Qualitative Analysis of Proteins in Two Snake Venoms, Gloydius Blomhoffii and Agkistrodon Acutus.

Objectives: Snake venom is a complex mixture of various pharmacologically active substances, such as small proteins, peptides, and organic and mineral components. This paper aims to identify and analyse the proteins in common venomous snakes, such as Gloydius blomhoffii (G. blomhoffii) and Agkistrodon acutus (A. acutus), in Korea. Methods: We used mass spectrometry, electrophoresis, N-terminal sequencing and in-gel digestion to analyse the proteins in these two snake venoms. Results: We identified eight proteins in G. blomhoffii venom and four proteins in A. acutus venom. The proteins detected in G. blomhoffii and A. acutus venoms were phospholipase A2, snake venom metalloproteinase and cysteine-rich secretory protein. Snake C-type lectin (snaclec) was unique to A. acutus venom. Conclusion: These data will contribute to the current knowledge of proteins present in the venoms of viper snakes and provide useful information for investigating their therapeutic potential.

NADPH Oxidase and Epidermal Growth Factor Receptor Are Promising Targets of Phytochemicals for Ultraviolet-Induced Skin Carcinogenesis.

The skin acts as the primary defense organ that protects the body from the external environment. Skin cancer is one of the most common cancers in the world. Skin carcinogenesis is usually caused by cell degeneration due to exposure to ultraviolet (UV) radiation, which causes changes in various signaling networks, disrupting the homeostasis of single skin cells. In this review, we summarize the roles of nicotinamide adenine dinucleotide phosphate oxidase (NOX) and epidermal growth factor receptor (EGFR) in UV-induced skin carcinogenesis. Furthermore, we describe the crosstalk that exists between NOX, EGFR, and protein tyrosine phosphatase κ and its oncogenic downstream signaling pathways. Chemoprevention is the use of chemical compounds to recover the healthy status of the skin or delay cancer development. Current evidence from in vitro and in vivo studies on chemopreventive phytochemicals that target NOX, EGFR, or both, as major regulators of skin carcinogenesis will also be discussed.

Efficacy and Safety of Yukgunja-Tang for Patients with Cancer-related Anorexia: A Randomized, Controlled Trial, Pilot Study.

OBJECTIVE: The purpose of this study is both to estimate the efficacy and the safety of Yukgunja-tang (YGJT) and to establish evidence for the use of herbal medicines in the management of patients with cancer-related anorexia. METHODS: We enrolled 40 patients with cancer-related anorexia. The enrolled participants were randomly allocated to 2 groups: the control group (n = 20), which received nutrition counseling, and the treatment group (n = 20), which received nutrition counseling and was administered YGJT at twice a day for 4 weeks (a total of 56 times @ 3.0 g each time). The primary outcome of this study was the score on the anorexia/cachexia subscale (ACS) of the Functional Assessment of Anorexia/Cachexia Therapy (FAACT). The secondary outcomes were the FAACT score with the ACS score excluded, the score on the Visual Analog Scale (VAS) for appetite, and the results on laboratory tests regarding appetite, such as leptin, tumor necrosis factors (TNF-α), interleukin-6 (IL-6), and ghrelin. All variables related to the safety assessment, such as vital signs, electrocardiography results, laboratory test results (complete blood cell count, chemistry, urine test), and adverse events, were documented on the case report form (CRF) at every visit. RESULT: The difference in the primary outcome, that is, the score on the anorexia/cachexia subscale (ACS) of the Functional Assessment of Anorexia/Cachexia Therapy (FAACT), between the control and the treatment groups was statistically significant (P = .023) as was the difference in the FAACT scores with the ACS score excluded, a secondary outcome, between the 2 groups; however, no statistically significant differences were noted in the scores on the VAS or the levels of leptin, TNF-α, IL-6, and ghrelin. In addition, no significant differences in the numbers and the types of adverse events or in the results on the laboratory tests between the control and the treatment groups were recorded. CONCLUSION: These results obtained in this research confirmed the efficacy and the safety of using YGJT as a herb-medicine treatment option for patients with cancer-related anorexia.

A randomized, multi-center, open-label study to compare the safety and efficacy between afatinib monotherapy and combination therapy of afatinib and HAD-B1 for the locally advanced or metastatic NSCLC patients with EGFR mutations.

BACKGROUND: Afatinib is an epidermal growth factor receptor - tyrosine kinase inhibitor (EGFR-TKI) with proven efficacy for treating patients with advanced or metastatic non-small cell lung cancer (NSCLC). Unfortunately, responses are limited by acquired resistance. Because traditional Korean medicine may have synergistic effects when combined with chemotherapy or radiotherapy, the aim of our study is to elucidate the efficacy and safety of afatinib plus HangAmDan-B1 (HAD-B1) combination therapy in the treatment of patients with NSCLC, as well as EGFR mutations, who need afatinib therapy. METHODS/DESIGN: This study is a randomized, multi-center, open clinical trial. A total of 178 eligible subjects, recruited at 8 centers, are randomly assigned to take Afatinib (20-40 mg) ±â€ŠHAD-B1 (0.972 g/day) for 48 weeks. In the test group, HAD-B1 and afatinib will be used in combination. The primary outcome is a comparison of progression-free survival (PFS) between afatinib monotherapy and afatinib plus HAD-B1 combination therapy in patients with local advanced or metastatic (Stage IIIA, B, C/IV) NSCLC. Secondary outcomes are the overall survival rates, clinical responses, tumor size reductions, health-related qualities of life, and safety. DISCUSSION: The result of this clinical trial will provide evidence for the efficacy and safety of using HAD-B1 in the treatment of EGFR-positive patients with locally advanced or metastatic NSCLC who require afatinib therapy. TRIAL REGISTRATION: Clinical Research Information Service (CRIS), Republic of Korea (ID: KCT0005414), on September 23, 2020.

Arctium lappa root extract containing L-arginine prevents TNF-α-induced early atherosclerosis in vitro and in vivo.

Atherosclerosis is a chronic inflammatory disease affecting the aorta and is a major cause of cardiovascular disease. Arctium lappa root is a plant widely used in traditional Chinese medicine (TCM), and Arctium lappa root extract (ALE) has been reported to exhibit anti-inflammatory capacity and to ameliorate endothelial dysfunction. Thus, we hypothesized that ALE would inhibit the early atherosclerotic stage. In this study, we evaluated the inhibitory effect of ALE on early arteriosclerosis and its mechanisms of action. ALE suppressed TNF-α-induced monocyte adhesion to the vascular endothelium by suppressing NF-κB signaling in HUVECs. In an acute mouse model of atherosclerosis, ALE suppressed TNF-α-induced monocyte infiltration of the vascular endothelium and the expression of genes encoding inflammatory cytokines including IL-1ß, IL-6, TNF-α, and MCP-1 in the mouse aorta. Moreover, inulin-type fructan and amino acids, especially L-aspartate and L-arginine (60.27 and 42.17 mg/g, respectively) were detected by NMR, MALDI-TOF MS, and HPLC analysis as the main components of ALE. Notably, L-arginine suppressed TNF-α-induced monocyte adhesion to HUVECs. Therefore, these results suggest that ALE may be a functional food for the suppression or prevention of early stages of atherosclerosis.

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.

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-κ 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-κ/EGFR axis. Syringic acid might serve as an effective chemopreventive and therapeutic agent against UVB-mediated skin cancer.

Ultrasonicated Lespedeza cuneata extract prevents TNF-α-induced early atherosclerosis in vitro and in vivo.

This study evaluated the use of ultrasonication to extract Lespedeza cuneata as a potential nutraceutical for preventing vascular inflammation. Ultrasonicated L. cuneata extract (ULCE) was prepared using 20% ethanol and 2 h of ultrasonication at room temperature, and its effects were investigated using relevant in vitro and in vivo models. ULCE suppressed tumor necrosis factor-alpha (TNF-α)-induced adhesion capacity, vascular cell adhesion protein 1 (VCAM-1) expression, and nuclear factor kappa-B (NF-κB) activity in human umbilical vein endothelial cells (HUVECs). ULCE also suppressed TNF-α-induced NF-κB signaling pathways and p65 translocation from the cytosol to the nucleus, as well as the mRNA expression of IL-1ß, IL-6, and TNF-α in HUVECs. Oral administration of ULCE suppressed TNF-α-induced monocyte infiltration into the intima and VCAM-1 expression, as well as the IL-1ß, IL-6, TNF-α, and monocyte chemoattractant protein-1 (MCP-1) mRNA expression in the main artery in mice. Among the compounds identified in the hydrolyzed ULCE, quercetin exhibited the strongest inhibitory effect against TNF-α-induced cell adhesion capacity. These results demonstrate that ULCE contains potent preventive factors against early atherosclerosis, which act by suppressing the NF-κB and VCAM-1 signaling axis.

Rice bran supplement prevents UVB-induced skin photoaging in vivo.

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.

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.

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.

Protective effect of Tremella fuciformis Berk extract on LPS-induced acute inflammation via inhibition of the NF-κB and MAPK pathways.

Tremella fuciformis Berk (TFB) has long been used as a traditional medicine in Asia. Although TFB exhibits antioxidant and anti-inflammatory effects, the mechanisms of action responsible have remained unknown. We confirmed the anti-inflammatory effects of Tremella fuciformis Berk extract (TFE) in RAW 264.7 cells and observed significantly suppressed LPS-induced iNOS/NO and COX-2/PGE2 production. TFE also suppressed LPS-induced IKK, IkB, and p65 phosphorylation, as well as LPS-induced translocation of p65 from the cytosol. Additionally, TFE inhibited LPS-induced phosphorylation of MAPKs. In an acute inflammation study, oral administration of TFE significantly inhibited LPS-induced IL-1ß, IL-6 and TNF-α production and iNOS and COX-2 expression. The major bioactive compounds from TFB extract were identified as gentisic acid, protocatechuic acid, 4-hydroxybenzoic acid, and coumaric acid. Among these compounds, protocatechuic acid showed the strongest inhibitory effects on LPS-induced NO production in RAW 264.7 cells. Overall, these results suggest that TFE is a promising anti-inflammatory agent that suppresses iNOS/NO and COX-2/PGE2 expression, as well as the NF-κB and MAPK signaling pathways.

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/cm(2)) 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 p90(RSK) 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.

Effect of Heat Treatment on Properties of Glass Nanocomposite Sealants.

The objective of this study was to investigate the effect of heat treatments on the viscosities and electrical conductivities of glass sealants to be used in solid oxide fuel cells. Glass-based sealants, both with and without an alumina nanopowder added as a nanofiller, were heat treated at temperatures ranging from 750 degrees C to 770 degrees C for periods of up to 240 h. The effects of heat treatments on the viscosities, electrical conductivities and phase transformations of the sealants were investigated. The results showed that alumina nanopowder added to the glass increased both high-temperature electrical conductivities and the viscosities of the sintered glass nanocomposite sealants. However, lengthy heat treatments decreased the electrical conductivities of the glass nanocomposite sealants. This decrease in the conductivities of the heat-treated glass nanocomposites was attributed to the crystallization of glass phase, owing to the dissolution of the alumina nanofiller in the sealing glass.

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.