Search

Objective: To evaluate the effects of Catalpa bignonioides fruit extract on the promotion of muscle growth and muscular capacity in vitro and in vivo. Methods: Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell proliferation was assessed using a 5-bromo-2’-deoxyuridine (BrdU) assay kit. Western blot analysis was performed to determine the protein expressions of related factors. The effects of Catalpa bignonioides extract were investigated in mice using the treadmill exhaustion test and whole-limb grip strength assay. Chemical composition analysis was performed using high-performance liquid chromatography (HPLC). Results: Catalpa bignonioides extract increased the proliferation of C2C12 mouse myoblasts by activating the Akt/mTOR signaling pathway. It also induced metabolic changes, increasing the number of mitochondria and glucose metabolism by phosphorylating adenosine monophosphate-activated protein kinase. In an in vivo study, the extract-treated mice showed improved motor abilities, such as muscular endurance and grip strength. Additionally, HPLC analysis showed that vanillic acid may be the main component of the Catalpa bignonioides extract that enhanced muscle strength. Conclusions: Catalpa bignonioides improves exercise performance through regulation of growth and metabolism in skeletal muscles, suggesting its potential as an effective natural agent for improving muscular strength.

Summary: Aging triggers spinal degeneration, including common spinal stenosis, which causes back and leg pain in older individuals, significantly impacting their quality of life. Here, we explored aging traits in turquoise killifish spines, potentially offering a model for age-linked spinal stenosis in humans. Aged turquoise killifish exhibited body shape deformation and increased vertebral collapse, which was further accelerated by spawning. High-resolution CT scans revealed suppressed cortical bone thickness and hemal arch area in vertebrae due to spawning, and osteophyte formation was observed in both aged and breeding fish populations. Scale mineralization mirrored these changes, increasing with age but being suppressed by spawning. The expression of sp7, sox9b, axin1, and wnt4a/b genes can be utilized to monitor age- and reproduction-dependent spine deformation. This study demonstrates that turquoise killifish and humans share certain phenotypes of age-related vertebral abnormalities, suggesting that turquoise killifish could serve as a potential model for studying human spinal stenosis.

The present study investigated the anti-inflammatory potential of phlorofucofuroeckol-A (PFF-A) from Ecklonia cava in TNF-α/IFN-γ-stimulated HaCaT keratinocytes and an acute ear edema mice model. Upon investigation, PFF-A had no cytotoxicity up to 62.5 μg/mL and indicated a dose-dependent-cytoprotective effect with a reduction of intracellular ROS in TNF-α/IFN-γ-stimulated HaCaT keratinocytes. PFF-A downregulated the mRNA expression of inflammatory cytokines (IL-25, IL-33, TSLP, IL-1β, IL-6, IL-8, IL-13, IFN-γ, and TNF-α) and chemokines (Eotaxin, MDC, RANTES, and TARC) in TNF-α/IFN-γ-stimulated HaCaT keratinocytes. Furthermore, PFF-A inhibited the activation of the NFκB/MAPK signaling whereas activated the Nrf2/HO-1 signaling. As an extended evaluation, PFF-A treatment significantly decreased the ear thickness and the expression of inflammatory mediators including iNOS and COX-2 in the TPA-induced ear edema mice model. This study suggests the anti-inflammatory potential of PFF-A in the skin and the potential of E. cava consisting of PFF-A to develop functional foods that have effects on abnormal immune responses.

We demonstrated the effect of Ishige okamurae extract (IOE) on the receptor activator of nuclear factor-κB ligand (RANKL)-promoted osteoclastogenesis in RAW 264.7 cells and confirmed that IOE inhibited RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity and osteoclast differentiation. IOE inhibited protein expression of TRAP, metallopeptidase-9 (MMP-9), the calcitonin receptor (CTR), and cathepsin K (CTK). IOE treatment suppressed the expression of activated T cell cytoplasmic 1 and activator protein-1, thus controlling the expression of osteoclast-related factors. Moreover, IOE significantly reduced RANKL-phosphorylated extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). It also reduced the RANKL-induced phosphorylation of NF-κB and nuclear translocation of p65. IOE inhibited Dex-induced bone loss and osteoclast-related gene expression in zebrafish larvae. HPLC analysis shows that IOE consists of 3.13% and 3.42% DPHC and IPA, respectively. Our results show that IOE has inhibitory effects on osteoclastogenesis in vitro and in vivo and is a potential therapeutic for osteoporosis.

Objective: To demonstrate the effect of dieckol from Eisenia bicyclis on osteoclastogenesis using RAW 264.7 cells. Methods: Murine macrophage RAW 264.7 cells were subjected to dieckol treatment, followed by treatment with receptor activator of nuclear factor kappa-B ligand (RANKL) to induce osteoclastogenesis. Tartrate-resistant acid phosphatase (TRAP) activity was examined using a TRAP activity kit. Western blotting analysis was conducted to examine the level of osteoclast- related factors, including TRAP and calcitonin receptor (CTR), transcriptional factors, including c-Fos, c-Jun, and nuclear factor of activated T cells cytoplasmic 1 (NFATc1), nuclear factor kappa-B (NF-κB), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Immunofluorescence staining was conducted to examine the expression of c-Fos, c-Jun, and NFATc1. Results: Among the four phlorotannin compounds present in Eisenia bicyclis, dieckol significantly hindered osteoclast differentiation and expression of RANKL-induced TRAP and CTR. In addition, dieckol downregulated the expression levels of c-Fos, c-Jun, NFATc1, ERK, and JNK, and suppressed NF-κB signaling. Conclusions: Dieckol can suppress RANKL-induced osteoclastogenesis. Therefore, it has therapeutic potential in treating osteoclastogenesis- associated diseases.

Presently, pertinent information on the ultra-small microbiome (USM) in fermented vegetables is still lacking. This study analyzed the metatranscriptome and metataxonome for the USM of kimchi. Tangential flow filtration was used to obtain a USM with a size of 0.2 μm or less from kimchi. The microbial diversity in the USM was compared with that of the normal microbiome (NM). Alpha diversity was higher in the USM than in NM, and the diversity of bacterial members of the NM was higher than that of the USM. At the phylum level, both USM and NM were dominated by Firmicutes. At the genus level, the USM and NM were dominated by Lactobacillus, Leuconostoc, and Weissella, belonging to lactic acid bacteria. However, as alpha diversity is higher in the USM than in the NM, the genus Akkermansia, belonging to the phylum Verrucomicrobia, was detected only in the USM. Compared to the NM, the USM showed a relatively higher ratio of transcripts related to “protein metabolism,” and the USM was suspected to be involved with the viable-but-nonculturable (VBNC) state. When comparing the sub-transcripts related to the “cell wall and capsule” of USM and NM, USM showed a proportion of transcripts suspected of being VBNC. In addition, the RNA virome was also identified, and both the USM and NM were confirmed to be dominated by pepper mild mottle virus (PMMoV). Additionally, the correlation between metataxonome and metatranscriptome identified USM and NM was estimated, however, only limited correlations between metataxonome and metatranscriptome were estimated. This study provided insights into the relationship between the potential metabolic activities of the USM of kimchi and the NM.

Abstract The present study aimed to evaluate the protective effect of a methanol extract of Sargassum horneri (SHM), which contains 6-hydroxy-4,4,7a-trimethyl-5,6,7,7a-tetrahydrobenzofuran-2(4H)-one (HTT) and apo-9′-fucoxanthinone, against ultraviolet B (UVB)-induced cellular damage in human keratinocytes and its underlying mechanism. SHM significantly improved cell viability of UVB-exposed human keratinocytes by reducing the generation of intracellular reactive oxygen species (ROS). Moreover, SHM inhibited UVB exposure-induced apoptosis by reducing the formation of apoptotic bodies and the populations of the sub-G1 hypodiploid cells and the early apoptotic cells by modulating the expression of the anti- and pro-apoptotic molecules, Bcl-2 and Bax, respectively. Furthermore, SHM inhibited NF-κB p65 activation by inducing the activation of Nrf2/HO-1 signaling. The cytoprotective and antiapoptotic activities of SHM are abolished by the inhibition of HO-1 signaling. In further study, SHM restored the skin dryness and skin barrier disruption in UVB-exposed human keratinocytes. Based to these results, our study suggests that SHM protects the cells against UVB-induced cellular damages through the Nrf2/HO-1/NF-κB p65 signaling pathway and may be potentially useful for the prevention of UVB-induced skin damage.

The balance between bone-resorbing osteoclasts and bone-forming osteoblasts is essential for the bone remodeling process. This study aimed to investigate the effect of Ishophloroglucin A (IPA) isolated from Ishige okamurae on the function of osteoclasts and osteoblasts in vitro. First, we demonstrated the effect of IPA on osteoclastogenesis in receptor activator of nuclear factor κB ligand (RANKL)-induced RAW 264.7 cells. IPA inhibited the tartrate-resistant acid phosphatase (TRAP) activity and osteoclast differentiation in RANKL-induced RAW 264.7 cells. Moreover, it inhibited the RANKL-induced osteoclast-related factors, such as TRAP, matrix metalloproteinase-9 (MMP-9), and calcitonin receptor (CTR), and transcription factors, such as nuclear factor of activated T cells 1 (NFATc1) and c-Fos. IPA significantly suppressed RANKL-activated extracellular signal-regulated kinase (ERK), and NF-κB in RAW 264.7 cells. Our data indicated that the ERK and NF-κB pathways were associated with the osteoclastogenesis inhibitory activity of IPA. Next, we demonstrated the effect of IPA on osteoblastogenesis in MG-63 cells. IPA significantly promoted alkaline phosphatase (ALP) activity in MG-63 cells, along with the osteoblast differentiation-related markers bone morphogenetic protein 2 (BMP2), type 1 collage (COL1), p-Smad1/5/8, and Runx2, by activating the MAPK signaling pathways. Taken together, the study indicated that IPA could be effective in treating bone diseases, such as osteoporosis.

Brown seaweed is a rich source of fucoidan, which exhibits a variety of biological activities. The present study discloses the protective effect of low molecular weight fucoidan (FSSQ) isolated from an edible brown alga, Sargassum siliquastrum, on lipopolysaccharide (LPS)-stimulated inflammatory responses in RAW 264.7 macrophages. The findings of the study revealed that FSSQ increases cell viability while decreasing intracellular reactive oxygen species production in LPS-stimulated RAW 264.7 macrophages dose-dependently. FSSQ reduced the iNOS and COX-2 expression, inhibiting the NO and prostaglandin E2 production. Furthermore, mRNA expression of IL-1β, IL-6, and TNF-α was downregulated by FSSQ via modulating MAPK and NF-κB signaling. The NLRP3 inflammasome protein complex, including NLRP3, ASC, and caspase-1, as well as the subsequent release of pro-inflammatory cytokines, such as IL-1β and IL-18, release in LPS-stimulated RAW 264.7 macrophages was inhibited by FSSQ. The cytoprotective effect of FSSQ is indicated via Nrf2/HO-1 signaling activation, which is considerably reduced upon suppression of HO-1 activity by ZnPP. Collectively, the study revealed the therapeutic potential of FSSQ against inflammatory responses in LPS-stimulated RAW 264.7 macrophages. Moreover, the study suggests further investigations on commercially viable methods for fucoidan isolation.

Objective: To investigate the anti-senescence effect of 3-bromo-4,5- dihydroxybenzaldehyde (BDB) from Polysiphonia morrowii Harvey in human dermal fibroblasts (HDF). Methods: HDF were subjected to treatment of BDB and then treated with hydrogen peroxide (H2O2) to induce premature senescence. Senescence-associated β-galactosidase (SA-β-gal) activity in HDF was determined using the SA-β-gal staining method. Intracellular reactive oxygen species (ROS) production was measured using the 2’,7’-dichlorodihydrofluorescein diacetate assay. Western blotting assay was performed to assess the level of antioxidant enzyme glutathione peroxidase 1 (GPX1). In addition, intracellular collagen and collagenase contents were analyzed using the respective ELISA kits. Elastase activity in HDF supernatants was measured from p-nitroaniline release and normalized using total protein content. Results: Treatment of HDF with H2O2 increased the activity of SAP-gal, but BDB pre-treatment resulted in the reduction of SA-β- gal activity. Moreover, BDB significantly reduced H2O2-induced intracellular ROS production. BDB also markedly increased the level of GPX1, which was inhibited by 400 µM of H2O2. Furthermore, in in vitro study, BDB significantly increased intracellular collagen content and decreased matrix metalloproteinase-1 and elastase activities in HDF. Conclusions: Our results demonstrate that BDB shows anti- senescence and anti-wrinkle activities in vitro.

Abstract Background Reactive oxygen species (ROS) produce different lesions in DNA by ROS-induced DNA damage. Detection and quantification of 8-oxo-7,8-dihydroguanine (8-oxoG) within cells are important for study. Human ribosomal protein S3 (hRpS3) has a high binding affinity to 8-oxoG. In this study, we developed an imaging probe to detect 8-oxoG using a specific peptide from hRpS3. Transactivator (TAT) proteins are known to have cell-penetrating properties. Therefore, we developed a TAT-S3 probe by attaching a TAT peptide to our imaging probe. Results A DNA binding assay was conducted to confirm that our probe bound to 8-oxoG and apurinic/apyrimidinic (AP) sites. We confirmed that the TAT-S3 probe localized in the mitochondria, without permeabilization, and fluoresced in H2O2-treated HeLa cells and zebrafish embryos. Treatment with Mitoquinone (MitoQ), a mitochondria-targeted antioxidant, reduced TAT-S3 probe fluorescence. Additionally, treatment with O8, an inhibitor of OGG1, increased probe fluorescence. A competition assay was conducted with an aldehyde reaction probe (ARP) and methoxyamine (MX) to confirm binding of TAT-S3 to the AP sites. The TAT-S3 probe showed competitive binding to AP sites with ARP and MX. Conclusions These results revealed that the TAT-S3 probe successfully detected the presence of 8-oxoG and AP sites in damaged cells. The TAT-S3 probe may have applications for the detection of diseases caused by reactive oxygen species.

Polysiphonia morrowii is a well-known red alga that has promising pharmacological characteristics. The current study evaluates the protective effect of 3-bromo-4,5-dihydroxybenzaldehyde (BDB) isolated from P. morrowii on tumor necrosis factor (TNF)-α/interferon (IFN)-γ-stimulated inflammation and skin barrier deterioration in HaCaT keratinocytes. The anti-inflammatory effect of BDB in TNF-α/IFN-γ-stimulated HaCaT keratinocytes is evaluated by investigating nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, inflammatory cytokines, and chemokines. Further, the interaction between BDB and the skin barrier functions in stimulated HaCaT keratinocytes is investigated. The findings of the study reveal that BDB dose-dependently increases cell viability while decreasing intracellular reactive oxygen species (ROS) production. BDB downregulates the expression of inflammatory cytokines, interleukin (IL)-6, -8, -13, IFN-γ, TNF-α, and chemokines, Eotaxin, macrophage-derived chemokine (MDC), regulated on activation, normal T cells expressed and secreted (RANTES), and thymus and activation-regulated chemokine (TARC) by modulating the MAPK and NF-κB signaling pathways in TNF-α/IFN-γ-stimulated HaCaT keratinocytes. Furthermore, BDB increases the production of skin hydration proteins and tight junction proteins in stimulated HaCaT keratinocytes by preserving skin moisturization and tight junction stability. These findings imply that BDB exhibits a protective ability against inflammation and deterioration of skin barrier via suppressing the expression of inflammatory signaling in TNF-α/IFN-γ-stimulated HaCaT keratinocytes.

Growth and maintenance of skeletal muscle is essential for athletic performance and a healthy life. Stimulating the proliferation and differentiation of muscle cells may help prevent loss of muscle mass. To discover effective natural substances enabling to mitigate muscle loss without side effects, we evaluated muscle growth with several compounds extracted from Catalpa bignonioides Walt. Among these compounds, pinoresinol and vanillic acid increased C2C12, a mouse myoblast cell line, proliferation being the most without cytotoxicity. These substances activated the Akt/mammalian target of the rapamycin (mTOR) pathway, which positively regulates the proliferation of muscle cells. In addition, the results of in silico molecular docking study showed that they may bind to the active site of insulin-like growth factor 1 receptor (IGF-1R), which is an upstream of the Akt/mTOR pathway, indicating that both pinoresinol and vanillic acid stimulate myoblast proliferation through direct interaction with IGF-1R. These results suggest that pinoresinol and vanillic acid may be a natural supplement to improve the proliferation of skeletal muscle via IGF-1R/Akt/mTOR signaling and thus strengthen muscles.

Summary: Systematic control of in vivo behavior of protein-based therapeutics is considered highly desirable for improving their clinical outcomes. Modulation of biochemical properties including molecular weight, surface charge, and binding affinity has thus been suggested to enhance their therapeutic effects. However, establishing a relationship between the binding affinity and tumor localization remains a debated issue. Here we investigate the influence of the binding affinity of proteins on tumor localization by using four repebodies having different affinities to EGFR. Biochemical analysis and molecular imaging provided direct evidence that optimal affinity with balanced target binding and dissociation can facilitate deep penetration and accumulation of protein binders in tumors by overcoming the binding-site-barrier effect. Our findings suggest that binding kinetics-based protein design can be implicated in the development of fine-tuned protein therapeutics for cancers.

We examined the protective effects of esculetin and fucoidan against the neurotoxicity of ZnO NPs in rats. Ninety rats were divided into nine groups and pre-treated with esculetin or fucoidan 1 h before ZnO NP administration on a daily basis for 2 weeks. Serum and brain homogenates were examined by enzyme-linked immunosorbent assay (ELISA), and neurons, microglia, and astrocytes in the hippocampal region were examined with immunohistochemical analysis. The serum levels of interleukin-1-beta (IL-1β), 3-nitrotyrosine (3-NT), superoxide dismutase (SOD), and 8-hydroxy-2′-deoxyguanosine (8-OHdG) were altered in the ZnO NP treatment groups. Brain IL-1β and TNF-α levels were elevated after ZnO NP administration, and these effects were inhibited by esculetin and fucoidan. SOD, 8-OHdG, and acetylcholinesterase (AChE) levels in the brain were decreased after ZnO NP administration. The brain levels of beclin-1 and caspase-3 were elevated after ZnO NP treatment, and these effects were significantly ameliorated by esculetin and fucoidan. The number of reactive astrocytes measured by counting glial fibrillary acidic protein (GFAP)-positive cells, but not microglia, increased following ZnO NP treatment, and esculetin and fucoidan ameliorated the changes. Esculetin and fucoidan may be beneficial for preventing ZnO NP-mediated autophagy and apoptosis by the modulation of reactive astrocyte and proinflammatory cytokines in the rat brain.

Pinus thunbergii is a black pine that has been widely used in health-promoting foods. This study was conducted to investigate the hepatoprotective effects of the alcohol-aqueous extract of Pinus thunbergii PARL leaves (PTE) in ethanol-induced liver-damaged mice. PTE increased the weight and survival rate of mice while reducing the levels of serum hepatic marker enzymes. PTE also restored the levels of antioxidant enzymes in liver tissues while drastically increasing lipid peroxidation concentrations. Histopathological examination of PTE-treated mice presented relatively less lipid droplets and hepatic tissue damage compared to ethanol-treated mice. Finally, to identify PTE components, Natural Products Application Solution with UNIFI along with an in-house library was processed and results revealed 35 compounds in the extract. These compounds were reported to exhibit significant antioxidant and/or hepatoprotective properties. Our results suggest that PTE improves liver function and can be developed as a food product with hepatoprotective effects.

In this study, we investigated the anti-allergic effects of 3,4-dihydroxybenzaldehyde (DHB) isolated from the marine red alga, Polysiphonia morrowii, in mouse bone-marrow-derived cultured mast cells (BMCMCs) and passive cutaneous anaphylaxis (PCA) in anti-dinitrophenyl (DNP) immunoglobulin E (IgE)-sensitized mice. DHB inhibited IgE/bovine serum albumin (BSA)-induced BMCMCs degranulation by reducing the release of β-hexosaminidase without inducing cytotoxicity. Further, DHB dose-dependently decreased the IgE binding and high-affinity IgE receptor (FcεRI) expression and FcεRI-IgE binding on the surface of BMCMCs. Moreover, DHB suppressed the secretion and/or the expression of the allergic cytokines, interleukin (IL)-4, IL-5, IL-6, IL-13, and tumor necrosis factor (TNF)-α, and the chemokine, thymus activation-regulated chemokine (TARC), by regulating the phosphorylation of IκBα and the translocation of cytoplasmic NF-κB into the nucleus. Furthermore, DHB attenuated the passive cutaneous anaphylactic (PCA) reaction reducing the exuded Evans blue amount in the mouse ear stimulated by IgE/BSA. These results suggest that DHB is a potential therapeutic candidate for the prevention and treatment of type I allergic disorders.

Tumor angiogenesis is enhanced in all types of tumors to supply oxygen and nutrients for their growth and metastasis. With the development of anti-angiogenic drugs, the importance of technology that closely monitors tumor angiogenesis has also been emerging. However, to date, the technology for observing blood vessels requires specialized skills with expensive equipment, thereby limiting its applicability only to the laboratory setting. Here, we used a preclinical optical imaging system for small animals and, for the first time, observed, in real time, the entire process of blood vessel development in tumor-bearing mice injected with indocyanine green. Time-lapse sequential imaging revealed blood vessel volume and blood flow dynamics on a microscopic scale. Upon analyzing fluorescence dynamics at each stage of tumor progression, vessel volume and blood flow were found to increase as the tumor developed. Conversely, these vascular parameters decreased when the mice were treated with angiogenesis inhibitors, which suggests that the effects of drugs targeting angiogenesis can be rapidly and easily screened. The results of this study may help evaluate the efficacy of angiogenesis-targeting drugs by facilitating the observation of tumor blood vessels easily in a laboratory unit without large and complex equipment.

Juglans mandshurica Maxim., a traditional folk medicinal plant, is widely distributed in Korea and China. In our previous study, we isolated a new phenylpropanoid compound, 4-((1R,2R)-3-hydroxy-1-(4-hydroxyphenyl)-1-methoxypropan-2-yl)-2-methoxyphenol (HHMP), from J. mandshurica. In the present study, we evaluated the anti-inflammatory activity of HHMP on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells and zebrafish larvae. HHMP significantly inhibited LPS-induced nitric oxide (NO) and prostaglandin E2 production in a dose-dependent manner. Moreover, HHMP treatment considerably suppressed LPS-induced expression of inducible nitric oxide synthase and cyclooxygenase-2. We also demonstrated the mechanisms of HHMP inhibition of inflammatory responses in LPS-stimulated RAW 264.7 cells via Western blot analysis and immunofluorescence staining. Furthermore, HHMP significantly inhibited NO production in LPS-stimulated zebrafish larvae. Consequently, we established that HHMP significantly inhibited the LPS-induced activation of NF-κB and MAPK and the nuclear translocation of p65 in RAW 264.7 cells. Taken together, our findings demonstrate the effect of HHMP on LPS-induced inflammatory responses in vitro and in vivo, suggesting its potential to be used as a natural anti-inflammatory agent.

Trimethyltin (TMT) is an environmental neurotoxin that mediates dopaminergic neuronal injury in the brain. In this study, we characterized the toxic mechanism and possible protective compounds against TMT-induced neurotoxicity in human dopaminergic neuroblastoma SH-SY5Y cells. Antioxidants such as melatonin, N-acetylcysteine (NAC), α-tocopherol, and allopurinol alleviated TMT toxicity. Apoptosis induced by TMT was identified by altered expression of cleaved caspase-3, Bax, Bcl-2, and Bcl-xL through Western blot analysis. The iron chelator deferoxamine ameliorated the alteration of apoptosis-related proteins through TMT exposure. TMT also induced delayed ultrastructural necrotic features such as mitochondrial swelling and cytoplasmic membrane rupture; NAC reduced these necrotic injuries. Esculetin, meloxicam, celecoxib, and phenidone decreased TMT toxicity. Elevation of the pro-inflammatory cytokines IL-1β, TNF-α, and NF-ĸB and reduction of the antioxidant enzymes catalase and glutathione peroxidase-1 (GPx-1) were induced by TMT and ameliorated by inhibitors of LOX and COX-2 enzymes. Both NMDA and non-NMDA antagonists attenuated TMT toxicity. The free calcium ion modulators nimodipine and BAPTA/AM contributed to neuronal survival against TMT toxicity. Inhibitors of the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathway, an autophagy regulator, decreased TMT toxicity. These results imply that TMT neurotoxicity is the chief participant in LOX- and COX-2-mediated apoptosis, partly via necrosis and autophagy in SH-SY5Y cells.

In this study, we investigated the anti-inflammatory effect of litsenolide A2 (LNA2) isolated from L. japonica fruit and its mechanism of action in LPS-induced RAW 264.7 cells. LPS-induced production of anti-inflammatory mediators and cytokines such as NO, PGE2, iNOS, COX-2, TNF-α, and IL-6 was significantly inhibited by LNA2. Next, western blot experiments were performed to investigate the mechanism of action of the anti-inflammatory effect of LNA2. The results indicated that LNA2 markedly reduced the LPS-induced activation of NF-κB and the nuclear translocation of NF-κB p65. Furthermore, LNA2 also inhibited the phosphorylation of MAPK by LPS. LNA2 was the major component in the ethanol extract of L. japonica at 59 mg/g according to high performance liquid chromatography (HPLC). These results indicate that LNA2 can be used as a functional and marker compound for standardization of the manufacturing process when L. japonica fruit is used as a functional food.

In this study, the effects of a polysaccharide purified from the Celluclast extract of Lactobacillus plantarum-fermented Ishige okamurae (CPFI) against gamma ray-irradiation-induced oxidative stress were investigated in a zebrafish model. CPFI showed an increased extraction yield and high 1,1-diphenyl-2-picrylhydrazyl and hydroxyl radical-scavenging activities. It was further purified using anion-exchange chromatography, and among the six fractions obtained, fraction 2 (AP2), with high glucose and mannose contents, exhibited the highest free radical scavenging activity. AP2 improved zebrafish survival and reduced malformations, such as yolk sac oedema and the occurrence of a bent tail. It also reduced cell death and the production of reactive oxygen species and nitric oxide in the zebrafish. Taken together, these results indicate that the AP2 polysaccharide has radioprotective and antioxidant effects, and is a value-added source of functional food ingredients.

Bioluminescence imaging has proven to be a highly sensitive technique for assessing in vitro transcriptional activity toward understanding gene regulation patterns; however, application of this technique is limited for brain research. In particular, the poor spatiotemporal resolution is a major hurdle for monitoring the dynamic changes of transcriptional activity in specific regions of the brain during longitudinal analysis of living animals. To overcome this limitation, in this study, we modified a lentivirus-based luciferase glucocorticoid receptor (GR) reporter by inserting destabilizing sequence genes, and then the reporter was stereotaxically injected in the mouse infralimbic prefrontal cortex (IL-PFC). Using this strategy, we could successfully pin-point and monitor the dynamic changes in GR activity in IL-PFC during normal stress adaptation. The modified reporter showed a 1.5-fold increase in temporal resolution for monitoring GR activity compared to the control, with respect to the intra-individual coefficients of variation. This novel in vivo method has broad applications, as it is readily adaptable to different types of transcription factor arrays as well spanning wide target regions of the brain to other organs and tissues.

Skeletal muscle is an important tissue in energy metabolism and athletic performance. The use of effective synthetic supplements and drugs to promote muscle growth is limited by various side effects. Moreover, their use is prohibited by anti-doping agencies; hence, natural alternatives are needed. Therefore, we evaluated the muscle growth effect of substances that can act like synthetic supplements from edible marine algae. First, we isolated six marine algal polyphenols belonging to the phlorotannin class, namely dieckol (DK), 2,7″-phloroglucinol-6,6′-bieckol (PHB), phlorofucofuroeckol A (PFFA), 6,6′-bieckol (6,6-BK), pyrogallol-phloroglucinol-6,6′-bieckol (PPB), and phloroglucinol (PG) from an edible brown alga, Ecklonia cava and evaluated their effects on C2C12 myoblasts proliferation and differentiation. Of the six phlorotannin isolates evaluated, DK and PHB induced the highest degree of C2C12 myoblast proliferation. In addition, DK and PHB regulates myogenesis by down-regulating the Smad signaling, a negative regulator, and up-regulating the insulin-like growth factor-1 (IGF-1) signaling, a positive regulator. Interestingly, DK and PHB bind strongly to myostatin, which is an inhibitor of myoblast proliferation, while also binding to IGF-1 receptors. Moreover, they bind to IGF-1 receptor. These results suggest that DK and PHB are potential natural muscle building supplements and could be a safer alternative to synthetic drugs.

In this study, the methanolic extract from seeds of Gardenia jasminoides exhibited strong antioxidant and enzyme inhibition activities with less toxicity to NIH3T3 and HepG2 cells at the concentration of 100 µg/mL. The antioxidant activities (DPPH and ABTS), α-amylase, and α-glucosidase inhibition activities were found higher in methanolic extract (MeOH-E) than H2O extract. Besides, 9.82 ± 0.62 µg and 6.42 ± 0.26 µg of MeOH-E were equivalent to 1 µg ascorbic acid for ABTS and DPPH scavenging, respectively while 9.02 ± 0.25 µg and 6.52 ± 0.15 µg of MeOH-E were equivalent to 1 µg of acarbose for inhibition of α-amylase and α-glucosidase respectively. Moreover, the cell assay revealed that the addition of MeOH-E (12.5 µg/mL) increased about 37% of glucose uptake in insulin resistant (IR) HepG2 as compared to untreated IR HepG2 cells. The LC- MS/MS and GC-MS analysis of MeOH-E revealed a total of 54 compounds including terpenoids, glycosides, fatty acid, phenolic acid derivatives. Among the identified compounds, chlorogenic acid and jasminoside A were found promising for anti-diabetic activity revealed by molecular docking study and these molecules are deserving further purification and molecular analysis.

The anti-inflammatory activities of the products of supercritical fluid extraction of Litsea japonica fruit (SFELJF) using supercritical carbon dioxide were investigated. The SFELJF extract dose-dependently inhibited the production of inflammatory markers [nitric oxide (NO), inducible nitric oxide synthase (iNOS), prostaglandin E2 (PGE2), and cyclooxygenase-2 (COX-2)] and proinflammatory cytokines [tumour necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6] induced by lipopolysaccharide (LPS) treatment. To further elucidate the mechanisms underlying these inhibitory effects, LPS-induced nuclear factor-κB (NF-κB) activation and mitogen-activated protein kinase (MAPK) phosphorylation were studied. The SFELJF extract inhibited NF-κB (p65 and p50) activation and MAPK [extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38] phosphorylation in a dose-dependent manner. These results suggest that the anti-inflammatory activities of SFELJF extracts are due to proinflammatory cytokines and mediators via suppression of NF-κB activation and MAPK phosphorylation.

Inflammation affects various organs of the human body, including skeletal muscle. Phlorotannins are natural biologically active substances found in marine brown algae and exhibit anti-inflammatory activities. In this study, we focused on the effects of phlorotannins on anti-inflammatory activity and skeletal muscle cell proliferation activity to identify the protective effects on the inflammatory myopathy. First, the five species of marine brown algal extracts dramatically inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW 264.7 cells without toxicity at all the concentrations tested. Moreover, the extracts collected from Ishige okamurae (I. okamurae) significantly increased cell proliferation of C2C12 myoblasts compared to the non-treated cells with non-toxicity. In addition, as a result of finding a potential tumor necrosis factor (TNF)-α inhibitor that regulates the signaling pathway of muscle degradation in I. okamurae-derived natural bioactive compounds, Diphlorethohydroxycarmalol (DPHC) is favorably docked to the TNF-α with the lowest binding energy and docking interaction energy value. Moreover, DPHC down-regulated the mRNA expression level of pro-inflammatory cytokines and suppressed the muscle RING-finger protein (MuRF)-1 and Muscle Atrophy F-box (MAFbx)/Atrgoin-1, which are the key protein muscle atrophy via nuclear factor-κB (NF-κB), and mitogen-activated protein kinase (MAPKs) signaling pathways in TNF-α-stimulated C2C12 myotubes. Therefore, it is expected that DPHC isolated from IO would be developed as a TNF-α inhibitor against inflammatory myopathy.

The anti-inflammatory effects of compounds isolated from the Litsea japonica fruit were investigated in LPS-stimulated murine macrophage (RAW 264.7) cells. The results indicated that litsenolide (LN)B2 significantly inhibited the LPS-induced release of pro-inflammatory mediators, such as NO and PGE2. LNC2, and, on the other hand, exhibited cytotoxicity at the same concentrations at which it exhibited an inhibitory property. Therefore, only LNB2 was used for further experimentation. LNB2 reduced the LPS-induced production of pro-inflammatory cytokines. We further investigated the mechanism by which LNB2 inhibits pro-inflammatory mediators and cytokines by examining the level of NF-κB and MAPKs phosphorylation, which all serve as key components in inflammation-induced signaling pathways in RAW 264.7 cells. LNB2 inhibited the LPS-induced phosphorylation of NF-κB and MAPKs. These results suggest that LNB2 has an anti-inflammatory effect, inhibiting the production of pro-inflammatory mediators and cytokines via inhibition of NF-κB and MAPK signaling in LPS-stimulated RAW 264.7 cells.

In the present study, the protective effects of polyphenol extract from Ecklonia cava (EPE) against ethanol-induced cell damage and oxidative stress were investigated in vitro and in vivo. E. cava was selected among twenty marine brown algae due to less cytotoxicity and higher cell viability against the ethanol-induced cell damage. The EPE extract showed the protective effect against the ethanol-mediated apoptosis, which was investigated via nuclear staining with Hoechst 33342 and flow cytometry. Furthermore, the treated EPE extract on zebrafish model was improved the ethanol induced survival rate and attenuated, oxidative stress and cell death. As well this result indicated that EPE extract rendered the protective effect of EPE extract against ethanol induced oxidative stress in vitro and zebrafish model.

The present study examined the potential toxic concentrations of zinc oxide nanoparticles (ZnO NPs) and associated autophagy and apoptosis-related injuries in primary neocortical astrocyte cultures. Concentrations of ZnO NPs ≥3 μg/mL induced significant toxicity in the astrocytes. At 24 h after exposure to the ZnO NPs, transmission electron microscopy revealed swelling of the endoplasmic reticulum (ER) and increased numbers of autophagolysosomes in the cultured astrocytes, and increased levels of LC3 (microtubule-associated protein 1 light chain 3)-mediated autophagy were identified by flow cytometry. Apoptosis induced by ZnO NP exposure was confirmed by the elevation of caspase-3/7 activity and 4′,6′-diamidino-2-phenylindole (DAPI) staining. Significant (p < 0.05) changes in the levels of glutathione peroxidase, superoxide dismutase, tumor necrosis factor (TNF-α), and interleukin-6 were observed by enzyme-linked immunoassay (ELISA) assay following the exposure of astrocyte cultures to ZnO NPs. Phosphatidylinositol 3-kinase (PI3K)/mitogen-activated protein kinase (MAPK) dual activation was induced by ZnO NPs in a dose-dependent manner. Additionally, the Akt (protein kinase B) inhibitor BML257 and the mTOR (mammalian target of rapamycin) inhibitor rapamycin contributed to the survival of astrocytes. Inhibitors of cyclooxygenase-2 and lipoxygenase attenuated ZnO NP-induced toxicity. Calcium-modulating compounds, antioxidants, and zinc/iron chelators also decreased ZnO NP-induced toxicity. Together, these results suggest that ZnO NP-induced autophagy and apoptosis may be associated with oxidative stress and the inflammatory process in primary astrocyte cultures.

Gliotoxin, a secondary metabolite produced by marine fungus Aspergillus sp., possesses various biological activities including anticancer activity. However, the mechanism underlying gliotoxin-induced cytotoxicity on human cervical cancer (Hela) and human chondrosarcoma (SW1353) cells remains unclear. In this study, we focused on the effect of gliotoxin induction on apoptosis, the activating expressions of caspase family enzymes in the cells. Apoptotic cell levels were measured through DAPI and Annexin V/Propidium Iodide (PI) double staining analysis. The apoptotic protein expression of Bcl-2 and caspase family was detected by Western blot in Hela and SW1353 cells. Our results showed that gliotoxin treatment inhibited cell proliferation and induced significant morphological changes. Gliotoxin induced apoptosis was further confirmed by DNA fragmentation, chromatin condensation and disrupted mitochondrial membrane potential. Gliotoxin-induced activation of caspase-3, caspase-8 and caspase-9, down-regulation of Bcl-2, up-regulation of Bax and cytochromec (cyt c) release showed evidence for the gliotoxin activity on apoptosis. These findings suggest that gliotoxin isolated from marine fungus Aspergillus sp. induced apoptosis in Hela and SW1353 cells via the mitochondrial pathway followed by downstream events leading to apoptotic mode of cell death.

The anti-cancer effect of diphlorethohydroxycarmalol (DC) isolated from Ishige okamurae, a brown alga, via the induction of apoptosis resulting from mitochondrial dysfunction was assessed in human promyelocytic leukemia (HL60) cells. The apoptotic mechanisms of DC induction on tumor cells were studied in this work for the first time. DCs were determined to evidence marked cytotoxicity on HL60 cells in a dose-dependent manner. The induction of apoptosis in HL60 cells by DC was evidenced by the accumulation of sub-G1 cell population and nuclear condensation. Further investigations into the depletion of mitochondrial membrane potential (ΔΨm) determined that DC treatment induced apoptosis via the regulation of the expression of pro-survival and pro-apoptotic Bcl-2 family members. It was demonstrated that the anti-cancer activity of DC was mediated by apoptotic induction resulting from mitochondrial dysfunction. Our study results also indicated dysfunction and that DCs may constitute a new and promising agent for the treatment of human promyelocytic leukemia cells.

Brown algae, Ecklonia cava has been regarded as a useful material in the prevention or treatment of several human diseases in Jeju Island, Korea. In this study, we investigated its antioxidant activity, and isolated a new antioxidative compound, which was identified as 2,7″-phloroglucinol-6,6′-bieckol (PHB) on the basis of LC/MS, as well as 1H and 13C NMR spectroscopy results. The antioxidant activity of PHB was evaluated via several in vitro assays, such as radical scavenging activities using electron spin resonance spectrometry, intracellular reactive oxygen species scavenging activity, and DNA damage assays. PHB evidenced profound scavenging activities on DPPH, alkyl, hydroxyl, and superoxide radicals (IC50: 0.51, 2.07, 75.64 and 57.19 μM for DPPH, alkyl, hydroxyl and superoxide radicals, respectively). The antioxidant activity of PHB was higher than that of ascorbic acid. Furthermore, PHB effectively inhibited H2O2-induced DNA damage. The results of this study indicate that PHB may prove useful as a novel natural marine antioxidant.

The cytoprotective effect of quercitrin (QR) against oxidative stress induced cell damage by hydrogen peroxide (H2O2) in Chinese hamster lung fibroblast (V79-4) cells was investigated. QR evidenced a scavenging effect of 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide, hydroxyl radicals and on intracellular ROS, and thus prevented lipid peroxidation. As a result, QR reduced H2O2-induced cell death and apoptosis in V79-4 cells. Moreover, H2O2 induced the cleavage of caspase-3, -9, and poly-ADP-ribose polymerase (PARP) and a reduction in Bcl-xL levels, whereas pretreatment with QR significantly inhibited caspase-3, -9, and PARP cleavage and the reduction in Bcl-xL levels, and ultimately ameliorated H2O2-induced apoptosis. Taken together, these results indicate that the treatment of V79-4 cells with QR can block H2O2-induced apoptosis via the regulation of Bcl-xL. QR may be exploited as a biopreservative in food applications or as a health supplement to alleviate oxidative stress.