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Aucklandia lappa Decne., a traditional herbal medicine, has been used to treat various diseases, including asthma, coughs, rheumatism, and diarrhea. In the present study, the effects of the oral administration of Aucklandia lappa Decne. extract on hair growth was investigated in hair-removed mice. A 70% ethanol extract of Aucklandia lappa Decne. (ALD) was prepared, and the extraction yield and total polyphenol content of ALD were measured as 27.30±0.01 and 28.39 mg gallic acid equivalent (GAE)/g, respectively. The oral administration of ALD to hair-removed CL57BL/6 mice for six weeks had no significant effects on food intake and body weight changes. Biochemical and histological examinations also showed that the oral administration of ALD for six weeks had no significant effect on the liver and kidney functions. On the other hand, hair growth was significantly higher in the ALD group than the control group and the Pancidil group (positive control). In addition, the number of hair follicles and the degree of collagen production in the dermis were significantly higher in the ALD group than in the control and pancidil groups. These results suggest that ALD is a potential source of nutricosmetics with hair growth-promoting effects.

Background We investigated the effects of tranilast on epithelial-to-mesenchymal transition (EMT) in an animal model and on the EMT signaling pathway in human peritoneal mesothelial cells (HPMCs). Methods We performed in vitro studies (cytotoxicity, cell morphology, and western blot analyses) on HPMCs from human omenta, along with in vivo studies (peritoneal membrane function and morphometric and immunohistochemical analyses) on Sprague Dawley rats. Thirty-two rats were divided into three groups: control (C) group (peritoneal dialysis [PD] catheter but not infused with dialysate), PD group (4.25% glucose-containing dialysate), and PD + tranilast group (4.25% glucose-containing dialysate along with tranilast). Results In in vitro experiments, transforming growth factor-beta 1 (TGF-β1) increased α-smooth muscle actin and Snail expression and reduced E-cadherin expression in HPMCs. TGF-β1 also reduced cell contact, induced a fibroblastoid morphology, and increased phosphorylation of Akt, Smad2, and Smad3 in HPMCs. Tranilast significantly inhibited TGF-β1-induced EMT and attenuated these morphological changes in HPMCs. In in vivo studies, after 6 weeks of experimental PD, the peritoneal membrane was significantly thicker in the PD group than in the C group. Tranilast protected against PD-induced glucose mass transfer change and histopathological changes in rats. Conclusion Tranilast prevented EMT both in HPMCs triggered with TGF-β1 and in rats with PD-induced peritoneal fibrosis. Thus, tranilast may be considered a therapeutic intervention that enables long-term PD by regulating TGF-β1 signaling pathways.

Angiogenesis should be precisely regulated because disordered neovascularization is involved in the aggravation of multiple diseases. The vascular endothelial growth factor (VEGF)-A/VEGF receptor 2 (VEGFR-2) axis is crucial for controlling angiogenic responses in vascular endothelial cells (ECs). Therefore, inactivating VEGFR-2 signaling may effectively suppress aberrant angiogenesis and alleviate related symptoms. In this study, we performed virtual screening, identified the synthetic disaccharide 6′-sialylgalactose (6SG) as a potent VEGFR-2-binding compound and verified its high binding affinity by Biacore assay. 6SG effectively suppressed VEGF-A-induced VEGFR-2 phosphorylation and subsequent in vitro angiogenesis in HUVECs without inducing cytotoxicity. 6SG also inhibited VEGF-A-induced extracellular-regulated kinase (ERK)/Akt activation and actin stress fiber formation in HUVECs. We demonstrated that 6SG inhibited retinal angiogenesis in a mouse model of retinopathy of prematurity and tumor angiogenesis in a xenograft mouse model. Our results suggest a potential therapeutic benefit of 6SG in inhibiting angiogenesis in proangiogenic diseases, such as retinopathy and cancer., Abnormal blood vessel formation: Synthetic disaccharide shows therapeutic promise Therapy based on a synthetic molecule can block abnormal blood vessel formation, limiting the progression of diabetic eye conditions and tumor growth in mice. The growth of new blood vessels from existing vessels, called angiogenesis, is critical to wound healing and embryonic development. The main angiogenesis signalling pathway involves growth factors, including one called VEGFR-2. Disruption to this pathway plays a significant part in the development of multiple diseases. A South Korean team led by Ki-Tae Ha at Pusan National University, Yangsan, and Young Jun Koh at Dongguk University, Seoul, identified and trialed a synthetic disaccharide capable of binding to and limiting the activity of VEGFR-2 during faulty signaling. Trials on mice with the diabetic eye condition retinopathy, and mice with implanted tumors, showed that the compound inhibited excessive angiogenesis and limited disease progression.

In recent years, a rapid increase in microbes that are resistant to conventional antibiotics has been observed (Goffeau, 2008). Especially, the frequency of infections provoked by opportunistic fungal strains has increased dramatically. Even though the majority of invasive fungal infections are still due to the Aspergillus or Candida species, the spectrum of fungal pathogens has changed and diversified (Denning, 1991; Ellis et al., 2000; Odds et al., 2003). Azoles that inhibit sterol formation and polyenes that bind to mature membrane sterols have been the mainstays regarding antifungal therapy for several decades (Kullberg & de Pauw, 1999; Sheehan et al., 1999). However, not only the emergence of fluconazole resistance among different pathogenic strains but also the high toxicity of amphotericin B (Alexander & Perfect, 1997; Mukherjee et al., 2003) has prompted research on new antifungal agents (Kontoyiannis et al., 2003). Bio-nanotechnology has emerged as an integration between biotechnology and nanotechnology for developing biosynthesis and environmental-friendly technology for synthesis of nanomaterials. We specifically regarded nanoparticles as clusters of atoms in the size of 1-100 nm. ‘Nano’ is a Greek word synonymous to dwarf meaning extremely small. The use of nanoparticles is gaining impetus in the present century as they posses defined chemical, optical and mechanical properties. Among them, the metallic nanoparticles are most promising as they contain remarkable antibacterial properties due to their large surface area to volume ratio, which is of interest to researchers due to the growing microbial resistance against metal ions, antibiotics, and the development of resistant strains (Rai et al., 2009; Gong et al., 2007). Different types of nanomaterials like copper, zinc, titanium (Schabes-Retchkiman et al., 2006), magnesium, gold (Gu et al., 2003), alginate (Ahmad et al., 2005) and silver have been developed but silver nanoparticles (Nano-Ag) have proved to be most effective as they exhibit potent antimicrobial efficacy against bacteria, viruses and eukaryotic micro-organisms. However, Nano-Ag used as a disinfectant drug also has some risks as the exposure to silver can cause argyrosis or argyria; it can be toxic to mammalian cells (Gong et al., 2007). 15

Fermented barley bran (FBB) using Bacillus spp. is the main ingredient of sigumjang, a traditional fermented food in South Korea. The aim of this study was to evaluate the gastro-protective effects of β-glucan-enriched FBB extracts in an experimental mouse model of the EtOH-induced gastric ulcer. The gastro-protective activity of FBB was determined using histopathological inspection, level of EtOH absorption from the gastrointestinal-tract and measurement of pro-inflammatory cytokine. Pretreatment of FBB showed less gastrointestinal bleeding and reduced EtOH absorption into the bloodstream with the gastric mucosa. Furthermore, FBB effectively reduced the EtOH-induced mRNA expression of tumor necrosis factor α in the gastric mucosa and its level in the blood. These results showed that FBB can prevent gastric mucosal damage induced by acute EtOH-administration in mice by reducing absorption of EtOH by gastric mucosa along with decreasing the inflammatory response. Altogether, this data provides useful information on how β-glucan-enriched food modulates the EtOH-induced gastric mucosal damage, and thus, it could lead to the development of formulations for protecting gastric mucosal injury with acute alcohol intake.

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.

Despite advances in assisted reproductive technology, treatment for deficient endometrial receptivity is a major clinical unmet need. In our previous study, the water extract of Paeonia lactiflora Pall. enhanced endometrial receptivity in vitro and in vivo via induction of leukemia inhibitory factor (LIF), an interleukin (IL)-6 family cytokine. In the present study, we found that paeoniflorin, a monoterpene glycoside, is the major active compound of P. lactiflora. Paeoniflorin significantly improved the embryo implantation rate in a murine model of mifepristone (RU486)-induced implantation failure. In addition, paeoniflorin increased the adhesion of human trophectoderm-derived JAr cells to endometrial Ishikawa cells through the expression of LIF in vitro. Moreover, using the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database of the human endometrium, we confirmed that LIF signaling is a key regulator for improving human endometrial receptivity. Therefore, these results suggest that paeoniflorin might be a potent drug candidate for the treatment of endometrial implantation failure by enhancing endometrial receptivity.

Mealworm and mealworm oil (MWO) have been reported to affect antioxidant, anti-coagulation, anti-adipogenic and anti-inflammatory activities. However, the function of MWO in wound healing is still unclear. In this study, we found that MWO induced the migration of fibroblast cells and mRNA expressions of wound healing factors such as alpha-smooth muscle actin (α-SMA), collagen-1 (COL-1) and vascular endothelial growth factor (VEGF) in fibroblast cells. The tube formation and migration of endothelial cells were promoted through the activation of VEGF/VEGF receptor-2 (VEGFR-2)-mediated downstream signals including AKT, extracellular signal-regulated kinase (ERK) and p38 by MWO-stimulated fibroblasts for angiogenesis. Moreover, we confirmed that MWO promoted skin wound repair by collagen synthesis, re-epithelialization and angiogenesis in an in vivo excisional wound model. These results demonstrate that MWO might have potential as a therapeutic agent for the treatment of skin wounds.

Ambient temperature can regulate the immune response and affect tumor growth. Although thermoneutral caging reduces tumor growth via immune activation, little attention has been paid to the tumorigenic effect of low temperature. In the present study, tumor growth was higher at low ambient temperature (4 &deg, C for 8 h/d) than at the standard housing temperature (22 &deg, C) in allograft models. Low temperature-stimulated tumor growth in mice was reduced by monocyte depletion using clodronate liposomes. Proliferation was considerably greater in cancer cells treated with 33 &deg, C-cultured RAW264.7 cell-conditioned media (33CM) than in cells treated with 37 &deg, C-cultured RAW264.7 cell-conditioned media (37CM). Additionally, glutamine levels were markedly higher in 33CM-treated cells than in 37CM-treated cells. We further confirmed that the addition of glutamine into 37CM enhanced its effects on cancer cell proliferation and glutamine uptake inhibition ameliorated the accelerated proliferation induced by 33CM. Consistently, the inhibition of glutamine uptake in the allograft model exposed to low temperature, effectively reduced tumor volume and weight. Collectively, these data suggest that the secretion and utilization of glutamine by macrophages and cancer cells, respectively, are key regulators of low temperature-enhanced cancer progression in the tumor microenvironment.

BackgroundTo characterize molecular mechanism underlying the regulation of sialylated glycan expression and its roles for endometrial receptivity and embryo implantation. Here, we characterized the role of a truncated form of sialylated O-glycan, sialyl-Tn, for endometrial receptivity.MethodsThe transcriptomes of human endometrium at mid-secretory phase were analyzed by Bioinformatics. Changes in gene expression, protein, and signal pathway were measured using RT-PCR and Western blot. The cell adhesion assay was visualized using a fluorescent microscope. In peri-implantation phase of mice, the expression of leukemia inhibitory factor (LIF) and sialyl-Tn were confirmed using immunohistochemistry and immnofluorescence analysis. The effect of sialy-Tn expression on embryo implantation was estimated by in vitro fertilization and embryo transfer using mice.ResultsIn in silico analysis, expression of O-glycosylation genes, especially ST6GalNAc1, was significantly increased in the human uterus of mid-secretory phase. Overexpression of the ST6GalNAc1 gene in non-receptive human endometrial AN3CA cells enhances the attachment of trophoblastic JAr cells. In an animal study, the results clearly indicated that sialyl-Tn was expressed on the surface of the mid-secretory uterus. In addition, blockade of the receptor using free sialyl-Tn epitope diminished the implantation rates of intrauterine transferred murine embryos.ConclusionFrom these results, here we suggest that sialyl-Tn expression might be a novel factor regulating the endometrial receptivity for successful embryo implantation.

본 연구는 전라도 장성지역의 편백나무 잎 추출물을 사용하여 in vitro에서 항염증 및 항알레르기 효과를 보고자 연구를 진행하였다. 편백나무 잎 추출물은 50°C에서 감압 건조하여 실험에 사용하였으며, 총 폴리페놀 함량을 측정한 결과 25.89±0.31mg GAE/g로 나타났다. 설정된 GC-MS 분석법으로 편백나무잎 추출물의 6종 성분에 대한 함량 분석을 실시한 결과, α-Terpinene 3.03 mg/g, α-Terpineol 9.48 mg/g, limonene 5.96mg/g, borneol 59.78 mg/g, myrcene 4.85 mg/g, sabinene 11.31 mg/g로 borneol이 가장 많은 것으로 나타났다. 편백나무 잎 추출물의 항산화 활성을 측정한 결과, H2O2와 ABTS+ 라디칼에 대한 추출물의 RC50이 각각 5.47±0.13 mg/mL와 4.00±0.01 mg/mL로 나타났다. 또한 마우스 유래의 대식세포 주인 RAW 264.7 세포에서 LPS 100 ng/mL을 처리를 통한 염증유도 군에서 주요 인자인 NO 생성이 28±0.38 μM까지 증가하였으나 편백나무 잎 추출물 150 μg/mL 처리 농도에서 IC50 으로 감소할 것으로 추정되므로, 편백나무 잎 추출물이 항염증 작용이 있음을 시사한다. 알레르기 주요 인자인 β-hexosaminidase의 경우 처리한 편백나무 잎 추출물의 농도 의존적으로 감소되어 항알레르기 효능이 있음을 알 수 있었으며, 인간 유래 섬유아세포인 CCD-986sk 세포에 대해 편백나무 잎 추출물의 농도 100∼800 μg/mL 범위에서 세포독성을 전혀 보이지 않았다. 따라서 편백나무 잎 추출물이 향후 기능성 화장품, 연고 등의 산업화에 광범위하게 이용될 수 있을 것으로 사료된다.

본 연구는 포도수확 후 버려지는 가지를 이용한 포도전정가지 추출물(grape pruning stem extracts, GPSE)에 함유된 polyphenol류가 UVB로 유도된 HR-1 hairless mice의 손상된 피부에 대한 피부증식 억제, 항 알레르기, 아포토시스 억제능을 분석하여 기능성 화장품 소재로서의 응용 가능성을 확인하고자 하였다. 포도전정가지에서 polyphenol 성분을 80% 에탄올로 추출하고 여과하여 농축한 후, 동결건조하여 -20°C에 보관하면서 사용하였다. GPSE의 유효성분 함량은 HPLC를 사용하여 선행연구논문에 보고하였다. 피부손상을 유도하기 위해 UVB를 HR-1 hairless mice에 조사하였고, GPSE가 피부 알레르기 반응을 일으키는지 확인하기 위하여 toluidine blue 염색을 통하여 비만세포의 수를 확인한 결과 포도전정가지 추출물(GPSE)를 도포한 그룹에서는 정상군과 유사한 결과를 나타내어 GPSE 도포군은 알레르기 반응이 없으며 suncream 대조군대비 현저하게 감소하는 것을 확인할 수 있었다. 표피의 증식표지자인 PCNA 면역조직화학적 염색을 시행한 결과 GPSE와 serum base 혼합물의 모든 농도에서 대조군 대비 평균 42%로 표피상피세포 증식 억제 효과를 나타내었다. 또한 GPSE와 serum base 혼합물이 자외선 조사로 인한 아포토시스 억제율은 대조군 대비 평균 27% 억제 한다는 것을 알 수 있었다. 이상의 결과로 포도전정가지로부터 추출한 페놀성 화합물이 화장품기초 원료인 serum base에 함유토록 제조된 GPSE는 피부증식억제, 알레르기 억제, 아포토시스 억제효과를 얻을 수 있었다. 본 연구의 의의는 지역포도농가에서 수확 후 버려지는 포도전정가지에서 폴리페놀류와 같은 유효성분을 확보하여 고부가가치를 창출할 수 있는 기능성화장품 원료로서의 활용 가치가 높을 것으로 기대된다.

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.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.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.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 αVβ3 and αVβ5. Furthermore, CR improved the number of implantation sites in pregnant mice despite RU injection.CR increased the expression of LIF-mediated integrins αVβ3 and αVβ5 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.

// Tae-Wook Chung 1 , Eun-Young Kim 1, 2 , Seok-Jo Kim 3 , Hee-Jung Choi 1 , Se Bok Jang 4 , Keuk-Jun Kim 5 , Sun-Hyung Ha 3 , Fukushi Abekura 3 , Choong-Hwan Kwak 3 , Cheorl-Ho Kim 3 and Ki-Tae Ha 1, 2 1 School of Korean Medicine, Healthy Aging Korean Medical Research Center, Pusan National University, Yangsan, Gyeongsangnam-do 50612, Republic of Korea 2 Graduate Training Program of Korean Medicine for Healthy-Aging, Pusan National University, Yangsan, Gyeongsangnam-do 50612, Republic of Korea 3 Department of Biological Science, Sungkyunkwan University, Suwon, Kyunggi-do 16419, Republic of Korea 4 Department of Molecular Biology, College of Natural Sciences, Pusan National University, Geumjeong-gu, Busan 46241, Republic of Korea 5 Department of Clinical Pathology, TaeKyeung University, Gyeongsan 38547, Republic of Korea Correspondence to: Cheorl-Ho Kim, email: chkimbio@skku.edu Ki-Tae Ha, email: hagis@pusan.ac.kr Keywords: sialyllactose, milk, oligosaccharides, angiogenesis, VEGF receptor-2 Received: May 31, 2016 Accepted: February 15, 2017 Published: March 14, 2017 ABSTRACT The oligosaccharides in human milk have various biological functions. However, the molecular mechanism(s) underlying the anti-angiogenic action of sialylated human milk oligosaccharides (HMOs) are still unclear. Here, we show that siallylactose (SL) found in human milk can inhibit the activation of vascular endothelial growth factor (VEGF)-mediated VEGF receptor-2 (VEGFR-2) by binding to its VEGF binding site (second and third IgG-like domains), thus blocking downstream signal activation. SL also inhibits growth of VEGF-stimulated endothelial cells. In endothelial cells treated with VEGF, SL diminished tube formation, migration, and the arrangement of actin filament. In addition, SL clearly suppressed VEGF-induced neovascularization in an in vivo Matrigel plug assay. Notably, SL prevented the growth of tumor cells, and angiogenesis on tumor tissues in in vivo mice models allotransplanted with Lewis lung carcinoma, melanoma, and colon carcinoma cells. Taken together, we have demonstrated that the sialylated milk oligosaccharide sialyllactose functions as an inhibitor of angiogenesis through suppression of VEGF-mediated VEGFR-2 activation in endothelial cells, Accordingly, it could be a novel candidate for the development of anti-angiogenic drugs without any side effects.

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.

The antifungal activity of polyhexamethylene guanidine hydrochloride (PHMGH) was studied against various pathogenic fungi. PHMGH had more potent antifungal activity than amphotericin B, which is a commonly used antifungal drug, and also showed no hemolytic and lactate dehydrogenase release activities in the range of 1.25–40.0 μg mL−1. PHMGH is a cationic polymer containing an amino group and a polymeric guanidine group. Based on its characteristics such as the cationic charge and hydrophobicity, the antifungal mechanism of PHMGH was investigated using Candida albicans, as a model organism. Flow cytometric contour-plot analysis and microscopy showed changes in the size and granularity of the cells after treatment with PHMGH. A membrane study using 1,6-diphenyl-1,3,5-hexatriene labelling indicated a great loss of phospholipid area in the plasma membrane following PHMGH treatment. To investigate the extent of the damage, fluorescein isothiocyanate-labelled dextran leakage from large unilamellar vesicles was observed, indicating that PHMGH acts on the fungal membranes by inducing pore formation, with the majority of pore size being between 2.3 and 3.3 nm. This mechanism was confirmed with ion transition assays using 3,3′-dipropylthiacarbocyanine iodide and an ion-selective electrode meter, which indicated that membrane depolarization involving K+ leakage was induced. Taken together, these results show that PHMGH exerts its fungicidal effect by forming pores in the cell membrane.

// Tae-Wook Chung 1, 2, * , Hee-Jung Choi 1, 2, * , Mi-Ju Park 1 , Hee-Jin Choi 1, 2 , Syng-Ook Lee 3 , Keuk-Jun Kim 4 , Cheorl-Ho Kim 5 , Changwan Hong 6 , Kyun-Ha Kim 2 , Myungsoo Joo 1, 2 , Ki-Tae Ha 1, 2 1 Korean Medical Research Center for Healthy Aging and Yangsan, Gyeongsangnam-do, Republic of Korea 2 School of Korean Medicine, Pusan National University, Yangsan, Gyeongsangnam-do, Republic of Korea 3 Department of Food Science and Technology, Keimyung University, Daegu, Republic of Korea 4 Department of Clinical Pathology, TaeKyeung University, Gyeongsan, Gyeongsangbuk-do, Republic of Korea 5 Department of Biological Science, Sungkyunkwan University, Suwon, Kyunggi-do, Republic of Korea 6 Department of Anatomy, School of Medicine, Pusan National University, Yangsan, Gyeongsangnam-do, Republic of Korea * These authors contributed equally to this work Correspondence to: Ki-Tae Ha, email: hagis@pusan.ac.kr Keywords: ganglioside, tumor-associated macrophage, macrophage mannose receptor, monocyte chemoattractant protein-1, angiogenesis Received: March 15, 2016 Accepted: December 05, 2016 Published: December 10, 2016 ABSTRACT Tumor-derived gangliosides in the tumor microenvironment are involved in the malignant progression of cancer. However, the molecular mechanisms underlying the effects of gangliosides shed from tumors on macrophage phenotype remain unknown. Here, we showed that ganglioside GM1 highly induced the activity and expression of arginase-1 (Arg-1), a major M2 macrophage marker, compared to various gangliosides in bone marrow-derived macrophages (BMDM), peritoneal macrophages and Raw264.7 macrophage cells. We found that GM1 bound to macrophage mannose receptor (MMR/CD206) and common gamma chain (γc). In addition, GM1 increased Arg-1 expression through CD206 and γc-mediated activation of Janus kinase 3 (JAK3) and signal transducer and activator of transcription- 6 (STAT-6). Interestingly, GM1-stimulated macrophages secreted monocyte chemoattractant protein-1 (MCP-1/CCL2) through a CD206/γc/STAT6-mediated signaling pathway and induced angiogenesis. Moreover, the angiogenic effect of GM1-treated macrophages was diminished by RS102895, an MCP-1 receptor (CCR2) antagonist. From these results we suggest that tumor-shed ganglioside is a secretory factor regulating the phenotype of macrophages and consequently enhancing angiogenesis.

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, β3, and β5 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.

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, β3, and β5 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.

Alcohol consumption exacerbates alcoholic liver disease by attenuating the activity of AMP-activated protein kinase (AMPK). AMPK is activated by fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, and inhibited by direct interaction with cereblon (CRBN), a component of an E3 ubiquitin ligase complex. Based on these preliminary findings, we investigated that CRBN would be up-regulated in the liver by alcohol consumption and that CRBN deficiency would ameliorate hepatic steatosis and pro-inflammatory responses in alcohol-fed mice by increasing AMPK activity. Wild-type, CRBN and PPARα null mice were fed an alcohol-containing liquid diet and administered with fenofibrate. Gene expression profiles and metabolic changes were measured in the liver and blood of these mice. Expression of CRBN, cytochrome P450 2E1 (CYP2E1), lipogenic genes, pro-inflammatory cytokines, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were increased in the Lieber–DeCarli alcohol-challenged mice. Fenofibrate attenuated the induction of CRBN and reduced hepatic steatosis and pro-inflammatory markers in these mice. Ablation of the gene encoding CRBN produced the same effect as fenofibrate. The increase in CRBN gene expression by alcohol and the reduction of CRBN expression by fenofibrate were negated in PPARα null mice. Fenofibrate increased the recruitment of PPARα on CRBN gene promoter in WT mice but not in PPARα null mice. Silencing of AMPK prevented the beneficial effects of fenofibrate. These results demonstrate that activation of PPARα by fenofibrate alleviates alcohol-induced hepatic steatosis and inflammation by reducing the inhibition of AMPK by CRBN. CRBN is a potential therapeutic target for the alcoholic liver disease.

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. 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. 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. 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.

Embryo implantation is the crucial step for a successful pregnancy. Diverse factors, including adhesion molecules, growth factors, and cytokines are important for embryo implantation through improving endometrial receptivity. Benzoic acid (BA), a component of various plants, has been shown to have antifungal and antioxidant effects. However, the effect of BA on embryo implantation remains unknown. Here, we showed the contribution of BA for the enhancement of endometrial receptivity through the leukemia inhibitory factor (LIF)-dependent increase of integrin αV, β3, and β5 expression. Furthermore, in vivo study using a mifepristone-induced implantation failure model showed that BA definitely improves the numbers of implantation embryos. Taken together, we suggest that BA has a novel function for embryo implantation through the up-regulation of LIF-mediated integrins, and may be a candidate for therapeutic medicine to increase the pregnancy rate.

Background Several new biomarkers for the detection of early tubular injury have been investigated in kidney transplant recipients. We recently identified day 2 urinary neutrophil gelatinase-associated lipocalin (NGAL) as a predictor of slow graft function and adverse 1-year outcome. In the present study, we further investigated the value of urinary NGAL and liver-type fatty acid binding protein (L-FABP) for predicting long-term graft outcomes up to 2 years. Methods This study was a single-center, prospective observational study. Serial urinary NGAL and L-FABP levels at 0 hours, 2 days, and 6 days after kidney transplantation (KT) were measured, and the clinical data were assessed during the 2-year period after KT. Results During the 2-year follow-up period, 13 (18.8%), 5 (7.2%), and 4 (5.8%) patients were diagnosed with acute T-cell–mediated rejection, acute antibody-mediated rejection (AMR) and chronic AMR, respectively. In addition, 10 patients (14.3%) developed calcineurin inhibitor toxicity and 6 (8.7%) developed BK viremia. The mean estimated glomerular filtration rates (eGFR) at 1 and 2 years after KT were 65.1 ± 19.1 and 58.5 ± 22.6 mL/min/1.73 m2, respectively, When poor long-term graft function was defined as eGFR of less than 50 mL/min/1.73 m2 at 2 years, elderly donors, acute rejection, and high 0-hour urinary L-FABP levels were significant risk factors. Furthermore, in rejection-free patients, L-FABP was strongly associated with poor long-term graft function (P = .006). Multivariate logistic regression analysis showed that high 0-hour L-FABP (P = .015) and acute rejection (P = .006) were independent factors predicting poor long-term graft function. Receiver operating characteristic analysis showed that the area under the curve for urinary L-FABP was 0.692 (P = .036). Conclusions Our results suggest that urinary L-FABP may be a useful predictor of adverse long-term outcomes in KT patients.

Aerobic glycolysis is one of the important metabolic characteristics of many malignant tumors. Pyruvate dehydrogenase kinase (PDHK) plays a key role in aerobic glycolysis by phosphorylating the E1&alpha, subunit of pyruvate dehydrogenase (PDH). Hence, PDHK has been recognized as a molecular target for cancer treatment. Here, we report that huzhangoside A (Hu.A), a triterpenoid glycoside compound isolated from several plants of the Anemone genus, acts as a novel PDHK inhibitor. Hu.A was found to decrease the cell viability of human breast cancer MDA-MB-231, hepatocellular carcinoma Hep3B, colon cancer HT-29, DLD-1, and murine lewis lung carcinoma LLC cell lines. The activity of PDHK1 was decreased by Hu.A in both in vitro assays and in vivo assays in DLD-1 cells. Hu.A significantly increased the oxygen consumption and decreased the secretory lactate levels in DLD-1 cells. In addition, Hu.A interacted with the ATP-binding pocket of PDHK1 without affecting the interaction of PDHK1 and pyruvate dehydrogenase complex (PDC) subunits. Furthermore, Hu.A significantly induced mitochondrial reactive oxygen species (ROS) and depolarized the mitochondrial membrane potential in DLD-1 cells. Consistently, when Hu.A was intraperitoneally injected into LLC allograft mice, the tumor growth was significantly decreased. In conclusion, Hu.A suppressed the growth of tumors in both in vitro and in vivo models via inhibition of PDHK activity.

Sex hormones have long been considered to play an important role in bone turnover rate, periodontal diseases, and wound healing. We have studied the effect of porcine testis steroid extract (PTSE), an extract of porcine testes, which holds a good ratio of 19-nortestosterone (nandrolone), testosterone, androstenedione, 17β-estradiol, and estrone, on the healing rate of a standardized full-thickness linear wound on the back of the rat. Skin punch or carbon dioxide (CO2) laser methods were used to create the deep skin injury in two groups of animals. The animals were treated with the PTSE cream, control cream and Vaseline (control) to find out the effect in re-epithelialization, contraction, and formation of granulation and scar tissues. Histological examination after 21 days showed 100, 87.4, and 80.5% recovery of epidermis, dermis, and hypodermis, respectively in the PTSE-treated animals. Similarly, on the 15th day of treatment, complete healing of intact skin was observed in the PTSE cream-treate...

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-β-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.

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.

CD4+ T cells are essential in inflammation and autoimmune diseases. Interferon-γ (IFN-γ) secreting T helper (Th1) and IL-17 secreting T helper (Th17) cells are critical for several autoimmune diseases. To assess the inhibitory effect of a given compound on autoimmune disease, we screened many compounds with an in vitro Th differentiation assay. BJ-3105, a 6-alkoxypyridin-3-ol analog, inhibited IFN-γ and IL-17 production from polyclonal CD4+ T cells and ovalbumin (OVA)-specific CD4+ T cells which were activated by T cell receptor (TCR) engagement. BJ-3105 ameliorated the experimental autoimmune encephalomyelitis (EAE) model by reducing Th1 and Th17 generation. Notably, Th cell differentiation was significantly suppressed by BJ-3105 treatment without inhibiting in vitro proliferation of T cells or inducing programmed cell death. Mechanistically, BJ-3105 inhibited the phosphorylation of JAK and its downstream signal transducer and activator of transcription (STAT) that is critical for Th differentiation. These results demonstrated that BJ-3105 inhibits the phosphorylation of STAT in response to cytokine signals and subsequently suppressed the differentiation of Th cell responses.

Ambient cold temperature, as an abiotic stress, regulates the survival, stability, transmission, and infection of pathogens. However, the effect of cold temperature on the host receptivity to the pathogens has not been fully studied. In this study, the expression of terminal α-2,3- and α-2,6-sialic acids were increased in murine lung tissues, especially bronchial epithelium, by exposure to cold condition. The expression of several sialyltransferases were also increased by exposure to cold temperature. Furthermore, in human bronchial epithelial BEAS-2B cells, the expressions of α-2,3- and α-2,6-sialic acids, and mRNA levels of sialyltransferases were increased in the low temperature condition at 33 °C. On the other hand, the treatment of Lith-Gly, a sialyltransferase inhibitor, blocked the cold-induced expression of sialic acids on surface of BEAS-2B cells. The binding of influenza H1N1 hemagglutinin (HA) toward BEAS-2B cells cultured at low temperature condition was increased, compared to 37 °C. In contrast, the cold-increased HA binding was blocked by treatment of lithocholicglycine and sialyl-N-acetyl-D-lactosamines harboring α-2,3- and α-2,6-sialyl motive. These results suggest that the host receptivity to virus at cold temperature results from the expressions of α-2,3- and α-2,6-sialic acids through the regulation of sialyltransferase expression.

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.

List of murine primers used for real time RT-PCR. (DOCX 17Â kb)

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. Electronic supplementary material The online version of this article (doi:10.1186/s12906-016-0997-0) contains supplementary material, which is available to authorized users.