Su Jeong Ha

Naringenin targets ERK2 and suppresses UVB‐induced photoaging

A number of natural phytochemicals have anti‐photoaging properties that appear to be mediated through the inhibition of matrix metalloproteinase‐1 (MMP‐1) expression, but their direct target molecule(s) and mechanism(s) remain unclear. We investigated the effect of naringenin, a major flavonoid found in citrus, on UVB‐induced MMP‐1 expression and identified its direct target. The HaCaT human skin keratinocyte cell line and 3‐dimensional (3‐D) human skin equivalent cultures were treated or not treated with naringenin for 1 hr before exposure to UVB. The mechanism and target(s) of naringenin were analysed by kinase assay and multiplex molecular assays. Dorsal skins of hairless mice were exposed to UVB 3 times per week, with a dose of irradiation that was increased weekly by 1 minimal erythema dose (MED; 45 mJ/cm2) to 4 MED over 15 weeks. Wrinkle formation, water loss and water content were then assessed. Naringenin suppressed UVB‐induced MMP‐1 expression and AP‐1 activity, and strongly suppressed UVB‐induced phosphorylation of Fos‐related antigen (FRA)‐1 at Ser265. Importantly, UVB irradiation‐induced FRA1 protein stability was reduced by treatment with naringenin, as well as with a mitogen‐activated protein kinase (MEK) inhibitor. Naringenin significantly suppressed UVB‐induced extracellular signal‐regulated kinase 2 (ERK2) activity and subsequently attenuated UVB‐induced phosphorylation of p90RSK by competitively binding with ATP. Constitutively active MEK (CA‐MEK) increased FRA1 phosphorylation and expression and also induced MMP‐1 expression, whereas dominant‐negative ERK2 (DN‐ERK2) had opposite effects. U0126, a MEK inhibitor, also decreased FRA1 phosphorylation and expression as well as MMP‐1 expression. The photoaging data obtained from mice clearly demonstrated that naringenin significantly inhibited UVB‐induced wrinkle formation, trans‐epidermal water loss and MMP‐13 expression. Naringenin exerts potent anti‐photoaging effects by suppressing ERK2 activity and decreasing FRA1 stability, followed by down‐regulation of AP‐1 transactivation and MMP‐1 expression.

MLK3 is a novel target of dehydroglyasperin D for the reduction in UVB-induced COX-2 expression in vitro and in vivo

Dehydroglyasperin D (DHGA‐D), a compound present in licorice, has been found to exhibit anti‐obesity, antioxidant and anti‐aldose reductase effects. However, the direct molecular mechanism and molecular targets of DHGA‐D during skin inflammation remain unknown. In the present study, we investigated the effect of DHGA‐D on inflammation and its mechanism of action on UVB‐induced skin inflammation in HaCaT human keratinocytes and SKH‐1 hairless mice. DHGA‐D treatment strongly suppressed UVB‐induced COX‐2 expression, PGE2 generation and AP‐1 transactivity in HaCaT cells without affecting cell viability. DHGA‐D also inhibited phosphorylation of the mitogen‐activated protein kinase kinase (MKK) 3/6/p38, MAPK/Elk‐1, MKK4/c‐Jun N‐terminal kinase (JNK) 1/2/c‐Jun/mitogen, and stress‐activated protein kinase (MSK), whereas phosphorylation of the mixed‐lineage kinase (MLK) 3 remained unaffected. Kinase and co‐precipitation assays with DHGA‐D Sepharose 4B beads showed that DHGA‐D significantly suppressed MLK3 activity through direct binding to MLK3. Knockdown of MLK3 suppressed COX‐2 expression as well as phosphorylation of MKK4/p38 and MKK3/6/JNK1/2 in HaCaT cells. Furthermore, Western blot assay and immunohistochemistry results showed that DHGA‐D pre‐treatment significantly inhibits UVB‐induced COX‐2 expression in vivo. Taken together, these results indicate that DHGA‐D may be a promising anti‐inflammatory agent that mediates suppression of both COX‐2 expression and the MLK3 signalling pathway through direct binding and inhibition of MLK3.

Protein Extraction from Porcine Myocardium Using Ultrasonication

Porcine myocardium is regarded as a byproduct in slaughterhouses and is rarely used as a food source due to its unsuitability for processing and consumption. In this study, we sought to develop an efficient ultrasonication method to extract protein from porcine myocardium. Comparisons of protein yield using various ultrasonication conditions with porcine myocardium revealed that treatment with 0.2 M NaCl, with pH 8.0, at an extraction temperature of less than 40 °C and an amplitude of 60% to 80% was optimal, yielding an extraction rate of 90%. In addition, SDS-PAGE analysis showed that increasing the time interval for ultrasonication increased the presence of myosin heavy chain and actin protein content. Functional analysis of the physiological properties of the isolated proteins using an ATPase assay showed that Ca and Mg ATPase activity was virtually undetectable in the early stages of ultrasonic treatment and that the proteins denatured rapidly. An analysis of protein digestion also showed that the digestive capacity of proteins treated by ultrasonication methods was greater. These results demonstrate that the ultrasonication method is effective for high-yield protein extraction from cardiac myofibrils of porcine myocardium with low salt concentrations, low Ca and Mg ATPase activities, and high digestive capacities.

Rice bran supplement prevents UVB-induced skin photoaging in vivo

Abstract Although rice bran consumption is reportedly has numerous beneficial effects on human health, the relationship between rice bran and the prevention of photoaging has not been investigated in detail. We sought to investigate whether consumption of rice bran supplement (RBS) can elicit preventive effects against UVB-induced photoaging in vivo. Dorsal skin sections of hairless mice were exposed to UVB over 16 weeks. RBS consumption suppressed UVB-induced wrinkle formation and inhibited the loss of water content and epidermal thickening in the mouse skin. Western blot and immunohistochemical analyses revealed that repeated exposure to UVB upregulated matrix metalloproteinase-13 (MMP-13) and cyclooxygenase-2 (COX-2) expression, while consumption of RBS suppressed MMP-13 and COX-2 expression, as well as mitogen-activated protein kinase (MAPK) signaling pathways. These findings suggest that RBS could be a potential bioactive ingredient in nutricosmetics to inhibit wrinkle formation and water content loss via the suppression of COX-2 and MMP-13 expression. Effect of RBS on UVB-irradiated COX-2 and MMP-13 expression in SKH-1 hairless mouse skin.

1,8-cineole prevents UVB-induced skin carcinogenesis by targeting the aryl hydrocarbon receptor

1,8-cineole is a natural monoterpene cyclic ether present in Eucalyptus, and has been reported to exhibit anti-inflammatory and antioxidant effects. However, the preventive effect of 1,8-cineole on skin carcinogenesis and the molecular mechanism of action responsible remains unknown. In the present study, we investigated the effect of 1,8-cineole on UVB-induced skin carcinogenesis. 1,8-cineole inhibited UVB-induced cyclooxygenase-2 (COX-2) protein and mRNA expression and prostaglandin E2 (PGE2) generation in HaCaT cells. 1,8-cineole also inhibited phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, and phosphorylation of its upstream kinases, c-Src and epidermal growth factor receptor (EGFR). Quantitative real-time RT-PCR (qRT-PCR) and drug affinity responsive target stability (DARTS) assay results showed that 1,8-cineole suppressed UVB-induced expression of a target gene of the aryl hydrocarbon receptor (AhR), cyp1a1, and directly binds to AhR. Knockdown of AhR suppressed COX-2 expression as well as phosphorylation of ERK1/2 in HaCaT cells. Furthermore, topical treatment of 1,8-cineole on mouse skin delayed tumor incidence and reduced tumor numbers, while inhibiting COX-2 expression in vivo. Taken together, these results suggest that 1,8-cineole is a potent chemopreventive agent that inhibits UVB-induced COX-2 expression by targeting AhR to suppress UVB-induced skin carcinogenesis.

Preventive effect of Ephedra sinica extract on UVB-induced COX-2 and MMP-1 expression

Ultraviolet B (UVB)-induced cyclooxygenase (COX)-2 and matrix metalloproteinase (MMP)-1 are representative markers for skin inflammation and photoaging, respectively. To evaluate compounds that may counteract the effects of UVB-induced skin damage, we developed an immortalized human keratinocyte (HaCaT) cell line with an MMP-1 reporter construct. Among the 30 botanical extracts screened, we selected Ephedra sinica extract (ESE) as a promising candidate and confirmed that ESE significantly suppresses UVB-induced COX-2 and MMP-1 expression in HaCaT cells. Treatment with ESE also potently suppressed UVB-induced ERK1/2 phosphorylation, as well as UVB-induced MEK1/2 and Raf phosphorylation in HaCaT cells. These findings suggest that our MMP-1 reporter system can be used to evaluate compounds with anti-inflammatory and anti-photoaging effects. We also report that ESE has potent suppressive effects against COX-2 and MMP-1 expression, which occurs via downregulation of Raf/MEK1/2/ERK1/2 phosphorylation.

Syringic acid prevents skin carcinogenesis via regulation of NoX and EGFR signaling

Graphical abstract Figure. No Caption available. ABSTRACT Validation of nutraceutical and pharmaceutical targets is essential for the prediction of physiological and side effects. Epidemiologic evidence and molecular studies suggest that non‐melanoma skin cancer is directly associated with excessive exposure to ultraviolet (UV) radiation. The aim of the present study was to evaluate the inhibitory effects of syringic acid on UVB‐induced signaling and skin carcinogenesis, and determine the molecular targets. Treatment of human epidermal keratinocytes (HaCaT) cells with syringic acid resulted in the suppression of UVB‐induced cyclooxygenase‐2, matrix metalloproteinase‐1, and prostaglandin E2 expression as well as activator protein‐1 activity. Moreover, syringic acid inhibited the UVB‐induced phosphorylation of mitogen‐activated protein kinases and Akt signaling pathways as well as epidermal growth factor receptor (EGFR). Syringic acid treatment further inhibited intracellular reactive oxygen species and protein‐tyrosine phosphatase‐&kgr; activity, a regulator of EGFR activation. Syringic acid and the antioxidant N‐acetyl‐l‐cysteine inhibited UVB‐induced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. In vivo, pretreatment of mouse skin with syringic acid significantly suppressed UVB‐induced skin tumor incidence in a dose‐dependent manner. Overall, these results indicate that syringic acid exerts potent chemopreventive activity in skin carcinogenesis mainly by inhibition of the Nox/PTP‐&kgr;/EGFR axis. Syringic acid might serve as an effective chemopreventive and therapeutic agent against UVB‐mediated skin cancer.