Critical roles of tubular mitochondrial ATP synthase dysfunction in maleic acid-induced acute kidney injury.

Maleic acid (MA) induces renal tubular cell dysfunction directed to acute kidney injury (AKI). AKI is an increasing global health burden due to its association with mortality and morbidity. However, targeted therapy for AKI is lacking. Previously, we determined mitochondrial-associated proteins are MA-induced AKI affinity proteins. We hypothesized that mitochondrial dysfunction in tubular epithelial cells plays a critical role in AKI. In vivo and in vitro systems have been used to test this hypothesis. For the in vivo model, C57BL/6 mice were intraperitoneally injected with 400 mg/kg body weight MA. For the in vitro model, HK-2 human proximal tubular epithelial cells were treated with 2 mM or 5 mM MA for 24 h. AKI can be induced by administration of MA. In the mice injected with MA, the levels of blood urea nitrogen (BUN) and creatinine in the sera were significantly increased (p < 0.005). From the pathological analysis, MA-induced AKI aggravated renal tubular injuries, increased kidney injury molecule-1 (KIM-1) expression and caused renal tubular cell apoptosis. At the cellular level, mitochondrial dysfunction was found with increasing mitochondrial reactive oxygen species (ROS) (p < 0.001), uncoupled mitochondrial respiration with decreasing electron transfer system activity (p < 0.001), and decreasing ATP production (p < 0.05). Under transmission electron microscope (TEM) examination, the cristae formation of mitochondria was defective in MA-induced AKI. To unveil the potential target in mitochondria, gene expression analysis revealed a significantly lower level of ATPase6 (p < 0.001). Renal mitochondrial protein levels of ATP subunits 5A1 and 5C1 (p < 0.05) were significantly decreased, as confirmed by protein analysis. Our study demonstrated that dysfunction of mitochondria resulting from altered expression of ATP synthase in renal tubular cells is associated with MA-induced AKI. This finding provides a potential novel target to develop new strategies for better prevention and treatment of MA-induced AKI.

The Role of Oxidative Stress and Autophagy in Blue-Light-Induced Damage to the Retinal Pigment Epithelium in Zebrafish In Vitro and In Vivo.

Age-related macular degeneration (AMD) is the progressive degeneration of the retinal pigment epithelium (RPE), retina, and choriocapillaris among elderly individuals and is the leading cause of blindness worldwide. Thus, a better understanding of the underlying mechanisms in retinal tissue activated by blue light exposure is important for developing novel treatment and intervention strategies. In this study, blue-light-emitting diodes with a wavelength of 440 nm were applied to RPE cells at a dose of 3.7 ± 0.75 mW/cm2 for 24 h. ARPE-19 cells were used to investigate the underlying mechanism induced by blue light exposure. A trypan blue exclusion assay was used for the cell viability determination. Flow cytometry was used for apoptosis rate detection and autophagy analysis. An immunofluorescence microscopy analysis was used to investigate cellular oxidative stress and DNA damage using DCFDA fluorescence staining and an anti-γH2AX antibody. Blue light exposure of zebrafish larvae was established to investigate the effect on retinal tissue development in vivo. To further demonstrate the comprehensive effect of blue light on ARPE-19 cells, next-generation sequencing (NGS) was performed for an ingenuity pathway analysis (IPA) to reveal additional related mechanisms. The results showed that blue light exposure caused a decrease in cell proliferation and an increase in apoptosis in ARPE-19 cells in a time-dependent manner. Oxidative stress increased during the early stage of 2 h of exposure and activated DNA damage in ARPE-19 cells after 8 h. Furthermore, autophagy was activated in response to blue light exposure at 24-48 h. The zebrafish larvae model showed the unfavorable effect of blue light in prohibiting retinal tissue development. The RNA-Seq results confirmed that blue light induced cell death and participated in tissue growth inhibition and maturation. The current study reveals the mechanisms by which blue light induces cell death in a time-dependent manner. Moreover, both the in vivo and NGS data uncovered blue light's effect on retinal tissue development, suggesting that exposing children to blue light could be relatively dangerous. These results could benefit the development of preventive strategies utilizing herbal medicine-based treatments for eye diseases or degeneration in the future.

Eicosapentaenoic acids enhance chemosensitivity through connexin 43 upregulation in murine melanoma models.

Chemotherapy is now in common use for the treatment of tumors; however, with tumor growth retardation comes the severe side effects that occur after a chemotherapy cycle. Eicosapentaenoic acids (EPA) used in combination with chemotherapy has an additive effects and provides a rationale for using EPA in tandem with chemotherapy. To improve the efficacy and safety of this combination therapy, a further understanding that EPA modulates with the tumor microenvironment is necessary. Connexin 43 (Cx43) is involved in enhancing chemosensitivity that was suppressed in a tumor microenvironment. We aim to investigate the role of EPA in chemosensitivity in murine melanoma by inducing Cx43 expression. The dose-dependent upregulation of Cx43 expression and gap junction intercellular communication were observed in B16F10 cells after EPA treatment. Furthermore, EPA significantly increased the expression levels of mitogen-activated protein kinases (MAPK) signaling pathways. The EPA-induced Cx43 expression was reduced after MAPK inhibitors. Knockdown Cx43 in B16F10 cells reduced the therapeutic effects of combination therapy (EPA plus 5-Fluorouracil). Our results demonstrate that the treatment of EPA is a tumor induced Cx43 gap junction communication and enhances the combination of EPA and chemotherapeutic effects.

LY303511 displays antiproliferation potential against oral cancer cells in vitro and in vivo.

LY303511 was developed as a negative control of LY294002 without pan-phosphoinositide 3-kinase (PI3K) inhibition. We hypothesize LY303511 generate reactive oxygen species (ROS) to induce apoptosis for killing oral cancer cells. In MTS assay, LY303511 dose-responsively decreases survival in three kinds of oral cancer cells but little damage to normal oral cells (HGF-1). Two oral cancer cells (CAL 27 and SCC-9) with highly sensitivity to LY303511 were used. In 7-aminoactinomycin D (7AAD) assay, LY303511 slightly increases subG1 population in oral cancer cells. In annexin V/7AAD and/or pancaspase assays, LY303511 induces apoptosis in oral cancer cells but HGF-1 cells remains in basal level. In oxidative stress, LY303511 induces ROS and mitochondrial superoxide in oral cancer cells. In 8-oxo-2'-deoxyguanosine assay, LY303511 induces oxidative DNA damage in oral cancer cells. In zebrafish model, LY303511 inhibits CAL 27-xenografted tumor growth. Therefore, LY303511 displays antiproliferation potential against oral cancer cells in vitro and in vivo.

Sandensolide Induces Oxidative Stress-Mediated Apoptosis in Oral Cancer Cells and in Zebrafish Xenograft Model.

Presently, natural sources and herbs are being sought for the treatment of human oral squamous cell carcinoma (OSCC) in order to alleviate the side effects of chemotherapy. This study investigates the effect of sandensolide, a cembrane isolated from Sinularia flexibilis, to inhibit human OSCC cell growth with the aim of developing a new drug for the treatment of oral cancer. In vitro cultured human OSCC models (Ca9.22, SCC9 and HSC-3 cell lines) and oral normal cells (HGF-1), as well as a zebrafish xenograft model, were used to test the cytotoxicity of sandensolide (MTT assay), as well as to perform cell cycle analysis and Western blotting. Both the in vitro bioassay and the zebrafish xenograft model demonstrated the anti-oral cancer effect of sandensolide. Moreover, sandensolide was able to significantly suppress colony formation and induce apoptosis, as well as cell cycle arrest, in OSCC by regulating multiple key proteins. Induction of reactive oxygen species (ROS) was observed in sandensolide-treated oral cancer cells. However, these apoptotic changes were rescued by NAC pretreatment. These findings contribute to the knowledge of the model of action of sandensolide, which may induce oxidative stress-mediated cell death pathways as a potential agent in oral cancer therapeutics.

Ethyl Acetate Extract of Scindapsus cf. hederaceus Exerts the Inhibitory Bioactivity on Human Non-Small Cell Lung Cancer Cells through Modulating ER Stress.

Unfolded protein response (UPR) is a cytoprotective mechanism that alleviates the protein-folding burden in eukaryotic organisms. Moderate activation of UPR is required for maintaining endoplasmic reticulum (ER) homeostasis and profoundly contributes to tumorigenesis. Defects in UPR signaling are implicated in the attenuation of various malignant phenotypes including cell proliferation, migration, and invasion, as well as angiogenesis. This suggests UPR as a promising target in cancer therapy. The pharmacological effects of the plant Scindapsus cf. hederaceus on human cancer cell lines is not understood. In this study, we identified an ethyl acetate extract from Scindapsus cf. hederaceus (SH-EAE), which markedly altered the protein expression of UPR-related genes in human non-small cell lung cancer (NSCLC) cells. Treatment with the SH-EAE led to the dose-dependent suppression of colony forming ability of both H1299 and H460 cells, but not markedly in normal bronchial epithelial BEAS-2B cells. SH-EAE treatment also attenuated the migration and invasion ability of H1299 and H460 cells. Moreover, SH-EAE strikingly suppressed the protein expression of two ER stress sensors, including inositol requiring enzyme-1α (IRE-1α) and protein kinase R-like ER kinase (PERK), and antagonized the induction of C/EBP homologous protein (CHOP) expression by thapsigargin, an ER stress inducer. SH-EAE induced the formation of massive vacuoles which are probably derived from ER. Importantly, SH-EAE impaired the formation of intersegmental vessels (ISV) in zebrafish larvae, an index of angiogenesis, but had no apparent effect on the rate of larval development. Together, our findings demonstrate, for the first time, that the ability of SH-EAE specifically targets the two sensors of UPR, with significant anti-proliferation and anti-migration activities as a crude extract in human NSCLC cells. Our finding also indicates potential applications of SH-EAE in preventing UPR activation in response to Tg-induced ER stress. We suggest that SH-EAE attenuates UPR adaptive pathways for rendering the NSCLC cells intolerant to ER stress.

Inducible liver-specific overexpression of gankyrin in zebrafish results in spontaneous intrahepatic cholangiocarcinoma and hepatocellular carcinoma formation.

Liver cancer is the second leading cause of death worldwide. As such, establishing animal models of the disease is important for both basic and translational studies that move toward developing new therapies. Gankyrin is a critical oncoprotein in the genetic control of liver pathology. In order to evaluate the oncogenic role of gankyrin without cancer cell inoculation and drug treatment, we overexpressed gankyrin under the control of the fabp10a promoter. A Tet-Off system was used to drive expression in hepatocytes. At seven to twelve months of age, gankyrin transgenic fish spontaneously incurred persistent hepatocyte damage, steatosis, cholestasis, cholangitis, fibrosis and hepatic tumors. The tumors were both hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). ICC is the second most frequent primary liver cancer in human patients and the first to develop in this tumor model. We further investigated the role of complement C3, a central molecule of the complement system, and found the expression levels of both in mRNA and protein are decreased during tumorigenesis. Together, these findings suggest that gankyrin can promote malignant transformation of liver cells in the context of persistent liver injury. This transformation may be related to compensatory proliferation and the inflammatory microenvironment. The observed decrease in complement C3 may allow transforming cells to escape coordinated induction of the immune response. Herein, we demonstrate an excellent zebrafish model for liver cancers that will be useful for studying the molecular mechanisms of tumorgenesis.

Ftr82 Is Critical for Vascular Patterning during Zebrafish Development.

Cellular components and signaling pathways are required for the proper growth of blood vessels. Here, we report for the first time that a teleost-specific gene ftr82 (finTRIM family, member 82) plays a critical role in vasculature during zebrafish development. To date, there has been no description of tripartite motif proteins (TRIM) in vascular development, and the role of ftr82 is unknown. In this study, we found that ftr82 mRNA is expressed during the development of vessels, and loss of ftr82 by morpholino (MO) knockdown impairs the growth of intersegmental vessels (ISV) and caudal vein plexus (CVP), suggesting that ftr82 plays a critical role in promoting ISV and CVP growth. We showed the specificity of ftr82 MO by analyzing ftr82 expression products and expressing ftr82 mRNA to rescue ftr82 morphants. We further showed that the knockdown of ftr82 reduced ISV cell numbers, suggesting that the growth impairment of vessels is likely due to a decrease of cell proliferation and migration, but not cell death. In addition, loss of ftr82 affects the expression of vascular markers, which is consistent with the defect of vascular growth. Finally, we showed that ftr82 likely interacts with vascular endothelial growth factor (VEGF) and Notch signaling. Together, we identify teleost-specific ftr82 as a vascular gene that plays an important role for vascular development in zebrafish.

A 4-Phenoxyphenol Derivative Exerts Inhibitory Effects on Human Hepatocellular Carcinoma Cells through Regulating Autophagy and Apoptosis Accompanied by Downregulating α-Tubulin Expression.

Hepatocellular carcinoma (HCC) is a leading cancer worldwide. Advanced HCCs are usually resistant to anticancer drugs, causing unsatisfactory chemotherapy outcomes. In this study, we showed that a 4-phenoxyphenol derivative, 4-[4-(4-hydroxyphenoxy)phenoxy]phenol (4-HPPP), exerts an inhibitory activity against two HCC cell lines, Huh7 and Ha22T. We further investigated the anti-HCC activities of 4-HPPP, including anti-proliferation and induction of apoptosis. Our results showed that higher dosage of 4-HPPP downregulates the expression of α-tubulin and causes nuclear enlargement in both the Huh-7 and Ha22T cell lines. Interestingly, the colony formation results showed a discrepancy in the inhibitory effect of 4-HPPP on HCC and rat liver epithelial Clone 9 cells, suggesting the selective cytotoxicity of 4-HPPP toward HCC cells. Furthermore, the cell proliferation and apoptosis assay results illustrated the differences between the two HCC cell lines. The results of cellular proliferation assays, including trypan blue exclusion and colony formation, revealed that 4-HPPP inhibits the growth of Huh7 cells, but exerts less cytotoxicity in Ha22T cells. Furthermore, the annexin V assay performed for detecting the apoptosis showed similar results. Western blotting results showed 4-HPPP caused the increase of pro-apoptotic factors including cleaved caspase-3, Bid and Bax in HCC cells, especially in Huh-7. Furthermore, an increase of autophagy-associated protein microtubule-associated protein-1 light chain-3B (LC3B)-II and the decrease of Beclin-1 and p62/SQSTM1 were observed following 4-HPPP treatment. Additionally, the level of γH2A histone family, member X (γH2AX), an endogenous DNA damage biomarker, was dramatically increased in Huh7 cells after 4-HPPP treatment, suggesting the involvement of DNA damage pathway in 4-HPPP-induced apoptosis. On the contrary, the western blotting results showed that treatment up-regulates pro-survival proteins, including the phosphorylation of protein kinase B (Akt) and the level of survivin on Ha22T cells, which may confer a resistance toward 4-HPPP. Notably, the blockade of extracellular signal-regulated kinases (ERK), but not Akt, enhanced the cytotoxicity of 4-HPPP against Ha22T cells, indicating the pro-survival role of ERK in 4-HPPP-induced anti-HCC effect. Our present work suggests that selective anti-HCC activity of 4-HPPP acts through induction of DNA damage. Accordingly, the combination of ERK inhibitor may significantly enhance the anti-cancer effect of 4-HPPP for those HCC cells which overexpress ERK in the future.

Enriched Astaxanthin Extract from Haematococcus pluvialis Augments Growth Factor Secretions to Increase Cell Proliferation and Induces MMP1 Degradation to Enhance Collagen Production in Human Dermal Fibroblasts.

Among many antioxidants that are used for the repairing of oxidative stress induced skin damages, we identified the enriched astaxanthin extract (EAE) from Haematococcus pluvialis as a viable ingredient. EAE was extracted from the red microalgae through supercritical fluid carbon dioxide extraction. To compare the effectiveness, EAE wastreated on human dermal fibroblasts with other components, phorbol 12-myristate 13-acetate (PMA), and doxycycline. With sirius red staining and quantitative real-time polymerase chain reaction (qRT-PCR), we found that PMA decreased the collagen concentration and production while overall the addition of doxycycline and EAE increased the collagen concentration in a trial experiments. EAE increased collagen contents through inhibited MMP1 and MMP3 mRNA expression and induced TIMP1, the antagonists of MMPs protein, gene expression. As for when tested for various proteins through western blotting, it was seen that the addition of EAE increased the expression of certain proteins that promote cell proliferation. Testing those previous solutions using growth factor assay, it was noticeable that EAE had a positive impact on cell proliferation and vascular endothelial growth factor (VEGF) than doxycycline, indicating that it was a better alternative treatment for collagen production. To sum up, the data confirmed the possible applications as medical cosmetology agentsand food supplements.

RIG-I specifically mediates group II type I IFN activation in nervous necrosis virus infected zebrafish cells.

The type I interferon (IFN) response has been shown to be crucial for the survival of zebrafish larvae infected with nervous necrosis virus (NNV). Teleost type I IFNs can be divided into two groups, based on their cysteine content. While teleost group I IFNs have been extensively studied in terms of their regulation and anti-viral properties, the characteristics of teleost group II IFNs have been relatively unexplored. In this study, we describe the mechanism by which group II IFNs are activated in response to NNV infection in a zebrafish cell line, by focusing on the relationship between type I IFNs and pattern recognition receptors. Expression profile analysis of infected cells by microarray and qPCR revealed signaling activation of two pattern recognition receptors (PRRs): RIG-I like receptors (RLRs) and MyD88-dependent Toll-like receptors (TLRs). Knockdown of retinoic acid-inducible gene I (RIG-I) specifically repressed induction of group II IFNs (IFNϕ2, IFNϕ3) by NNV infection. Furthermore, Ingenuity Pathway Analysis (IPA) was used to demonstrate that RIG-I knockdown results in down-regulation of the inflammatory response in NNV-infected cells. Taken together, our results indicate that RIG-I plays an essential role in zebrafish group II type I IFN induction and the inflammatory response to NNV infection.

4-(Phenylsulfanyl)butan-2-One Suppresses Melanin Synthesis and Melanosome Maturation In Vitro and In Vivo.

In this study, we screened compounds with skin whitening properties and favorable safety profiles from a series of marine related natural products, which were isolated from Formosan soft coral Cladiella australis. Our results indicated that 4-(phenylsulfanyl)butan-2-one could successfully inhibit pigment generation processes in mushroom tyrosinase platform assay, probably through the suppression of tyrosinase activity to be a non-competitive inhibitor of tyrosinase. In cell-based viability examinations, it demonstrated low cytotoxicity on melanoma cells and other normal human cells. It exhibited stronger inhibitions of melanin production and tyrosinase activity than arbutin or 1-phenyl-2-thiourea (PTU). Also, we discovered that 4-(phenylsulfanyl)butan-2-one reduces the protein expressions of melanin synthesis-related proteins, including the microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (Trp-1), dopachrome tautomerase (DCT, Trp-2), and glycoprotein 100 (GP100). In an in vivo zebrafish model, it presented a remarkable suppression in melanogenesis after 48 h. In summary, our in vitro and in vivo biological assays showed that 4-(phenylsulfanyl)butan-2-one possesses anti-melanogenic properties that are significant in medical cosmetology.

Genome sequences characterizing five mutations in RNA polymerase and major capsid of phages ϕA318 and ϕAs51 of Vibrio alginolyticus with different burst efficiencies.

BACKGROUND: The burst size of a phage is important prior to phage therapy and probiotic usage. The efficiency for a phage to burst its host bacterium can result from molecular domino effects of the phage gene expressions which dominate to control host machinery after infection. We found two Podoviridae phages, ϕA318 and ϕAs51, burst a common host V. alginolyticus with different efficiencies of 72 and 10 PFU/bacterium, respectively. Presumably, the genome sequences can be compared to explain their differences in burst sizes. RESULTS: Among genes in 42.5 kb genomes with a GC content of 43.5%, 16 out of 47 open-reading frames (ORFs) were annotated to known functions, including RNA polymerase (RNAP) and phage structure proteins. 11 strong phage promoters and three terminators were found. The consensus sequence for the new vibriophage promoters is AATAAAGTTGCCCTATA, where the AGTTG bases of -8 through -12 are important for the vibriophage specificity, especially a consensus T at -9 position eliminating RNAP of K1E, T7 and SP6 phages to transcribe the genes. ϕA318 and ϕAs51 RNAP shared their own specific promoters. In comparing ϕAs51 with ϕA318 genomes, only two nucleotides were deleted in the RNAP gene and three mutating nucleotides were found in the major capsid genes. CONCLUSION: Subtle analyses on the residue alterations uncovered the effects of five nucleotide mutations on the functions of the RNAP and capsid proteins, which account for the host-bursting efficiency. The deletion of two nucleotides in RNAP gene truncates the primary translation due to early stop codon, while a second translational peptide starting from GTG just at deletion point can remediate the polymerase activity. Out of three nucleotide mutations in major capsid gene, H53N mutation weakens the subunit assembly between capsomeres for the phage head; E313K reduces the fold binding between ß-sheet and Spine Helix inside the peptide.

Oxygenated eremophilane- and neolemnane-derived sesquiterpenoids from the soft coral Lemnalia philippinensis.

Five sesquiterpene-related metabolites (1-5), including two new eremophilane-type compounds, philippinlins C and D (1 and 2) and a 4,5-seconeolemnane philippinlin E (3), were isolated from the organic extract of a Taiwanese soft coral Lemnalia philippinensis. The structures of the new metabolites were determined on the basis of extensive spectroscopic analysis and by comparison of NMR data with those of related metabolites. Compound 3 was suggested to be derived from the neolemnane skeleton.

New nitrogenous bisabolene-type sesquiterpenes from a formosan sponge Axinyssa sp.

Chemical investigation of a Formosan sponge Axinyssa sp. has led to the isolation of five nitrogenous bisabolene-type sesquiterpenes 1-5, including two new compounds axinysalines A (1) and B (2). The structures of new compounds were elucidated by analysis of high resolution (HR)-MS and two dimensional (2D)-NMR spectra and comparison of its NMR data with those of known analogues. Compound 1 exhibited moderate to weak cytotoxicity against Molt 4 and K562 cancer cell lines.

Burning down the house: Pyroptosis in the tumor microenvironment of hepatocellular carcinoma.

A high mortality rate makes hepatocellular carcinoma (HCC) a difficult cancer to treat. When surgery is not possible, liver cancer patients are treated with chemotherapy. However, HCC management and treatment are difficult. Sorafenib, which is a first-line treatment for hepatocellular carcinoma, initially slows disease progression. However, sorafenib resistance limits patient survival. Recent studies have linked HCC to programmed cell death, which has increased researcher interest in therapies targeting cell death. Pyroptosis, which is an inflammatory mode of programmed cell death, may be targeted to treat HCC. Pyroptosis pathways, executors, and effects are examined in this paper. This review summarizes how pyroptosis affects the tumor microenvironment (TME) in HCC, including the role of cytokines such as IL-1ß and IL-18 in regulating immune responses. The use of chemotherapies and their ability to induce cancer cell pyroptosis as alternative treatments and combining them with other drugs to reduce side effects is also discussed. In conclusion, we highlight the potential of inducing pyroptosis to treat HCC and suggest ways to improve patient outcomes. Studies on cancer cell pyroptosis may lead to new HCC treatments.

Exploring the Relationship between Gut Microbiome Composition and Blood Indole-3-acetic Acid in Hemodialysis Patients.

Indole-3-acetic acid (IAA), a protein-bound uremic toxin resulting from gut microbiota-driven tryptophan metabolism, increases in hemodialysis (HD) patients. IAA may induce endothelial dysfunction, inflammation, and oxidative stress, elevating cardiovascular and cognitive risk in HD patients. However, research on the microbiome-IAA association is limited. This study aimed to explore the gut microbiome's relationship with plasma IAA levels in 72 chronic HD patients aged over 18 (August 2016-January 2017). IAA levels were measured using tandem mass spectrometry, and gut microbiome analysis utilized 16s rRNA next-generation sequencing. Linear discriminative analysis effect size and random forest analysis distinguished microbial species linked to IAA levels. Patients with higher IAA levels had reduced microbial diversity. Six microbial species significantly associated with IAA levels were identified; Bacteroides clarus, Bacteroides coprocola, Bacteroides massiliensi, and Alisteps shahii were enriched in low-IAA individuals, while Bacteroides thetaiotaomicron and Fusobacterium varium were enriched in high-IAA individuals. This study sheds light on specific gut microbiota species influencing IAA levels, enhancing our understanding of the intricate interactions between the gut microbiota and IAA metabolism.

A zebrafish model of intrahepatic cholangiocarcinoma by dual expression of hepatitis B virus X and hepatitis C virus core protein in liver.

UNLABELLED: The mechanisms that mediate the initiation and development of intrahepatic cholangiocarcinoma (ICC) associated with hepatitis B and C virus (HBV and HCV, respectively) infection remain largely unclear. In this study we conditionally coexpressed hepatitis B virus X (HBx) and hepatitis C virus core (HCP) proteins in zebrafish livers, which caused fibrosis and consequently contributed to ICC formation at the age of 3 months. Suppressing the transgene expression by doxycycline (Dox) treatment resulted in the loss of ICC formation. The biomarker networks of zebrafish ICC identified by transcriptome sequencing and analysis were also frequently involved in the development of human neoplasms. The profiles of potential biomarker genes of zebrafish ICC were similar to those of human cholangiocarcinoma. Our data also showed that the pSmad3L oncogenic pathway was activated in HBx and HCP-induced ICC and included phosphorylation of p38 mitogen-activated proteinbase (MAPK) and p44/42 mitogen-activated protein kinase (ERK1/2), indicating the association with transforming growth factor beta 1 (TGF-ß1) signaling pathway in ICC. Bile duct proliferation, fibrosis, and ICC were markedly reduced by knockdown of TGF-ß1 by in vivo morpholinos injections. CONCLUSION: These results reveal that TGF-ß1 plays an important role in HBx- and HCP-induced ICC development. This in vivo model is a potential approach to study the molecular events of fibrosis and ICC occurring in HBV and HCV infection.

Grape Seed Proanthocyanidins Inhibit Replication of the Dengue Virus by Targeting NF-kB and MAPK-Mediated Cyclooxygenase-2 Expression.

Dengue virus (DENV) infection is a serious global health issue as it causes severe dengue hemorrhagic fever and dengue shock syndrome. Since no approved therapies are available to treat DENV infection, it is necessary to develop new agents or supplements that can do this. In this study, grape seed proanthocyanidins extract (GSPE), which is widely consumed as a dietary supplement, dose-dependently suppressed the replication of four DENV serotypes. The inhibitory mechanism demonstrated that GSPE downregulated DENV-induced aberrant cyclooxygenase-2 (COX-2) expression, revealing that the inhibitory effect of the GSPE on DENV replication involved targeting DENV-induced COX-2 expression. Mechanistic studies on signaling regulation have demonstrated that GSPE significantly reduced COX-2 expression by inactivating NF-κB and ERK/P38 MAPK signaling activities. Administrating GSPE to DENV-infected suckling mice reduced virus replication, mortality, and monocyte infiltration of the brain. In addition, GSPE substantially reduced the expression of DENV-induced inflammatory cytokines associated with severe dengue disease, including tumor necrosis factor-α, nitric oxide synthase, interleukin (IL)-1, IL-6, and IL-8, suggesting that GSPE has potential as a dietary supplement to attenuate DENV infection and severe dengue.

Antiangiogenic potential of Lepista nuda extract suppressing MAPK/p38 signaling-mediated developmental angiogenesis in zebrafish and HUVECs.

The medicinal properties of natural/edible plant products and their use are popular in traditional practice owing to their nutritional contents with little to no side effects. Lepista nuda (L. nuda), an edible mushroom (Clitocybe nuda, commonly known as blewit), has attracted researchers to evaluate its contents and the mechanism of its activities. In the current study, we focused on evaluating the antiangiogenic effects of L. nuda water extract on zebrafish development and in vitro human umbilical vein endothelial cell (HUVEC) tube formation. Bioactive components such as ergothioneine, eritadenine, and adenosine were identified and quantified by HPLC analysis. The L. nuda extract showed antiangiogenic properties and inhibited intersegmental vessel (ISV), caudal vein plexus (CVP), hyaloid vessel (HV), and subintestinal vessel (SIV) development in Tg (fli1: EGFP) zebrafish embryos. The expression of angiogenesis-related genes (vegfaa, kdrl, vegfba, flt1, kdr) was affected following L. nuda extract treatment. L. nuda extract attenuated in vitro HUVEC tube formation, migration, and invasion. Furthermore, inhibition of MAPK/p38 signaling and depletion of proangiogenic genes, including growth factors (fgf, ang2, and vegfa); primary and accessory receptors (tie2, vegfr2, and eng); MMPs (mmp1 and mmp2); and cytokines (il-1α, il-1ß, il-6, and tnf-α) was observed in HUVECs following L. nuda treatment. An in vivo zebrafish xenograft assay showed that L. nuda extract inhibited HuCCT1 cell-induced SIV sprouting in HuCCT1-injected embryos. Collectively, the results suggest that L. nuda could be a potential inhibitor of angiogenesis limiting cancer progression.