Eunsun Jung

Isorhamnetin Represses Adipogenesis in 3T3-L1 Cells

Adipocyte dysfunction is strongly associated with the development of obesity, which is a major risk factor for many disorders including diabetes, hypertension, and heart disease. It is generally accepted that the regulation of adipogenesis or adipokines expression prevents obesity. In this study, we show that isorhamnetin inhibits adipocyte differentiation, as evidenced by reduced triglyceride (TG) accumulation and glycerol‐3‐phosphate dehydrogenase (GPDH) activity. At the molecular level, the mRNA expression levels of peroxidase proliferator‐activated receptor‐γ (PPAR‐γ) and CCAAT/enhancer‐binding protein‐α (C/EBP‐α), which are the major adipogenic transcription factors, were markedly reduced by isorhamnetin. However, the mRNA levels of C/EBP‐β and ‐δ, the upstream regulators of PPAR‐γ and C/EBP‐α, were not reduced by isorhamnetin. Moreover, the mRNA levels of PPAR‐γ target genes such as lipoprotein lipase (LPL), CD36, aP2, and liver X receptor‐α (LXR‐α) were downregulated by isorhamnetin. We also showed that isorhamnetin inhibits the expression and secretion of adiponectin, and the results of adiponectin promoter assays suggest the inhibition of PPAR‐γ expression as a possible mechanism underlying the isorhamnetin‐mediated effects. Taken together, these results indicate that isorhamnetin inhibits adipogenesis through downregulation of PPAR‐γ and C/EBP‐α.

Effect of rosmarinic acid on atopic dermatitis

Rosmarinic acid is known to have anti‐inflammatory and immunomodulatory activities. This study was performed to evaluate the effect of rosmarinic acid on atopic dermatitis (AD), one of the inflammatory disorders of the skin. Twenty‐one subjects (14 women and seven men, 5–28 years of age) with mild AD participated in this study. Rosmarinic acid (0.3%) emulsion was topically applied to the elbow flexures of AD patients twice a day (once in the morning and once in the evening). All subjects were evaluated for skin conditions before treatment at the first visit, and then at 4 and 8 weeks after treatment. According to local Severity Scoring of Atopic Dermatitis index results, erythema on antecubital fossa was significantly reduced at 4 and 8 weeks (P < 0.05). Transepidermal water loss of the antecubital fossa was significantly reduced at 8 weeks compared to before treatment (P < 0.05). The results from self‐questionnaires on the efficacy of rosmarinic acid indicated that dryness, pruritus and general AD symptoms improved. Our investigation into the AD‐mitigating effect of rosmarinic acid through in vivo experiments demonstrated the possible clinical use of rosmarinic acid as a therapeutic agent for AD.

Rosmarinic acid as a downstream inhibitor of IKK-β in TNF-α-induced upregulation of CCL11 and CCR3

1 Tumor necrosis factor (TNF)‐α is known to induce the expression of CCL11 and CCR3 via the activation of NF‐κB. CCL11 (eotaxin), the C–C chemokine, is a potent chemoattractant for eosinophils and Th2 lymphocytes, and CCR3 is the receptor for CCL11. 2 In order to determine the effects of rosmarinic acid on the TNF‐α‐induced upregulation of CCL11 and CCR3 in human dermal fibroblasts, we performed an enzyme‐linked immunosorbent assay for CCL11 and a Western blot assay for CCR3. The TNF‐α‐induced expression of CCL11 and CCR3 genes was attenuated by rosmarinic acid. 3 In our NF‐κB luciferase reporter system, TNF‐α‐induced NF‐κB activation was observed to be reduced by rosmarinic acid. In accordance with this result, rosmarinic acid also inhibited TNF‐α‐induced phosphorylation and degradation of IκB‐α, as well as nuclear translocation of NF‐κB heterodimer induced by TNF‐α. This suggests that rosmarinic acid downregulates the expression of CCL11 and CCR3 via the inhibition of NF‐κB activation signaling. 4 Using the NF‐κB luciferase reporter system, Western blot analysis, and IKK‐β activity assay, we determined that rosmarinic acid inhibits IKK‐β activity in NF‐κB signaling, which upregulates the expression of CCL11 and CCR3. Additionally, TNF‐α‐induced secretion of soluble intercellular adhesion molecule‐1 and soluble vascular cell adhesion molecule‐1 molecules was found to be attenuated by rosmarinic acid. 5 Our results show that rosmarinic acid inhibits the expression of CCL11 and CCR3 by suppressing the IKK‐β activity in NF‐κB activation signaling. Further, these results suggest that rosmarinic acid might inhibit the expression of NF‐κB promoter‐related genes.

Inhibitory effects of (-)-α-bisabolol on LPS-induced inflammatory response in RAW264.7 macrophages.

Although (-)-α-bisabolol, a natural monocyclic sesquiterpene alcohol, is often used as a cosmetic soothing supplement, little is known about its mechanisms of anti-inflammatory effects. Therefore, this study was designed to investigate anti-inflammatory effects of (-)-α-bisabolol and its mechanisms of action. In this study, we found that (-)-α-bisabolol inhibited lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) in RAW264.7 cells. In addition, expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) genes was reduced, as evidenced by Western blot and luciferase reporter assays for COX-2 and iNOS. To assess the mechanism of the anti-inflammatory property of (-)-α-bisabolol, its effects on the activity of AP-1 and NF-κB promoters were examined. LPS-induced activation of AP-1 and NF-κB promoters was significantly reduced by (-)-α-bisabolol. Consistently, (-)-α-bisabolol reduced LPS-induced phosphorylation of IκBα. In addition, while LPS-induced phosphorylation of ERK and p38 was attenuated by (-)-α-bisabolol, significant changes in the level of phosphorylated JNK were not observed. Our results indicate that (-)-α-bisabolol exerts anti-inflammatory effects by downregulating expression of iNOS and COX-2 genes through inhibition of NF-κB and AP-1 (ERK and p38) signaling.

7,8-Dihydroxyflavone protects human keratinocytes against oxidative stress-induced cell damage via the ERK and PI3K/Akt-mediated Nrf2/HO-1 signaling pathways.

This study investigated the effect of 7,8-dihydroxyflavone (DHF) on the expression and activity of heme oxygenase-1 (HO-1), an enzyme with potent antioxidant properties, as well as the molecular mechanisms involved. DHF markedly upregulated HO-1 mRNA and protein expression in human keratinocytes (HaCaT cells), resulting in increased HO-1 activity. DHF also increased the protein level of transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which regulates HO-1 expression by binding to the antioxidant response element (ARE) within the HO-1 gene promoter, in a time-dependent manner. Moreover, DHF decreased the expression of Kelch-like ECH-associated protein 1, a repressor of Nrf2 activity, and induced the translocation of Nrf2 from the cytosol into the nucleus, thereby allowing its association with the ARE site. DHF activated extracellular-regulated kinase (ERK) and protein kinase B (PKB, Akt) in keratinocytes, while the ERK and Akt inhibitors attenuated DHF-enhanced Nrf2 and HO-1 expression. DHF also protected the keratinocytes against hydrogen peroxide- and ultraviolet B-induced oxidative damage, while HO-1, ERK and Akt inhibitors markedly suppressed DHF-mediated cytoprotection. Taken together, the results suggested that DHF activates ERK- and Akt-Nrf2 signaling cascades in HaCaT cells, leading to the upregulation of HO-1 and cytoprotection against oxidative stress.

Ultraviolet A Regulates Adipogenic Differentiation of Human Adipose Tissue-derived Mesenchymal Stem Cells via Up-regulation of Kruppel-like Factor 2

Adipocyte dysfunction is strongly associated with the development of obesity, which is a major risk factor for many disorders, including diabetes, hypertension, and heart disease. This study shows that ultraviolet A (UVA) inhibits adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells and its action mechanisms. The mRNA levels of peroxidase proliferator-activated receptor (PPAR) γ and CCAAT/enhancer-binding protein α (C/EBPα), but not CCAAT/enhancer-binding protein ((C/EBP) β and δ, were reduced by UVA. Moreover, the mRNA levels of PPAR γ target genes (lipoprotein lipase (LPL), CD36, adipocyte protein (aP2), and liver X receptor α (LXR)) were down-regulated by UVA. Additionally, attempts to elucidate a possible mechanism underlying the UVA-mediated effects revealed that UVA induced migration inhibitory factor (MIF) gene expression, and this was mediated through activation of AP-1 (especially JNK and p42/44 MAPK) and nuclear factor-κB. In addition, reduced adipogenesis by UVA was recovered upon the treatment with anti-MIF antibodies. AMP-activated protein kinase phosphorylation and up-regulation of Kruppel-like factor 2 (KLF2) were induced by UVA. Taken together, these findings suggest that the inhibition of adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells by UVA occurs primarily through the reduced expression of PPAR γ, which is mediated by up-regulation of KLF2 via the activation of MIF-AMP-activated protein kinase signaling.

Mechanisms of carvacrol-induced expression of type I collagen gene.

BACKGROUND Skin aging is accompanied by wrinkle formation and appears to be principally related to decreases in the levels of type I collagen, the primary component of the dermal layer of skin. OBJECTIVE To investigate the effect of carvacrol on collagen gene expression and its mechanisms of action. METHODS To elucidate the effect of carvacrol on collagen expression and its mechanism, several experiments were performed in human dermal fibroblasts. Collagen production, small interference RNA, Ca(2+) mobilization, COL1A2/AP-1 luciferase reporter assays and Western blots for proteins that are involved in collagen gene expression were used in this study. RESULTS Carvacrol activated both the human COL1A2 promoter activity and the synthesis of human type I procollagen. Additionally, we attempted to characterize the mechanism of action of carvacrol in type I procollagen synthesis. In a human COL1A2 promoter luciferase assay, the small interference RNA for SP-1 did not reduce the carvacrol-induced promoter activation. Also, Smad 2 phosphorylation was not induced by carvacrol. However, in the AP-1 (activator protein-1) luciferase reporter assay and in the Western blot analysis for mitogen-activated protein kinases (MAPKs), carvacrol induced the activation of the AP-1 promoter and the phosphorylation of JNK and ERK1/2 (p42/44 MAPK), but did not induce the phosphorylation of p38 MAPK. Carvacrol also induced intracellular Ca(2+) mobilization and phosphorylation of phospholipase Cgamma1 (PLCgamma1), a molecule upstream of Ca(2+). In addition, PAO, a PLCgamma1 inhibitor, attenuated the carvacrol-induced production of collagen. CONCLUSION Our study suggests that the PLCgamma1 signaling pathway may be involved in the carvacrol-induced expression of the type I collagen gene.

Isorhamnetin-induced anti-adipogenesis is mediated by stabilization of β-catenin protein

AIMS Previous studies have shown that isorhamnetin has anti-adipogenic effects in mouse 3T3-L1 cells. This study was conducted to elucidate the inhibitory mechanisms of isorhamnetin during adipogenic differentiation of human adipose tissue-derived stem cells (hAMSCs). MAIN METHODS The effect of isorhamnetin on adipogenic differentiation of hAMSCs was quantified by Oil Red O staining and a triglyceride assay. In addition, real-time PCR and Western blot were used to determine the expression of adipogenesis-related genes. KEY FINDINGS Isorhamnetin inhibited the adipocyte differentiation of hAMSCs. Additionally, when the effects of Wnt antagonists that promote adipogenesis were evaluated, isorhamnetin was found to down-regulate the mRNA levels of sFRP1 and Dkk1, but had no effect on the mRNA levels of sFRP2, sFRP3, sFRP4 and Dkk3. Isorhamnetin also inhibited the expression of Wnt receptor and co-receptor genes. Furthermore, isorhamnetin increased the protein levels of beta-catenin, an effector molecule of Wnt signaling, but had no effect on the mRNA levels of beta-catenin. The phosphorylation level of GSK 3beta was also increased by isorhamnetin. These results were confirmed by the fact that the expression of c-myc, cyclin D1 and PPARdelta, which are target genes of beta-catenin, was upregulated by isorhamnetin. Moreover, isorhamnetin reduced the mRNA expression levels of C/EBPalpha and PPARgamma, which are known to be inhibited by c-myc or by cyclin D1 and PPARdelta, respectively. SIGNIFICANCE Our results indicate that isorhamnetin inhibits the adipogenic differentiation of hAMSCs and that its mechanisms are mediated by the stabilization of beta-catenin.

Antagonizing effects and mechanisms of afzelin against UVB-induced cell damage.

Ultraviolet (UV) radiation induces DNA damage, oxidative stress, and inflammatory processes in human keratinocytes, resulting in skin inflammation, photoaging, and photocarcinogenesis. Adequate protection of skin against the harmful effects of UV irradiation is essential. Therefore, in this study, we investigated the protective effects of afzelin, one of the flavonoids, against UV irradiation in human keratinocytes and epidermal equivalent models. Spectrophotometric measurements revealed that the afzelin extinction maxima were in the UVB and UVA range, and UV transmission below 376 nm was <10%, indicating UV-absorbing activity of afzelin. In the phototoxicity assay using the 3T3 NRU phototoxicity test (3T3-NRU-PT), afzelin presented a tendency to no phototoxic potential. In addition, in order to investigate cellular functions of afzelin itself, cells were treated with afzelin after UVB irradiation. In human keratinocyte, afzelin effectively inhibited the UVB-mediated increase in lipid peroxidation and the formation of cyclobutane pyrimidine dimers. Afzelin also inhibited UVB-induced cell death in human keratinocytes by inhibiting intrinsic apoptotic signaling. Furthermore, afzelin showed inhibitory effects on UVB-induced release of pro-inflammatory mediators such as interleukin-6, tumor necrosis factor-α, and prostaglandin-E2 in human keratinocytes by interfering with the p38 kinase pathway. Using an epidermal equivalent model exposed to UVB radiation, anti-apoptotic activity of afzelin was also confirmed together with a photoprotective effect at the morphological level. Taken together, our results suggest that afzelin has several cellular activities such as DNA-protective, antioxidant, and anti-inflammatory as well as UV-absorbing activity and may protect human skin from UVB-induced damage by a combination of UV-absorbing and cellular activities.

Artemisinic acid is a regulator of adipocyte differentiation and C/EBP δ expression

Adipocyte dysfunction is associated with the development of obesity. In this study, artemisinic acid, which was isolated from Artemisia annua L., inhibited adipogenic differentiation of human adipose tissue‐derived mesenchymal stem cells (hAMSCs) and its mechanism of action was determined. The mRNA levels of peroxidase proliferation‐activated receptor (PPAR) γ and CCAAT/enhancer binding protein (C/EBP) α, late adipogenic factors, were reduced by artemisinic acid. Moreover, the mRNA levels of the PPAR γ target genes lipoprotein lipase, CD36, adipocyte protein, and liver X receptor were down‐regulated by artemisinic acid. Artemisinic acid reduced expression of the C/EBP δ gene without impacting C/EBP β. In addition, attempts to elucidate a possible mechanism underlying the artemisinic acid‐mediated effects revealed that reduced expression of the C/EBP δ gene was mediated by inhibiting Jun N‐terminal kinase (JNK). Additionally, artemisinic acid also reduced the expression of the adipogenesis‐associated genes glucose transporter‐4 and vascular endothelial growth factor. In addition to the interference of artemisinic acid with adipogenesis, artemisinic acid significantly attenuated tumor necrosis factor‐α‐induced secretion of interleukin‐6 by undifferentiated hAMSCs, thus influencing insulin resistance and the inflammatory state characterizing obesity. Taken together, these findings indicate that inhibiting adipogenic differentiation of hAMSCs by artemisinic acid occurs primarily through reduced expression of C/EBP δ, which is mediated by the inhibition of JNK and suggest that aremisinic acid may be used as a complementary treatment option for obesity associated with metabolic syndrome. J. Cell. Biochem. 113: 2488–2499, 2012.