Enhancement of Endometrial Receptivity by Cnidium officinale through Expressing LIF and Integrins.

Improvement of endometrial receptivity is necessary for successful embryo implantation, and its impairment is associated with female infertility. In this study, we investigated the effect of the roots of Cnidium officinale Makino (CoM) on endometrial receptivity in both in vitro and in vivo model of embryo implantation. We found that CoM enhanced the adhesion of JAr cells to Ishikawa cells by stimulating expression of leukemia inhibitory factor (LIF) and integrins. In addition, blocking of LIFR using hLA or neutralization of integrins αV, ß3, and ß5 using antibodies significantly reduced the enhanced adhesion between JAr cell and CoM-treated Ishikawa cells, indicating that LIF and integrin play an important role in trophoblast-endometrium adhesion for embryo implantation. Furthermore, we identified that CoM significantly improved the implantation rate of blastocysts in the mouse model of RU-induced implantation failure. By collecting these results, here, we suggest that CoM has a therapeutic potential against female infertility associated with decreased endometrial receptivity.

Dual targeting of Nur77 and AMPKα by isoalantolactone inhibits adipogenesis in vitro and decreases body fat mass in vivo.

BACKGROUND: Suppression of adipogenesis has been considered as a potential target for the prevention and treatment of obesity and associated metabolic disorders, and the nuclear receptor 4A1 (NR4A1/Nur77) and AMPKα are known to play important roles during early and intermediate stages of adipogenesis. Therefore, we hypothesized that dual targeting Nur77 and AMPKα would show strong inhibitory effect on adipogenesis. METHODS: We screened a herbal medicine-based small molecule library to identify novel natural compounds dual targeting Nur77 and AMPKα, and the antiadipogenic effects and mechanisms of action of a "hit" compound were studied in 3T3-L1 cells. In vivo antiobesity effects of the compound were also investigated in high-fat diet (HFD)-induced obese mice. RESULTS: We identified isoalantolactone (ISO) as a new NR4A1 inactivator that also activates AMPKα in 3T3-L1 cells. ISO, as expected, inhibited adipogenic differentiation of 3T3-L1 preadipocytes, accompanied by reduced mitotic clonal expansion (MCE) which occurs in the early stage of adipogenesis and decreased expression of genes required for MCE and cell cycle markers including cyclin A, cyclin D1. Furthermore, ISO reduced body weight gain and fat mass (epididymal, subcutaneous, perirenal, and inguinal white adipose tissues) in the high-fat diet-fed C57BL/6 N mice. Serum levels of triglycerides, aspartate transaminase, and alanine transaminase and hepatic steatosis were also significantly improved in the ISO-treated group compared to the high-fat diet control group. CONCLUSIONS: These results suggest that ISO dual targeting Nur77 and AMPKα during adipogenesis represents a novel class of mechanism-based antiadipogenic agents for treatment of obesity and associated metabolic disorders, including hyperlipidemia and fatty liver.

Deoxypodophyllotoxin in Anthriscus sylvestris alleviates fat accumulation in the liver via AMP-activated protein kinase, impeding SREBP-1c signal.

Deoxypodophyllotoxin (DPT) is a naturally occurring flavolignan in Anthriscus sylvestris known as cow parsley or wild chervil, and has been reported to have inhibitory effects against several pathological processes including cancer, inflammation and infection. Here, we report the effects of DPT in the fatty liver induced by high fat diet in vivo as well as its regulatory mechanism related with the transcription factor for lipogenic genes such as sterol regulatory element binding protein-1c (SREBP-1c) in vitro. C57BL/6 mice were fed high fat diet for 10 weeks and also orally administrated with DPT for additional 4 weeks. 5 and 10 mg/kg of DPT decreased lipid accumulation in the liver induced by high fat diet, as indicated by histological parameters such as Oil Red O staining and hematoxylin & eosin as well as the contents of hepatic triglyceride and cholesterol. In hepatocytes, DPT inhibited the liver X receptor α-mediated SREBP-1c induction and expression of the lipogenic genes, including fatty acid synthase, acetyl-CoA carboxylase and stearoyl-CoA desaturase-1. Moreover, DPT induced AMP-activated protein kinase (AMPK) activation, which has been known to inhibit the expression of SREBP-1c in hepatocyte. Also this compound restored the dysregulation of AMPK and SREBP-1c induced by high fat diet in mice. In conclusion, we demonstrated that DPT significantly inhibited fatty liver by adjusting lipid metabolism coordinated with AMPK activation and SREBP-1c inhibition.

Water-extracted tubers of Cyperus rotundus L. enhance endometrial receptivity through leukemia inhibitory factor-mediated expression of integrin αVß3 and αVß5.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyperus rotundus L. (CR) has been traditionally used as an herbal medicine in Asian countries to treat diverse gynecological disorders. However, the potential therapeutic effect of CR on endometrial receptivity for successful embryo implantation to treat female infertility has not been fully studied. AIM OF STUDY: The aim of this study was to evaluate the effect of water-extracted CR on endometrial receptivity by investigating the expression of leukemia inhibitory factor (LIF) and integrins, cell adhesion, and embryo implantation using mifepristone (RU486; RU)-induced implantation failure model. MATERIALS AND METHODS: The water extract of CR was prepared and fingerprinted using high-performance liquid chromatography (HPLC). For the expression and regulation of LIF, reverse transcription polymerase chain reaction (RT-PCR) and western blotting were performed in CR-stimulated Ishikawa cells. To evaluate LIF-mediated integrin expression, knockdown of LIF by shRNA was performed in Ishikawa cells. The effect of CR on endometrial receptivity was determined by an in vitro adhesion assay between JAr cells and CR-induced Ishikawa cells. In vivo, C57BL/6 female mice (n = 7 per group) orally received CR (31.68mg/kg/day), a similar dose as used clinically. Seven days after CR treatment, all female mice were caged with male mice until pregnancy was verified. On day 4 of pregnancy, RU (4mg/kg) was injected subcutaneously to induce embryo implantation failure. RESULT: CR increased the expression of LIF through the phosphatidylinositol-3-kinase/ protein kinase B (PI-3K/AKT) signaling pathway in Ishikawa cells. In addition, CR enhanced adhesion of JAr cells onto Ishikawa cells by inducing the expression of LIF-dependent integrins αVß3 and αVß5. Furthermore, CR improved the number of implantation sites in pregnant mice despite RU injection. CONCLUSION: CR increased the expression of LIF-mediated integrins αVß3 and αVß5 on the surface of endometrial cells, which is associated with adhesion of trophoblastic cells to endometrial cells for blastocyst implantation. Our findings provide evidence that CR has therapeutic potential against poor endometrial receptivity.

Inhibition of lung cancer growth by HangAmDan-B is mediated by macrophage activation to M1 subtype.

Re-education of tumor-associated macrophages (TAMs) toward antitumor effectors may be a promising therapeutic strategy for the successful treatment of cancer. HangAmDan-B (HAD-B), a herbal formula, has been used for stimulating immune function and activation of vital energy to cancer patients in traditional Korean Medicine. Previous studies have reported the anti-angiogenic and anti-metastatic effects of HAD-B; however, evidence on the immunomodulatory action of HAD-B was not demonstrated. In the present study, immunocompetent mice were used to demonstrate the suppression of the in vivo growth of allograft Lewis lung carcinoma (LLC) cells, by HAD-B. In addition, HAD-B inhibited the in vitro growth of LLC cells by driving macrophages toward M1 polarization, but not through direct inhibition of tumor cell growth. Furthermore, culture media transfer of HAD-B-treated macrophages induced apoptosis of LLC cells. Results of the present study suggest that the antitumor effect of HAD-B may be explained by stimulating the antitumor function of macrophages. Considering the importance of re-educating TAMs in the regulation of the tumor microenvironment, the present study may confer another option for anti-cancer therapeutic strategy, using herbal medicines such as HAD-B.

Liqustri lucidi Fructus inhibits hepatic injury and functions as an antioxidant by activation of AMP-activated protein kinase in vivo and in vitro.

Medicinal herbs are used to treat or prevent various diseases, and function to regulate protective mechanisms as nutraceuticals. Fructus Ligustri lucidi is the fruit of Ligustrum lucidum and has been used for its tonic effects on the liver. This study was designed to examine the effects of Fructus Ligustri lucidi water extract (FLL) against severe oxidative stress and mitochondrial impairment in vivo and in vitro and to elucidate its cellular mechanisms of action. Treatment of HepG2 cells with arachidonic acid (AA) + iron successfully induced oxidative stress and apoptosis, as indicated by depletion of glutathione, formation of ROS, decreses in mitochondrial membrane potential (Δψm), and altered expression of apoptosis-related proteins, such as procaspase-3 and Bcl-xL. FLL treatment significantly blocked these pathological changes and the mitochondrial dysfunction caused by AA + iron, which were similar with the effect of aminoimidazole-carboxamide-ß-d-ribofuranoside (AICAR). Moreover, FLL induced the activation of AMP-activated protein kinase (AMPK), which was mediated by its upstream kinase LKB1. Inhibition or activation of AMPK revealed the role of AMPK in cellular protection conferred by FLL in LKB1-deficient cells. In mice, oral administration of 100 mg/kg FLL activated AMPK in the liver, and protected against oxidative stress and liver injury induced by CCl4 injection. Among the components of FLL, chlorogenic acid was found to be responsible for the protection of hepatocytes against AA + iron-induced cellular damage. Overall, our results confirmed that FLL has the ability to protect hepatocytes against oxidative injury through regulation of the AMPK signaling pathway.

Myristica fragrans Suppresses Tumor Growth and Metabolism by Inhibiting Lactate Dehydrogenase A.

Most cancer cells predominantly produce ATP by maintaining a high rate of lactate fermentation, rather than by maintaining a comparatively low rate of tricarboxylic acid cycle, i.e., Warburg's effect. In the pathway, the pyruvate produced by glycolysis is converted to lactic acid by lactate dehydrogenase (LDH). Here, we demonstrated that water extracts from the seeds of Myristica fragrans Houtt. (MF) inhibit the in vitro enzymatic activity of LDH. MF effectively suppressed cell growth and the overall Warburg effect in HT29 human colon cancer cells. Although the expression of LDH-A was not changed by MF, both lactate production and LDH activity were decreased in MF-treated cells under both normoxic and hypoxic conditions. In addition, intracellular ATP levels were also decreased by MF treatment, and the uptake of glucose was also reduced by MF treatment. Furthermore, the experiment on tumor growth in the in vivo mice model revealed that MF effectively reduced the growth of allotransplanted Lewis lung carcinoma cells. Taken together, these results suggest that MF effectively inhibits cancer growth and metabolism by inhibiting the activity of LDH, a major enzyme responsible for regulating cancer metabolism. These results implicate MF as a potential candidate for development into a novel drug against cancer through inhibition of LDH activity.

KOTMIN13, a Korean herbal medicine alleviates allergic inflammation in vivo and in vitro.

BACKGROUND: The ethanol extract of KOTMIN13, composed of Inula japonica Flowers, Trichosanthes kirilowii Semen, Peucedanum praeruptorum Radix, and Allium macrostemon Bulbs, was investigated for its anti-asthmatic and anti-allergic activities. METHODS: The anti-asthmatic effects of KOTMIN13 were evaluated on ovalbumin (OVA)-induced murine asthma model. Anti-allergic properties of KOTMIN13 in bone-marrow derived mast cells (BMMC) and passive cutaneous anaphylaxis (PCA) in vivo were also examined. RESULTS: In asthma model, KOTMIN13 effectively suppressed airway hyperresponsiveness induced by aerosolized methacholine when compared to the levels of OVA-induced mice. KOTMIN13 treatment reduced the total leukocytes, eosinophil percentage, and Th2 cytokines in the bronchoalveolar lavage fluids in OVA-induced mice. The increased levels of eotaxin and Th2 cytokines in the lung as well as serum IgE were decreased by KOTMIN13. The histological analysis shows that the increased inflammatory cell infiltration and mucus secretion were also reduced. In addition, the degranulation and leukotriene C4 production were inhibited in BMMC with IC50 values of 3.9 µg/ml and 1.7 µg/ml, respectively. Furthermore, KOTMIN13 treatment attenuated mast-mediated PCA reaction. CONCLUSIONS: These results demonstrate that KOTMIN13 has anti-asthmatic and anti-allergic effects in vivo and in vitro models.

Britanin attenuates ovalbumin-induced airway inflammation in a murine asthma model.

We previously demonstrated the alleviation of ovalbumin (OVA)-induced airway inflammation by Inulae flos. In the present study, the effects of britanin, a sesquiterpene compound isolated from Inulae flos, were evaluated in an in vivo animal model for anti-asthma activity through observation of airway hyperresponsiveness (AHR), eosinophil recruitment, Th2 cytokine and IgE levels, and lung histopathology. Britanin administration effectively reduced AHR induced by aerosolized methacholine, airway eosinophilia, Th2 cytokines in bronchoalveolar lavage fluids and the supernatant of cultured splenocytes compared with OVA-induced mice. Histological studies showed that increased inflammatory cell infiltration and mucus secretion were reduced by britanin administration. Thus, britanin may have therapeutic potential for treating allergic asthma.

Paeonia lactiflora Enhances the Adhesion of Trophoblast to the Endometrium via Induction of Leukemia Inhibitory Factor Expression.

In the present study, we investigated the role of Paeonia lactiflora Pall. extract on embryo implantation in vitro and in vivo. A polysaccharides depleted-water extract of P. lactiflora (PL-PP) increased LIF expression in human endometrial Ishikawa cells at non-cytotoxic doses. PL-PP significantly increased the adhesion of the human trophectoderm-derived JAr spheroids to endometrial Ishikawa cells. PL-PP-induced LIF expression was decreased in the presence of a p38 kinase inhibitor SB203580 and an MEK/ERK inhibitor U0126. Furthermore, endometrial LIF knockdown by shRNA reduced the expression of integrins ß3 and ß5 and adhesion of JAr spheroids to Ishikawa cells. In vivo administration of PL-PP restored the implantation of mouse blastocysts in a mifepristone-induced implantation failure mice model. Our results demonstrate that PL-PP increases LIF expression via the p38 and MEK/ERK pathways and favors trophoblast adhesion to endometrial cells.

Hepatoprotective effect of licorice, the root of Glycyrrhiza uralensis Fischer, in alcohol-induced fatty liver disease.

BACKGROUND: Our previous study suggested that licorice has anti-inflammatory activity in lipopolysaccharide-stimulated microglial cells and anti-oxidative activity in tert-butyl hydroperoxide-induced oxidative liver damage. In this study, we evaluated the effect of licorice on chronic alcohol-induced fatty liver injury mediated by inflammation and oxidative stress. METHODS: Raw licorice was extracted, and quantitative and qualitative analysis of its components was performed by using LC-MS/MS. Mice were fed a liquid alcohol diet with or without licorice for 4 weeks. RESULTS: We have standardized 70% fermented ethanol extracted licorice and confirmed by LC-MS/MS as glycyrrhizic acid (GA), 15.77 ± 0.34 µg/mg; liquiritin (LQ), 14.55 ± 0.42 µg/mg; and liquiritigenin (LG), 1.34 ± 0.02 µg/mg, respectively. Alcohol consumption increased serum alanine aminotransferase and aspartate aminotransferase activities and the levels of triglycerides and tumor necrosis factor (TNF)-α. Lipid accumulation in the liver was also markedly induced, whereas the glutathione level was reduced. All these alcohol-induced changes were effectively inhibited by licorice treatment. In particular, the hepatic glutathione level was restored and alcohol-induced TNF-α production was significantly inhibited by licorice. CONCLUSION: Taken together, our data suggests that protective effect of licorice against alcohol-induced liver injury may be attributed to its anti-inflammatory activity and enhancement of antioxidant defense.

A novel mechanism for the antibacterial effect of silver nanoparticles on Escherichia coli.

Silver nanoparticles are known to have antimicrobial properties and have been used extensively in medicine, although the mechanism(s) of action have not yet been clearly established. In the present study, the findings suggest a novel mechanism for the antibacterial effect of silver nanoparticles on Escherichia coli, namely, the induction of a bacterial apoptosis-like response. We propose a possible mechanism for the bacterial apoptosis-like response that includes the following: accumulation of reactive oxygen species (ROS) (detected with H2DCFDA staining), increased intracellular calcium levels (detected with Fura-2 AM), phosphatidylserine exposure in the outer membrane (detected with Annexin V) which is the hallmarks of early apoptosis, disruption of the membrane potential [detected with DiBAC4(3)], activation of a bacterial caspase-like protein (detected by FITC-VAD-FMK staining) and DNA degradation (detected with TUNEL assay) which is the hallmarks of late apoptosis in bacterial cells treated with silver nanoparticles. We also performed RecA expression assay with western blotting and observed activation of SOS response to repair the damaged DNA. To summarize, silver nanoparticles are involved in the apoptosis-like response in E. coli and the novel mechanisms which were identified in this study, suggest that silver nanoparticles may be an effective antimicrobial agent with far lower propensity for inducing microbial resistance than antibiotics.

Anti-allergic effect of a Korean traditional medicine, Biyeom-Tang on mast cells and allergic rhinitis.

BACKGROUND: Biyeom-Tang, a medicine prescribed by oriental clinics, has been used for the treatment of the allergic rhinitis (AR). In the present study, an ethanol extract of Biyeom-Tang (EBT) was investigated for anti-allergic properties on bone-marrow derived mast cells (BMMC) and in vivo models. METHODS: The anti-allergic properties of EBT were evaluated by measuring ß-Hex release and the production of prostaglandin D2 (PGD2) and leukotriene C4 (LTC4) on BMMC in vitro and PCA and OVA-induced AR models in vivo. RESULTS: EBT strongly inhibited a degranulation reaction in a dose dependent manner with an IC50 value of 35.6 µg/ml. In addition, the generation of PGD2 and LTC4 was inhibited in BMMC in a concentration-dependent manner with IC50 values of 7.0 µg/ml and 10.9 µg/ml, respectively. When administrated orally, EBT ameliorated the mast cell-mediated PCA reaction. In the OVA-induced AR model, the increased levels of IgE were reduced by EBT. The levels of cytokines, such as IL-4, IL-5, IL-10, and IL-13 decreased in the splenocytes of EBT-treated mice. The histological analysis shows that the infiltration of inflammatory cells increased by OVA-sensitization was also reduced. CONCLUSIONS: Taken together, these results suggested that EBT has anti-allergic and anti-inflammatory effects in vitro and in vivo models.

Inhibitory Effect of Nelumbo nucifera (Gaertn.) on the Development of Atopic Dermatitis-Like Skin Lesions in NC/Nga Mice.

Atopic dermatitis (AD) is a chronic inflammatory skin disease which has a complex etiology that encompasses immunologic responses. The study was carried out to examine the effect of Nelumbo nucifera (Gaertn.) leaf (NL) on the AD-like skin lesion induced by repeated epicutaneous application of 2,4-dinitrochlorobenzene (DNCB) on the dorsal skin of NC/Nga mice. Three different doses of NL (5, 25, and 50 mg/mice/day) were administered orally from the day of sensitization with DNCB for 4 weeks. The efficacy of NL was judged by histopathological examination, blood IgE level, measurement of transepidermal water loss (TEWL), scratching behavior, and skin severity score. NL resulted in the suppression of clinical severity score, TEWL, scratching behavior, and blood IgE level. Histopathologic analyses revealed that thickening of the epidermis and mast cell degranulation was significantly reduced in NL group. These results suggest that NL may be a useful natural resource for the management of AD.

Alleviation of OVA-induced airway inflammation by flowers of Inula japonica in a murine model of asthma.

The flowers of Inula japonica (Inulae Flos) have long been used in traditional medicine for treating inflammatory diseases. The effects on OVA-induced asthmatic mice of an Inulae Flos extract (IFE) were evaluated in this study. The anti-asthmatic effects of IFE were determined by observing eosinophil recruitment, airway hyper-responsiveness (AHR), Th2 cytokine and IgE levels, and lung histopathology. The IFE treatment effectively reduced the percentage of eosinophils and Th2 cytokines in the bronchoalveolar lavage fluid (BALF) when compared to the levels in OVA-induced mice. IFE also suppressed AHR induced by aerosolized methacholine in OVA-induced mice. The results of the histopathological studies indicate that inflammatory cell infiltration and mucus hypersecretion were both inhibited by the IFE administration when compared to the effect on OVA-induced mice. The IFE treatment also suppressed the serum IgE levels and decreased Th2 cytokines in the supernatant of cultured splenocytes. These results suggest that IFE may have therapeutic potential against asthma.

Effect of different ingredients in traditional Korean medicine for human uterine leiomyoma on normal myometrial and leiomyomal smooth muscle cell proliferation.

Crude water extracts of 13 traditional Korean medicinal ingredients used for leiomyomal treatment were prepared and used to treat human uterine normal myometrial and leiomyomal cell cultures. All the ingredients inhibited proliferation and altered the morphology of both myometrial and leiomyomal cells. Among the 13 ingredients, n-hexane-, chloroform-, and ethylacetate-soluble fractions were extracted from seven ingredients that potently inhibited cell proliferation in their water extract form. Among these, the ethylacetate-fraction of Phlomis umbrosa and Spatholobus suberectus, and the chloroform-fraction of Curcuma zedoaria and S. suberectus inhibited leiomyomal cell proliferation significantly compared to myometrial cell proliferation. Similarly, immunohistochemical analysis showed the inhibition of transforming growth factor-beta receptor 2 in leiomyomal tissue after treatment with the fractions of the ingredients. Moreover, the chloroform-fraction of C. zedoaria was subfractionated by open column chromatography. Two of the eight subfractions (fractions 6 and 7) potently inhibited cell proliferation in leiomyoma compared to myometrium. Further study will be performed with the goal of isolating specific compounds from two effective subfractions of C. zedoaria, ethylacetate-fraction of P. umbrosa, and the ethylacetate and chloroform-fractions of S. suberectus. The present study may be helpful in developing an alternative remedy to leiomyoma with minimal side-effects compared to the current treatments.

Deoxypodophyllotoxin (DPT) inhibits eosinophil recruitment into the airway and Th2 cytokine expression in an OVA-induced lung inflammation.

The effect of deoxypodophyllotoxin (DPT) isolated from Anthriscus sylvestris Hoffm. was evaluated in an IN VIVO animal model for antiasthmatic activity. DPT (1.0 to 5 mg/kg) was given orally to ovalbumin (OVA)/alum-induced asthmatic mice. DPT reduced the number of infiltrated eosinophils in bronchoalveolar lavage (BAL) fluid in a dose-dependent manner. Dexamethasone (5 mg/kg), which was used as a positive control, also strongly inhibited the number of infiltrated eosinophils. The effect of DPT on a transcript profile in a murine asthma model was determined by RT-PCR, which showed that DPT decreased the mRNA levels of the Th2 cytokines. Northern blot analysis showed that DPT also reduced both the eotaxin and arginase I mRNA levels in a dose-dependent manner.