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// Kyoung Mi Moon 1, 5, * , Bonggi Lee 1, 5, * , Ji Won Jeong 1 , Dae Hyun Kim 1 , Yun Jung Park 2 , Hye Rim Kim 2 , Ji Young Park 2 , Min Jo Kim 1 , Hye Jin An 1 , Eun Kyeong Lee 1 , Young Mi Ha 3 , Eunok Im 1 , Pusoon Chun 4 , Jin Yeul Ma 5 , Won-Kyung Cho 5 , Hyung Ryong Moon 2 and Hae Young Chung 1 1 Molecular Inflammation Research Center for Aging Intervention, College of Pharmacy, Pusan National University, Busan, Korea 2 Laboratory of Medicinal Chemistry, College of Pharmacy, Pusan National University, Busan, Republic of Korea 3 Department of Chemistry, Dong-A University, Busan, Republic of Korea 4 College of Pharmacy, Inje University, Inje-ro, Gyeongnam, Korea 5 Korean Medicine Application Center, Korea Institute of Oriental Medicine, Daegu, Republic of Korea * These authors have contributed equally to this work Correspondence to: Hae Young Chung, email: hyjung@pusan.ac.kr Hyung Ryong Moon, email: mhr108@pusan.ac.kr Keywords: oxidative stress, antioxidant, compound 2d, compound 2l, inflammation Received: May 24, 2017 Accepted: July 12, 2017 Published: October 10, 2017 ABSTRACT Liver inflammation is closely associated with metabolic syndrome. Oxidative stress plays a synergistic role in inflammation by activating nuclear factor kappa B (NF-κB) signaling in the liver. Therefore, substantial efforts have been made to develop compounds that inhibit the generation of oxidative stress and activation of NF-κB. We synthesized twenty-six novel 5-(substituted benzyl)-2-oxo- and 5-(substituted benzyl)-2-thioxo-dihydropyrimidine-4,6(1 H ,5 H )-dione derivatives for the development of potential antioxidants and examined their biological activities in vitro and in vivo . Thio-barbiturate-derived compounds 5-[4-hydroxy-3-methoxybenzy]-2-thioxodihydropyrimidine-4,6[1 H ,5 H ]-dione (2d) and 5-[4-hydroxy-3,5-methoxybenzy]-2-thioxodihydropyrimidine-4,6[1 H ,5 H ]-dione (2l) had the strongest inhibitory effect on reactive oxygen species and peroxynitrite generation in vitro . Furthermore, oral administration of compounds 2d and 2l in mice notably suppressed lipopolysaccharide (LPS)-induced oxidative stress and NF-κB activation in the liver. Because macrophages play an essential role in liver inflammation, we investigated the effects of these compounds on inflammatory signaling in LPS-induced RAW264.7 macrophages. LPS-induced NF-κB activation and protein expression of cyclooxygenase 2 and inducible nitric oxide synthase were inhibited by pretreatment of these compounds in macrophages. In parallel with this finding, the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and AKT signalings, which are upstream activators of p65, were decreased by these compounds in macrophages. Our study suggests that compounds 2d and 2l inhibit oxidative stress and NF-кB-mediated inflammation, at least partially, through suppressing PTEN/AKT signaling. Therefore, these compounds may be useful as therapeutic agents for the amelioration of inflammatory diseases.

// Bonggi Lee 1, 5, * , Kyoung Mi Moon 2, 5, * , Jong Seung Lim 2 , Yeojin Park 2 , Do Hyun Kim 1, 2 , Sujin Son 1, 2 , Hyoung Oh Jeong 2 , Dae Hyun Kim 1, 2 , Eun Kyeong Lee 2 , Ki Wung Chung 2 , Hye Jin An 2 , Pusoon Chun 3 , Arnold Y. Seo 4 , Ju-Hye Yang 5 , Bong-Seon Lee 5 , Jin Yeul Ma 5 , Won-Kyung Cho 5 , Hyung Ryong Moon 1, 2 and Hae Young Chung 1, 2 1 Molecular Inflammation Research Center for Aging Intervention (MRCA), Pusan National University, Busan, Republic of Korea 2 College of Pharmacy, Pusan National University, Busan, Republic of Korea 3 College of Pharmacy, Inje University, Gimhae, Gyeongnam, Republic of Korea 4 Janelia Research Campus, Howard Huge Medical Institute, Ashburn, VA, USA 5 Korean Medicine (KM)-Application Center, Korea Institute of Oriental Medicine (KIOM), Dong-gu, Daegu, Korea * These authors have contributed equally to this work Correspondence to: Hae Young Chung, email: hyjung@pusan.ac.kr Hyung Ryong Moon, email: mhr108@pusan.ac.kr Keywords: skin, melanogenesis, tyrosinase inhibitor, pigmentation, photoaging Received: May 27, 2017 Accepted: July 13, 2017 Published: September 06, 2017 ABSTRACT Tyrosinase is a key player in ultraviolet-induced melanogenesis. Because excessive melanin accumulation in the skin can induce hyperpigmentation, the development of tyrosinase inhibitors has attracted attention in cosmetic-related fields. However, side effects including toxicity and low selectivity have limited the use of many tyrosinase inhibitors in cosmetics. We synthesized 12 novel 2-(substituted benzylidene)malononitrile derivatives and investigated their anti-melanogenic activities. Of these 12 compounds, 2-(3, 4-dihydroxy benzylidene)malononitrile (BMN11) exhibited the strongest inhibitory activity against tyrosinase (IC 50 = 17.05 μM). In parallel with this, BMN11 treatment notably decreased alpha-melanocyte-stimulating hormone-induced melanin accumulation in B16F10, cells without toxicity and also decreased melanin accumulation in a human skin model. As a mechanism underlying the BMN11-mediated anti-melanogenic effect, docking simulation showed that BMN11 can directly bind to tyrosinase by forming two hydrogen bonds with GLY281 and ASN260 residues, and via three hydrophobic interactions with VAL283, PHE264, and ALA286 residues in the tyrosinase binding pocket, and this likely contributes to its inhibitory effect on tyrosinase. Consistently, Lineweaver-Burk and Cornish-Bowden plots showed that BMN11 is a competitive inhibitor of tyrosinase. We concluded that BMN11 may be a novel tyrosinase inhibitor that could be used in cosmetics.

// Ha Ram Bae 1,* , Dae Hyun Kim 1,* , Min Hi Park 1 , Bonggi Lee 1 , Min Jo Kim 1 , Eun Kyeong Lee 1 , Ki Wung Chung 1 , Seong Min Kim 1 , Dong Soon Im 1 and Hae Young Chung 1 1 Molecular Inflammation Research Center for Aging Intervention, College of Pharmacy, Pusan National University, Geumjeong-gu, Busan, Republic of Korea * These authors have contributed equally to this work Correspondence to: Hae Young Chung, email: // Keywords : β-hydroxybutyrate; endoplasmic reticulum; aging; inflammasome; AMPK; Gerotarget Received : May 10, 2016 Accepted : August 26, 2016 Published : September 19, 2016 Abstract β-Hydroxybutyrate, a ketone body that is used as an energy source in organs such as the brain, muscle, and heart when blood glucose is low, is produced by fatty acid oxidation in the liver under the fasting state. Endoplasmic reticulum (ER) stress is linked with the generation of intracellular reactive oxygen species and the accumulation of misfolded protein in the ER. ER stress is known to induce the NOD-like receptor protein 3 inflammasome, which mediates activation of the proinflammatory cytokine interleukin-1β, whose maturation is caspase-1-dependent. We investigated whether β-hydroxybutyrate modulates ER stress, inflammasome formation, and insulin signaling. Sprague Dawley rats (6 and 24 months of age) that were starved for 3 d and rats treated with β-hydroxybutyrate (200 mg·kg -1 ·d -1 i.p., for 5 d) were used for in vivo investigations, whereas human hepatoma HepG2 cells were used for in vitro studies. Overexpression of AMPK in cultured cells was performed to elucidate the molecular mechanism. The starvation resulted in increased serum β-hydroxybutyrate levels with decreased ER stress (PERK, IRE1, and ATF6α) and inflammasome (ASC, caspase-1, and NLRP3) formation compared with non-fasted 24-month-old rats. In addition, β-hydroxybutyrate suppressed the increase of ER stress- and inflammasome-related marker proteins. Furthermore, β-hydroxybutyrate treatment increased the expression of manganese superoxide dismutase and catalase via the AMP-activated protein kinase-forkhead box protein O3α transcription factor pathway both in vivo and in vitro . The significance of the current study was the discovery of the potential therapeutic role of β-hydroxybutyrate in suppressing ER-stress-induced inflammasome formation.

// Eun Kyeong Lee 1 , Hyoung Oh Jeong 1 , Eun Jin Bang 1 , Chul Hong Kim 2 , Ji Young Mun 3,4 , Seulgi Noh 4 , Jeong-An Gim 5 , Dae Hyun Kim 1 , Ki Wung Chung 1 , Byung Pal Yu 6 and Hae Young Chung 1 1 Molecular Inflammation Research Center for Aging Intervention, College of Pharmacy, Pusan National University, Busan, Republic of Korea 2 Genomictree Inc., Daejeon, Republic of Korea 3 Department of Biomedical Laboratory Science, College of Health Science, Eulji University, Seongnam-Si, Gyeonggi-Do, Republic of Korea 4 BK21 Plus Program, Department of Senior Healthcare, Graduate School, Eulji University, Seongnam-Si, Gyeonggi-Do, Republic of Korea 5 Department of Biological Sciences, College of Natural Sciences, Pusan National University, Busan, Republic of Korea 6 Department of Physiology, The University of Texas Health Science Center at San Antonio, San Antonio, TX, USA Correspondence to: Hae Young Chung, email: // Keywords : aging; calorie restriction; exosome; miR-500-3p; miR-770-3p; Gerotarget Received : September 04, 2017 Accepted : November 16, 2017 Published : December 24, 2017 Abstract Recent studies have shown a role for miRNAs in aging and age-related diseases, and the modulation of miRNA expression by diet attracts attention as a new therapeutic strategy. Here, we focused on identifying specific exosomal miRNAs derived from serum of aged rats and the effect of short-term calorie restriction (CR) on their expression. Exosomes from serum of young (7-month), old (22-month), and old-CR Sprague Dawley rats were isolated and characterized by transmission electron microscopy analyses, dynamic light scattering measurements, and Western blotting. A total of 12 significantly expressed miRNAs in serum exosomes of young and old rats were identified by next generation sequencing. After analysis of qRT-PCR, we found that miR-500-3p and miR-770-3p expression was significantly upregulated by aging and downregulated by CR. Furthermore, receiver operating characteristic (ROC) curve revealed that the selected miRNAs represented high accuracy in discriminating old rats from young rats. Finally, PANTHER analysis predicted selected miRNAs targets genes involved in Wnt/chemokines and cytokines -related inflammatory signaling pathway and function as transcription factor. In conclusion, our results suggest that the expression of serum exosomal miR-500-3p and miR-770-3p was significantly increased with aging, whereas these were decreased by CR, and age-/CR-modulated exosomal miR-500-3p and miR-770-3p could potentially be used as informative biomarkers candidates for aging.

// Seong Min Kim 1, 2 , Bonggi Lee 1, 3 , Hye Jin An 1 , Dae Hyun Kim 1 , Kyung Chul Park 1 , Sang-Gyun Noh 1 , Ki Wung Chung 1 , Eun Kyeong Lee 1 , Kyung Mok Kim 1 , Do Hyun Kim 1 , Su Jeong Kim 1 , Pusoon Chun 4 , Ho Jeong Lee 2 , Hyung Ryong Moon 1 and Hae Young Chung 1 1 Molecular Inflammation Research Center for Aging Intervention (MRCA), College of Pharmacy, Pusan National University, Busan 46241, Republic of Korea 2 Research Institute of Life Science and College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Republic of Korea 3 Korean Medicine (KM)-Application Center, Korea Institute of Oriental Medicine (KIOM), Daegu 41062, Republic of Korea 4 College of Pharmacy, Inje University, Gimhae 621-749, Republic of Korea Correspondence to: Hae Young Chung, email: hyjung@pusan.ac.kr Hyung Ryong Moon, email: mhr108@pusan.ac.kr Keywords: MHY553, PPARα agonist, hepatic steatosis, fatty acid oxidation, aging Received: February 08, 2017 Accepted: March 26, 2017 Published: May 08, 2017 ABSTRACT Hepatic steatosis is frequently observed in obese and aged individuals. Because hepatic steatosis is closely associated with metabolic syndromes, including insulin resistance, dyslipidemia, and inflammation, numerous efforts have been made to develop compounds that ameliorate it. Here, a novel peroxisome proliferator-activated receptor (PPAR) α agonist, 4-(benzo[d]thiazol-2-yl)benzene-1,3-diol (MHY553) was developed, and investigated its beneficial effects on hepatic steatosis using young and old Sprague-Dawley rats and HepG2 cells. Docking simulation and Western blotting confirmed that the activity of PPARα, but not that of the other PPAR subtypes, was increased by MHY553 treatment. When administered orally, MHY553 markedly ameliorated aging-induced hepatic steatosis without changes in body weight and serum levels of liver injury markers. Consistent with in vivo results, MHY553 inhibited triglyceride accumulation induced by a liver X receptor agonist in HepG2 cells. Regarding underlying mechanisms, MHY553 stimulated PPARα translocation into the nucleus and increased mRNA levels of its downstream genes related to fatty acid oxidation, including CPT-1A and ACOX1, without apparent change in lipogenesis signaling. Furthermore, MHY553 significantly suppresses inflammatory mRNA expression in old rats. In conclusion, MHY553 is a novel PPARα agonist that improved aged-induced hepatic steatosis, in part by increasing β-oxidation signaling and decreasing inflammation in the liver. MHY553 is a potential pharmaceutical agent for treating hepatic steatosis in aging.

// Ki Wung Chung 1 , Hyeong Oh Jeong 1 , Bonggi Lee 1 , Daeui Park 1,2 , Dae Hyun Kim 1 , Yeun Ja Choi 1 , Eun Kyeong Lee 1 , Kyung Mok Kim 1 , June Whoun Park 1 , Byung Pal Yu 3 and Hae Young Chung 1 1 Department of Pharmacy, College of Pharmacy, Pusan National University, Busan, 46241, Republic of Korea 2. Systems Toxicology Research Center, Korea Institute of Toxicology, Daejeon, 34114, Republic of Korea 3 Department of Physiology, The University of Texas Health Science Center at San Antonio, San Antonio, TX, 78229, USA Correspondence to: Hae Young Chung, email: // Keywords : NF-κBIZ, aging, inflammation, renal fibrosis Received : July 13, 2016 Accepted : January 04, 2017 Published : January 12, 2017 Abstract Chronic inflammation is a major contributor to age-related nephropathic changes, including renal fibrosis. In this study, various experimental paradigms were designed to delineate the role played by NF-κBIZ (also known as IκBζ) in age-associated renal fibrosis. Analyses based on RNA-sequencing findings obtained by next generation sequencing (NGS) revealed the upregulations of NF-κBIZ and of IL-6 and MCP-1 (both known to be regulated by NF-κBIZ) during aging. The up-regulation of NF-κBIZ in aged rat kidneys coincided with increased macrophage infiltration. In LPS-treated macrophages, oxidative stress was found to play a pivotal role in NF-κBIZ expression, suggesting age-related oxidative stress is associated with NF-κBIZ activation. Furthermore, these in vitro findings were confirmed in LPS-treated old rats, which showed higher levels of oxidative stress and NF-κBIZ in kidneys than LPS-treated young rats. Additional in vitro experiments using macrophages and kidney fibroblasts demonstrated NF-κBIZ and related cytokines participate in fibrosis. In particular, increased levels of NF-κBIZ-associated cytokines in macrophages significantly up-regulated TGF-β induced kidney fibroblast activation. Moreover, experiments with NF-κBIZ knocked down macrophages showed reduced TGF-β-induced kidney fibroblast activation. The findings of the present study provide evidence regarding an involvement of NF-κBIZ in age-associated progressive renal fibrosis and provides potential targets for its prevention.

// Eun Ji Jang 1,* , Dae Hyun Kim 1,* , Bonggi Lee 1 , Eun Kyeong Lee 1 , Ki Wung Chung 1 , Kyoung Mi Moon 1 , Min Jo Kim 1 , Hye Jin An 1 , Ji Won Jeong 1 , Ye Ra Kim 1 , Byung Pal Yu 2 and Hae Young Chung 1 1 Department of Pharmacy, Molecular Inflammation Research Center for Aging Intervention (MRCA), Pusan National University, Busan, Republic of Korea 2 Department of Physiology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States of America * These authors have contributed equally to this work Correspondence to: Hae Young Chung, email: // Keywords : 4-HNE; Src; aged kidney; inflammation; cell senescence; Gerotarget Received : November 06, 2015 Accepted : July 17, 2016 Published : July 26, 2016 Abstract In our previous study, reactive 4-hydroxy-2-nonenal (4-HNE) was shown to activate Src (a non-receptor tyrosine kinase) by forming an adduct on binding with a specific residue of Src, leading to the activation of proinflammatory signaling pathways in cultured cells. However, to date, the deleterious roles of 4-HNE in inflammatory signaling activation in kidneys during aging have not been explored. The purpose of the present study was to document the mechanisms by which 4-HNE induces inflammation in the kidney during aging. Initial experiments revealed that activated nuclear factor-κB (NF-κB) expression was caused by 4-HNE activation, which suppressed transcriptional activity in the aged kidney. Treatment of human umbilical vein endothelial cells with 4-HNE revealed that Src caused senescence via NF-κB activation. Furthermore, our immunohistochemistry data showed that 4-HNE-adducted Src significantly increased in aged kidney tissues. The data showed age-related upregulation of downstream signaling molecules such as mitogen activated protein kinases (MAPKs), activator protein-1 (AP-1), NF-κB, and COX-2 in a cell culture cell system. Taken together, the results of this study show that the formation of adducts between 4-HNE and Src activates inflammatory signaling pathways in the aged kidney, contributing to age-related nephropathy.

The beneficial role of FoxO during aging has been proposed for its promotion of resistance to oxidative stress and inhibition of pro-inflammatory mediators. On the other hand, NF-κB is a pro-inflammatory transcription factor which is a key mediator of inflammatory cytokine generation. However, the correlation between FoxO6 and NF-κB during aging has not fully been explored. The main purpose of the present study was to elucidate mechanisms underlying the protective role of FoxO6 in the maintenance of cellular homeostasis under potent pro-inflammatory conditions induced by LPS. Initial experimentation revealed that reduced FoxO6 activity during aging was caused by its phosphorylation, which suppressed its transcriptional activity in aged livers. Transfection with FoxO6-wt virus and FoxO6-siRNA in HepG2 cells revealed that FoxO6 phosphorylation by LPS leads to NF-κB activation via Akt and Pak1 pathways. Furthermore, Pak1 activity was increased in a phosphatidylinositol 3-kinase independent manner, and LPS-induced FoxO6 phosphorylation and FoxO6 inactivation were Pak1-dependent in nuclear fractions of cells. Further revealed Pak1 phosphorylation by LPS permitted interaction between FoxO6 and Akt. Current study suggests FoxO6 phosphorylation facilitates the nuclear translocation of NF-κB via Akt and Pak1 pathways induced by LPS in aged rats.

Recently, agonists targeting multiple peroxisome proliferator-activated receptors (PPARs) have been developed to improve metabolic disorders and minimize the side effects of selective PPAR agonists such as weight gain and dyslipidemia. We newly synthesized six 2-methyl-2-(o-tolyloxy)propanoic acid derivatives based on the structure of a well-known PPAR pan agonist, bezafibrate. Of six compounds, MHY2013 was screened as the strongest activator of three PPAR subtypes based on protein docking simulation and luciferase assays. When treated orally in db/db mice, MHY2013 ameliorated obesity-induced insulin resistance, dyslipidemia, and hepatic steatosis without changes of the body weight and levels of liver and kidney injury markers. MHY2013 decreased the serum triglyceride and fatty acid levels, which is associated with an increase in fatty acid oxidation signaling in the liver and thermogenic signaling on white adipose tissue, respectively. Furthermore, MHY2013 markedly increased serum levels of insulin-sensitizing hormones including fibroblast growth factor 21 (FGF21) and adiponectin. In conclusion, this study suggests that, MHY2013 is a novel PPAR pan agonist that improves obesity-induced insulin resistance, dyslipidemia and hepatic steatosis and elevates insulin-sensitizing hormones in the blood.