African swine fever virus B175L inhibits the type I interferon pathway by targeting STING and 2'3'-cGAMP.

IMPORTANCE: African swine fever virus (ASFV), the only known DNA arbovirus, is the causative agent of African swine fever (ASF), an acutely contagious disease in pigs. ASF has recently become a crisis in the pig industry in recent years, but there are no commercially available vaccines. Studying the immune evasion mechanisms of ASFV proteins is important for the understanding the pathogenesis of ASFV and essential information for the development of an effective live-attenuated ASFV vaccines. Here, we identified ASFV B175L, previously uncharacterized proteins that inhibit type I interferon signaling by targeting STING and 2'3'-cGAMP. The conserved B175L-zf-FCS motif specifically interacted with both cGAMP and the R238 and Y240 amino acids of STING. Consequently, this interaction interferes with the interaction of cGAMP and STING, thereby inhibiting downstream signaling of IFN-mediated antiviral responses. This novel mechanism of B175L opens a new avenue as one of the ASFV virulent genes that can contribute to the advancement of ASFV live-attenuated vaccines.

The African Swine Fever Virus Virulence Determinant DP96R Suppresses Type I IFN Production Targeting IRF3.

DP96R of African swine fever virus (ASFV), also known as uridine kinase (UK), encodes a virulence-associated protein. Previous studies have examined DP96R along with other genes in an effort to create live attenuated vaccines. While experiments in pigs have explored the impact of DP96R on the pathogenicity of ASFV, the precise molecular mechanism underlying this phenomenon remains unknown. Here, we describe a novel molecular mechanism by which DP96R suppresses interferon regulator factor-3 (IRF3)-mediated antiviral immune responses. DP96R interacts with a crucial karyopherin (KPNA) binding site within IRF3, disrupting the KPNA-IRF3 interaction and consequently impeding the translocation of IRF3 to the nucleus. Under this mechanistic basis, the ectopic expression of DP96R enhances the replication of DNA and RNA viruses by inhibiting the production of IFNs, whereas DP96R knock-down resulted in higher IFNs and IFN-stimulated gene (ISG) transcription during ASFV infection. Collectively, these findings underscore the pivotal role of DP96R in inhibiting IFN responses and increase our understanding of the relationship between DP96R and the virulence of ASFV.

African Swine Fever Virus EP364R and C129R Target Cyclic GMP-AMP To Inhibit the cGAS-STING Signaling Pathway.

African swine fever virus (ASFV) is a highly pathogenic swine DNA virus with high mortality that causes African swine fever (ASF) in domestic pigs and wild boars. For efficient viral infection, ASFV has developed complex strategies to evade key components of antiviral innate immune responses. However, the immune escape mechanism of ASFV remains unclear. Upon ASFV infection, cyclic GMP-AMP (2',3'-cGAMP) synthase (cGAS), a cytosolic DNA sensor, recognizes ASFV DNA and synthesizes the second messenger 2',3'-cGAMP, which triggers interferon (IFN) production to interfere with viral replication. In this study, we demonstrated a novel immune evasion mechanism of ASFV EP364R and C129R, which blocks cellular cyclic 2',3'-cGAMP-mediated antiviral responses. ASFV EP364R and C129R with nuclease homology inhibit IFN-mediated responses by specifically interacting with 2',3'-cGAMP and exerting their phosphodiesterase (PDE) activity to cleave 2',3'-cGAMP. Particularly notable is that ASFV EP364R had a region of homology with the stimulator of interferon genes (STING) protein containing a 2',3'-cGAMP-binding motif and point mutations in the Y76S and N78A amino acids of EP364R that impaired interaction with 2',3'-cGAMP and restored subsequent antiviral responses. These results highlight a critical role for ASFV EP364R and C129R in the inhibition of IFN responses and could be used to develop ASFV live attenuated vaccines. IMPORTANCE African swine fever (ASF) is a highly contagious hemorrhagic disease in domestic pigs and wild boars caused by African swine fever virus (ASFV). ASF is a deadly epidemic disease in the global pig industry, but no drugs or vaccines are available. Understanding the pathogenesis of ASFV is essential to developing an effective live attenuated ASFV vaccine, and investigating the immune evasion mechanisms of ASFV is crucial to improve the understanding of its pathogenesis. In this study, for the first time, we identified the EP364R and C129R, uncharacterized proteins that inhibit type I interferon signaling. ASFV EP364R and C129R specifically interacted with 2',3'-cGAMP, the mammalian second messenger, and exerted phosphodiesterase activity to cleave 2',3'-cGAMP. In this study, we discovered a novel mechanism by which ASFV inhibits IFN-mediated antiviral responses, and our findings can guide the understanding of ASFV pathogenesis and the development of live attenuated ASFV vaccines.

Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering of Leuconostoc citreum and Its Application for Engineering Riboflavin Biosynthesis.

Leuconostoccitreum, a hetero-fermentative type of lactic acid bacteria, is a crucial probiotic candidate because of its ability to promote human health. However, inefficient gene manipulation tools limit its utilization in bioindustries. We report, for the first time, the development of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference (CRISPRi) system for engineering L. citreum. For reliable expression, the expression system of synthetic single guide RNA (sgRNA) and the deactivated Cas9 of Streptococcus pyogenes (SpdCas9) were constructed in a bicistronic design (BCD) platform using a high-copy-number plasmid. The expression of SpdCas9 and sgRNA was optimized by examining the combination of two synthetic promoters and Shine-Dalgarno sequences; the strong expression of sgRNA and the weak expression of SpdCas9 exhibited the most significant downregulation (20-fold decrease) of the target gene (sfGFP), without cell growth retardation caused by SpdCas9 overexpression. The feasibility of the optimized CRISPRi system was demonstrated by modulating the biosynthesis of riboflavin. Using the CRISPRi system, the expression of ribF and folE genes was downregulated (3.3-fold and 5.6-fold decreases, respectively), thereby improving riboflavin production. In addition, the co-expression of the rib operon was introduced and the production of riboflavin was further increased up to 1.7 mg/L, which was 1.53 times higher than that of the wild-type strain.

Cytoprotective effect of eckol against oxidative stress-induced mitochondrial dysfunction: involvement of the FoxO3a/AMPK pathway.

This study investigated the cytoprotective effect of Ecklonia cava-derived eckol against H2O2-induced mitochondrial dysfunction in Chang liver cells. While H2O2 augmented levels of mitochondrial reactive oxygen species (ROS), eckol decreased it. Eckol also attenuated high intracellular Ca(2+) levels stimulated by H2O2 and recovered H2O2-diminished ATP levels and succinate dehydrogenase activity. Eckol time-dependently increased the expression of manganese superoxide dismutase (Mn SOD), a mitochondrial antioxidant enzyme with cytoprotective effect against oxidative stress. Eckol recovered Mn SOD expression and activity that were decreased by H2O2. Finally, eckol induced Mn SOD through phosphorylated AMP-activated protein kinase (AMPK) and forkhead box O3a (FoxO3a). Specific silencing RNAs (siRNAs) against FoxO3a and AMPK reduced eckol-stimulated Mn SOD expression, and diethyldithiocarbamate (Mn SOD inhibitor) and siRNA against Mn SOD reduced the cytoprotective effect of eckol against H2O2-provoked cell death. These results demonstrate that eckol protects cells from mitochondrial oxidative stress by activating AMPK/FoxO3a-mediated induction of Mn SOD.

Fucoxanthin enhances the level of reduced glutathione via the Nrf2-mediated pathway in human keratinocytes.

Fucoxanthin, a natural carotenoid, is abundant in seaweed with antioxidant properties. This study investigated the role of fucoxanthin in the induction of antioxidant enzymes involved in the synthesis of reduced glutathione (GSH), synthesized by glutamate-cysteine ligase catalytic subunit (GCLC) and glutathione synthetase (GSS), via Akt/nuclear factor-erythroid 2-related (Nrf2) pathway in human keratinocytes (HaCaT) and elucidated the underlying mechanism. Fucoxanthin treatment increased the mRNA and protein levels of GCLC and GSS in HaCaT cells. In addition, fucoxanthin treatment promoted the nuclear translocation and phosphorylation of Nrf2, a transcription factor for the genes encoding GCLC and GSS. Chromatin immune-precipitation and luciferase reporter gene assays revealed that fucoxanthin treatment increased the binding of Nrf2 to the antioxidant response element (ARE) sequence and transcriptional activity of Nrf2. Fucoxanthin treatment increased phosphorylation of Akt (active form), an up-regulator of Nrf2 and exposure to LY294002, a phosphoinositide 3-kinase (PI3K)/Akt inhibitor, suppressed the fucoxanthin-induced activation of Akt, Nrf2, resulting in decreased GCLC and GSS expression. In accordance with the effects on GCLC and GSS expression, fucoxanthin induced the level of GSH. In addition, fucoxanthin treatment recovered the level of GSH reduced by ultraviolet B irradiation. Taken together, these findings suggest that fucoxanthin treatment augments cellular antioxidant defense by inducing Nrf2-driven expression of enzymes involved in GSH synthesis via PI3K/Akt signaling.

The ethyl acetate fraction of Sargassum muticum attenuates ultraviolet B radiation-induced apoptotic cell death via regulation of MAPK- and caspase-dependent signaling pathways in human HaCaT keratinocytes.

CONTEXT: Our previous work demonstrated that an ethyl acetate extract derived from Sargassum muticum (Yendo) Fenshol (SME) protected human HaCaT keratinocytes against ultraviolet B (UVB)-induced oxidative stress by increasing antioxidant activity in the cells, thereby inhibiting apoptosis. OBJECTIVE: The aim of the current study was to further elucidate the anti-apoptotic mechanism of SME against UVB-induced cell damage. MATERIALS AND METHODS: The expression levels of several apoptotic-associated and mitogen-activated kinase (MAPK) signaling proteins were determined by western blot analysis of UVB-irradiated HaCaT cells with or without prior SME treatment. In addition, the loss of mitochondrial membrane potential (Δψm) was detected using flow cytometry or confocal microscopy and the mitochondria membrane-permeate dye, JC-1. Apoptosis was assessed by quantifying DNA fragmentation and apoptotic body formation. Furthermore, cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RESULTS: SME absorbed electromagnetic radiation in the UVB range (280-320 nm) of the UV/visible light spectrum. SME also increased Bcl-2 and Mcl-1 expression in UVB-irradiated cells and decreased the Bax expression. Moreover, SME inhibited the UVB-induced disruption of mitochondrial membrane potential and prevented UVB-mediated increases in activated caspase-9 and caspase-3 (an apoptotic initiator and executor, respectively) levels. Notably, treatment with a pan-caspase inhibitor enhanced the anti-apoptotic effects of SME in UVB-irradiated cells. Finally, SME reduced the UVB-mediated phosphorylation of p38 MAPK and JNK, and prevented the UVB-mediated dephosphorylation of Erk1/2 and Akt. DISCUSSION AND CONCLUSION: The present results indicate that SME safeguards HaCaT keratinocytes from UVB-mediated apoptosis by inhibiting a caspase-dependent signaling pathway.

CA-CAS-01-A: A Permissive Cell Line for Isolation and Live Attenuated Vaccine Development Against African Swine Fever Virus.

African swine fever virus (ASFV) is the causative agent of the highly lethal African swine fever disease that affects domestic pigs and wild boars. In spite of the rapid spread of the virus worldwide, there is no licensed vaccine available. The lack of a suitable cell line for ASFV propagation hinders the development of a safe and effective vaccine. For ASFV propagation, primary swine macrophages and monocytes have been widely studied. However, obtaining these cells can be time-consuming and expensive, making them unsuitable for mass vaccine production. The goal of this study was to validate the suitability of novel CA-CAS-01-A (CAS-01) cells, which was identified as a highly permissive cell clone for ASFV replication in the MA-104 parental cell line for live attenuated vaccine development. Through a screening experiment, maximum ASFV replication was observed in the CAS-01 cell compared to other sub-clones of MA-104 with 14.89 and log10 7.5 ± 0.15 Ct value and TCID50/ml value respectively. When CAS-01 cells are inoculated with ASFV, replication of ASFV was confirmed by Ct value for ASFV DNA, HAD50/ml assay, TCID50/ml assay, and cytopathic effects and hemadsoption were observed similar to those in primary porcine alveolar macrophages after 5th passage. Additionally, we demonstrated stable replication and adaptation of ASFV over the serial passage. These results suggest that CAS-01 cells will be a valuable and promising cell line for ASFV isolation, replication, and development of live attenuated vaccines.

Production of Cinnamaldehyde through Whole-Cell Bioconversion from trans-Cinnamic Acid Using Engineered Corynebacterium glutamicum.

Cinnamaldehyde (CAD) has various applications in foods and pharmaceuticals and has gained prominence as a potent nematicide in agricultural research owing to its nematicidal activity. However, conventional methods of CAD production, including extraction from plants or organic chemical synthesis, are environmentally hazardous and limit its utilization for downstream applications. Here, we engineered Corynebacterium glutamicum as a whole-cell biocatalyst for the efficient bioconversion of trans-cinnamic acid (t-CA) into CAD. An expression module of Mycobacterium phlei carboxylic acid reductase was constructed for the conversion of t-CA to CAD. Additionally, the putative dehydrogenase-related genes (dkgA, adhC, and cg1176) responsible for the conversion of CAD to cinnamyl alcohol were deleted from the engineered C. glutamicum strain to prevent the loss of CAD. Furthermore, as the conversion is NADPH-dependent, we investigated the conversion efficiency by exchanging the putative promoter region for the zwf gene, which encodes glucose-6-phosphate dehydrogenase, with a strong promoter to increase the NADPH pool. Finally, a bioconversion platform using C. glutamicum as a whole-cell biocatalyst was developed by deleting the vdh gene, which is involved in the reverse conversion of CAD to t-CA. Taken together, a 100% conversion yield of 1.1 g/L CAD from 1.2 g/L t-CA was obtained within 30 min.

Development of bicistronic expression system for the enhanced and reliable production of recombinant proteins in Leuconostoc citreum.

The lactic acid bacteria (LAB) Leuconostoc citreum are non-sporulating hetero-fermentative bacteria that play an important role in the fermented food industry. In this study, for the enhanced and reliable production of recombinant proteins in L. citreum, we developed a bicistronic design (BCD) expression system which includes a short leader peptide (1st cistron) followed by target genes (2nd cistron) under the control of a single promoter. Using superfolder green fluorescent protein (sfGFP) as a reporter, the functionality of BCD in L. citreum was verified. Further, to improve the expression in BCD, we tried to engineer a Shine-Dalgarno sequence (SD2) for the 2nd cistron and a promoter by FACS screening of random libraries, and both strong SD2 (eSD2) and promoter (P710V4) were successfully isolated. The usefulness of the engineered BCD with P710V4 and eSD2 was further validated using three model proteins-glutathione-s-transferase, human growth hormone, and α-amylase. All examined proteins were successfully produced with levels highly increased compared with those in the original BCD as well as the monocistronic design (MCD) expression system.

Galangin (3,5,7-trihydroxyflavone) shields human keratinocytes from ultraviolet B-induced oxidative stress.

Most skin damage caused by ultraviolet B (UVB) radiation is owing to the generation of reactive oxygen species. Phytochemicals can act as antioxidants against UVB-induced oxidative stress. This study investigated the protective effects of the flavone galangin against UVB-induced oxidative damage in human keratinocytes. Galangin efficiently scavenged free radicals and reduced UVB-induced damage to cellular macromolecules, such as DNA, lipids, and proteins. Furthermore, galangin rescued cells undergoing apoptosis induced by UVB radiation via recovering mitochondrial polarization and down-regulating apoptotic proteins. These results showed that galangin protects human keratinocytes against UVB radiation-induced cellular damage and apoptosis via its antioxidant effects.

Phloroglucinol enhances the repair of UVB radiation-induced DNA damage via promotion of the nucleotide excision repair system in vitro and in vivo.

Exposure to solar UVB radiation can lead to the formation of DNA lesions such as cyclobutane pyrimidine dimers (CPDs). Nucleotide excision repair (NER) is critical for the repair of CPDs induced by UV radiation. The purpose of this study was to investigate the ability of phloroglucinol to protect against the formation of UVB-induced CPDs in vitro and in vivo. Exposure to UVB radiation increased the number of CPDs in both HaCaT keratinocytes and mouse skin; however, these increases were reduced by treatment with phloroglucinol. Expression levels of xeroderma pigmentosum complementation group C (XPC) and excision repair cross-complementation 1 (ERCC1), which are essential components of the NER pathway, were reduced following UVB exposure, although phloroglucinol treatment recovered these levels in both HaCaT keratinocytes and mouse skin. Phloroglucinol also inhibited UVB-induced reductions in binding of the transcription factors specificity protein 1 to the XPC promoter. These results demonstrate that phloroglucinol can protect cells against UVB-induced DNA damage by inducing NER.

Fisetin attenuates hydrogen peroxide-induced cell damage by scavenging reactive oxygen species and activating protective functions of cellular glutathione system.

Hydrogen peroxide (H2O2) can induce cell damage by generating reactive oxygen species (ROS), resulting in DNA damage and cell death. The aim of this study is to elucidate the protective effects of fisetin (3,7,3',4',-tetrahydroxy flavone) against H2O2-induced cell damage. Fisetin reduced the level of superoxide anion, hydroxyl radical in cell free system, and intracellular ROS generated by H2O2. Moreover, fisetin protected against H2O2-induced membrane lipid peroxidation, cellular DNA damage, and protein carbonylation, which are the primary cellular outcomes of H2O2 treatment. Furthermore, fisetin increased the level of reduced glutathione (GSH) and expression of glutamate-cysteine ligase catalytic subunit, which is decreased by H2O2. Conversely, a GSH inhibitor abolished the cytoprotective effect of fisetin against H2O2-induced cells damage. Taken together, our results suggest that fisetin protects against H2O2-induced cell damage by inhibiting ROS generation, thereby maintaining the protective role of the cellular GSH system.

Photo-protective effect of americanin B against ultraviolet B-induced damage in cultured human keratinocytes.

Excessive ultraviolet (UV) radiation, a constituent of sunlight, can induce multiple types of skin damage. We recently demonstrated that americanin B, a lignin compound, protected cells against hydrogen peroxide (H2O2)-induced damage by exerting antioxidant effects and inhibiting apoptosis. In this study, we investigated the ability of americanin B to protect against cell injury induced by UVB (280-320nm), the most harmful UV wavelengths, in human HaCaT keratinocytes. Americanin B absorbed UVB, eliminated UVB-induced intracellular reactive oxygen species (ROS), and decreased the extent of UVB-induced oxidative modification of lipids, proteins, and DNA. In addition, americanin B inhibited UVB-induced apoptosis, as indicated by reductions in apoptotic body formation and DNA fragmentation. Furthermore, americanin B reversed the depolarization of the mitochondrial membrane induced by UVB exposure. These protective activities were associated with down-regulation of apoptosis-promoting proteins, Bax, caspase-9, and caspase-3 and up-regulation of an apoptosis inhibitor, Bcl-2. These results suggest that americanin B can protect human keratinocytes against UVB-induced cell damage.

Fucodiphlorethol G Purified from Ecklonia cava Suppresses Ultraviolet B Radiation-Induced Oxidative Stress and Cellular Damage.

Fucodiphlorethol G (6'-[2,4-dihydroxy-6-(2,4,6-trihydroxyphenoxy)phenoxy]biphenyl-2,2',4,4',6-pentol) is a compound purified from Ecklonia cava, a brown alga that is widely distributed offshore of Jeju Island. This study investigated the protective effects of fucodiphlorethol G against oxidative damage-mediated apoptosis induced by ultraviolet B (UVB) irradiation. Fucodiphlorethol G attenuated the generation of 2, 2-diphenyl-1-picrylhydrazyl radicals and intracellular reactive oxygen species in response to UVB irradiation. Fucodiphlorethol G suppressed the inhibition of human keratinocyte growth by UVB irradiation. Additionally, the wavelength of light absorbed by fucodiphlorethol G was close to the UVB spectrum. Fucodiphlorethol G reduced UVB radiation-induced 8-isoprostane generation and DNA fragmentation in human keratinocytes. Moreover, fucodiphlorethol G reduced UVB radiation-induced loss of mitochondrial membrane potential, generation of apoptotic cells, and active caspase-9 expression. Taken together, fucodiphlorethol G protected human keratinocytes against UVB radiation-induced cell damage and apoptosis by absorbing UVB radiation and scavenging reactive oxygen species.

The Polyphenol Chlorogenic Acid Attenuates UVB-mediated Oxidative Stress in Human HaCaT Keratinocytes.

We investigated the protective effects of chlorogenic acid (CGA), a polyphenol compound, on oxidative damage induced by UVB exposure on human HaCaT cells. In a cell-free system, CGA scavenged 1,1-diphenyl-2-picrylhydrazyl radicals, superoxide anions, hydroxyl radicals, and intracellular reactive oxygen species (ROS) generated by hydrogen peroxide and ultraviolet B (UVB). Furthermore, CGA absorbed electromagnetic radiation in the UVB range (280-320 nm). UVB exposure resulted in damage to cellular DNA, as demonstrated in a comet assay; pre-treatment of cells with CGA prior to UVB irradiation prevented DNA damage and increased cell viability. Furthermore, CGA pre-treatment prevented or ameliorated apoptosis-related changes in UVB-exposed cells, including the formation of apoptotic bodies, disruption of mitochondrial membrane potential, and alterations in the levels of the apoptosis-related proteins Bcl-2, Bax, and caspase-3. Our findings suggest that CGA protects cells from oxidative stress induced by UVB radiation.

Phloroglucinol inhibits ultraviolet B radiation-induced oxidative stress in the mouse skin.

PURPOSE: Previously we demonstrated that phloroglucinol (1,3,5-trihydroxybenzene) protected human HaCaT keratinocytes against ultraviolet B (UVB, 280-320 nm)-induced oxidative stress in vitro by scavenging intracellular reactive oxygen species (ROS). The current study investigated whether phloroglucinol could similarly protect the mouse skin against UVB-induced oxidative tissue damage in vivo. MATERIALS AND METHODS: Male 7-week-old Balb/c mice were divided into the following untreated normal control, phloroglucinol only-treated, vehicle plus UVB (30 or 60 mJ/cm(2))-exposed, and phloroglucinol (10 or 50 mg/ml) plus UVB (30 or 60 mJ/cm(2))-treated groups. Following UVB exposure, phloroglucinol or phosphate buffered saline vehicle was applied to the dorsal skin of each mouse daily for 3 days. Studies were conducted at 24 h after the last of the UVB exposures. Histopathological analyses of dorsal skin lesions were performed on all mice. In addition, the levels of UVB-provoked injury to cellular components, including DNA, proteins, and lipids were detected by levels of 8-oxoguanine (8-oxoG), protein carbonyls, and 8-isoprostane. Apoptosis were assessed by using western blot for B-cell lymphoma-2-associated X protein (Bax) and activated caspase-3 expression, by using immunohistochemistry. RESULTS: UVB radiation increased the thickness of the epidermis and the dermis, and also stimulated the accumulation of mast cells in the irradiated skin. However, treatment with phloroglucinol significantly decreased all of these parameters. Furthermore, phloroglucinol decreased UVB-provoked injury to cellular components, including DNA, proteins, and lipids; down-regulated the expression of phospho-histone H2A.X in the injured skin; and reduced the UVB-generated levels of 8-oxoG, protein carbonyls, and 8-isoprostane, which are all markers of oxidative stress. In addition, phloroglucinol attenuated the UVB-induced expression of the pro-apoptotic proteins, Bax protein, and activated caspase-3. CONCLUSION: These results suggest that phloroglucinol safeguards the mouse skin against UVB-induced oxidative stress and apoptosis.

Cytoprotective effects of 6'-O-galloylpaeoniflorin against ultraviolet B radiation-induced cell damage in human keratinocytes.

The cytoprotective effects of 6'-O-galloylpaeoniflorin against injury and death of human HaCaT keratinocytes resulting from ultraviolet B radiation were investigated. 6'-O-galloylpaeoniflorin exhibited the capacity to scavenge intracellular reactive oxygen species (ROS) generated by ultraviolet B radiation. 6'-O-galloylpaeoniflorin also attenuated ultraviolet B-induced oxidative macromolecular damage to DNA, lipids, and proteins, decreasing the number of DNA strand breaks, the level of 8-isoprostane (a biomarker of lipid peroxidation), and the level of protein carbonylation. Moreover, 6'-O-galloylpaeoniflorin rescued HaCaT cells from ultraviolet induced cell death, by downregulating the mitochondrial apoptotic pathway. Taken together, these results indicate that 6'-O-galloylpaeoniflorin has the potential to be developed as a medical agent against ROS-mediated skin diseases.

Americanin B protects cultured human keratinocytes against oxidative stress by exerting antioxidant effects.

We evaluated the cytoprotective effects of americanin B, a lignan compound, against hydrogen peroxide (H2O2)-induced cell damage. Americanin B decreased the level of DPPH radicals, superoxide anions, hydroxyl radicals, and intracellular reactive oxygen species. Americanin B also attenuated DNA damage induced by H2O2 treatment, as shown by the inhibition of formation of comet tails, indicative of DNA strand breakage, and prevented the oxidation of protein and peroxidation of lipid, as determined by protein carbonyls and 8-isoprostane. Furthermore, americanin B protected against H2O2-induced apoptotic cell death, as determined by a reduction in the numbers of apoptotic bodies stained with Hoechst 33342. These findings suggest that americanin B protects cells against oxidative damage by exerting antioxidant effects and inhibiting apoptosis.

Effect of 7, 8-dihydroxyflavone on the up-regulation of Nrf2-mediated heme oxygenase-1 expression in hamster lung fibroblasts.

The cytoprotective mechanism of 7, 8-dihydroxyflavone (DHF) against oxidative stress-induced cell damage with respect to its stimulatory effect on the expression of heme oxygenase-1 (HO-1), a potent antioxidant enzyme, was investigated in the present study. Up-regulation of HO-1 expression by DHF was both dose and time dependent in lung fibroblast V79-4 cells. DHF also increased the protein expression level of the transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2), and induced the translocation of Nrf2 from the cytosol into the nucleus, leading to elevated HO-1 expression. The siNrf2 RNA-transfection attenuated HO-1 expression induced by DHF treatment. In addition, DHF induced the activation of extracellular signal-regulated kinase (ERK), while U0126 (a specific pharmacological inhibitor of ERK kinase) abrogated DHF-activated Nrf2 and HO-1 expression. This suggests that DHF increased the levels of Nrf2 and HO-1 via ERK-dependent pathways. Furthermore, DHF significantly prevented the reduction of cell viability in response to oxidative stress; however, U0126 attenuated the protective effect of DHF. Taken together, these results demonstrate that DHF protected cells from oxidative stress via the activation of an ERK/Nrf2/HO-1 signaling pathway.