Rhein: A potent immunomodulator empowering largemouth bass against MSRV infection.

Micropterus salmoides rhabdovirus (MSRV) is a significant viral pathogen in largemouth bass aquaculture, causing substantial annual economic losses. However, effective prevention methods remain elusive for various reasons. Medicinal plant extracts have emerged as valuable tools in preventing and managing aquatic animal diseases. Thus, the search for immunomodulators with straightforward, safe structures in plant extracts is imperative to ensure the continued health and growth of the largemouth bass industry. In our research, we employed epithelioma papulosum cyprinid (EPC) cells and largemouth bass as models to assess the anti-MSRV properties and immunomodulatory effects of ten plant-derived bioactive compounds. Among them, rhein demonstrated noteworthy potential, exhibiting a 75 % reduction in viral replication in vitro at a concentration of 50 mg/L. Furthermore, rhein pre-treatment significantly inhibited MSRV genome replication in EPC cells, with the highest inhibition rate reaching 64.8 % after 24 h, underscoring rhein's preventive impact against MSRV. Likewise, rhein displayed remarkable therapeutic effects on EPC cells during the early stages of MSRV infection, achieving a maximum inhibition rate of 85.6 % in viral replication. Subsequent investigations unveiled that rhein, with its consistent activity, effectively mitigated cytopathic effects (CPE) and nuclear damage induced by MSRV infection. Moreover, it restrained mitochondrial membrane depolarization and reduced the apoptosis rate by 38.8 %. In vivo experiments reinforced these findings, demonstrating that intraperitoneal injection of rhein enhanced the expression levels of immune related genes in multiple organs, hindered virus replication, and curtailed the mortality rate of MSRV-infected largemouth bass by 29 %. Collectively, our study endorses the utility of rhein as an immunomodulator to combat MSRV infections in largemouth bass. This not only underscores the potential of rhein as a broad-spectrum antiviral and means to bolster the immune response but also highlights the role of apoptosis as an immunological marker, making it an invaluable addition to the armamentarium against aquatic viral pathogens.

Isolation and identification of Chryseobacterium indologenes and its pathological changes in Pelodiscus sinensis.

The Chinese revered a species of aquatic reptile known as Pelodiscus sinensis as both an edible and medicinal species. When artificially breeding, many deaths occurred at the farmed P. sinensis, mainly due to excessive breeding density, water contamination, and turtles biting each other secondary to bacterial infections. In this study, an isolate of gram-negative bacteria WH0623 was isolated from the liver and kidney of diseased P. sinensis to trace the potential pathogen of this disease. Based on biochemical characteristics and 16S rRNA gene sequencing analyses, this isolated strain of WH0623 was identified as Chryseobacterium indologenes. The strain's median lethal dose (LD50 ) was 3.3 × 105 colony-forming units (CFU)/g per fish weight tested using artificial infection. Histopathological analysis revealed pathological changes, including cell swelling, hyperaemia, and necrosis in many tissues. Antibiotic susceptibility tests revealed that the bacteria WH0623 was susceptible to doxycycline, sulphonamides, ceftazidime, norfloxacin, and ciprofloxacin. These antibiotics could treat the disease. In conclusion, the pathogen causing the death of farmed P. sinensis was isolated and identified, and a drug-sensitive test was conducted. Our findings contribute to the future diagnosis and treatment of the disease.

Isolation, identification and characterization of Aeromonas jandaei from diseased Chinese soft-shell turtles.

Aeromonas jandaei is a gram-negative bacterium commonly found in aquatic environments and can induce illnesses in amphibians, reptiles and aquatic animals. In this study, a strain of bacteria was isolated from the diseased Chinese soft-shell turtle (Pelodiscus sinensis), then named strain JDP-FX. This isolate was identified as A. jandaei after analysis of morphological, physiological and biochemical characteristics, as well as 16S rRNA and gyrB gene sequences. Virulence genetic testing further detected temperature-sensitive protease (eprCAI), type III secretion system (TTSS) (ascv), nuclease (nuc), cytotonic enterotoxin (alt) and serine proteinase (ser) in JDP-FX. Compared with healthy Chinese soft-shell turtle, the serum levels of total protein (TP), albumin (ALB) and globulin (GLB) were significantly decreased in the diseased Chinese soft-shell turtle, while, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were significantly increased. Histopathological observations showed that multiple tissues, including intestinal mucosa, liver and kidney, were severely damaged in the diseased Chinese soft-shell turtle. Moreover, the diseased Chinese soft-shell turtle had significant cell degeneration, necrosis, sloughing and interstitial inflammatory cell infiltration. The pathogenicity of JDP-FX was tested via artificial infection. The median lethal dosage (LD50 ) of the strain was 1.05 × 105 colony forming units (CFU/g) per weight of Chinese soft-shell turtle. Drug susceptibility analysis revealed that JDP-FX was susceptible to ceftazidime, minocycline, cefoperazone, ceftriaxone and piperacillin. In addition, JDP-FX was resistant to doxycycline, florfenicol, sulfonamides, gentamicin, ampicillin and neomycin. Therefore, this study may provide guidance for further research into the diagnosis, prevention and treatment of JDP-FX infection.

Hypolipidemic Effect of Rice Bran Oil Extract Tocotrienol in High-Fat Diet-Induced Hyperlipidemia Zebrafish (Danio Rerio) Induced by High-Fat Diet.

In recent years, the potent influence of tocotrienol (T3) on diminishing blood glucose and lipid concentrations in both Mus musculus (rats) and Homo sapiens (humans) has been established. However, the comprehensive exploration of tocotrienol's hypolipidemic impact and the corresponding mechanisms in aquatic species remains inadequate. In this study, we established a zebrafish model of a type 2 diabetes mellitus (T2DM) model through high-fat diet administration to zebrafish. In the T2DM zebrafish, the thickness of ocular vascular walls significantly increased compared to the control group, which was mitigated after treatment with T3. Additionally, our findings demonstrate the regulatory effect of T3 on lipid metabolism, leading to the reduced synthesis and storage of adipose tissue in zebrafish. We validated the expression patterns of genes relevant to these processes using RT-qPCR. In the T2DM model, there was an almost two-fold upregulation in pparγ and cyp7a1 mRNA levels, coupled with a significant downregulation in cpt1a mRNA (p < 0.01) compared to the control group. The ELISA revealed that the protein expression levels of Pparγ and Rxrα exhibited a two-fold elevation in the T2DM group relative to the control. In the T3-treated group, Pparγ and Rxrα protein expression levels consistently exhibited a two-fold decrease compared to the model group. Lipid metabolomics showed that T3 could affect the metabolic pathways of zebrafish lipid regulation, including lipid synthesis and decomposition. We provided experimental evidence that T3 could mitigate lipid accumulation in our zebrafish T2DM model. Elucidating the lipid-lowering effects of T3 could help to minimize the detrimental impacts of overfeeding in aquaculture.

Metabolomics in rare minnow (Gobiocypris rarus) after infection by attenuated and virulent grass carp reovirus genotype â ¡.

Grass carp reovirus genotype Ⅱ (GCRV Ⅱ) causes hemorrhagic disease in a variety fish, seriously affecting the aquaculture industry in China. However, the pathogenesis of GCRV Ⅱ is unclear. Rare minnow is an ideal model organism to study the pathogenesis of GCRV Ⅱ. Herein, we applied liquid chromatography-tandem mass spectrometry metabolomics to investigate metabolic responses in the spleen and hepatopancreas of rare minnow injected with virulent GCRV Ⅱ isolate DY197 and attenuated isolate QJ205. Results indicated that marked metabolic changes were identified in both the spleen and hepatopancreas after GCRV Ⅱ infection, and the virulent DY197 strain induced more significantly different metabolites (SDMs) than the attenuated QJ205 strain. Moreover, most SDMs were downregulated in the spleen and tend to be upregulated in hepatopancreas. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that tissue-specific metabolic responses were identified after viruses infection, and the virulent DY197 strain induced more SDMs involved in amino acid metabolism in the spleen, especially the tryptophan metabolism, cysteine and methionine metabolism, which were essential for immune regulation in host; Meanwhile, nucleotide metabolism, protein synthesis and metabolism related pathways were enriched in the hepatopancreas by both virulent and attenuated strains. Our findings revealed the large scale metabolic alterations in rare minnow in response to attenuated and virulent GCRV Ⅱ infection, which will lead to a better understanding of the pathogenesis of viruses and host-pathogens interactions.

Characteristics and expression profiles of MHC class â molecules in Carassius auratus.

Major histocompatibility complex class Ⅰ (MHC Ⅰ) molecules play a vital role in adaptive immune systems in vertebrates by presenting antigens to effector T cells. Understanding the expression profiling of MHC Ⅰ molecules in fish is essential for improving our knowledge of the relationship between microbial infection and adaptive immunity. In this study, we conducted a comprehensive analysis of MHC Ⅰ gene characteristics in Carassius auratus, an important freshwater aquaculture fish in China that is susceptible to Cyprinid herpesvirus 2 (CyHV-2) infection. We identified approximately 20 MHC Ⅰ genes discussed, including U, Z, and L lineage genes. However, only U and Z lineage proteins were identified in the kidney of Carassius auratus using high pH reversed-phase chromatography and mass spectrometry. The L lineage proteins were either not expressed or present at an extremely low level in the kidneys of Carassius auratus. We also used targeted proteomics to analyze changes in protein MHC Ⅰ molecules abundance in healthy and CyHV-2-infected Carassius auratus. We observed that five MHC Ⅰ molecules were upregulated, and Caau-UFA was downregulated in the diseased group. This study is the first to reveal the expression of MHC Ⅰ molecules at a large scale in Cyprinids, which enhances our understanding of fish adaptive immune systems.

Anti-inflammatory and antioxidant properties of rice bran oil extract in copper sulfate-induced inflammation in zebrafish (Danio rerio).

Tocotrienols have strong antioxidant properties; however, tocotrienol has not been investigated in detail in aquatic products. In this study, the anti-inflammatory and antioxidant activities of the tocotrienol-rich fraction from rice bran oil and its potential mechanism were verified in a zebrafish CuSO4 inflammation model. The in vitro antioxidant activity was evaluated using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) stable radical method. The copper chelating activity was determined using the pyrocatechol violet method. Intracellular reactive oxygen species in zebrafish were detected using a fluorescent ROS probe. Transgenic Tg (lyz: DsRed2) zebrafish were used for neutrophil transmigration assays. The mRNA expression levels of antioxidant and pro-inflammatory factor genes were measured using quantitative real-time reverse transcription PCR. In the concentration range tested, 100 µg/mL TRF had the highest copper chelating activity (10%). TRF showed DPPH-free radical scavenging ability, which was 53% at 100 µg/mL TRF. TRF effectively repressed ROS generation and inhibited neutrophil migration to the inflamed site. Moreover, TRF upregulated the expression of antioxidant genes sod and gpx4b, inhibited the expression of pro-inflammatory factors tnfa and il8, and suppressed CuSO4-induced inflammation. In conclusion, TRF has significant anti-inflammatory and antioxidant properties, which supports the use of TRF as an aquatic feed additive to improve the anti-inflammatory and antioxidant capacity of aquatic products.

Development of a droplet digital PCR assay for the sensitive detection of iridovirus in Andrias davidianus.

Chinese giant salamander iridovirus (GSIV) is the first known and causative viral pathogen in Andrias davidianus. Developing a sensitive, accurate and specific assay to detect GSIV in samples is essential to prevent the further spread of the pathogen. In this study, we established a droplet digital PCR (ddPCR) assay that targeted the mcp gene of GSIV, enabling rapid and quantitative detection of the virus. We determined that the optimal annealing temperature, primer concentration and probe concentration were 57.1°C, 50 nM and 500 nM, respectively. We analysed the specificity and sensitivity of the ddPCR assay and found that five common aquatic animal viruses, including Cyprinid herpesvirus 2 (CyHV-2), infectious spleen and kidney necrosis virus (ISKNV), Koi herpesvirus (KHV) and Carp Edema Virus (CEV) displayed negative results based on this GSIV ddPCR assay. The assay can detect GSIV with the lowest detection limit of 3.7 copies per reaction. To evaluate the sensitivity and accuracy of the ddPCR assay, we tested different infected tissue samples with both the ddPCR and TaqMan real-time PCR assays. Our results showed that the ddPCR assay detected GSIV in all samples with 100% positivity, while the TaqMan real-time PCR assay detected GSIV in only 82.1% of samples. The established ddPCR method provided several advantages in detecting GISV, including high sensitivity, high precision and absolute quantification, making it a powerful tool for detection of possible and potential GSIV infection, even in samples with low viral load.

Development and application of a sensitive droplet digital PCR-based method to detect tilapia parvovirus.

Tilapia parvovirus (TiPV) causes severe mortality rates in cultured tilapia, resulting in substantial losses to the fish industry. Droplet digital PCR (ddPCR) is a sensitive, accurate, and absolute quantitation method, plus it does not require a standard curve. Herein we report the development and application of a sensitive ddPCR-based method to rapidly detect and quantify TiPV. Optimal annealing temperature was determined to be 59.3°C, and optimal primer and probe concentrations were 900 nmol/L and 250 nmol/L, respectively. Our ddPCR method was highly specific to TiPV and showed no cross-reactivity with other viruses. Further, the detection limit of ddPCR was 0.07 copies/µl, being lower than that of real-time PCR (qPCR, 4.63 copies/µl). We also investigated the ability of ddPCR to detect TiPV in 50 samples and compared the outcome with qPCR data in terms of sensitivity and accuracy. The results showed that the positive detection rate of ddPCR (32%) was higher than that of qPCR (18%). To conclude, our ddPCR method was effective at detecting TiPV in samples with low viral loads. We believe that its application can facilitate the surveillance of sources and transmission routes of TiPV.

Development of a droplet digital PCR method for the sensitive detection and quantification of largemouth bass ranavirus.

Largemouth bass ranavirus (LMBRaV), also known as largemouth bass virus (LMBV), is a high mortality pathogen in largemouth bass. A rapid, sensitive, specific and convenient diagnosis method is an urgent requirement for the prevention of virus transmission. In the present study, a droplet digital PCR (ddPCR) method based on the major capsid protein (mcp) gene was established to detect and quantify the virus genome copy number. Oligonucleotide primers were designed based on the LMBRaV mcp gene sequence. The specificity and sensitivity of ddPCR assay were analysed. The other aquatic virus including Chinese giant salamander iridovirus (GSIV), Cyprinid herpesvirus II (CyHV-2) and infectious spleen and kidney necrosis virus could not be detected by LMBRaV ddPCR assay. The detection limit of ddPCR assay was 2 ± 0.37 copies/µl DNA sample. And this ddPCR assay had great repeatability and reproducibility. In clinical diagnosis of 50 largemouth bass, 43 positive samples were detected by ddPCR, whereas only 34 positive samples were detected by quantitative PCR (qPCR). This LMBRaV detection assay provided a specific and sensitive method for the rapid diagnosis of LMBRaV infection in largemouth bass as well as quantification of the virus load.

Anti-Inflammatory and Antioxidant Properties of Squalene in Copper Sulfate-Induced Inflammation in Zebrafish (Danio rerio).

Long-term or excessive oxidative stress can cause serious damage to fish. Squalene can be added to feed as an antioxidant to improve the body constitution of fish. In this study, the antioxidant activity was detected by 2,2-diphenyl-1-acrylhydrazyl (DPPH) test and fluorescent probe (dichloro-dihydro-fluorescein diacetate). Transgenic Tg (lyz: DsRed2) zebrafish were used to evaluate the effect of squalene on CuSO4-induced inflammatory response. Quantitative real-time reverse transcription polymerase chain reaction was used to examine the expression of immune-related genes. The DPPH assay demonstrated that the highest free radical scavenging exerted by squalene was 32%. The fluorescence intensity of reactive oxygen species (ROS) decreased significantly after 0.7% or 1% squalene treatment, and squalene could exert an antioxidative effect in vivo. The number of migratory neutrophils in vivo was significantly reduced after treatment with different doses of squalene. Moreover, compared with CuSO4 treatment alone, treatment with 1% squalene upregulated the expression of sod by 2.5-foldand gpx4b by 1.3-fold to protect zebrafish larvae against CuSO4-induced oxidative damage. Moreover, treatment with 1% squalene significantly downregulated the expression of tnfa and cox2. This study showed that squalene has potential as an aquafeed additive to provide both anti-inflammatory and antioxidative properties.

Proteomic and Phosphoproteomic Analyses Reveal Gibel Carp Responses to Cyprinid Herpesvirus 2 Infection.

Cyprinid herpesvirus 2 (CyHV-2) is a typical linear double-stranded DNA virus, which can induce severe herpesviral hematopoietic necrosis disease (HVHND) in gibel carp. However, the CyHV-2 infection mechanisms still remain unresolved till now. Here, we combined the isobaric tag for relative absolute quantitation (iTRAQ)-labeled quantitative proteomic and phosphoproteomic analysis enriched by Ti4+-immobilized titanium ion affinity chromatography (IMAC) to uncover the host responses to CyHV-2 infection in the kidneys of symptomatic and diseased gibel carp. We totally identified 192 differential expression proteins and 951 high-confident phosphopeptides involved in 657 proteins. After being infected with CyHV-2, the proteins involved in energy generation and ion balance were significantly downregulated in the host, and the phosphorylated proteins induced by viral infection mainly participated in the regulation for RNA processing, translation, cytoskeleton organization, immunoreaction, etc. Furthermore, 11 phosphorylated CyHV-2 viral proteins were found in the diseased group by the host proteome. The virus-host protein-protein interactions were investigated, in which the potential host kinases casein kinase II (CK-II) and cyclin-dependent kinase (CDK) that interacted with viral ORF88 or ORF89 were identified and can serve as candidate targets for disease treatment in the future. Overall, our study provides a comprehensive understanding of CyHV-2-induced perturbations at the protein and phosphorylation levels in gibel carp, forming a base for the treatment of HVHND.

Susceptibility of a cell line derived from the kidney of Chinese rice-field eel, Monopterus albus to the infection of rhabdovirus, CrERV.

Chinese rice-field eels rhabdovirus (CrERV), belonging to the genus Perhabdovirus in the family Rhabdoviridae, is the causative agent of the haemorrhagic disease of Chinese rice-field eels, Monopterus albus. The present study aims to establish a cell line derived from the kidney of Chinese rice-field eel (CrEK) for the further study of the pathogenic virus. CrEK cells were epithelioid-like and grew well in M199 medium supplemented with 10% foetal bovine serum at 28°C, and the cell line has been subcultured for more than 80 times. Karyotyping analysis of CrEK cells at 25th passage indicated a modal chromosome number of 24. Significant cytopathic effect (CPE) was observed in CrEK cells after infection with CrERV, and the virus titre reached 107.8 ± 0.45 TCID50 /mL. The transmission electron microscopy revealed that there were a large number of virus particles in the cytoplasm of cells. The virus infection in cells was also assayed by using indirect immunofluorescence assay (IFA), fluorescence in situ hybridization (FISH), reverse transcription PCR (RT-PCR) and quantitative real-time reverse transcription-PCR (qRT-PCR). In experimental infection, CrERV cultured by cells could cause over 90% mortality in fish. CrEK represents the first kidney cell line originated from Chinese rice-field eels and be a potential material for investigating the mechanism of virus infection in this fish and the control methods for the disease.

Rag1 and Rag2 Gene Expressions Identify Lymphopoietic Tissues in Larvae of Rice-Field Eel (Monopterus albus).

In immature lymphocytes, recombination activating genes 1 and 2 are necessary for antigen receptor V (D) J recombination, representing immature lymphocyte biomarkers. Herein, we cloned and sequenced rice-field eel rag1 and rag2 genes. Their expressions in the thymus, liver, and kidney were significant from 0 days post hatching (dph) to 45 dph, peaking at 45 dph in these three tissues. In situ hybridization detected high rag1 and rag2 expressions in the liver, kidney, and thymus of rice-field eel from 2 to 45 dph, suggesting that multiple tissues of rice-field eel contain lymphocyte lineage cells and undergo lymphopoiesis. Tissue morphology was used to observe lymphopoiesis development in these three tissues. The thymus primordium began to develop at 2 dph, while the kidney and liver have generated. Our findings verified that the thymus is the primary lymphopoietic tissue and suggested that, in rice-field eel, lymphocyte differentiation also occurs in the liver and kidney.

Chinese Giant Salamander (Andrias davidianus) Iridovirus Infection Leads to Apoptotic Cell Death through Mitochondrial Damage, Caspases Activation, and Expression of Apoptotic-Related Genes.

Chinese giant salamander iridovirus (GSIV) is the causative pathogen of Chinese giant salamander (Andrias davidianus) iridovirosis, leading to severe infectious disease and huge economic losses. However, the infection mechanism by GSIV is far from clear. In this study, a Chinese giant salamander muscle (GSM) cell line is used to investigate the mechanism of cell death during GSIV infection. Microscopy observation and DNA ladder analysis revealed that DNA fragmentation happens during GSIV infection. Flow cytometry analysis showed that apoptotic cells in GSIV-infected cells were significantly higher than that in control cells. Caspase 8, 9, and 3 were activated in GSIV-infected cells compared with the uninfected cells. Consistently, mitochondria membrane potential (MMP) was significantly reduced, and cytochrome c was released into cytosol during GSIV infection. p53 expression increased at an early stage of GSIV infection and then slightly decreased late in infection. Furthermore, mRNA expression levels of pro-apoptotic genes participating in the extrinsic and intrinsic pathway were significantly up-regulated during GSIV infection, while those of anti-apoptotic genes were restrained in early infection and then rose in late infection. These results collectively indicate that GSIV induces GSM apoptotic cell death involving mitochondrial damage, caspases activation, p53 expression, and pro-apoptotic molecules up-regulation.

Ammonia exposure alters the expression of immune-related and antioxidant enzymes-related genes and the gut microbial community of crucian carp (Carassius auratus).

Chronic exposure of ammonia in fish can affect the activities of antioxidant enzymes but few studies investigate the influence of ammonia exposure on the expression of immune-related and antioxidant enzymes-related genes. Also, there is no study demonstrates the effect of ammonia exposure on gut microbial community of fish. In this study, crucian carp (Carassius auratus) were exposed to the ammonia concentrations, 0 (control), 10 mg L-1 (low) or 50 mg L-1 (high) for consecutive 30 days at 25 ± 1 °C temperature, respectively, and after that, the fish from all exposure groups were maintained in control conditions for another 15 days. The results showed that low concentration ammonia increased the expression of immune-related genes and antioxidant enzymes-related genes, but high concentration ammonia inhibited the expression of immune-related genes and antioxidant enzymes-related genes. After a 15-day treatment without ammonia, the expression of antioxidant enzymes-related genes and immune-related genes showed no significant changes compared with control. The results of high-throughput sequencing showed that gut microbial communities were significantly differentiated following ammonia exposure. The abundance of Bacteroides and Cetobacterium (two kinds of potential probiotics) increased while fish exposed to 10 mg L-1 ammonia. The Flavobacterium (a potential fish pathogen) showed increasing trends when the exposure dose reached 50 mg L-1, while the Bacteroides and Cetobacterium showed almost no abundance. The results also revealed that ammonia exposure concentration or time can alter the intestinal microbial community. In conclusion, ammonia exposure could induce the immune response in crucian carp, and alter the gut microbial community. The results may help us understand the correlations of gut microbial community shift and ammonia exposure and extend our knowledge to comprehend the effects of environmental factors on intestinal microbial community.

ß-Sitosterol Reduces the Content of Triglyceride and Cholesterol in a High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease Zebrafish (Danio rerio) Model.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is strongly associated with hyperlipidemia, which is closely related to high levels of sugar and fat. ß-sitosterol is a natural product with significant hypolipidemic and cholesterol-lowering effects. However, the underlying mechanism of its action on aquatic products is not completely understood. METHODS: A high-fat diet (HFD)-induced NAFLD zebrafish model was successfully established, and the anti-hyperlipidemic effect and potential mechanism of ß-sitosterol were studied using oil red O staining, filipin staining, and lipid metabolomics. RESULTS: ß-sitosterol significantly reduced the accumulation of triglyceride, glucose, and cholesterol in the zebrafish model. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that differential lipid molecules in ß-sitosterol mainly regulated the lipid metabolism and signal transduction function of the zebrafish model. ß-sitosterol mainly affected steroid biosynthesis and steroid hormone biosynthesis in the zebrafish model. Compared with the HFD group, the addition of 500 mg/100 g of ß-sitosterol significantly inhibited the expression of Ppar-γ and Rxr-α in the zebrafish model by at least 50% and 25%, respectively. CONCLUSIONS: ß-sitosterol can reduce lipid accumulation in the zebrafish model of NAFLD by regulating lipid metabolism and signal transduction and inhibiting adipogenesis and lipid storage.

Aflatoxin B1 Induced Oxidative Stress and Gut Microbiota Disorder to Increase the Infection of Cyprinid Herpesvirus 2 in Gibel Carp (Carassius auratus gibelio).

Aflatoxin contamination of food and water is a serious problem worldwide. This study investigated the defensive ability of gibel carp exposed to aflatoxin B1 (AFB1) by challenging it with cyprinid herpesvirus 2 (CyHV-2) infection. The data showed that AFB1 exposure significantly increased the mortality of CyHV-2-infected gibel carp, and enhanced the viral load in the fish liver, kidney, and spleen. The oxidative-antioxidant balance suggested that AFB1 induced severe oxidative stress, including increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels in the AFB1 exposed group, and the reduced activity of superoxide dismutase (SOD), glutathione-S-transferase (GST) and catalase (CAT) in the AFB1 exposed group. Meanwhile, the related expression of nuclear factor erythroid 2-related factor 2 (Nrf2), interferon regulatory factor 3 (IRF3) and the type 1 interferon (IFN1) were noticeably down-regulated, but caspase-1 was up-regulated, after exposure to AFB1, demonstrating that fish are unable to avoid the virus infection. It should be noted that the intestinal microbiota diversity and richness were lower in the AFB1 exposed group, and the composition of intestinal microbiota was affected by AFB1, resulting in the higher abundance of bacteria (such as Aeromonas and Bacteroides) and the lower abundance of potentially beneficial bacteria (such as Cetobacterium and Clostridium) in the AFB1 exposed group. This research provides insight into the possibility that AFB1 may increase the susceptibility of C. gibelio to CyHV-2 infection, and thus amplify the viral outbreak to endanger ecological safety in aquatic environment.

Anti-Inflammatory and Antioxidant Properties of ß-Sitosterol in Copper Sulfate-Induced Inflammation in Zebrafish (Danio rerio).

ß-Sitosterol, which is used extensively in pharmaceuticals, nutraceuticals, and cosmetics, has high nutritional value along with immunomodulatory and anti-inflammatory properties. In this study, we investigated the antioxidant and anti-inflammatory effects of ß-sitosterol in zebrafish and explored the associated molecular mechanisms. In an in vivo antioxidant experiment, zebrafish (Danio rerio) larvae were treated with different concentrations of ß-sitosterol and then exposed to a nonlethal concentration of CuSO4 to induce oxidative stress. Treatment with ß-sitosterol at 70 or 100 µg/mL significantly reduced CuSO4-induced oxidative stress in the zebrafish, demonstrating the strong antioxidant activity of ß-sitosterol. Treatment with ß-sitosterol protected zebrafish larvae against oxidative damage from CuSO4 by upregulating the expressions of sod and gpx4b. In a zebrafish model of inflammation, pretreatment with ß-sitosterol before CuSO4 exposure inhibited neutrophil recruitment and damage to lateral line neuromasts, indicating a potent anti-inflammatory effect derived from reductions in the expressions of il-8 and myd88. The results demonstrate the antioxidative and anti-inflammatory activities of ß-sitosterol and suggest that ß-sitosterol may be useful for the treatment of various inflammatory diseases.

Elder and booster vaccination associates with decreased risk of serious clinical outcomes in comparison of Omicron and Delta variant: A meta-analysis of SARS-CoV-2 infection.

Background: The COVID-19 pandemic brings great pressure to the public health systems. This meta-analysis aimed to compare the clinical outcomes among different virus variants, to clarify their impact on medical resources and to provide evidence for the formulation of epidemic prevention policies. Methods: A systematic literature search was performed in the PubMed, Embase, and Cochrane Library databases using the key words "Omicron" and "Delta." The adjusted Risk ratios (RRs), Odds ratios (ORs) and Hazard ratios (HRs) were extracted, and RRs and Rate difference % (RD%) were used to interpret the risk estimates of the outcomes ultimately. Results: Forty-three studies were included, with 3,812,681 and 14,926,841 individuals infected with SARS-CoV-2 Delta and Omicron variant, respectively. The relative risks of hospitalization, death, ICU admission, and mechanical ventilation use after infection with the Omicron variant were all significantly reduced compared those after infection with the Delta variant (RRhospitalization = 0.45, 95%CI: 0.40-0.52; RRdeath = 0.37, 95%CI: 0.30-0.45; RRICU = 0.35, 95%CI: 0.29-0.42; RRmechanical ventilation = 0.33, 95%CI: 0.25-0.44). The change of both absolute and relative risks for hospitalization was more evident (RR = 0.47, 95%CI: 0.42-0.53；RD% =10.61, 95%CI: 8.64-12.59) and a significant increase was observed for the absolute differences in death in the elderly (RD% = 5.60, 95CI%: 4.65-6.55); the change of the absolute differences in the risk of hospitalization and death were most markedly observed in the patients with booster vaccination (RD%hospitalization = 8.60, 95CI%: 5.95-11.24; RD%death = 3.70, 95CI%: 0.34-7.06). Conclusion: The ability of the Omicron variant to cause severe clinical events has decreased significantly, as compared with the Delta variant, but vulnerable populations still need to be vigilant. There was no interaction between the vaccination doses and different variants.