Sargachromenol Attenuates Inflammatory Responses by Regulating NF-κB and Nrf2 Pathways in RAW 264.7 Cells and LPS-treated Mice.

This study aims to explore the anti-inflammatory mechanisms of sargachromenol in both RAW 264.7 cells and lipopolysaccharide (LPS)-treated mice, as previous reports have suggested that sargachromenol possesses anti-aging, anti-inflammatory, antioxidant, and neuroprotective properties. Although the precise mechanism behind its anti-inflammatory activity remains unclear, pretreatment with sargachromenol effectively reduced the production of nitric oxide, prostaglandin E2, and interleukin (IL)-1ß in LPS-stimulated RAW 264.7 cells by inhibiting cyclooxygenase-2. Moreover, sargachromenol inhibited the activation of nuclear factor-κB (NF-κB) by preventing the degradation of the inhibitor of κB-α (IκB-α) and inhibiting protein kinase B (Akt) phosphorylation in LPS-stimulated cells. We also found that sargachromenol induced the production of heme oxygenase-1 (HO-1) by activating the nuclear transcription factor erythroid-2-related factor 2 (Nrf2). In LPS-treated mice, oral administration of sargachromenol effectively reduced the levels of IL-1ß, IL-6, and tumor necrosis factor-α (TNF-α) in the serum, suggesting its ability to suppress the production of inflammatory mediators by inhibiting the Akt/NF-κB pathway and upregulating the Nrf2/HO-1 pathway.

Decreased retinal and choroidal endothelial surface molecules in spontaneously hypertensive rats.

Hypertension is associated with changes in the retina and choroid, with resulting consequences of increased vascular permeability and microhemorrhages. To date, very little information is available regarding the changes in the retinal and choroidal endothelial surface layer. In this study, we have examined changes in protein expression of several molecules including platelet endothelial cell adhesion molecule-1 (PECAM-1), vascular endothelial cadherin (VE-cadherin), glypican-1, and syndecan-1, in spontaneously hypertensive rats (SHR) compared to control normotensive Wistar Kyoto (WKY) rats. In male SHR vs WKY rat retinas, decreases were found for VE-cadherin and syndecan-1; whereas in female retinas, decreases were found for PECAM-1, glypican-1, and syndecan-1. In male SHR vs WKY rat choroid, we found an increase in glypican-1, but choroidal syndecan-1 was decreased in SHR in both males and females. Therefore, decreases in SHR of both retinal and choroidal syndecan-1 were found in both males and females. These losses of syndecan-1 were accompanied by an increase in plasma levels of the proteoglycan, indicating possible systemic endothelial shedding. In contrast, plasma levels of glypican-1 decreased. Interestingly, in normotensive WKY rats, retinal levels of all four endothelial surface molecules were higher in females than in males, in some cases, by substantial amounts. In summary, a number of changes occur in endothelial surface molecules in SHR, with some changes being sex-dependent; it is possible that the loss of these molecules contributes to the vascular dysfunction that occurs in hypertensive retina and choroid.

Retinal hypoxia and angiogenesis with methamphetamine.

Central retinal artery occlusion, retinopathy, and retinal neovascularization have been reported in methamphetamine (METH) abusers. In the current study, we investigated whether METH induces retinal neovascularization in a mouse model, and if so, whether the neovascularization is associated with increased hypoxia, hypoxia-inducible factor 1α (HIF-1α), and vascular endothelial growth factor (VEGF). Mice were administrated METH by intraperitoneal injection over a 26-day period, or injected with saline as a vehicle control. The number of retinal arterioles and venules were counted using in vivo live imaging following infusion with fluorescein isothiocyanate-dextran. Excised retinas were stained with griffonia simplicifolia lectin I and flat mounted for a measurement of vascularity (length of vessels per tissue area) with AngioTool. Retinal hypoxia was examined by formation of pimonidazole adducts with an anti-pimonidazole antibody, and HIF-1α and VEGFa protein levels in the retina were detected by immunoblot. METH administration increased vascularity (including the number of arterioles) measured on Day 26. Retinal VEGFa protein level was not changed in METH-treated mice on Day 5, but was increased on Day 12 and Day 26. Hypoxia (pimonidazole adduct formation) was increased in retinas of METH-treated mice on Day 12 and Day 26, as were HIF-1α protein expression levels. These results indicate that METH administration induces hypoxia, HIF-1α, VEGFa, and angiogenesis in the retina.

Acute changes in the retina and central retinal artery with methamphetamine.

Methamphetamine (METH), an addictive stimulant of neurotransmitters, is associated with cardiovascular and neurological diseases. METH-induced ophthalmic complications are also present but have been insufficiently investigated. The purpose of this study is to investigate the retinal effects of METH. C57BL/6 mice were administrated progressively increasing doses of METH (0-6 mg/kg) by repetitive intraperitoneal injections for 5 days (4 times per day). Retinal degeneration was examined by morphological changes and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assay. Norepinephrine levels were measured by ELISA, protein expression levels were determined by immunoblot and immunostaining, and gelatinase activity was examined by zymography. The thickness of the retina and the number of nuclei in the inner and outer nuclear layers were decreased by METH. Retinal cell death and astrocyte activation by METH treatment were confirmed by TUNEL assay and glial fibrillary acidic protein expression, respectively. Increased tumor necrosis factor-α protein in the retina and elevated norepinephrine levels in plasma were found in METH-treated mice. Platelet endothelial cell adhesion molecule-1 (PECAM-1) protein expression level was decreased in the retina and central retinal artery (CRA) by METH treatment, along with the endothelial proteoglycans glypican-1 and syndecan-1. Moreover, a regulator of the extracellular matrix, matrix metalloproteinase-14 (MMP-14) in the retina, and MMP-2 and MMP-9 in plasma, were increased by METH treatment. In conclusion, METH administration is involved in retinal degeneration with a vascular loss of PECAM-1 and the glycocalyx in the CRA and retina, and an increase of MMPs.

Hypopigmenting Effects of Brown Algae-Derived Phytochemicals: A Review on Molecular Mechanisms.

There is a rapid increase in the demand for natural hypopigmenting agents from marine sources for cosmeceutical and pharmaceutical applications. Currently, marine macroalgae are considered as a safe and effective source of diverse bioactive compounds. Many research groups are exploring marine macroalgae to discover and characterize novel compounds for cosmeceutical, nutraceutical, and pharmaceutical applications. Many types of bioactive secondary metabolites from marine algae, including phlorotannins, sulfated polysaccharides, carotenoids, and meroterpenoids, have already been documented for their potential applications in the pharmaceutical industry. Among these metabolites, phlorotannins from brown algae have been widely screened for their pharmaceutical and hypopigmenting effects. Unfortunately, the majority of these articles did not have detailed investigations on molecular targets, which is critical to fulfilling the criteria for their cosmeceutical and pharmaceutical use. Very recently, a few meroterpenoids have been discovered from Sargassum sp., with the examination of their anti-melanogenic properties and mechanisms. Despite the scarcity of in vivo and clinical investigations of molecular mechanistic events of marine algae-derived hypopigmenting agents, identifying the therapeutic targets and their validation in humans has been a major challenge for future studies. In this review, we focused on available data representing molecular mechanisms underlying hypopigmenting properties of potential marine brown alga-derived compounds.

Quercetin Mitigates Inflammatory Responses Induced by Vascular Endothelial Growth Factor in Mouse Retinal Photoreceptor Cells through Suppression of Nuclear Factor Kappa B.

Retinal vascular endothelial growth factor (VEGF) increased by neovascularization is well known as a pathogenic factor in ocular neovascular diseases. However, it is still unclear how retinal neurons are damaged by VEGF. The aims of this study are to demonstrate the inflammatory protein expression regulated by VEGF using mouse photoreceptor-derived cells and the protective effect of quercetin against VEGF-induced inflammatory response. Expression and phosphorylation of protein and expression of mRNA were detected by immunoblot and reverse transcriptase polymerase chain reaction. VEGF-induced degradation of limiting membrane and translocation of nuclear factor kappa B (NF-κB) were analyzed by immunocytochemistry. VEGF treatment activated angiogenic signaling pathway in photoreceptor cells. In addition, adhesion molecules and matrix metalloproteinases were increased in VEGF-treated photoreceptor cells. All these events were reversed by quercetin. Zona occludins-1 and ß-catenin decreased by VEGF were recovered by quercetin. NF-κB signaling pathway regulated by VEGF through phosphorylations of mitogen-activated protein kinases (MAPK) and protein kinase B (Akt) was suppressed by quercetin. These results suggest that quercetin suppressed VEGF-induced excessive inflammatory response in retinal photoreceptor cells by inactivation of NF-κB signals through inhibition of MAPKs and Akt. These data may provide a basic information for development of pharmaceuticals or nutraceuticals for treatment of retinal diseases caused by excessive VEGF.

Chondrocyte-derived extracellular matrix suppresses pathogenesis of human pterygium epithelial cells by blocking the NF-κB signaling pathways.

PURPOSE: We previously have reported that chondrocyte-derived extracellular matrix (CDECM) suppresses the growth of pterygium in athymic nude mice. The aim of this study is to demonstrate the effect of CDECM on the pterygium epithelial cells and molecular signaling pathways in human primary pterygium epithelial cells (hPECs). METHODS: Human conjunctival epithelial cells (hConECs) were used for identification of the effect of CDECM on normal conjunctiva. The effects of CDECM on proliferation were measured with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfenyl)-2H-tetrazolium (MTS) assay. Cell migration was evaluated according to the scratch wound closure assay and the Transwell invasion assay. Pterygium-related angiogenesis, inflammation, and extracellular matrix remodeling were analyzed with immunoblot and enzyme-linked immunosorbent assay (ELISA). The level of oxidative stress was detected with 2',7'-dichlorofluorescein diacetate (DCFH-DA). Protein kinase signaling was also analyzed with immunoblot. RESULTS: CDECM did not show cytotoxicity until 1 mg/ml in the hConECs and hPECs. Cell migration and invasion were markedly reduced by treatment of 1 mg/ml CDECM in the hPECs to 34% of the control, but not in the hConECs. CDECM significantly downregulated matrix metallopeptidase 9 (MMP-9) and fibronectin and upregulated tissue inhibitor of metalloprotease 1 (TIMP-1) and -2 in the hPECs. Angiogenic factors, such as vascular endothelial growth factor (VEGF), antivascular cellular adhesion molecule 1 (VCAM-1), and cluster of differentiation 31 (CD31), and proinflammatory factors, including tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (Cox2), interleukin 6 (IL-6), and prostaglandin E2 (PGE2), were dramatically reduced by CDECM in the hPECs. Furthermore, CDECM significantly inhibited the generation of intracellular reactive oxygen species and the expression of NADPH oxidase subunits, Nox2 and p47phox. CDECM induced nuclear factor erythroid-2 related factor 2 (Nrf2) mediated-antioxidant enzyme heme oxygenase-1 (HO-1). CDECM also suppressed nuclear factor-kappa B (NF-κB) activation and the phosphorylation of p38 mitogen-activated protein kinase (MAPK), protein kinase C alpha (PKCα), and PKCθ. CONCLUSIONS: CDECM was markedly effective in pathogenesis of hPECs. CDECM-suppressed migration of hPECs resulted from the inhibition of NF-κB activation and the improvement of Nrf2 induction by blocking the p38 MAPK and PKC signaling pathways.

Molecular cloning and characterization of a lysozyme cDNA from the mole cricket Gryllotalpa orientalis (Orthoptera: Gryllotalpidae).

A full-length lysozyme cDNA from Gryllotalpa orientalis was cloned and sequenced. The deduced amino acid sequence of the lysozyme protein was 143 amino acids in length, with a calculated molecular mass of 15.84 kDa and an isoelectric point of 4.74. Sequence motifs, together with alignment and phylogenetic results, confirmed that G. orientalis lysozyme belongs to the C (chicken)-type lysozyme family of proteins. The protein sequence of lysozyme from G. orientalis showed high identity to that of Drosophila melanogaster (51.7 %); however, in contrast to D. melanogaster lysozyme, G. orientalis lysozyme was immune inducible and expressed in a wide range of tissues. Expression of G. orientalis lysozyme mRNA was highest at 8 h post-infection and subsequently decreased with time after bacterial infection. We also expressed G. orientalis lysozyme protein in vitro using the pET expression system. Compared with the negative control, over-expressed G. orientalis lysozyme showed antimicrobial activity against Gram-negative bacteria Escherichia coli and Gram-positive bacteria Bacillus subtilis by radial diffusion assay, with minimal inhibitory concentration values of 30.3 and 7.55 µM, respectively. These results indicate that G. orientalis lysozyme may have stronger antimicrobial activity than other lysozymes against a broad range of microorganisms.

Anti-inflammatory effects of sargachromenol-rich ethanolic extract of Myagropsis myagroides on lipopolysaccharide-stimulated BV-2 cells.

BACKGROUND: Excessive pro-inflammatory cytokine production from activated microglia contributes to neurodegenerative diseases, thus, microglial inactivation may delay the progress of neurodegeneration by attenuating the neuroinflammation. Among 5 selected brown algae, we found the highest antioxidant and anti-neuroinflammatory activities from Myagropsis myagroides ethanolic extract (MME) in lipopolysaccharide (LPS)-stimulated BV-2 cells. METHODS: The levels of nitric oxide (NO), prostaglandin E2 (PGE2), and pro-inflammatory cytokines were measured by Griess assay and enzyme linked immunesorbent assay. The levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), mitogen-activated protein kinases (MAPKs), and Akt were measured using Western blot. Nuclear translocation and transcriptional activation of nuclear factor-κB (NF-κB) were determined by immunefluorescence and reporter gene assay, respectively. RESULTS: MME inhibited the expression of iNOS and COX-2 at mRNA and protein levels, resulting in reduction of NO and PGE2 production. As a result, pro-inflammatory cytokines were reduced by MME. MME also inhibited the activation and translocation of NF-κB by preventing inhibitor κB-α (IκB-α) degradation. Moreover, MME inhibited the phosphorylation of extracellular signal regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs). Main anti-inflammatory compound in MME was identified as sargachromenol by NMR spectroscopy. CONCLUSIONS: These results indicate that the anti-inflammatory effect of sargachromenol-rich MME on LPS-stimulated microglia is mainly regulated by the inhibition of IκB-α/NF-κB and ERK/JNK pathways.

eckol enhances heme oxygenase-1 expression through activation of Nrf2/JNK pathway in HepG2 cells.

Eckol isolated from Ecklonia stolonifera was previously reported to exhibit cytoprotective activity with its intrinsic antioxidant activity in in vitro studies. In this study, we characterized the mechanism underlying the eckol-mediated the expression of heme oxygenase-1 (HO-1). Eckol suppressed the production of intracellular reactive oxygen species and increased glutathione level in HepG2 cells. Eckol treatment enhanced the expression of HO-1 at the both level of protein and mRNA in HepG2 cells. Enhanced expression of HO-1 by eckol was presumed to be the activation of the nuclear factor erythroid-derived 2-like 2 (Nrf2) demonstrated by its nuclear translocation and increased transcriptional activity. c-Jun NH2-terminal kinases (JNKs) and PI3K/Akt contributed to Nrf2-mediated HO-1 expression. These results demonstrate that the eckol-mediated expression of HO-1 in HepG2 cells is regulated by Nrf2 activation via JNK and PI3K/Akt signaling pathways, suggesting that eckol may be used as a natural antioxidant and cytoprotective agent.

Anthocyanins inhibit lipogenesis during adipocyte differentiation of 3T3-L1 preadipocytes.

Anthocyanins have been shown to suppress body weight and fat mass in animal studies. However, the effect of anthocyanins on the process of lipid accumulation during adipocyte differentiation is not fully understood and the lipogenic transcription factors regulated by anthocyanins have not been identified. We investigated the effects of anthocyanins on lipogenesis pathways during adipocyte differentiation in 3T3-L1 cells. Anthocyanins reduced triglyceride (TG) accumulation in a dose-dependent manner during adipocyte differentiation. Accumulation of TG was rapidly reversed by anthocyanin withdrawal. Anthocyanins markedly reduced gene and protein expression levels of lipogenic transcription factors such as liver X receptor α, sterol regulatory element-binding protein-1c, peroxisome proliferators-activated receptor-γ, and CCAAT enhancer-binding protein-α. In addition, the target gene and protein expression of these lipogenic transcription factors such as fatty acid synthase, stearoyl-CoA desaturase-1, and acetyl-CoA carboxylase α were markedly suppressed by anthocyanins. Thus, anthocyanins suppress lipid accumulation in adipocytes due to broad inhibition of the transcription factors regulating lipogenesis. This may partially explain the mechanism by which anthocyanins exert their anti-obesity effect.

High Glucose Induces Oxidative Stress That Alters Glycocalyx Proteoglycan Levels in Primary Rat Retinal Microvascular Endothelial Cells and in Isolated Ophthalmic Arteries.

Our purpose in this study was to identify the role played by oxidative stress in the changes to proteoglycans that occur under hyperglycemic conditions, using primary rat retinal microvascular endothelial cells (RRMEC) and cultured ophthalmic arteries. The cells and blood vessels obtained from rats were cultured in normal glucose (5.6 mM) and high glucose (25 mM) with or without N-acetylcysteine (NAC), an antioxidant. Intracellular oxidative stress was determined by measuring dihydroethidium (DHE) fluorescence and malondialdehyde (MDA)-modified protein levels. mRNA and protein levels were evaluated using quantitative real-time polymerase chain reaction and immunoblot, respectively. High glucose increased levels of glypican-1 mRNA and protein. The level of syndecan-1 mRNA also was increased, but its protein level was decreased, by high glucose. Evaluation of DHE and MDA showed that high glucose increased oxidative stress. These changes caused by high glucose were significantly reversed by NAC treatment. Matrix metalloproteinase-9 (MMP-9) levels, which increased under high glucose conditions, were suppressed by NAC treatment. Oxidative stress caused by hyperglycemia may be responsible for significant changes to the ocular endothelial glycocalyx.

Enhanced antibacterial activity of an attacin-coleoptericin hybrid protein fused with a helical linker.

Previously, we isolated and characterized attacin from Spodoptera exigua and a coleoptericin-like protein from Protaetia brevitarsis seulensis. In this study, we fused these two genes encoding antimicrobial proteins to obtain a hybrid protein with enhanced antimicrobial activity. To fuse the two antimicrobial proteins, we employed helical and non-helical linker sequences that function as inter-domain linkers in proteins. We used the Gly-Gly-Gly-Gly-Ser peptide as a non-helical linker. The hybrid protein produced using this linker showed less antimicrobial activity against Escherichia coli, Bacillus subtilis, Burkholderia glumae, Pseudomonas corrugate, and Erwinia rhapontici than either of the two parental antimicrobial proteins. In addition, the MIC value of the hybrid protein was 23.1 µM, which indicates poor activity against E. coli. When we used three Glu-Ala-Ala-Ala-Lys (EAAAK) peptide sequences as a helical linker to fuse the two proteins, the resultant hybrid protein had much higher antimicrobial activity than the parental antimicrobial proteins. In particular, this hybrid protein had strong antimicrobial activity against P. corrugate. These results indicate that the EAAAK motif can be used to effectively separate two antimicrobial proteins and produce a hybrid protein with more antimicrobial activity than either of the parent proteins.

Hexane fraction from Laminaria japonica exerts anti-inflammatory effects on lipopolysaccharide-stimulated RAW 264.7 macrophages via inhibiting NF-kappaB pathway.

PURPOSE: Laminaria japonica is a representative marine brown alga used as a culinary item in East Asia. L. japonica extract was shown to exert various biological activities; however, its anti-inflammatory activity has not been reported. The aim of this study is to investigate the molecular mechanisms underlying its anti-inflammatory action. METHODS: Anti-inflammatory mechanisms of L. japonica n-hexane fraction (LHF) were assessed using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. An anti-inflammatory compound isolated from LHF by reverse-phase chromatography was identified using nuclear magnetic resonance (NMR) spectroscopy. RESULTS: Our results indicate that LHF significantly inhibited LPS-stimulated nitric oxide (NO) and prostaglandin E(2) (PGE(2)) secretion in a dose-dependent manner and suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) with no cytotoxicity. As results, levels of pro-inflammatory cytokines were significantly reduced by pretreatment of LHF in LPS-stimulated RAW 264.7 cells. Treatment of LHF strongly suppressed nuclear factor-κB (NF-κB) promoter-driven expression and nuclear translocation of NF-κB by preventing proteolytic degradation of inhibitor of κB (IκB)-α in LPS-stimulated RAW 264.7 cells. Moreover, LHF inhibited the phosphorylation of Akt and mitogen-activated protein kinase (MAPK) in LPS-stimulated RAW 264.7 cells. One of the anti-inflammatory compounds was isolated from LHF and identified as fucoxanthin. CONCLUSIONS: These results indicate that the LHF-mediated inhibition of NO and PGE(2) secretion in LPS-stimulated macrophages is regulated by NF-κB inactivation through inhibition of IκB-α, MAPKs, and Akt phosphorylation. LHF may be considered as a functional food candidate for the prevention or treatment of inflammatory diseases.

Skin-Whitening Effect of a Callus Extract of Nelumbo nucifera Isolate Haman.

The sacred lotus (Nelumbo nucifera Gaertn. Isolate Haman, in the family Nelumbonaceae) used in this study originated from the Haman region of Korea, and lotus seeds dating back to the Goryeo Dynasty (650-760 years ago) were accidentally discovered. Lotus is known to possess antioxidant, anti-inflammatory, and soothing properties. Instead of using the lotus alone, we obtained extracts using Haman region lotus-derived callus (HLC), which allowed for a controlled, quantitative, and infinite supply. Based on the reported effects of the lotus, we formulated a hypothesis to investigate the skin-whitening effect of the HLC extract (HLCE). The HLCE was first obtained by extraction with distilled water and using 5% propanediol as a solvent and subsequently verified for the whitening effect (melanin content tests) using mammalian cells in vitro. Its efficacy at the molecular level was confirmed through real-time polymerase chain reaction (PCR) using melanin-related genes. Furthermore, clinical trials with 21 volunteers confirmed the significant whitening effect of cosmetics containing the HLCE. In conclusion, we found that the HLCE not only has anti-inflammatory, antioxidant, and skin-soothing properties but also plays an essential role in skin whitening. Therefore, we propose that the HLCE has the potential to become a new raw material for the cosmetic industry.

Dioxinodehydroeckol inhibits melanin synthesis through PI3K/Akt signalling pathway in α-melanocyte-stimulating hormone-treated B16F10 cells.

Antimelanogenic activity has previously been reported in ethyl acetate fraction of Ecklonia stolonifera. In this study, using the isolated dioxinodehydroeckol from the fraction, we sought to investigate an antimelanogenic signalling pathway in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16F10 melanoma cells. Treatment with dioxinodehydroeckol inhibited the cellular melanin contents and expression of melanogenesis-related proteins, including microphthalmia-associated transcription factor (MITF), tyrosinase and tyrosinase-related proteins TRP-1 and TRP-2. Moreover, dioxinodehydroeckol stimulated phosphorylation of Akt in a dose-dependent manner without affecting phosphorylation of ERK. These data suggest that dioxinodehydroeckol reduces melanin synthesis through the MITF regulation dependent upon PI3K/Akt signalling pathway.

Anti-inflammatory effect of ethanolic extract from Myagropsis myagroides on murine macrophages and mouse ear edema.

BACKGROUND: This study aims to investigate anti-inflammatory effect of ethanolic extract of Myagropsis myagroides (EMM) in the lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and the phorbol 12-myristate 13-acetate (PMA)-induced ear edema in mice, and to clarify its underlying molecular mechanisms. METHODS: The levels of nitric oxide (NO), prostaglandin E2 (PGE2), and pro-inflammatory cytokines were measured by Griess assay and enzyme linked immunosorbent assay. The expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), mitogen-activated protein kinases (MAPKs), and Akt were measured using Western blotting. Nuclear translocation and transcriptional activation of nuclear factor-κB (NF-κB) were determined by immunocytochemistry and reporter gene assay, respectively. PMA-induced mouse ear edema was used as the animal model of inflammation. Anti-inflammatory compounds in EMM were isolated using high-performance liquid chromatography and identified by nuclear magnetic resonance. RESULTS: EMM significantly inhibited the production of NO, PGE2, and pro-inflammatory cytokines in a dose-dependent manner and suppressed the expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells. EMM strongly suppressed nuclear translocation of NF-κB by preventing degradation of inhibitor of κB-α as well as by inhibiting phosphorylation of Akt and MAPKs. EMM reduced ear edema in PMA-induced mice. One of the anti-inflammatory compounds in EMM was identified as 6,6'-bieckol. CONCLUSIONS: These results suggest that the anti-inflammatory properties of EMM are associated with the down-regulation of iNOS, COX-2, and pro-inflammatory cytokines through the inhibition of NF-κB pathway in LPS-stimulated macrophages.

Retinal Physiology and Circulation: Effect of Diabetes.

In this article, we present a discussion of diabetes and its complications, including the macrovascular and microvascular effects, with the latter of consequence to the retina. We will discuss the anatomy and physiology of the retina, including aspects of metabolism and mechanisms of oxygenation, with the latter accomplished via a combination of the retinal and choroidal blood circulations. Both of these vasculatures are altered in diabetes, with the retinal circulation intimately involved in the pathology of diabetic retinopathy. The later stages of diabetic retinopathy involve poorly controlled angiogenesis that is of great concern, but in our discussion, we will focus more on several alterations in the retinal circulation occurring earlier in the progression of disease, including reductions in blood flow and a possible redistribution of perfusion that may leave some areas of the retina ischemic and hypoxic. Finally, we include in this article a more recent area of investigation regarding the diabetic retinal vasculature, that is, the alterations to the endothelial surface layer that normally plays a vital role in maintaining physiological functions. © 2020 American Physiological Society. Compr Physiol 10:933-974, 2020.

Protective effects of flavonoids isolated from Korean milk thistle Cirsium japonicum var. maackii (Maxim.) Matsum on tert-butyl hydroperoxide-induced hepatotoxicity in HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Milk thistle leaves and flowers have been traditionally used as herbal remedy to alleviate liver diseases for decades. Korean milk thistle, Cirsium japonicum var. maackii (Maxim.) Matsum has been employed in traditional folk medicine as diuretic, antiphlogistic, hemostatic, and detoxifying agents. AIM OF THE STUDY: The aim of current investigation was to evaluate hepatoprotective properties of the MeOH extract of the roots, stems, leaves and flowers of Korean milk thistle as well as four isolated flavonoids, luteolin, luteolin 5-O-glucoside, apigenin and apigenin 7-O-glucuronide during t-BHP-induced oxidative stress in HepG2 cells. MATERIALS AND METHODS: Hepatoprotective potential of the MeOH extracts and flavonoids derived from Korean milk thistle against t-BHP-induced oxidative stress in HepG2 cells were evaluated following MTT method. Incubating HepG2 cells with t-BHP markedly decreased the cell viability and increased the intracellular ROS generation accompanied by depleted GSH levels. Protein expression of heme oxygenase (HO-1) and nuclear factor-E2-related factor 2 (Nrf-2) was determined by Western blot. RESULTS: Our findings revealed that pretreating HepG2 cells with MeOH extracts and bioactive flavonoids significantly attenuated the t-BHP-induced oxidative damage, followed by increased cell viability in a dose-dependent manner. The results illustrate that excess ROS generation was reduced and GSH levels increased dose-dependently when HepG2 cells were pretreated with four flavonoids. Moreover, Western blotting analysis demonstrated that protein expressions of Nrf-2 and HO-1 were also up-regulated by flavonoids treatment. CONCLUSIONS: These results clearly demonstrate that the MeOH extracts and flavonoids from Korean milk thistle protected HepG2 cells against oxidative damage triggered by t-BHP principally by modulating ROS generation and restoring depleted GSH levels in addition to the increased Nrf-2/HO-1 signaling cascade. These flavonoids are potential natural antioxidative biomarkers against oxidative stress-induced hepatotoxicity.

Interphotoreceptor Retinoid-Binding Protein Mitigates Cellular Oxidative Stress and Mitochondrial Dysfunction Induced by All-trans-Retinal.

PURPOSE: Point and null mutations in interphotoreceptor retinoid-binding protein (IRBP) cause retinal dystrophy in affected patients and IRBP-deficient mice with unknown mechanism. This study investigated whether IRBP protects cells from damages induced by all-trans-retinal (atRAL), which was increased in the Irbp(-/-) retina. METHODS: Wild-type and Irbp(-/-) mice retinal explants in buffer with or without purified IBRP were exposed to 800 lux light for different times and subjected to retinoid analysis by high-performance liquid chromatography. Purity of IRBP was determined by Coomassie Brilliant Blue staining and immunoblot analysis. Cellular damages induced by atRAL in the presence or absence of IRBP were evaluated in the mouse photoreceptor-derived 661W cells. Cell viability and death were measured by 3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and TUNEL assays. Expression and modification levels of retinal proteins were determined by immunoblot analysis. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) were detected with fluorogenic dyes and confocal microscopy. Mitochondrial membrane potential was analyzed by using JC-1 fluorescent probe and a flow cytometer. RESULTS: Content of atRAL in Irbp(-/-) retinal explants exposed to light for 40 minutes was significantly higher than that in wild-type retinas under the same light conditions. All-trans-retinal caused increase in cell death, tumor necrosis factor activation, and Adam17 upregulation in 661W cells. NADPH oxidase-1 (NOX1) upregulation, ROS generation, NO-mediated protein S-nitrosylation, and mitochondrial dysfunction were also observed in 661W cells treated with atRAL. These cytotoxic effects were significantly attenuated in the presence of IRBP. CONCLUSIONS: Interphotoreceptor retinoid-binding protein is required for preventing accumulation of retinal atRAL, which causes inflammation, oxidative stress, and mitochondrial dysfunction of the cells.