the NGF receptor p75(NTR) signaling pathway between vehicle and all CKJ treated groups. In Tg2576 mice showing early stage of AD, the concentrations of NGF in the serum and brain were reduced compared with those in Non-Tg mice. Treatment of Tg2576 mice with SH-CKJ, which contains high concentrations of total flavonoids and phenolic compounds, for 8 weeks dramatically recovered the NGF level to that of Non-Tg mice. Furthermore, the low phosphorylation levels of TrkA and Erk in the NGF receptor TrkA signaling pathway were rapidly recovered to those of Non-Tg mice after SH-CKJ treatment in vehicle treated Tg2576 mice, whereas the phosphorylation level of Akt was maintained at a constant level. These results suggest that CKJ may stimulate NGF secretion ability as well as the NGF receptor TrkA signaling pathway in PC12 cells and Tg2576 mice.

The aim of the present study was to investigate the protective effects of gomisin A, a lignan compound isolated from Schisandra chinensis, against liver and kidney damage induced by CCl(4) exposure. We assessed alterations in organ weights, levels of serum biochemical indicators, and activation of the caspase-3 and MAPK signaling pathways and carried out histological analysis of liver and kidney tissue in rats pretreated with gomisin A for four days. In the gomisin A/CCl(4)-treated group, only the liver experienced a significant increase in weight, whereas the other organs did not undergo any changes. Five biochemical indicators in serum indicated that liver and kidney toxicity dramatically decreased upon gomisin A pretreatment, although the decrease in ratios varied. Upon histological analysis, the gomisin A/CCl(4)-treated group showed less hepatocellular necrosis, a poorly dilated central vein in the liver section, decreased diameter of the glomerulus, a lower number of capillaries, and a convoluted tubule in the kidney section. Furthermore, the formation of active caspase-3 was inhibited by gomisin A pretreatment in the gomisin A/CCl(4)-treated group, whereas the expression level of Bax protein was slightly increased. Western blot analysis revealed that there were differences between the liver and kidney in terms of activation of the MAPK signaling pathway. In the liver, gomisin A pretreatment increased phosphorylation of three members of the MAPK pathway when compared to that in the vehicle pretreatment group. However, in the kidney, only the phosphorylation level of p38 was elevated upon gomisin A pretreatment, whereas levels of the other two members were decreased. These results suggest that gomisin A induces marked protective effects against hepatic and renal injury induced by CCl(4) exposure through differential regulation of the MAPK signal transduction pathway.

Red Liriope platyphylla (RLP) produced by steaming process has been reported to enhance the secretion of insulin and nerve growth factor (NGF). However, there has been no report on the toxicity of RLP in the specific organs of mice. To investigate the toxic effect of RLP, we tried to observe a significant alteration on body weight, food/water intake, organ weight, liver pathology and kidney pathology in female ICR mice received 12.5, 25.0 and 50.0 mg/kg body weight/day of RLP via gavage for 10 days. Out of seven organs including brain, heart, lung, liver, kidney, spleen and ovary, two organs (heart and lung) showed significantly decreased weights in the medium dosage RLP-treated group, whereas weights of other organs were maintained at constant levels in all dosage groups. In the liver toxicity analysis, no significant increase of alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate amino-transferase (AST) were detected in any RLP-treated group compared to vehicle-treated group. The specific pathological changes induced by most of toxic compounds were not observed in the liver in microscopic examination. Furthermore, in the kidney toxicological analysis, a significant enhancement of the blood urea nitrogen (BUN) concentration was detected in the high dosage RLP-treated group compared to the vehicle-treated group. However, the serum creatinine (CA) concentration on the serum biochemistry as well as the pathological changes in microscopic examination were not significantly different between the vehicle- and RLP-treated groups. Therefore, these results suggest that RLP does not induce any specific toxicity in liver or kidney tissues of mice, although the BUN level slightly increased in 50.0 mg/kg of RLP-treated group.

Several natural products containing tannins are used as traditional medicines for treatment of constipation; however, their pharmacological mechanism is not well understood. The laxative effects of gallotannin-enriched extract isolated from Galla Rhois (GEGR) were investigated using a constipation model induced by loperamide (Lop) injection. After analysis for antioxidant activity of GEGR, alterations in the excretion parameters, histological structure, mucin secretion, and related protein levels were measured in the transverse colon of Sprague Dawley (SD) rats with Lop-induced constipation following treatment with 250, 500 and 1,000 mg/ml of GEGR. The number and weight of feces increased significantly by 48–79% and 128–159%, respectively, in the Lop+GEGR treated group relative to the Lop+vehicle treated group, while food intake and water consumption were maintained at a constant level. The thickness of mucosa, muscle and flat luminal surface, as well as the number of goblet cells and crypt of lieberkuhn were enhanced in the Lop+GEGR treated group. Moreover, mucin secretion increased significantly in a dose dependent manner in the Lop+GEGR treated group. Furthermore, the downstream signaling pathway of the muscarinic acetylcholine receptors (mAChR) M2 and M3 was recovered by GEGR treatment, although the expression level varied. The levels of Gα expression and inositol triphosphate (IP3) concentration were also recovered in the Lop+GEGR treated group relative to the Lop+vehicle treated group. The results of the present study provide strong evidence that tannins distributed in various medicinal plants are important candidates for improving chronic constipation induced by Lop treatment in animal models.

Animal models of constipation induced with drugs and diet have been widely employed to investigate therapeutic effects and the action mechanism of drugs against this disease. ICR mice were selected to produce this disease model through oral administration of loperamide (Lop), even though SD rats are commonly utilized in studies of constipation. To compare the responses of ICR mice obtained from three different sources to constipation inducers, alterations in stool number, histopathological structure, mucin secretion and opioid-receptor downstream signaling pathway were measured in KorhlCR (Korea FDA source), A:ICR (USA source) and B:ICR (Japan source) injected with low and high concentrations of Lop (LoLop and HiLop). The number, weight and moisture content of stools decreased significantly in the Lop treated group of all ICR relative to the Vehicle treated group. Additionally, decreased mucosa layer thickness, muscle thickness, and mucin secretion were observed in the transverse colon of Lop treated ICR mice, while a similar number of goblet cells and crypt of lieberkuhn were detected in the same group. Furthermore, a similar change in the level of Ca expression and PKC phosphorylation was detected in the Lop treated group relative to the vehicle treated group, while some differences in the change pattern were observed in the B:ICR group. Therefore, these results of the present study provide strong additional evidence that KorhlCR, A:ICR and B:ICR derived from different sources have a similar overall response to constipation induced by Lop injection, although there were a few differences in the magnitude of their responses.

To investigate the beneficial effects of diosgenin (DC) on the multiple types of brain damage induced by Aβ-42 peptides and neurotoxicants, alterations in the specific aspects of brain functions were measured in trimethyltin (TMT)-injected transgenic 2576 (TG) mice that had been pretreated with DC for 21 days. Multiple types of damage were successfully induced by Aß-42 accumulation and TMT injection into the brains of TG mice. However, DC treatment significantly reduced the number of Aß-stained plaques and dead cells in the granule cells layer of the dentate gyrus. Significant suppression of acetylcholinesterase (AChE) activity and Bax/Bcl-2 expression was also observed in the DC treated TG mice (TG+DG group) when compared with those of the vehicle (VC) treated TG mice (TG+VC group). Additionally, the concentration of nerve growth factor (NGF) was dramatically enhanced in TG+DG group, although it was lower in the TG+VC group than the non-transgenic (nTG) group. Furthermore, the decreased phosphorylation of downstream members in the TrkA high affinity receptor signaling pathway in the TG+VC group was significantly recovered in the TG+DG group. A similar pattern was observed in p75NTR expression and JNK phosphorylation in the NGF low affinity receptor signaling pathway. Moreover, superoxide dismutase (SOD) activity was enhanced in the TG+DG group, while the level of malondialdehyde (MDA), a marker of lipid peroxidation, was lower in the TG+DG group than the TG+VC group. These results suggest that DC could exert a wide range of beneficial activities for multiple types of brain damage through stimulation of NGF biosynthesis.

A combination of selenium (Se) with other trace element is associated with partially modulate fatty acid distribution as well as reduction of the body weight and feed efficiency. To investigate whether or not Se treatment has an impact on lipid metabolism, we examined the levels of lipid metabolism-related factors, including abdominal fat, adiponectin, cholesterol, very long chain dehydrogenase (VLCAD), and medium chain acyl-CoA dehydrogenase (MCAD) in 20-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats following sodium selenite treatment for 2 weeks. Herein, we observed that (a) Se treatment induced insulin-like effects by lowering the serum glucose level in rats; (b) Se-treated rats showed significance values decreases in abdominal fat mass, adipocyte size, and adiponectin, which are associated with lipid metabolism; (c) Se treatment led to reduced levels of cholesterol, triglycerides, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) cholesterol; (d) fat tissue in Se-treated rats displayed significantly lower expression of adipocyte marker genes along with increased expression of VLCAD and MCAD; and (e) fatty liver formation and β-oxidation gene expression were both significantly reduced in liver tissue of Se-treated rats. Therefore, our results suggest that Se may induce inhibition of adipocyte hypertrophy and abdominal fat accumulation along with suppression of fatty liver formation by the differential regulation of the gene expression for fatty acid β-oxidation in the OLETF model.

The production and secretion of growth hormone (GH) in the anterior pituitary gland can be induced by several natural products to control cell proliferation, differentiation, and migration. To investigate whether Chungkookjang (CKJ) produced by the fermentation process affects GH-related metabolism, the secretion and the response of GH were observed in pituitary cells and GH target cells. Among six CKJs manufactured by different strains of glycine max, only three CKJs, including Daewon (DW), Daepung (DP), and Taegwang (TG), induced GH secretion from GH3 cells at 5.0 mg/ml concentration. There were no significant changes detected in the viability of any of the cells treated with these CKJs. In addition, the increase in GH secretion from the GH3 cells was dependent on the concentration of the three types of CKJs. The proliferation of cell lines, including MG63 and HepG2 cells, that originated from those derived from the GH target organs was significantly activated by treatment with the GH-containing conditional medium (GCM) harvested from the three CKJ-treated GH3 cells, although their induction rate was different from each other. In these cells, p-STAT5 was maximally translocated into the nucleus of MG63 cells 30 min after DW treatment, while it was translocated in HepG2 cells at 60 min. These results suggest that these three types of CKJ could enhance the secretion of GH, as well as the GCM-derived response, in the two target organs.

Red Liriope platyphylla (RLP) manufactured by two repeated steps (steaming and drying) stimulates the insulin secretion ability and glucose receptor signaling pathway in an animal model for type I diabetes. This study examined the levels of glucose and lipid metabolism-related factors in a useful animal model for type II diabetes with obesity following RLP treatment for 3 weeks to determine if RLP treatment affects the glucose concentration, insulin secretion and fatty acid oxidation. The following results were obtained: i) RLP contained a large amount of polyphenolic compounds; ii) insulin secretion was induced in RLP-treated OLETF rats, although there were no significant differences in body weight, glucose tolerance test and glucose concentration; iii) the RLP-treated OLETF rats showed a significant increase in adiponectin concentration but the concentration of triglyceride and LDL decreased compared to the vehicle-treated rats; iv) although the abdominal fat mass and adipocyte size did not change with RLP treatment, expression of the adipocyte marker genes and β-oxidation genes in fat tissue was recovered to the level of the LETO rats; v) fatty liver formation was reduced dramatically in the liver of the RLP-treated group compared to the vehicle-treated group; vi) the expression of adipocyte marker genes and the β-oxidation gene in the liver tissue were generally similar to those of the abdominal fat but PPAR-γ showed a reverse pattern in the RLP- and vehicle-treated OLETF rats. These results suggest that RLP may stimulate insulin secretion and a decrease in lipid in serum, and may also suppress fatty liver formation through the regulation of fatty acid oxidation. The data presented here highlight the possibility that RLP can be considered a candidate for the prevention or alleviation of obesity-related diseases.

Altered expression of neurotrophic factors as well as neuroinflammation is commonly associated with Major depressive disorder (MDD) and Alzheimer's disease (AD). To investigate whether or not reserpine-induced MDD affects the expression of AD-related proteins, the expression of γ-secretase components and substrate were measured in brains of ICR mice following reserpine treatment for 15 days. In active avoidance test, total response time and peak slightly increased in the 2 mg/kg reserpine (RSP2)-treated group compared to vehicle-treated group (P

It was reported that the novel compounds (LP9M80-H) of Liriope platyphylla regulate glucose transporter (Glut) biosynthesis by activating the insulin-signaling pathway in the liver and brain of ICR mice. To investigate the therapeutic effects of LP9M80-H on the pathology of diabetes and obesity, alterations of key factors related to symptoms were analyzed in the Otsuka Long Evans Tokushima Fatty (OLETF) rats treated with LP9M80-H for 2 weeks. The abdominal fat masses in the LP9M80-H-treated group were lower than the vehicle-treated group, although there was no difference in body weight between the two groups. Additionally, when compared to the vehicle-treated group, LP9M80-H treatment induced a significant decrease in glucose levels and an increase in the insulin concentration in the blood of OLETF rats. A high level of insulin protein was also detected in pancreatic β cells of LP9M80-H-treated OLETF rats. A significant reduction in the concentration of lipids and adiponectin was detected only in LP9M80-H-treated OLETF rats. Furthermore, the expression of insulin receptor β and the insulin receptor substrate (IRS) was dramatically decreased in LP9M80-H-treated OLETF rats compared to the vehicle-treated group. Of the glucose transporters located downstream of the insulin-signaling pathway, glucose transporters (Glut) -2 and -3 were significantly decreased in LP9M80-H-treated OLETF rats, while the level of Glut-4 was maintained under all conditions. Therefore, these results suggest that LP9M80-H may contribute to relieving symptoms of diabetes and obesity through glucose homeostasis and regulation of lipid concentration.

Liriope platyphylla has been used in oriental medicine as an effective medical plant to improve symptoms of cough, sputum production, neurodegenerative disorders, obesity and diabetes for long time. In order to investigate the effects of novel extracts on nerve growth factors (NGF)-stimulated neuritic outgrowth, the alteration of NGF expression and NGF receptor signaling pathway were detected in neuroblastoma cells and C57BL/6 mice. Of a total of 13 novel extracts, 4 extracts (LP-E, LP-M, LP-M50, LP2E17PJ) showed high viability on MTT assay. Also, all of these extracts induced NGF secretion and NGF mRNA expression in neuroblastoma cells. However, the NGF-induced neuritic outgrowth from PC12 cells was only stimulated by LP-E, LP-M and LP-M50. Furthermore, we selected LP-M as a best candidate, based on method and amounts of extraction, in order to verify its effect in mice. C57BL/6 mice were treated with 50 mg/kg of LP-M for 2 weeks and the effects on NGF regulation were analyzed with various methods. The expression of NGF mRNA was significantly increased in LP-M treated mice compared to vehicle treated mice. Also, the signaling pathway of p75NTR was inhibited in the cortex by LP-M treatment, with no change in the hippocampus of brain. However, the signaling pathway of TrkA was dramatically activated in only hippocampus via LP-M treatment. Therefore, these results suggest that the novel four extracts of L. platyphylla may contribute to the regulation of NGF expression and secretion in neuronal cells. LP-M was especially considered to be an excellent candidate for a neurodegenerative disease-therapeutic drug.

Liriope platyphylla is a medical herb that has long been used in Korea and China to treat cough, sputum, neurodigenerative disorders, obesity and diabetes. The aims of this study were to study the antidiabetic effects of the aqueous extract of L. platyphylla (AEtLP) through pancreatic and extrapancreatic actions. AEtLP were orally administrated to ICR mice once a day for 7 days. Of three different concentrations of AEtLP, only 10% AEtLP were low toxic to liver, based on body weight and serum biochemical analyses. However, 10% AEtLP-treated mice displayed signifi cant reduction of the glucose concentration and increased insulin concentration; no changes were noted using 5% and 15% AEtLP. Also, the increase of glucose transporter (Glut)-1 expression in liver was dependent on the concentration of AEtLP, and was regulated by the phosphorylation of Akt. The lowest expression of Glut-3 was observed in 15% AEtLP treated mice, followed by 10% AEtLP- and 5% AEtLP-treated mice. This pattern of Glut-3 expression was roughly in accord with the phosphorylation of c-Jun N-teminal kinase (JNK) in the mitogen-activated protein kinase (MAPK) pathway. Furthermore, a signifi cant rise of the superoxide dismutase activity (SOD) was detected in AEtLP-treated mice. The fi ndings suggest that AEtLP should be considered as a diabetes therapeutic candidate to induce insulin secretion from pancreatic β-cells and glucose uptake in liver cells.

맥문동(Liriope platyphylla)은 한국과 중국에서 전통적으로 당뇨, 비만, 뇌신경질환, 천식 등의 치료를 위해 사용 해온 치료제이다. 최근에 이들 맥문동으로부터 새로운 치료제를 개발하려는 노력이 활발히 진행 중이지만 아직도 유력한 치료후보제는 확보되지 않았다. 따라서 본 연구에서는 맥문동의 새로운 추출물을 이용하여 당뇨치료 제로서의 가능성을 평가하기 위하여 새로운 방법으로 10가지 후보물질을 추출하고 이들의 독성과 효능을 평가하였다. 그 결과 10가지 추출물 중에서 LP9M80-H가 인슐린 분비를 가장 많이 촉진하였고 다음으로는 LP-H, LP-M, LP-E과 LP9M80-C 등의 순서로 촉진을 하였으나 나머지는 인슐린 분비를 촉지하지 못하였다. 그러나 이들 물질은 인슐린 분비를 촉진하는 농도에서 강한 세포 독성을 나타내었다. 따라서 이들 물질 중에서 가장 효능이 좋은 LP9M80-H의 치료용 최적농도를 설정하였으며, 대략 100-25 ug/ml가 최적농도로 결정되었다. 이러한 결과는 맥문동 추출물 중에서 LP9M80-H가 췌장 $\beta$ -세포의 인슐린 분비능을 유도하는 새로운 당뇨치료 후보물질로서 향후에 사용될 가능성을 시사하고 있다. 【Liriope platyphylla has traditionally been used in Korea and China as a therapeutic drug for the treatment of coughing, sputum, neurodegenerative disorders, obesity, and diabetes. In an effort to assess the functions of a novel extract from Liriope platyphylla in diabetes therapy, the insulin secretion abilities of 10 extracts were screened via measurements of insulin concentration in the culture supernatant using an Insulin ELISA kit. The results of this assay showed the highest levels of insulin in the LP9M80-H treated group, followed by the LP-H, LP-M, LP-E and LP9M80-C treated groups, whereas other extracts did not induce insulin secretion in the HIT-T15 cells. However, the extracts capable of stimulating insulin secretion simultaneously evidenced high apoptotic activity as compared with other extracts. Therefore, one of these extracts, LP9M80-H, was initially selected as the optimal candidate for a therapeutic drug and its optimal concentration was determined. The results of the ELISA and MTT assay demonstrated that a concentration of approximately 100-125 ug/ml of LP9M80-H was optimal with regards to cell viability and insulin secretion in the HIT-T15 cells. These results suggest that LP9M80-H could be considered as an excellent candidate for a diabetes-therapeutic drug that could induce insulin secretion in pancreatic $\beta$ -cells.】

A few clues about correlation between endoplasmic reticulum (ER) stress and mulberry (Morus alba) leaves were investigated in only the experimental autoimmune myocarditis and streptozotocin-induced diabetes. To investigate whether a novel extract of mulberry leaves fermented with Cordyceps militaris (EMfC) could suppress ER in fatty liver, alterations in the key parameters for ER stress response were measured in high fat diet (HFD)-induced obese C57L/6 mice treated with EMfC for 12 weeks. The area of adipocytes in the liver section were significantly decreased in the HFD+EMfC treated group as compared to the HFD+Vehicle treated group, while their level was higher in HFD+Vehicle treated group than No treated group. The level of the eukaryotic initiation factor 2 alpha (eIF2α) and inositol-requiring enzyme 1 beta (IRE1α) phosphorylation and CCAAT-enhancer-binding protein homologous protein (CHOP) expression were remarkably enhanced in the HFD+Vehicle treated group. However, their levels were restored in the HFD+EMfC treated group, although some differences were detected in the decrease rate. Similar recovery was observed on the ER stress-induced apoptosis. The level of Caspase-3, Bcl-2 and Bax were decreased in the HFD+EMfC and HFD+orlistat (OT) treated group compared to the HFD+Vehicle treated group. The results of the present study therefore provide first evidence that EMfC with the antiobesity effects can be suppressed ER stress and ER stress-induced apoptosis in the hepatic steatosis of HFD-induced obesity model.

Non-alcoholic fatty liver disease (NAFLD) is believed to be the most prevalent liver disease worldwide and a major cause of chronic liver injury. It is characterized by lipid accumulation in the absence of significant alcohol consumption and frequently progresses to steatohepatitis, liver fibrosis, and hepatocellular carcinoma. Although many studies have been conducted to better understand NAFLD since it was first recognized, there are still many gaps in knowledge of etiology, prognosis, prevention and treatment. Methionine-choline deficient (MCD) diet, a well-established experimental model of NAFLD in rodents, rapidly and efficiently produces the clinical pathologies including macrovesicular steatosis and leads to disease progression. In this study, we measured the response to MCD diet in C57BL/6N mice obtained from three different sources; Korea NIFDS, USA, and Japan. We evaluated changes in body weight, food consumption, and relative weights of tissues such as liver, kidney, gonadal white adipose tissue, inguinal white adipose tissue, and brown adipose tissue. These basic parameters of mice with an MCD diet were not significantly different among the sources of mice tested. After 3 weeks on an MCD diet, histopathological analyses showed that the MCD diet induced clear fat vacuoles involving most area of the acinus in the liver of all mice. It was accompanied by increased serum activities of alanine aminotransferase and aspartate aminotransferase, and decreased levels of serum triglyceride and cholesterol. In conclusion, the response of C57BL6N mice originating from different sources to the MCD diet showed no significant differences as measured by physiological, biochemical, and histopathological parameters.