Chloroform Fraction of Prasiola japonica Ethanolic Extract Alleviates UPM 1648a-Induced Lung Injury by Suppressing NF-κB Signaling

Prasiola japonica is an edible alga, and the ethanol extract of P. japonica (Pj-EE) possesses various biological activities. Interestingly, in a recent study, we observed the potent anti-inflammatory activity of the chloroform fraction of Pj-EE (Pj-EE-CF). Thus, to extend the application of Pj-EE-CF, we further studied its effects on lung injury. To establish an experimental model of lung injury, we nasally administered urban particulate matter UPM 1648a (50 mg/kg) to mice. In addition, BEAS-2B cells were treated with 300 µg/mL of UPM 1648a for in vitro analysis. Intranasal administration of UPM 1648a increased lung injury score, macrophage infiltration, and upregulation of the inflammatory enzyme inducible nitric oxide synthase (iNOS) in lung tissues. On the other hand, oral administration of Pj-EE-CF (25, 50, and 100 mg/kg) alleviated these pathological features as assessed by lung wet/dry ratio, lung injury score, bronchoalveolar lavage fluid (BALF) protein amount in the lung tissues up to 70%, 95%, and 99%, respectively. In addition, Pj-EE-CF down-regulated the release of inflammatory cytokines, interleukins (ILs), tumor necrosis factor (TNF)-α, and interferon (IFN)-γ elevated by UPM 1648a in the lung tissues and lung BALF up to 95%. According to Western blot and luciferase assay, Pj-EE-CF (100 mg/kg in vivo or 50 and 100 µg/mL in vitro) significantly reduced the nuclear factor-κB (NF-κB) signal activated by UPM 1648a. Finally, UPM 1648a increased cellular reactive oxygen species (ROS) levels in BEAS-2B cells, while Pj-EE-CF reduced them. These results suggest that Pj-EE-CF alleviates UPM 1648a-induced lung damage via anti-inflammatory and antioxidant activities and by suppressing NF-κB signaling. In conclusion, these observations imply that Pj-EE-CF could be a practical component of food supplements to mitigate air pollution-derived lung damage.

Anti-Inflammatory Activities of the Ethanol Extract of Prasiola japonica, an Edible Freshwater Green Algae, and Its Various Solvent Fractions in LPS-Induced Macrophages and Carrageenan-Induced Paw Edema via the AP-1 Pathway.

Prasiola japonica possesses several biological activities. However, reports on the anti-inflammatory activities and molecular mechanisms of its different solvent fractions remain limited. In this study, we investigated the potential anti-inflammatory activities of P. japonica ethanol extract (Pj-EE) and four solvent fractions of Pj-EE made with hexane (Pj-EE-HF), chloroform (Pj-EE-CF), butanol (Pj-EE-BF), or water (Pj-EE-WF) in both in vitro (LPS-induced macrophage-like RAW264.7 cells) and in vivo (carrageenan-induced acute paw edema mouse models) experiments. The most active solvent fraction was selected for further analysis. Various in vitro and in vivo assessments, including nitric oxide (NO), cytokines, luciferase assays, real-time polymerase chain reactions, and immunoblotting analyses were performed to evaluate the underlying mechanisms. In addition, the phytochemical constituents were characterized by Liquid chromatography-tandem mass spectrometry. In in vitro studies, the highest inhibition of NO production was observed in Pj-EE-CF. Further examination revealed that Pj-EE-CF decreased the expression of inflammation-related cytokines in LPS-induced RAW264.7 cells and suppressed subsequent AP-1-luciferase activity by inhibition of phosphorylation events in the AP-1 signaling pathway. Pj-EE-CF treatment also demonstrated the strongest reduction in thickness and volume of carrageenan-induced paw edema, while Pj-EE-BF showed the lowest activity. Furthermore, Pj-EE-CF also reduced gene expression and cytokines production in tissue lysates of carrageenan-induced paw edema. These findings support and validate the evidence that Pj-EE, and especially Pj-EE-CF, could be a good natural source for an anti-inflammatory agent that targets the AP1 pathway.

Anti-Inflammatory Functions of Alverine via Targeting Src in the NF-κB Pathway.

Alverine, a smooth muscle relaxant, is used to relieve cramps or spasms of the stomach and intestine. Although the effects of alverine on spontaneous and induced contractile activity are well known, its anti-inflammatory activity has not been fully evaluated. In this study, we investigated the anti-inflammatory effects of alverine in vitro and in vivo. The production of nitric oxide (NO) in RAW264.7 cells activated by lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly (I:C)) was reduced by alverine. The mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and tumor necrosis factor-α (TNF-α) was also dose-dependently inhibited by treatment with alverine. In reporter gene assays, alverine clearly decreased luciferase activity, mediated by the transcription factor nuclear factor κB (NF-κB) in TIR-domain-containing adapter-inducing interferon-ß (TRIF)- or MyD88-overexpressing HEK293 cells. Additionally, phosphorylation of NF-κB subunits and upstream signaling molecules, including p65, p50, AKT, IκBα, and Src was downregulated by 200 µM of alverine in LPS-treated RAW264.7 cells. Using immunoblotting and cellular thermal shift assays (CETSAs), Src was identified as the target of alverine in its anti-inflammatory response. In addition, HCl/EtOH-stimulated gastric ulcers in mice were ameliorated by alverine at doses of 100 and 200 mg/kg. In conclusion, alverine reduced inflammatory responses by targeting Src in the NF-κB pathway, and these findings provide new insights into the development of anti-inflammatory drugs.

Anti-Apoptotic and Anti-Inflammatory Activities of Edible Fresh Water Algae Prasiola japonica in UVB-Irradiated Skin Keratinocytes.

Skin is the outer tissue layer and is a barrier protecting the body from various external stresses. The fresh water green edible algae Prasiola japonica has antiviral, antimicrobial, and anti-inflammatory properties; however, few studies of its effects on skin-protection have been reported. In this study, Prasiola japonica ethanol extract (Pj-EE) was prepared, and its skin-protective properties were investigated in skin keratinocytes. Pj-EE inhibited ROS production in UVB-irradiated HaCaT cells without cytotoxicity. Pj-EE also suppressed the apoptotic death of UVB-irradiated HaCaT cells by decreasing the generation of apoptotic bodies and the proteolytic activation of apoptosis caspase-3, -8, and -9. Moreover, Pj-EE downregulated the mRNA expression of the inflammatory gene cyclooxygenase-2 (COX-2), the pro-inflammatory cytokine genes interleukin (IL)-1ß, IL-8, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ, and the tissue remodeling genes matrix metalloproteinase (MMP)-1, -2, -3, and -9. The Pj-EE-induced anti-inflammatory effect was mediated by suppressing the activation of nuclear factor-kappa B (NF-κB) signaling pathway in the UVB-irradiated HaCaT cells. Taken together, these results suggest that Pj-EE exerts skin-protective effects through anti-oxidant, anti-apoptotic, and anti-inflammatory activities in skin keratinocytes.

Loliolide Presents Antiapoptosis and Antiscratching Effects in Human Keratinocytes.

Loliolide is a monoterpenoid hydroxylactone present in freshwater algae that has anti-inflammatory and antiaging activity; however, its effects on ultraviolet-damaged skin have yet to be elucidated. This study investigated the antiapoptosis and wound-healing effects of loliolide using HaCaT cells (a human keratinocyte cell line). Loliolide inhibited the expression of reactive oxygen species (ROS) induced by ultraviolet radiation as well as wrinkle formation-related matrix metalloproteinase genes and increased the expression of the damage repair-related gene SIRT1. The apoptosis signaling pathway was confirmed by Western blot analysis, which showed that loliolide was able to reduce the expression of caspases 3, 8, and 9, which are related to ROS-induced apoptosis. In addition, Western blotting, reverse-transcription polymerase chain reaction (PCR), and real-time PCR analyses showed that loliolide enhanced the expression of the epidermal growth factor receptor signaling pathway (PI3K, AKT) and migration factors, such as K6, K16, and K17; keratinocyte growth factor; and inflammatory cytokines, such as interleukin (IL)-1, IL-17, and IL-22 expressed during the cellular scratching process, suggesting a putative wound-healing ability. Because of the antiapoptosis and antiscratching effects on skin of both loliolide and loliolide-rich Prasiola japonica ethanol extract, we consider the former to be an important compound used in the cosmeceutical industry.

Oxidative Stress-Protective and Anti-Melanogenic Effects of Loliolide and Ethanol Extract from Fresh Water Green Algae, Prasiola japonica.

Loliolide is a monoterpenoid hydroxylactone found in many algae, including fresh water green algae, Prasiola japonica. To date, loliolide and compounds in P. japonica have not been studied systematically with respect to skin pharmacology. In this study, we investigated oxidative stress-protective and anti-melanogenic effects of loliolide and P. japonica ethanol extract (Pj-EE), known to contain loliolide, in human keratinocyte (HaCaT) cells and mouse melanoma (B16F10) cells. Loliolide suppressed the transcription of genes encoding matrix metalloproteinases (MMPS), which were induced in HaCaT cells by hydrogen peroxide (H2O2) treatment. Loliolide and Pj-EE not only reduced the melanin secretion and content in B16F10 cells but also increased the expression of the antioxidant proteins nuclear factor (erythroid-derived 2)-like 2 (NRF2) and heme oxygenase-1 (HO-1) in HaCaT cells subjected to H2O2 treatment. Furthermore, loliolide and Pj-EE decreased expression of the anti-melanogenic protein microphthalmia-associated transcription factor (MITF) and tyrosinase in B16F10 cells subjected to α-melanocyte-stimulating hormone (α-MSH) treatment. Our findings demonstrate that loliolide and Pj-EE have antioxidant and anti-melanogenic effects on skin.

Melanogenesis inhibitory activity of Korean Undaria pinnatifida in mouse B16 melanoma cells.

A number of seaweed species are used as traditional foods and medicine in different parts of the world, including Asian countries. However, very few data on the anti-melanogenic effect of seaweed have been published. Undaria pinnatifida (Dolmiyeok), a brown alga, is a traditional food in Jeju Island, the southern regions of the Korea peninsula. In this study, ethylacetate extracts of U. pinnatifida (UPE) were examined for their anti-melanogenic potentials. Our results supports the finding that UPE down-regulated melanin content in a dose-dependent pattern. To clarify the target of UPE action in melanogenesis, we performed Western blotting for tyrosinase and microphthalmia-associated transcription factor (MITF), which are key melanogenic enzymes. UPE inhibited tyrosinase and MITF expressions in a dose-dependent manner. These results indicate that treatment with UPE significantly inhibits the melanogenesis in B16 cells, and may be effective in the whitening agent for the skin.

Protective effect of the ethyl acetate fraction of Sargassum muticum against ultraviolet B-irradiated damage in human keratinocytes.

The aim of this study was to investigate the cytoprotective properties of the ethyl acetate fraction of Sargassum muticum (SME) against ultraviolet B (UVB)-induced cell damage in human keratinocytes (HaCaT cells). SME exhibited scavenging activity toward the 1,1-diphenyl-2-picrylhydrazyl radicals and hydrogen peroxide (H(2)O(2)) and UVB-induced intracellular reactive oxygen species (ROS). SME also scavenged the hydroxyl radicals generated by the Fenton reaction (FeSO(4) + H(2)O(2)), which was detected using electron spin resonance spectrometry. In addition, SME decreased the level of lipid peroxidation that was increased by UVB radiation, and restored the level of protein expression and the activities of antioxidant enzymes that were decreased by UVB radiation. Furthermore, SME reduced UVB-induced apoptosis as shown by decreased DNA fragmentation and numbers of apoptotic bodies. These results suggest that SME protects human keratinocytes against UVB-induced oxidative stress by enhancing antioxidant activity in cells, thereby inhibiting apoptosis.

Inhibitory effect of Jeju endemic seaweeds on the production of pro-inflammatory mediators in mouse macrophage cell line RAW 264.7.

Seaweed has been used in traditional cosmetics and as a herbal medicine in treatments for cough, boils, goiters, stomach ailments, and urinary diseases, and for reducing the incidence of tumors, ulcers, and headaches. Despite the fact that seaweeds are frequently used in the practice of human health, little is known about the role of seaweed in the context of inflammation. This study aimed to investigate the influence of Jeju endemic seaweed on a mouse macrophage cell line (RAW 264.7) under the stimulation of lipopolysaccharide (LPS). Ethyl acetate extracts obtained from 14 different kinds of Jeju seaweeds were screened for inhibitory effects on pro-inflammatory mediators. Our results revealed that extracts from five seaweeds, Laurencia okamurae, Grateloupia elliptica, Sargassum thunbergii, Gloiopeltis furcata, and Hizikia fusiformis, were potent inhibitors of the production of pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E(2) (PGE(2)), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha). Based on these results, the anti-inflammatory effects and low cell toxicity of these seaweed extracts suggest potential therapeutic applications in the regulation of the inflammatory response.