갈화 (Flos puerariae) 의 항당뇨 효과

학위논문 (석사) -- 서울대학교 대학원 : 수의과대학 수의학과, 2021. 2. 백승준.Pueraria lobata (Wild.) Ohwi. (P. lobata) flowers known as ‘Kudzu flower’ contain isoflavonoids and essential oil components. It has a wide range of biological and pharmacological activities, including protective effect in non-alcoholic fatty liver disease, hyperglycemia, and hypolipidemia, anti-mutagenic effects, and benefits for weight loss. However, the molecular mechanism of these effects remains unclear. Our study aimed to systematically examine the effects of Flos Puerariae crude extract (FPE) as an anti-diabetic agent using in vitro assays. The cytotoxicity of FPE was evaluated using MTS assay in L6 rat myocyte and 3T3-L1 murine fibroblast cell lines. PPARγ binding activity and adipogenesis were examined using dual-luciferase and differentiation assays, respectively. For investigating the anti-diabetic activity, glucose utilization, including GLUT4 protein expression, glucose uptake assay, and GLUT4 translocation using immunofluorescence microscopy were conducted in L6 cells. Furthermore, we assessed the anti-oxidant and anti-inflammatory activities of FPE. Our results demonstrated the ability to augment glucose uptake in L6 cells and enhance glucose utilization activity by increasing the expression of glucose transporter type 4 (GLUT4). In summary, our findings suggest that FPE may be a potential anti-diabetic substance for the treatment of diabetic patients and can prevent inflammatory or oxidation-related diseases.Pueraria lobata (Wild.) Ohwi. (P. lobata) '쿠즈 꽃'으로 알려진 꽃에는 이소플라보노이드와 필수 오일 성분이 함유되어 있다. 무알코올성 지방간 질환, 고혈당, 저혈당증 등에 대한 보호효과, 항돌연변이 효과, 체중 감소에 대한 혜택 등 생물학적·약리학적 활동 범위가 넓다. 그러나 이러한 효과의 분자 메커니즘은 여전히 불분명하다. 우리의 연구는 in vitro를 이용하여 FPE (Flos Puerariae 원유 추출물)가 항당뇨제로서 어떤 영향을 미치는지 체계적으로 검사하는 것을 목표로 했다. FPE의 세포독성은 L6 rat 근세포와 3T3-L1 murine pre-adipocytes 세포 라인의 MTS 검사를 사용하여 평가되었다. 각각 이중 루시페라제 및 분화측정법을 사용하여 PPAR* 바인딩 활성과 지방생식을 검사했다. 당뇨병 예방 활동을 조사하기 위해 L6 세포에서 GLUT4 단백질 발현, 포도당 흡수측정, 형광 현미경을 이용한 GLUT4 번역 등 포도당 활용을 실시했다. 또한, 우리는 FPE의 항산화 및 항염증 활동을 평가했다. 우리의 결과는 L6 세포에서 포도당 흡수를 증가시키고 Glucose transporter type 4 (GLUT4)의 발현을 증가시킴으로써 포도당 이용 활동을 향상시키는 능력을 보여주었다. 요약하자면, 본 연구결과는 FPE가 당뇨병 환자들의 치료를 위한 잠재적인 항당뇨 물질일 수 있고 염증이나 산화 관련 질병을 예방할 수 있다는 것을 시사한다.Chapter 1. Introduction 1 Chapter2. Materials and methods 7 2.1. Sample preparation 7 2.2. HPLC analysis of FPE 8 2.3. Cell culture 9 2.4. Cytotoxicity assay 9 2.5. Dual-luciferase assay 10 2.6. 3T3-L1 cell differentiation 11 2.7. Immunofluorescence analysis 12 2.8. Glucose uptake assay 13 2.9. Immunoblotting analysis 14 2.10. Nitric oxide assay 15 2.11. In vitro radical scavenging assay 16 2.12. Statistical analysis 17 Chapter 3. Results 18 3.1. Screening of PPARγ ligand activity 18 3.2. Effect of FPE on 3T3-L1 pre-adipocytes 21 3.3. Effect of FPE on glucose utilization 25 3.4. Anti-oxidant activity of FPE 30 3.5. Anti-inflammatory activity of FPE 33 Chapter 4. Discussion 37 Chapter 5. Conclusion 41 Bibliography 43 Abstract in Korean 49Maste

Anti-proliferative activity of A. Oxyphylla and its bioactive constituent nootkatone in colorectal cancer cells

Background A. oxyphylla extract is known to possess a wide range of pharmacological activites. However, the molecular mechanism of A. oxyphylla and its bioactive compound nootkatone in colorectal cancer is unknown. Methods Our study aims to examine the role of A. oxyphylla and its bioactive compound nootkatone, in tumor suppression using several in vitro assays. Results Both A. oxyphylla extract and nootkatone exhibited antiproliferative activity in colorectal cancer cells. A. oxyphylla displayed antioxidant activity in colorectal cancer cells, likely mediated via induction of HO-1. Furthermore, expression of pro-apoptotic protein NAG-1 and cell proliferative protein cyclin D1 were increased and decreased respectively in the presence of A. oxyphylla. When examined for anticancer activity, nootkatone treatment resulted in the reduction of colony and spheroid formation. Correspondingly, nootkatone also led to increased NAG-1 expression and decreased cyclin D1 expression. The mechanism by which nootkatone suppresses cyclin D1 involves protein level regulation, whereas nootkatone increases NAG-1 expression at the transcriptional level. In addition to having PPARγ binding activity, nootkatone also increases EGR-1 expression which ultimately results in enhanced NAG-1 promoter activity. Conclusion In summary, our findings suggest that nootkatone is an anti-tumorigenic compound harboring antiproliferative and pro-apoptotic activity.This work was supported by the Research Institute for Veterinary Science, and BK21 PLUS Program for Creative Veterinary Science Research Center, Seoul National University, and by a National Research Foundation of Korea (NRF) grant funded by the Korean government (2018R1A2B2002923) to S.J.B. This work was also partially supported by a clinical research grant (NCC1810150) provided by the National Cancer Center to J.R. and S.J.B. The Fig. 7 Nootkatone controls the NAG-1 expression at the transcriptional level. a Nootkatone increases NAG-1 promoter activity. HCT-116 cells were transfected with pNAG-1 − 1086/+ 41 luciferase and pRL-null plasmid. The cells were treated with EtOH or various concentrations of nootkatone for 24 h, and luciferase activity was measured. The y-axis refers to the ratio of firefly luciferase over renillar luciferase activity. The EtOH-treated cells were set as 1.0. Statistical significance was displayed as *p < 0.05, ***p < 0.001 versus EtOH-treated cells. The data represent mean ± SD from three independent experiments. b Three deletion NAG-1 promoter constructs were co-transfected with pRL-null vector into HCT-116 cells. The cells were treated with EtOH or 100 μM of nootkatone for 24 h, and luciferase activity was measured. Fold induction refers to the ratio of luciferase activity in nootkatone-treated cells versus EtOH-treated cells. Statistical significance was displayed as **p < 0.01 and ***p < 0.001 versus EtOH-treated cells. The data represent mean ± SD from three independent experiments. c HCT-116 cells were co-transfected with wild type pNAG-1 − 133/+ 41 in the presence of empty or EGR-1 expression vector. Cells were subsequently treated with 100 μM nootkatone for 24 h. The results are presented as means ± S.D. of three independent transfections. d Western blot of EGR-1 protein in the presence of nootkatone. β-actin was used as loading control. e Luciferase activity of EGR-1 promoter-luciferase construct (pEGR-1260-LUC). The cells were treated with EtOH or nootkatone for 24 h prior to measurement of luciferase activity. Fold induction refers to the ratio of luciferase activity in nootkatone-treated cells compared to EtOH-treated cells. Statistical significance represented as *p < 0.05, ***p < 0.001 versus EtOH-treated cells. n.s. represents not significant. The data represent mean ± SD from four independent experiments Yoo et al. BMC Cancer (2020) 20:881 Page 10 of 12 funding agency did not have any influence in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript

Potential Anti-Diabetic Activity of Pueraria lobata Flower (Flos Puerariae) Extracts

Pueraria lobata (Wild.) Ohwi. (P. lobata) flowers known as ‘Kudzu flower’ contain isoflavonoids and essential oil components. They have a wide range of biological and pharmacological activities, including protective effects against non-alcoholic fatty liver disease, hyperglycemia, and hypolipidemia, anti-mutagenic effects, and benefits for weight loss. However, the molecular mechanism of these effects remains unclear. Our study aimed to systematically examine the effects of flos puerariae crude extract (FPE) as an anti-diabetic agent using in vitro assays. The cytotoxicity of FPE was evaluated using MTS assay in L6 rat myocyte and 3T3-L1 murine fibroblast cell lines. PPARγ binding activity and adipogenesis were examined using dual-luciferase and differentiation assays, respectively. For investigating the anti-diabetic activity, glucose utilization, including GLUT4 protein expression, glucose uptake assay, and GLUT4 translocation using immunofluorescence microscopy were conducted in L6 cells. Furthermore, we assessed the antioxidant and anti-inflammatory activities of FPE. Our results demonstrated the ability to augment glucose uptake in L6 cells and enhance glucose utilization activity by increasing the expression of glucose transporter type 4 (GLUT4). In summary, our findings suggest that FPE may be a potential anti-diabetic substance for the treatment of diabetic patients and can prevent inflammatory or oxidation-related diseases

Nano-encapsulated quercetin by soluble soybean polysaccharide/chitosan enhances anti-cancer, anti-inflammation, and anti-oxidant activities

Dietary polyphenols including quercetin are secondary plant metabolites that have been reported to prevent many chronic diseases; however, the high hydrophobicity of quercetin results in low water solubility and bioavailability, which limits the therapeutic efficacy and impedes further applications in aqueous systems. Encapsulation may be a potential solution to the aforementioned problems. Some polysaccharides, including soluble soybean polysaccharides (SSPS) and chitosan, were widely used as polymeric backbones for the formation of nanoparticles. We investigated the physicochemical property of encapsulated quercetin in SSPS with chitosan. Encapsulation of quercetin was confirmed using Dynamic light scattering, Zeta potential, Fouriertransform infrared spectroscopy, Differential scanning calorimetry, and Transmission electron microscopy. The biological activity of this nanoparticle showed that encapsulated quercetin exhibits better biological activity compared to free quercetin. Alongside the enhanced solubility of quercetin in an aqueous solution, our results illustrate the improved utility of this nanoparticle in the biomedical and food industry.N

Trans-chalcone suppresses tumor growth mediated at least in part by the induction of heme oxygenase-1 in breast cancer

Despite intensive research efforts in recent decades, cancer remains a leading cause of death worldwide. The chalcone family is a promising group of phytochemicals for therapeutic use against cancer development. Naturally-occurring chalcones, as well as synthetic chalcone analogues, have shown many beneficial biological properties, including anti-inflammatory, antioxidant, and anti-cancer activities. In this report, trans-chalcone (TChal) was found to increase cell death in breast cancer cells, assessed using high content screening. Subsequently, using antibody array analysis, TChal was found to increase heme oxygenase-1 (HO-1) expression in TChal-treated breast cancer cells. Blocking of HO-1 by siRNA in breast cancer cells diminished the effect of TChal on cell growth inhibition. TChal-fed mice also showed less tumor growth compared to vehicle-fed mice. Overall, we found that TChal increases HO-1 expression in breast cancer cells, thereby enhancing anti-tumorigenesis. Our results suggest that HO-1 expression could be a potential new target of TChal for anti-tumorigenesis in breast cancer.N

The anti-diabetic effects of NAG-1/GDF15 on HFD/STZ-induced mice

Nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) plays a role in various diseases. Here, the anti-diabetic effects of NAG-1 were evaluated using a high-fat diet/streptozotocin-induced diabetic mouse model. NAG-1-overexpressing transgenic (NAG-1 Tg) mice exhibited lower body weight, fasting blood glucose levels, and serum insulin levels than wild-type (WT) mice. The homeostatic model assessment of insulin resistance scores of NAG-1 Tg mice were lower than those of WT mice. Hematoxylin and eosin staining revealed a smaller lipid droplet size in the adipose tissues, lower lipid accumulation in the hepatocytes, and larger beta cell area in the pancreas of NAG-1 Tg mice than in those of WT mice. Immunohistochemical analysis revealed downregulated expression of cleaved caspase-3, an apoptosis marker, in the beta cells of NAG-1 Tg mice. Adiponectin and leptin mRNA levels were upregulated and downregulated in NAG-1 Tg mice, respectively. Additionally, the expression of IRS1/PI3K/AKT signaling pathway components, especially Foxo1, which regulates gluconeogenesis in the muscle and white adipose tissue, was downregulated in NAG-1 Tg mice. Furthermore, NAG-1 overexpression promoted the expression of As160 in both muscles and adipocytes, and the mRNA levels of the NLRP3 pathway members were downregulated in NAG-1 Tg mice. Our findings suggest that NAG-1 expression alleviates diabetes in mice.Y

Novel thrombospondin-1 transcript exhibits distinctive expression and activity in thyroid tumorigenesis

Thrombospondin 1 (TSP1) is known for its cell-specific functions in cancer progression, such as proliferation and migration. It contains 22 exons that may potentially produce several different transcripts. Here, we identified TSP1V as a novel TSP1-splicing variant produced by intron retention (IR) in human thyroid cancer cells and tissues. We observed that TSP1V functionally inhibited tumorigenesis contrary to TSP1 wild-type, as identified in vivo and in vitro. These activities of TSP1V are caused by inhibiting phospho-Smad and phospho-focal adhesion kinase. Reverse transcription polymerase chain reaction and minigene experiments revealed that some phytochemicals/non-steroidal anti-inflammatory drugs enhanced IR. We further found that RNA-binding motif protein 5 (RBM5) suppressed IR induced by sulindac sulfide treatment. Additionally, sulindac sulfide reduced phospho-RBM5 levels in a time-dependent manner. Furthermore, trans-chalcone demethylated TSP1V, thereby preventing methyl-CpG-binding protein 2 binding to TSP1V gene. In addition, TSP1V levels were significantly lower in patients with differentiated thyroid carcinoma than in those with benign thyroid nodule, indicating its potential application as a diagnostic biomarker in tumor progression.Y