Hye Jin An

Anti-Wrinkle and Anti-Inflammatory Effects of Active Garlic Components and the Inhibition of MMPs via NF-κB Signaling

Skin aging is a multisystem degenerative process caused by several factors, such as, UV irradiation, stress, and smoke. Furthermore, wrinkle formation is a striking feature of photoaging and is associated with oxidative stress and inflammatory response. In the present study, we investigated whether caffeic acid, S-allyl cysteine, and uracil, which were isolated from garlic, modulate UVB-induced wrinkle formation and effect the expression of matrix-metalloproteinase (MMP) and NF-κB signaling. The results obtained showed that all three compounds significantly inhibited the degradation of type І procollagen and the expressions of MMPs in vivo and attenuated the histological collagen fiber disorder and oxidative stress in vivo. Furthermore, caffeic acid and S-allyl cysteine were found to decrease oxidative stress and inflammation by modulating the activities of NF-κB and AP-1, and uracil exhibited an indirect anti-oxidant effect by suppressing cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expressions levels and downregulating transcriptional factors. These results suggest that the anti-wrinkle effects of caffeic acid, S-allyl cysteine, and uracil are due to anti-oxidant and/or anti-inflammatory effects. Summarizing, caffeic acid, S-allyl cysteine, and uracil inhibited UVB-induced wrinkle formation by modulating MMP via NF-κB signaling.

Anti-Wrinkle Effect of Magnesium Lithospermate B from Salvia miltiorrhiza BUNGE: Inhibition of MMPs via NF-kB Signaling

Skin is in direct contact with the environment and therefore undergoes aging as a consequence of environmentally induce damage. Wrinkle formation is a striking feature of intrinsic and photo-induced skin aging, which are both associated with oxidative stress and inflammatory response. The present study was undertaken to identify the mechanisms responsible for the anti-wrinkle effects of MLB, and thus, we investigated whether magnesium lithospermate B (MLB) from Salvia miltiorrhiza BUNGE associated with wrinkle formation caused by intrinsic and extrinsic skin aging using Sprague-Dawley rats aged 5 and 20 months and ultraviolet B (UVB)-irradiated human skin fibroblasts cells, respectively. The results obtained showed that the oral administration of MLB significantly upregulated the level of type I procollagen and downregulated the activities and expressions of matrix-metalloproteinases (MMPs) in rat skin. In fibroblasts, MLB suppressed the transactivation of nuclear factor-kB (NF-kB) and activator protein 1(AP-1), which are the two transcription factors responsible for MMP expression, by suppressing oxidative stress and the mitogen activated protein kinase (MAPK) pathway. Our results show that the antioxidant effect of MLB is due to the direct scavenging of reactive oxygen species (ROS) and its inhibitory effects on NF-kB-dependent inflammation genes, such as, cyclooxygenase-2 and inducible nitric oxide synthase. MLB was found to reverse both age- and UVB-related reductions in skin procollagen levels by suppressing the expressions and activities of NF-kB and AP-1-dependent MMPs by modulating ROS generation and the MAPK signaling pathway. We suggest that MLB potentially has anti-wrinkle and anti-skin aging effects.

Caffeic acid regulates LPS-induced NF-κB activation through NIK/IKK and c-Src/ERK signaling pathways in endothelial cells

The redox sensitive, proinflammatory nuclear transcription factor NF-κB plays a key role in inflammation. In a redox state disrupted by oxidative stress, pro-inflammatory genes are upregulated by the activation of NF-κB via diverse kinases. Thus, the search and characterization of new substances that modulate NF-κB are topics of considerable research interest. Caffeic acid is a component of garlic, some fruits, and coffee, and is widely used as a phenolic agent in beverages. In the present study, caffeic acid was examined with respect to the modulation of inflammatory NF-κB activation via the redox-related c-Src/ERK and NIK/IKK pathways via the reduction of oxidative stress. YPEN-1 cells (an endothelial cell line) were used to explore the molecular mechanism underlying the anti-inflammatory effect of caffeic acid by examining its modulation of NF-κB signaling pathway by LPS. Our results show that LPS-induced oxidative stress-related NF-κB activation upregulated pro-inflammatory COX-2, NF-κB targeting gene which were all inhibited effectively by caffeic acid. Our study shows that caffeic acid inhibits the activation of NF-κB via the c-Src/ERK and NIK/IKK signal transduction pathways. Our results indicate that antioxidative effect of caffeic acid and its restoration of redox balance are responsible for its anti-inflammatory action. Thus, the study provides new information regarding the anti-inflammatory properties of caffeic acid and the roles in the regulation of LPS-induced oxidative stress induces alterations in signal transduction pathways.

Molecular study of dietary heptadecane for the anti-inflammatory modulation of NF-kB in the aged kidney.

Heptadecane is a volatile component of Spirulina platensis, and blocks the de novo synthesis of fatty acids and ameliorates several oxidative stress-related diseases. In a redox state disrupted by oxidative stress, pro-inflammatory genes are upregulated by the activation of NF-kB via diverse kinases. Thus, the search and characterization of new substances that modulate NF-kB are lively research topics. In the present study, heptadecane was examined in terms of its ability to suppress inflammatory NF-kB activation via redox-related NIK/IKK and MAPKs pathway in aged rats. In the first part of the study, Fischer 344 rats, aged 9 and 20 months, were administered on average approximately 20 or 40 mg/Kg body weight over 10 days. The potency of heptadecane was investigated by examining its ability to suppress the gene expressions of COX-2 and iNOS (both NF-κB-related genes) and reactive species (RS) production in aged kidney tissue. In the second part of the study, YPEN-1 cells (an endothelial cell line) were used to explore the molecular mechanism underlying the anti-inflammatory effect of heptadecane by examining its modulation of NF-kB and NF-kB signal pathway. Results showed that heptadecane exhibited a potent anti-oxidative effect by protecting YPEN-1 cells from tert-butylhydroperoxide induced oxidative stress. Further molecular investigations revealed that heptadecane attenuated RS-induced NF-kB via the NIK/IKK and MAPKs pathways in YPEN-1 cells and aged kidney tissues. Based on these results, we conclude that heptadecane suppresses age-related increases in pro-inflammatory gene expressions by reducing NF-kB activity by upregulating the NIK/IKK and MAPKs pathways induced by RS. These findings provide molecular insight of the mechanisms by which heptadecane exerts its antiinflammatory effect in aged kidney tissues. We conclude that heptadecane suppresses age-related increases in pro-inflammatory gene expressions then travel upstream set by step by reducing NF-kB activity by downregulating the NIK/IKK and MAPKs pathways induced by RS.

The roles of FoxOs in modulation of aging by calorie restriction

FoxO activity and modifications, such as its phosphorylation, acetylation, and methylation, may help drive the expression of genes involved in combating oxidative stress by causing the epigenetic modifications, and thus, preserve cellular function during aging and age-related diseases, such as diabetes, cancer, and Alzheimer disease. Insulin signaling has been postulated to influence the aging process by increasing resistance to oxidative stress, and slowing the accumulation of oxidative damage. Some antioxidative effects are mediated by a conserved family of forkhead box transcription factors (FoxOs), which in the absence of insulin signaling freely bind to promoters of antioxidant enzymes, superoxide dismutase, and catalase. On the other hand, calorie restriction (CR) extends the lifespans of several species via the insulin pathway, and extends longevity and healthspan in diverse species via a conserved mechanism. CR enhances adaptive stress responses at the cellular and organism levels and extends lifespan in a FoxO-independent manner. Thus, increased modification of FoxO is modulated via the hyperinsulinemia-induced PI3K/Akt pathway during aging, and CR reverses this process. Accordingly, FoxO plays an important role in maintenance of metabolic homeostasis and removal of oxidative stress in the aging process and in the effect of CR on lifespan.

Short‐term calorie restriction ameliorates genomewide, age‐related alterations in DNA methylation

DNA methylation plays major roles in many biological processes, including aging, carcinogenesis, and development. Analyses of DNA methylation using next‐generation sequencing offer a new way to profile and compare methylomes across the genome in the context of aging. We explored genomewide DNA methylation and the effects of short‐term calorie restriction (CR) on the methylome of aged rat kidney. Whole‐genome methylation of kidney in young (6 months old), old (25 months old), and OCR (old with 4‐week, short‐term CR) rats was analyzed by methylated DNA immunoprecipitation and next‐generation sequencing (MeDIP‐Seq). CpG islands and repetitive regions were hypomethylated, but 5′‐UTR, exon, and 3′‐UTR hypermethylated in old and OCR rats. The methylation in the promoter and intron regions was decreased in old rats, but increased in OCR rats. Pathway enrichment analysis showed that the hypermethylated promoters in old rats were associated with degenerative phenotypes such as cancer and diabetes. The hypomethylated promoters in old rats related significantly to the chemokine signaling pathway. However, the pathways significantly enriched in old rats were not observed from the differentially methylated promoters in OCR rats. Thus, these findings suggest that short‐term CR could partially ameliorate age‐related methylation changes in promoters in old rats. From the epigenomic data, we propose that the hypermethylation found in the promoter regions of disease‐related genes during aging may indicate increases in susceptibility to age‐related diseases. Therefore, the CR‐induced epigenetic changes that ameliorate age‐dependent aberrant methylation may be important to CRs health‐ and life‐prolonging effects.

Age-related sensitivity to endotoxin-induced liver inflammation: Implication of inflammasome/IL-1β for steatohepatitis

Aging is associated with increased vulnerability to inflammatory challenge. However, the effects of altered inflammatory response on the metabolic status of tissues or organs are not well documented. In this study, we present evidence demonstrating that lipopolysaccharide (LPS)‐induced upregulation of the inflammasome/IL‐1β pathway is accompanied with an increased inflammatory response and abnormal lipid accumulation in livers of aged rats. To monitor the effects of aging on LPS‐induced inflammation, we administered LPS (2 mg kg−1) to young (6‐month old) and aged (24‐month old) rats and found abnormal lipid metabolism in only aged rats with increased lipid accumulation in the liver. This lipid accumulation in the liver was due to the dysregulation of PPARα and SREBP1c. We also observed severe liver inflammation in aged rats as indicated by increased ALT levels in serum and increased Kupffer cells in the liver. Importantly, among many inflammation‐associated factors, the aged rat liver showed chronically increased IL‐1β production. Increased levels of IL‐1β were caused by the upregulation of caspase‐1 activity and inflammasome activation. In vitro studies with HepG2 cells demonstrated that treatment with IL‐1β significantly induced lipid accumulation in hepatocytes through the regulation of PPARα and SREBP1c. In summary, we demonstrated that LPS‐induced liver inflammation and lipid accumulation were associated with a chronically overactive inflammasome/IL‐1β pathway in aged rat livers. Based on the present findings, we propose a mechanism of aging‐associated progression of steatohepatitis induced by endotoxin, delineating a pathogenic role of the inflammasome/IL‐1β pathway involved in lipid accumulation in the liver.

(Z)-5-(2,4-Dihydroxybenzylidene)thiazolidine-2,4-dione Prevents UVB-Induced Melanogenesis and Wrinkle Formation through Suppressing Oxidative Stress in HRM-2 Hairless Mice

Background. Uncontrolled melanogenesis and wrinkle formation are an indication of photoaging. Our previous studies demonstrated that (Z)-5-(2,4-dihydroxybenzylidene)thiazolidine-2,4-dione (MHY498) inhibited tyrosinase activity and melanogenesis in vitro. Objective. To examine in vivo effects of MHY498 as an antiaging compound on UVB-induced melanogenesis and wrinkle formation, we topically applied MHY498 on dorsal skin of HRM-2 hairless mice. Methods. Using histological analysis, we evaluated effects of MHY498 on melanogenesis and wrinkle formation after UVB exposure. In addition, related molecular signaling pathways were examined using western blotting, fluorometric assay, and enzyme-linked immunosorbent assay. Results. MHY498 suppressed UVB-induced melanogenesis by inhibiting phosphorylation of CREB and translocation of MITF protein into the nucleus, which are key factors for tyrosinase expression. Consistently, tyrosinase protein levels were notably reduced in the dorsal skin of the hairless mice by MHY498 treatment. Furthermore, MHY498 inhibited UVB-induced wrinkle formation and collagen fiber destruction by increasing type 1 procollagen concentration and decreasing protein expression levels of MMPs, which play an essential role in collagen fiber degradation. As a mechanism, MHY498 notably ameliorated UVB-induced oxidative stress and NF-κB activation in the dermal skin of the hairless mice. Conclusion. Our study suggests that MHY498 can be used as a therapeutic or cosmetic agent for preventing uncontrolled melanogenesis and wrinkle formation.

A PPAR Pan Agonist, MHY2013 Alleviates Age-Related Hepatic Lipid Accumulation by Promoting Fatty Acid Oxidation and Suppressing Inflammation

Nonalcoholic fatty liver disease (NAFLD) is frequently observed in obese and aged individuals. Peroxisome proliferator-activated receptors (PPARs) play a role in regulating hepatic lipid accumulation, a hallmark of NAFLD development. A PPAR pan agonist, 2-(4-(5,6-methylenedioxybenzo[d]thiazol-2-yl)-2-methylphenoxy)-2-methylpropanoic acid (MHY2013) has been shown to prevent fatty liver formation and insulin resistance in obese mice (db/db) model. However, the beneficial effects of MHY2013 in aged model remain unknown. In this study, we investigated whether MHY2013 alleviates hepatic lipid accumulation in aged Sprague-Dawley (SD) rats. We confirmed that MHY2013 increased the activities of three PPAR subtypes in HepG2 cells using luciferase assay. When administered orally in aged SD rats, MHY2013 markedly decreased the hepatic triglyceride levels without changes in body weight. Regarding underlying mechanisms, MHY2013 increased the mRNA levels of lipid oxidation-related genes, including carnitine palmitoyltransferase 1 (CPT1) and peroxisomal acyl-CoA oxidase 1 (ACOX1), without apparent change in the mRNA expression of lipogenesis-related genes. Furthermore, MHY2013 significantly increased systemic fibroblast growth factor 21 (FGF21) and adiponectin levels and suppressed inflammatory mRNA expression in the liver. In conclusion, MHY2013 alleviated age-related hepatic lipid accumulation, in part by upregulating β-oxidation signaling and suppressing inflammation in the liver. Therefore, MHY2013 is a potential pharmaceutical agent for treating age-related hepatic lipid accumulation.

The critical role played by endotoxin-induced liver autophagy in the maintenance of lipid metabolism during sepsis

ABSTRACT Macroautophagy/autophagy is a central mechanism by which cells maintain integrity and homeostasis, and endotoxin-induced autophagy plays important roles in innate immunity. Although TLR4 stimulation mediated by lipopolysaccharide (LPS) also upregulates autophagy in hepatocytes and liver, its physiological role remains elusive. The objective of this study was to determine the role of LPS-induced autophagy in the regulation of liver lipid metabolism. LPS treatment (5 mg/kg) increased autophagy, as detected by LC3 conversion and transmission electron microscopy (TEM) analysis in C57BL6 mouse livers. AC2F hepatocytes also showed increased autophagic flux after LPS treatment (1 μg/ml). To investigate the role of LPS-induced autophagy further, liver lipid metabolism changes in LPS-treated mice and fasted controls were compared. Interestingly, LPS-treated mice showed less lipid accumulation in liver than fasted mice despite increased fatty acid uptake and lipid synthesis-associated genes. In vitro analysis using AC2F hepatocytes demonstrated LPS-induced autophagy influenced the degradation of lipid droplets. Inhibition of LPS-induced autophagy using bafilomycin A1 or Atg7 knockdown significantly increased lipid accumulation in AC2F hepatocytes. In addition, pretreatment with chloroquine aggravated LPS-induced lipid accumulation and inflammation in C57BL6 mouse livers. The physiological importance of autophagy was verified in LPS-treated young and aged rats. Autophagic response was diminished in LPS-treated aged rats and lipid metabolism was impaired during sepsis, indicating autophagy response is important for regulating lipid metabolism after endotoxin challenge. Our findings demonstrate endotoxin-induced autophagy is important for the regulation of lipid metabolism, and suggest that autophagy helps maintain lipid metabolism homeostasis during sepsis.