제2형 당뇨마우스 모델에서 Nobiletin의 항당뇨 작용

정운주(Un Ju Jung) 한국식품영양과학회 2019 한국식품영양과학회지 Vol.48 No.5

본 연구에서는 nobiletin 보충이 렙틴수용체 결핍에 의해 제2형 당뇨환자와 유사한 증세를 보이는 인슐린 비의존형 당뇨병 모델동물인 db/db 마우스에서 당대사 및 지질대사 개선에 미치는 영향을 살펴보았다. db/db 마우스에 5주 동안의 nobiletin(0.02%, w/w) 보충은 당뇨로 인한 식이섭취 증가를 유의적으로 억제시켰고 체중감소 현상을 완화시키는 경향을 나타내었다. 또한 nobiletin은 당뇨로 인해 증가된 혈중 HbA1c 농도를 유의적으로 감소시켰고 고인슐린혈증, 내당능 및 인슐린저항성을 개선시켰으며 간조직의 당신생효소 활성도를 억제시켰다. 비록 혈장 총 콜레스테롤 농도가 nobiletin 보충에 의해 증가하였으나 HDL-콜레스테롤도 함께 증가하여 동맥경화지수 및 HTR 수치는 두 군 간에 유의적인 차이가 없었다. 또한 nobiletin 보충에 의한 혈장 유리지방산과 중성지질 농도 및 간조직 콜레스테롤 함량 변화는 관찰되지 않았으나, nobiletin은 간조직에서 지방산 합성 효소인 FAS 활성도를 억제시키고 지방산의 β-oxidation을 활성화시킴으로써 간조직의 중성지질 함량을 당뇨대조군보다 유의적으로 감소시켰다. 또한 체지방을 유의적으로 감소시켰고 지방조직의 MCP-1, TNF-α 및 IL-6 mRNA 발현과 이들 케모카인 및 사이토카인들의 혈중 농도를 유의적으로 감소시켰다. 이러한 연구 결과는 nobiletin이 염증반응 및 당대사와 지질대사 조절을 통해 인슐린저항성과 비알코올성 지방간을 완화함으로써 향후 당뇨병 및 관련 대사성 질환을 예방하고 개선시키는 건강기능성식품 및 천연물 기반 신약 소재로 이용될 수 있는 가능성이 있음을 제시하였다. This study investigated the anti-diabetic effect of nobiletin in type 2 diabetic mice and its underlying mechanism. Male mice were divided randomly into three groups: Control C57BL/KsJ-db/db mice, age-matched lean non-diabetic db/+ mice, and nobiletin-supplemented db/db mice. The control db/db mice and db/+ mice were fed a normal diet for five weeks, and the nobiletin group were fed a normal diet containing nobiletin (0.02%, w/w) for five weeks. As expected, the db/db mice showed higher food intake and lower body weight gain. Nobiletin tended to improve the weight loss and suppressed hyperphagia. Furthermore, nobiletin decreased the plasma insulin and blood HbA1c levels, improved the glucose tolerance and insulin resistance, and inhibited the hepatic gluconeogenic enzyme activity. Although the plasma total cholesterol concentration was increased by nobiletin, the HDL-cholesterol concentration was also increased in the nobiletin group, resulting in no significant difference in the atherogenic index and HDL-cholesterol to total cholesterol ratio. No significant differences in plasma free fatty acid and triglyceride concentrations as well as hepatic cholesterol content were observed between the control db/db mice and nobiletin-supplemented db/db mice. However, nobiletin significantly decreased the hepatic triglyceride content by inhibiting the fatty acid synthase activity and activating β-oxidation in the liver. Moreover, nobiletin significantly decreased the adipose tissue weight, mRNA expression of pro-inflammatory cytokines and chemokines in the adipose tissue and their circulating levels. These results suggest that nobiletin may be a useful phytochemical compound for improving the glucose intolerance, insulin resistance, hepatic steatosis, and inflammation in type 2 diabetes.

고혈당으로 유도된 신장 mesangial cell에서 nobiletin의 matrix accumulation과 TGF-β1-Src-caveolin-1 signaling에 의한 사구체 경화증 억제효과

김동연(Dong Yeon Kim),강영희(Young-Hee Kang),강민경(Min-Kyung Kang) 한국영양학회 2020 Journal of Nutrition and Health Vol.53 No.1

본 연구에서는 고혈당으로 인해 유발되는 당뇨병성 신장병증의 대표적인 증상인 사구체 경화증을 완화시키는 nobiletin의 효능에 대해 알아보고자 하였다. 신장 세포인 HRMC를 이용하여 고혈당에서의 세포외 기질 축적 단백질의 발현과 경화에 관여하는 신호 전달 억제 효능을 확인한 결과 nobiletin은 고혈당의 자극에 의해 증가하는 섬유화 단백질인 collagen IV, fibronectin 그리고 CTGF의 발현을 억제하였으며, 여기에 관여하는 TGF-β1-Src-caveolin-1 신호 전달 경로를 통해 사구체 경화증을 억제하는 것을 확인하였다. 따라서 nobiletin은 고혈당으로 유도된 당뇨병성 신장병증에 있어 사구체 경화증을 예방하는 기능성 성분으로서의 활용 가능성을 확인하였다. Purpose: Diabetic nephropathy is one of the most important diabetic complications prompted by chronic hyperglycemia, characterized by glomerulosclerosis, tubular fibrosis, and it eventually causes kidney failure. Nobiletin is a polymethoxyflavone present in tangerine and other citrus peels, and has anti-cancer and anti-inflammatory effects. This study investigated the effects of nobiletin on glomerular fibrosis through inhibition of the transforming growth factor (TGF)-β1-Src-caveolin-1 pathway. Methods: Human renal mesangial cells (HRMC) were incubated in media containing 33 mM glucose with or without 1–20 uM nobiletin for 3 day. The cellular expression levels of fibrogenic collagen IV, fibronectin, connective tissue growth factor (CTGF), TGF-β1, Src and caveolin-1 were all examined. In addition, TGF-β1, Src and caveolin-1 proteins were screened to reveal the relationship among TGF-β1-Src-caveolin-1 signaling in glomerular fibrosis. Results: High glucose promoted the production of collagen IV, fibronectin and CTGF in HRMC, which was inhibited in a dose dependent manner by 1–20 uM nobiletin. The Western blot data showed that high glucose elevated the expression of TGF-β1, Src, caveolin-1 and Rho GTPase. When nobiletin was treated to the HRMC exposed to high glucose, the expression of TGF-β1-Src-caveolin-1 was dampened. Finally, TGF-β1-Src-caveolin-1 signaling pathway was activated in high glucose-exposed HRMC, and such activation was encumbered by nobiletin. Conclusion: These result demonstrated that nobiletin blunted high glucose-induced extracellular matrix accumulation via inhibition of the TGF-β1-Src-caveolin-1 related intracellular signaling pathway. Nobiletin may be a potent renoprotective agent to counteract diabetes-associated glomerular fibrosis that leads to kidney failure.

인간진피섬유아세포에서 Nobiletin이 Mitogen-Activated Protein Kinases를 통한 Matrix-Metalloprotease 1 발현조절에 미치는 영향

차화준,김영주 한국피부과학연구원 2014 대한피부미용학회지 Vol.12 No.6

Nobiletin is widely used for the treatment of cancer and inflammation disorder. In neurocytes andmelanocytes, nobiletin exposure induced dendrite formation. Recently, nobiletin is suggested as a newwhitening agent. However, in dermis, effects of nobiletin are not understood currently. Therefore, it isimportant to determine how nobiletin exerts its therapeutic action and its effect on the anti-wrinkle. Todetermine whether MMP1 gene expression is regulated by nobiletin, we measured MMP1 mRNA expressionin nobiletein-treated human dermal fibroblasts (HDFs). And we showed that transactivation activity of TPAresponsive element (TRE) which is a major regulation promoter element and mitogen-activated proteinkinase (MAPK) activity which main regulator of AP-1 complex. As shown results, nobiletin was decreaseexpression level of MMP1 mRNA in oxidative-stressed HDFs. In addition, nobiletin repressed MAPKphosphorylation and transcriptional activity of AP-1 complex in oxidative-stressed HDFs. Therefore, nobiletinprotects oxidative-stress through repressing MAPK phosphorylation and transcriptional activity of AP-1complex. In addition, these results suggest that nobiletin is a potential cosmetic ingredient repressed antiagingand anti-wrinkle in skin.

Nobiletin attenuates neurotoxic mitochondrial calcium overload through K⁺ influx and ∆Ψ_m across mitochondrial inner membrane

Lee, Ji Hyung,Amarsanaa, Khulan,Wu, Jinji,Jeon, Sang-Chan,Cui, Yanji,Jung, Sung-Cherl,Park, Deok-Bae,Kim, Se-Jae,Han, Sang-Heon,Kim, Hyun-Wook,Rhyu, Im Joo,Eun, Su-Yong The Korean Society of Pharmacology 2018 The Korean Journal of Physiology & Pharmacology Vol.22 No.3

Mitochondrial calcium overload is a crucial event in determining the fate of neuronal cell survival and death, implicated in pathogenesis of neurodegenerative diseases. One of the driving forces of calcium influx into mitochondria is mitochondria membrane potential (${\Delta}{\psi}_m$). Therefore, pharmacological manipulation of ${\Delta}{\psi}_m$ can be a promising strategy to prevent neuronal cell death against brain insults. Based on these issues, we investigated here whether nobiletin, a Citrus polymethoxylated flavone, prevents neurotoxic neuronal calcium overload and cell death via regulating basal ${\Delta}{\psi}_m$ against neuronal insult in primary cortical neurons and pure brain mitochondria isolated from rat cortices. Results demonstrated that nobiletin treatment significantly increased cell viability against glutamate toxicity ($100{\mu}M$, 20 min) in primary cortical neurons. Real-time imaging-based fluorometry data reveal that nobiletin evokes partial mitochondrial depolarization in these neurons. Nobiletin markedly attenuated mitochondrial calcium overload and reactive oxygen species (ROS) generation in glutamate ($100{\mu}M$)-stimulated cortical neurons and isolated pure mitochondria exposed to high concentration of $Ca^{2+}$ ($5{\mu}M$). Nobiletin-induced partial mitochondrial depolarization in intact neurons was confirmed in isolated brain mitochondria using a fluorescence microplate reader. Nobiletin effects on basal ${\Delta}{\psi}_m$ were completely abolished in $K^+-free$ medium on pure isolated mitochondria. Taken together, results demonstrate that $K^+$ influx into mitochondria is critically involved in partial mitochondrial depolarization-related neuroprotective effect of nobiletin. Nobiletin-induced mitochondrial $K^+$ influx is probably mediated, at least in part, by activation of mitochondrial $K^+$ channels. However, further detailed studies should be conducted to determine exact molecular targets of nobiletin in mitochondria.

Nobiletin promotes adipogenesis in 3T3-L1 cells through the activation of Akt

Huimin Peng,Xiayu Tian,Lu Gan,Xiangliang Yang 경희대학교 융합한의과학연구소 2023 Oriental Pharmacy and Experimental Medicine Vol.23 No.1

The objective of the study was to investigate the effect of nobiletin on adipogenesis in 3T3-L1 cells. Here, we found that nobiletin could promote adipogenesis in 3T3-L1 cells in the absence of adipogenic inducers such as insulin, 3-Isobutyl-1-methylxanthine and dexamethasone. In addition, Real time quantitative PCR showed that the expression of adipogenic genes such as CCAAT/enhancer binding proteins, peroxisome proliferator-activated receptors-γ and fatty acid-binding protein aP2 were up-regulated in 3T3-L1 cells in the presence of nobiletin. Next, we investigated the role of Akt in the effect of nobiletin on the adipogenesis in 3T3-L1 cells by LY294002 blocking PI3K/Akt pathway. The experimental results showed that the promotive effect of nobiletin on adipogenesis was attenuated, accompanied by the down-regulation of adipogenic genes expression, after the activity of Akt was inhibited, suggesting that Akt plays an important role in the effect of nobiletin on promoting adipogenesis in 3T3-L1 cells. Still, the inhibition of Akt pathway by LY294002 was attenuated in the presence of nobiletin. These findings provide a possibility that nobiletin may be a potential candidate agent for stimulating Akt pathway in 3T3-L1 cells.

Nobiletin attenuates neurotoxic mitochondrial calcium overload through K+ influx and ΔΨm across mitochondrial inner membrane

Ji Hyung Lee,Khulan Amarsanaa,Jinji Wu,Sang-Chan Jeon,Yanji Cui,Sung-Cherl Jung,Deok-Bae Park,Se-Jae Kim,Sang-Heon Han,Hyun-Wook Kim,Im Joo Rhyu,Su-Yong Eun 대한생리학회-대한약리학회 2018 The Korean Journal of Physiology & Pharmacology Vol.22 No.3

Mitochondrial calcium overload is a crucial event in determining the fate of neuronal cell survival and death, implicated in pathogenesis of neurodegenerative diseases. One of the driving forces of calcium influx into mitochondria is mitochondria membrane potential (ΔΨm). Therefore, pharmacological manipulation of ΔΨm can be a promising strategy to prevent neuronal cell death against brain insults. Based on these issues, we investigated here whether nobiletin, a Citrus polymethoxylated flavone, prevents neurotoxic neuronal calcium overload and cell death via regulating basal ΔΨm against neuronal insult in primary cortical neurons and pure brain mitochondria isolated from rat cortices. Results demonstrated that nobiletin treatment significantly increased cell viability against glutamate toxicity (100 μM,20 min) in primary cortical neurons. Real-time imaging-based fluorometry data reveal that nobiletin evokes partial mitochondrial depolarization in these neurons. Nobiletin markedly attenuated mitochondrial calcium overload and reactive oxygen species (ROS) generation in glutamate (100 μM)-stimulated cortical neurons and isolated pure mitochondria exposed to high concentration of Ca2+ (5 μM). Nobiletininduced partial mitochondrial depolarization in intact neurons was confirmed in isolated brain mitochondria using a fluorescence microplate reader. Nobiletin effects on basal ΔΨm were completely abolished in K+-free medium on pure isolated mitochondria. Taken together, results demonstrate that K+ influx into mitochondria is critically involved in partial mitochondrial depolarization–related neuroprotective effect of nobiletin. Nobiletin-induced mitochondrial K+ influx is probably mediated, at least in part, by activation of mitochondrial K+ channels. However, further detailed studies should be conducted to determine exact molecular targets of nobiletin in mitochondria.

Nobiletin Attenuates the Inflammatory Response Through Heme Oxygenase-1 Induction in the Crosstalk Between Adipocytes and Macrophages

남궁슬기,성지혜,양진우,최용민,정헌상,이준수 한국식품영양과학회 2017 Journal of medicinal food Vol.20 No.9

Crosstalk between adipocytes and macrophages has been suggested to play a crucial role in metabolic disorders such as obesity, insulin resistance, and type 2 diabetes. The objective of this study was to evaluate the effect of nobiletin on the interaction between adipocytes and macrophages. The results showed that nobiletin significantly and dose-dependently inhibited the secretion of inflammatory mediators, such as nitric oxide (NO), tumor necrosis factor (TNF-α), and monocyte chemoattractant protein (MCP)-1, in a coculture of adipocytes and macrophages. The expression of adipogenic transcription factors, including peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), in differentiated 3T3-L1 cells cocultured in transwell system was blocked by nobiletin. Nobiletin also downregulated the expression of inducible NO synthase in cocultured differentiated RAW264.7 cells. Furthermore, heme oxygenase-1 (HO-1) was significantly induced by nobiletin treatment in both cell types, and small interfering (si) RNA-mediated knockdown of HO-1 significantly recovered the inhibitory effects of nobiletin on the NO production in cocultured cells. These results suggest that nobiletin exerts anti-inflammatory effects on the crosstalk between adipocytes and macrophages by inducing HO-1. Nobiletin may have potential for the prevention of obesity-related metabolic diseases.

Nobiletin Inhibits Angiogenesis by Regulating Src/FAK/STAT3-Mediated Signaling through PXN in ER ⁺ Breast Cancer Cells

Sp, Nipin,Kang, Dong Young,Joung, Youn Hee,Park, Jong Hwan,Kim, Wan Seop,Lee, Hak Kyo,Song, Ki-Duk,Park, Yeong-Min,Yang, Young Mok MDPI AG 2017 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.18 No.5

<P>Tumor angiogenesis is one of the major hallmarks of tumor progression. Nobiletin is a natural flavonoid isolated from citrus peel that has anti-angiogenic activity. Steroid receptor coactivator (Src) is an intracellular tyrosine kinase so that focal adhesion kinase (FAK) binds to Src to play a role in tumor angiogenesis. Signal transducer and activator of transcription 3 (STAT3) is a marker for tumor angiogenesis which interacts with Src. Paxillin (PXN) acts as a downstream target for both FAK and STAT3. The main goal of this study was to assess inhibition of tumor angiogenesis by nobiletin in estrogen receptor positive (ER<SUP>+</SUP>) breast cancer cells via Src, FAK, and STAT3-mediated signaling through PXN. Treatment with nobiletin in MCF-7 and T47D breast cancer cells inhibited angiogenesis markers, based on western blotting and RT-PCR. Validation of in vitro angiogenesis in the human umbilical vein endothelial cells (HUVEC) endothelial cell line proved the anti-angiogenic activity of nobiletin. Electrophoretic mobility shift assay and the ChIP assay showed that nobiletin inhibits STAT3/DNA binding activity and STAT3 binding to a novel binding site of the <I>PXN</I> gene promoter. We also investigated the migration and invasive ability of nobiletin in ER<SUP>+</SUP> cells. Nobiletin inhibited tumor angiogenesis by regulating Src, FAK, and STAT3 signaling through PXN in ER<SUP>+</SUP> breast cancer cells.</P>

Nobiletin attenuates neurotoxic mitochondrial calcium overload through K+ influx and ΔΨm across mitochondrial inner membrane

이지형,Khulan Amarsanaa,우진지,전상찬,추연지,정성철,박덕배,김세재,한상헌,김현욱,류임주,은수용 대한약리학회 2018 The Korean Journal of Physiology & Pharmacology Vol.22 No.3

Mitochondrial calcium overload is a crucial event in determining the fate of neuronal cell survival and death, implicated in pathogenesis of neurodegenerative diseases. One of the driving forces of calcium influx into mitochondria is mitochondria membrane potential (ΔΨm). Therefore, pharmacological manipulation of ΔΨm can be a promising strategy to prevent neuronal cell death against brain insults. Based on these issues, we investigated here whether nobiletin, a Citrus polymethoxylated flavone, prevents neurotoxic neuronal calcium overload and cell death via regulating basal ΔΨm against neuronal insult in primary cortical neurons and pure brain mitochondria isolated from rat cortices. Results demonstrated that nobiletin treatment significantly increased cell viability against glutamate toxicity (100 μM, 20 min) in primary cortical neurons. Real-time imaging-based fluorometry data reveal that nobiletin evokes partial mitochondrial depolarization in these neurons. Nobiletin markedly attenuated mitochondrial calcium overload and reactive oxygen species (ROS) generation in glutamate (100 μM)-stimulated cortical neurons and isolated pure mitochondria exposed to high concentration of Ca2+ (5 μM). Nobiletininduced partial mitochondrial depolarization in intact neurons was confirmed in isolated brain mitochondria using a fluorescence microplate reader. Nobiletin effects on basal ΔΨm were completely abolished in K+-free medium on pure isolated mitochondria. Taken together, results demonstrate that K+ influx into mitochondria is critically involved in partial mitochondrial depolarization–related neuroprotective effect of nobiletin. Nobiletin-induced mitochondrial K+ influx is probably mediated, at least in part, by activation of mitochondrial K+ channels. However, further detailed studies should be conducted to determine exact molecular targets of nobiletin in mitochondria.

Nobiletin Protects Dopaminergic Neurons in the 1-Methyl-4-Phenylpyridinium-Treated Rat Model of Parkinson's Disease

정경훈,전민태,김흥덕,정운주,장민철,주진우,양승준,최일윤,최명숙,김상룡 한국식품영양과학회 2015 Journal of medicinal food Vol.18 No.4

This study investigated the effect of nobiletin, a flavonoid found in citrus fruits, on the degeneration of dopaminergic (DA) neurons in a neurotoxin model of Parkinson’s disease (PD). 1-Methyl-4-phenylpyridinium (MPP+) was unilaterally injected into the median forebrain bundle of rat brains (to generate a neurotoxin model of PD) with or without daily intraperitoneal injection of nobiletin. Our results showed that nobiletin treatment at 10 mg/kg bw, but not at 1 or 20 mg/kg bw, significantly protected DA neurons in the substantia nigra (SN) of MPP+ -treated rats. In parallel to the neuroprotection, nobiletin treatment at 10 mg/kg inhibited microglial activation and preserved the expression of the glial cell line-derived neurotrophic factor, which is a therapeutic agent against PD, in the SN. These results suggest that the proper supplementation with nobiletin may protect against the neurodegeneration involved in PD.