Integrated multi-omics analysis reveals liver metabolic reprogramming by fish iridovirus and antiviral function of alpha-linolenic acid.

Iridovirus poses a substantial threat to global aquaculture due to its high mortality rate; however, the molecular mechanisms underpinning its pathogenesis are not well elucidated. Here, a multi-omics approach was applied to groupers infected with Singapore grouper iridovirus (SGIV), focusing on the roles of key metabolites. Results showed that SGIV induced obvious histopathological damage and changes in metabolic enzymes within the liver. Furthermore, SGIV significantly reduced the contents of lipid droplets, triglycerides, cholesterol, and lipoproteins. Metabolomic analysis indicated that the altered metabolites were enriched in 19 pathways, with a notable down-regulation of lipid metabolites such as glycerophosphates and alpha-linolenic acid (ALA), consistent with disturbed lipid homeostasis in the liver. Integration of transcriptomic and metabolomic data revealed that the top enriched pathways were related to cell growth and death and nucleotide, carbohydrate, amino acid, and lipid metabolism, supporting the conclusion that SGIV infection induced liver metabolic reprogramming. Further integrative transcriptomic and proteomic analysis indicated that SGIV infection activated crucial molecular events in a phagosome-immune depression-metabolism dysregulation-necrosis signaling cascade. Of note, integrative multi-omics analysis demonstrated the consumption of ALA and linoleic acid (LA) metabolites, and the accumulation of L-glutamic acid (GA), accompanied by alterations in immune, inflammation, and cell death-related genes. Further experimental data showed that ALA, but not GA, suppressed SGIV replication by activating antioxidant and anti-inflammatory responses in the host. Collectively, these findings provide a comprehensive resource for understanding host response dynamics during fish iridovirus infection and highlight the antiviral potential of ALA in the prevention and treatment of iridoviral diseases.

Dual-specificity phosphatase 1 inhibits Singapore grouper iridovirus replication via regulating apoptosis in Epinephelus coioides.

The dual-specificity phosphatase (DUSP) family plays key roles in the maintenance of cellular homeostasis and apoptosis etc. In this study, the DUSP member DUSP1 of Epinephelus coioides was characterized: the length was 2371 bp including 281 bp 5' UTR, 911 bp 3' UTR, and a 1125 bp open reading frame encoding 374 amino acids. E. coioides DUSP1 has two conserved domains, a ROHD and DSPc along with a p38 MAPK phosphorylation site, localized at Ser308. E. coioides DUSP1 mRNA can be detected in all of the tissues examined, and the subcellular localization showed that DUSP1 was mainly distributed in the nucleus. Singapore grouper iridovirus (SGIV) infection could induce the differential expression of E. coioides DUSP1. Overexpression of DUSP1 could inhibit SGIV-induced cytopathic effect (CPE), the expressions of SGIV key genes, and the viral titers. Overexpression of DUSP1 could also regulate SGIV-induced apoptosis, and the expression of apoptosis-related factor caspase 3. The results would be helpful to further study the role of DUSP1 in viral infection.

Identification of circRNAs and circRNA-mRNA network of Epinephelus coioides during Singapore grouper iridovirus infection.

Circular RNA (circRNA), one of the important non-coding RNA molecules with a closed-loop structure, plays a key regulatory role in cell processing. In this study, circRNAs of Epinephelus coioides, an important marine cultured fish in China, were isolated and characterized, and the network of circRNAs and mRNA was explored during Singapore grouper iridovirus (SGIV) infection, one of the most important double stranded DNA virus pathogens of marine fish. 10 g of raw data was obtained by high-throughput sequencing, and 2599 circRNAs were classified. During SGIV infection, 123 and 37 circRNAs occurred differential expression in spleen and spleen cells, indicating that circRNAs would be involved in the viral infection. GO annotation and KEGG demonstrated that circRNAs could target E. coioides genes to regulate cell activity and the activation of immune factors. The results provide some insights into the circRNAs mediated immune regulatory network during bony fish virus infection.

Curcumin Alleviates Singapore Grouper Iridovirus-Induced Intestine Injury in Orange-Spotted Grouper (Epinephelus coioides).

Singapore grouper iridovirus (SGIV) is a new ranavirus species in the Iridoviridae family, whose high lethality and rapid spread have resulted in enormous economic losses for the aquaculture industry. Curcumin, a polyphenolic compound, has been proven to possess multiple biological activities, including antibacterial, antioxidant, and antiviral properties. This study was conducted to determine whether curcumin protected orange-spotted grouper (Epinephelus coioides) from SGIV-induced intestinal damage by affecting the inflammatory response, cell apoptosis, oxidative stress, and intestinal microbiota. Random distribution of healthy orange-spotted groupers (8.0 ± 1.0 cm and 9.0 ± 1.0 g) into six experimental groups (each group with 90 groupers): Control, DMSO, curcumin, SGIV, DMSO + SGIV, and curcumin + SGIV. The fish administered gavage received DMSO dilution solution or 640 mg/L curcumin every day for 15 days and then were injected intraperitoneally with SGIV 24 h after the last gavage. When more than half of the groupers in the SGIV group perished, samples from each group were collected for intestinal health evaluation. Our results showed that curcumin significantly alleviated intestine damage and repaired intestinal barrier dysfunction, which was identified by decreased intestine permeability and serum diamine oxidase (DAO) activity and increased expressions of tight junction proteins during SGIV infection. Moreover, curcumin treatment suppressed intestinal cells apoptosis and inflammatory response caused by SGIV and protected intestinal cells from oxidative injury by enhancing the activity of antioxidant enzymes, which was related to the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. Moreover, we found that curcumin treatment restored the disruption of the intestinal microbiota caused by SGIV infection. Our study provided a theoretical basis for the functional development of curcumin in aquaculture by highlighting the protective effect of curcumin against SGIV-induced intestinal injury.

Relationship of environmental factors in pond water and dynamic changes of gut microbes of sea bass Lateolabrax japonicus.

The effect of structure of gut microbes on the health of host has attracted increasing attention. Sea bass Lateolabrax japonicus is an important farmed fish in China. The relationship of the dynamic changes of intestinal bacterial communities in L. japonicus and the cultural water environment is very important for healthy culture. Here, the diversity and abundance of the gut microbial communities of L. japonicus were evaluated during the culture using 16S rRNA Illumina sequencing. Both the opportunistic pathogens Aeromonas (1.68%), Vibrio (1.59%), and Acinetobacter (1.22%); and the potential probiotics Lactobacillus (2.27%), Bacillus (1.16%), and Lactococcus (0.37%) were distributed in the gut of L. japonicus. The increasing concentration of nitrogen of water environments with the increase of culture time significantly correlated with shifts in the microbial community structure: 40.04% of gut microbial changes due to nitrogen concentration. Higher concentrations of nitrogen showed a significantly negative correlation with intestinal probiotics in L. japonicus. The results indicate that the abundance of intestinal bacteria of L. japonicus is mainly driven by the changes of environmental factors (e.g., nitrogen), and it's very important that the linking environmental parameters with bacterial data of guts could be used as an early warning indicator in L. japonicus heath culture.

The extract of Curcumae Longae Rhizoma suppresses angiogenesis via VEGF-induced PI3K/Akt-eNOS-NO pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Curcumae Longae Rhizoma (CLR) is a safe natural herbal medicine, and which has been widely used for centuries as functional food and health products, but its effects on angiogenesis and related underlying mechanism remain unclear. AIM OF THE STUDY: The abnormal angiogenesis is closely related with various diseases, and therefore the precise control of angiogenesis is of great importance. The well-known angiogenic factor, vascular endothelial growth factor (VEGF), mediates angiogenesis and induces multiple signalling pathways via binding to VEGF receptor (VEGFR). The attenuation of VEGF-triggered angiogenic-related signalling pathways may relieve various diseases through suppression of angiogenesis. Here, we aimed to elucidate that CLR extract could exert striking anti-angiogenic activities both in vitro and in vivo. MATERIALS AND METHODS: The viability of human umbilical vascular endothelial cell (HUVEC) was examined by LDH and MTT assays. Migrative and invasive ability of the endothelial cells were independently evaluated by wound healing and transwell assays. The activities of CLR extract on in vitro angiogenesis was tested by tube formation assay. In vivo vascularization was determined by using zebrafish embryo model in the present of CLR extract. Western blotting was applied to determine the phosphorylated levels of VEGFR2, PI3K, AKT and eNOS. Besides, the levels of nitric oxide (NO) and reactive oxygen species (ROS) were separately evaluated by Griess assay and 2'7'-dichlorofluorescein diacetate reaction. In addition, the cell migrative ability of cancer cell was estimated by using cultured human colon carcinoma cells (HT-29 cell line), and immunofluorescence assay was applied to evaluate the effect of CLR extract on nuclear translocation of NF-κB p65 subunit in the VEGF-treated HT-29 cultures. RESULTS: CLR extract significantly suppressed a series of VEGF-mediated angiogenic responses, including endothelial cell proliferation, migration, invasion, and tube formation. Moreover, CLR extract reduced in vivo sub-intestinal vessel formation in zebrafish embryo model. Mechanistically, the extract of CLR attenuated the VEGF-triggered signalling, as demonstrated by decreased level of phosphorylated VEGFR2 and subsequently inactivated its downstream regulators, e.g. phospho-PI3K, phospho-AKT and phospho-eNOS. The production of NO and formation of ROS were markedly inhibited in HUVECs. Furthermore, CLR extract suppressed cell migration and NF-κB translocation in cultured HT-29 cells. CONCLUSIONS: These preclinical findings demonstrate that the extract of CLR remarkably attenuates angiogenesis and which has great potential as a natural drug candidate with excellent anti-angiogenic activity.

Molecular characterization, expression and function analysis of Epinephelus coioides PKC-ɑ response to Singapore grouper iridovirus (SGIV) infection.

Protein kinase C (PKC) constitutes the main signal transduction pathway, and participates in the signal pathway of cell proliferation and movement in mammals. In this study, PKC-ɑ was obtained from Epinephelus coioides, an important marine fish cultivated in the coastal areas of southern China and Southeast Asia. The full length cDNA of PKC-ɑ was 3362 bp in length containing a 23 bp 5'UTR, a 1719 bp 3'UTR, and a 1620 bp open reading frame encoding 539 amino acids. It contains three conservative domains including protein kinase C conserved region 2 (C2), Serine/Threonine protein kinases, catalytic domain (S_TKc) and ser/thr-type protein kinases (S_TK_X). Its mRNA can be detected in all 11 tissues examined of E. coioides, and the expression was significantly upregulated response to Singapore grouper iridovirus (SGIV) infection, one of the important pathogens of marine fish. Upregulated E. coioides PKC-ɑ significantly inhibited the activation of nuclear factor kappa-B (NF-κB) and activator protein-1 (AP-1), and SGIV-induced cell apoptosis. The results indicated that the PKC-ɑ may play an important role in pathogenic stimulation.

Identification and characterization of lncRNAs and the interaction of lncRNA-mRNA in Epinephelus coioides induced with Singapore grouper iridovirus infection.

Singapore grouper iridovirus (SGIV) is a highly pathogenic double-stranded DNA virus, and the fatality rate of SGIV-infected grouper is more than 90%. Up to now, there is no effective methods to control the disease. Long non-coding RNAs (lncRNAs) might play an important role in individual growth and development, immune regulation and other life processes. In this study, lncRNAs were identified in Epinephelus coioides, an important economic aquaculture marine fish in China and Southeast Asia, and the regulatory relationships of lncRNAs and mRNA response to SGIV infection were analyzed. A total of 11,678 lncRNAs were identified and classified from the spleen and GS (grouper spleen) cells. 105 differentially expressed lncRNAs (DElncRNAs) were detected during SGIV infection. The lncRNAs and the regulated mRNAs were analyzed using co-expression network, lncRNA target gene annotation and GO enrichment. At 24 and 48 h after SGIV infection, 118 and 339 lncRNA-mRNA pairs in GS cells were detected, and 728 and 688 differentially expressed lncRNA-mRNA pairs in spleen were obtained, respectively. GO and KEGG were used to predict the DE lncRNAs' target genes, and deduce the DE lncRNAs-affected signaling pathways. In GS cells, lncRNAs might participate in cell part, binding and catalytic activity; and lncRNAs might be involved in immune system process and transcription factor activity in spleen. These data demonstrated that lncRNAs could regulate the expression of immune-related genes response to viral infection, and providing a new insight into understanding the complexity of immune regulatory networks mediated by lncRNAs during viral infection in teleost fish.

The inclusion of extract from aerial part of Scutellaria baicalensis in feeding of pearl gentian grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceo-latus♂) promotes growth and immunity.

The root of Scutellaria baicalensis (Scutellaria Radix) has been used as herbal medicine for years in China; however, its stem and leaf (aerial part) are considered as waste. The water extract of aerial part of S. baicalensis, named as SBA, having anti-microbial property has been applied in fish aquaculture. To extend the usage of SBA in fish feeding, SBA was employed to feed pearl gentian grouper (a hybrid of Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂), and subsequently the total fish output, the levels of digestive enzymes and inflammatory cytokines were determined. Feeding the fish with different doses of SBA for two months, the body length and weight were significantly increased by 5%-10%. In parallel, the expressions of alkaline phosphatase and growth-related factors in bone, liver and muscle of SBA-fed fish were doubled, which could account the growth promoting effect of SBA. Besides, the activity of digestive enzyme, lipase, and the expressions of anti-inflammatory cytokines were markedly stimulated by 2-3 times under the feeding of 3% SBA-containing diet. The results indicated the growth promoting activity of SBA in culture of pearl gentian grouper, as well as the effect of SBA in strengthening the immunity. These beneficial effects of SBA feeding can increase the total yield of pearl gentian grouper in aquaculture. Thus, the re-cycle of waste products during the farming of S. baicalensis herb in serving as fish feeding should be encouraged.

Resveratrol, an Inhibitor Binding to VEGF, Restores the Pathology of Abnormal Angiogenesis in Retinopathy of Prematurity (ROP) in Mice: Application by Intravitreal and Topical Instillation.

Retinopathy of prematurity (ROP) is a severe eye disease leading to blindness. Abnormal vessel formation is the pathological hallmark of neovascular ROP. In forming vessels, vascular endothelial growth factor (VEGF) is an important stimulator. The current anti-ROP therapy has focused on bevacizumab, a monoclonal antibody against VEGF, and pazopanib, a tyrosine kinase inhibitor on the VEGF receptor (VEGFR). Several lines of evidence have proposed that natural compounds may be more effective and safer for anti-VEGF function. Resveratrol, a common natural compound, binds to VEGF and blocks its interaction with VEGFR, thereafter suppressing angiogenesis. Here, we evaluate the efficacy of intravitreal injection, or topical instillation (eye drops), of resveratrol into the eyes of mice suffering from oxygen-induced retinopathy, i.e., developing ROP. The treatment of resveratrol significantly relieved the degree of vascular distortion, permeability and hyperplasia; the efficacy could be revealed by both methods of resveratrol application. In parallel, the treatments of resveratrol inhibited the retinal expressions of VEGF, VEGFR and CD31. Moreover, the applied resveratrol significantly relieved the damage caused by oxygen radicals through upregulating the level of superoxide dismutase (SOD) and downregulating the level of malondialdehyde (MDA) in the retina. Taken together, the potential therapeutic benefit of resveratrol in pro-angiogenic diseases, including retinopathy, can be considered.

Epinephelus coioides PCSK9 affect the infection of SGIV by regulating the innate immune response.

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in mammals is a multifunctional protein. In this study, PCSK9 of marine fish Epinephelus coioides was characterized. The full-length cDNA of E. coioides PCSK9 was 2458 bp in length containing 185 bp 5' UTR, 263 bp 3' UTR and 2010 bp open reading frame (ORF) encoding 669 amino acids with the predicted molecular weight of 71 kDa and the theoretical PI of 6.6. Similar to other members of PCSK9 family, E. coioides PCSK9 has three conserved domains: Inhibitor_ I9 super family, Peptidases_ S8_ PCSK9_ Proteinase K_ like, and PCSK9_ C-CRD super family. E. coioides PCSK9 mRNA could be detected in all the tissues examined by real-time quantitative PCR, with the highest expression in the brain, followed by skin, trunk kidney, head kidney, intestine, blood, liver, spleen, gill, muscle and heart. E. coioides PCSK9 was distributed in both the cytoplasm and nucleus. The expression of E. coioides PCSK9 was significantly upregulated during Singapore grouper iridovirus (SGIV) infection. Upregulated PCSK9 could significantly affect the activities of nuclear factor kappaB (NF-κB) promoter, SGIV-induced apoptosis, and the expressions of the key SGIV genes (ICP18, LITAT, MCP, and VP19) and the E. coioides proinflammatory factors (IL-6, IL-1ß, IL-8, and TNF-α). The results illustrated that E. coioides PCSK9 might be involved in the pathogen infection by regulating the innate immune response.

Molecular cloning, inducible expression and function analysis of Epinephelus coioides Sec6 response to SGIV infection.

Exocyst complex component 3 Sec6 of mammals, one of the components of the exocyst complex, participates in numerous cellular functions, such as promoting cell migration and inhibiting apoptosis. In this study, the Sec6 was obtained from Epinephelus coioides, an economically important cultured fish. The full length of E. coioides Sec6 was 2655 bp including a 245 bp 5' UTR, a 154 bp 3' UTR, and a 2256 bp open reading frame (ORF) encoding 751 amino acids, with a molecular mass of 86.76 kDa and a theoretical pI of 5.57. Sec6 mRNA was detected in all the tissues examined, but the expression level is different in these tissues. Using fluorescence microscopy, Sec6 were distributed in both the nucleus and the cytoplasm. After SGIV infection, the expression of E. coioides Sec6 was significantly up-regulated in both trunk kidney and spleen response to Singapore grouper iridovirus (SGIV), an important pathogens of E. coioides. Sec6 could increase the SGIV-induced cytopathic effects (CPE), the expression of the SGIV genes VP19, LITAF, MCP, ICP18 and MCP, and the viral titers. Besides, E. coioides Sec6 significantly downregulated the promoter of NF-κB and AP-1, and inhibited the SGIV-induced apoptosis. The results demonstrated that E. coioides Sec6 might play important roles in SGIV infection.

Epinephelus coioides Hsp27 negatively regulates innate immune response and apoptosis induced by Singapore grouper iridovirus (SGIV) infection.

Heat shock proteins (Hsps) are important for maintaining protein homeostasis and cell survival. In this study, Hsp27 of Epinephelus coioides, an economically important marine fish in China and Southeast Asian countries, was characterized. E. coioides Hsp27 contains the consered ACD_HspB1_like domain and three p38 MAPK phosphorylation sites, located at Thr-13, Thr-60 and Ser-167. E. coioides Hsp27 was distributed in both the cytoplasm and nucleus, its mRNA was detected in all 14 tissues examined, and its expression was up-regulated after challenge with Singapore grouper iridovirus (SGIV), an important E. coioides pathogen. Over-expression of E. coioides Hsp27 significantly upregulated the expressions of the key SGIV genes (VP19, LITAF, MCP, and ICP18), downgraded the expressions of the E. coioides immune factors (IRF3, IRF7, ISG15, and TRAF6) and proinflammatory factors (TNF-α, IL-8), downgraded the activation of nuclear factor kappa-B (NF-κB) and activator protein-1 (AP-1), and substantially inhibited the cell apoptosis induced by SGIV infection. These data illustrated that E. coioides Hsp27 might be involved in SGIV infection by negatively regulating the innate immune response.

MicroRNA-124 Promotes Singapore Grouper Iridovirus Replication and Negatively Regulates Innate Immune Response.

Viral infections seriously affect the health of organisms including humans. Now, more and more researchers believe that microRNAs (miRNAs), one of the members of the non-coding RNA family, play significant roles in cell biological function, disease occurrence, and immunotherapy. However, the roles of miRNAs in virus infection (entry and replication) and cellular immune response remain poorly understood, especially in low vertebrate fish. In this study, based on the established virus-cell infection model, Singapore grouper iridovirus (SGIV)-infected cells were used to explore the roles of miR-124 of Epinephelus coioides, an economically mariculture fish in southern China and Southeast Asia, in viral infection and host immune responses. The expression level of E. coioides miR-124 was significantly upregulated after SGIV infection; miR-124 cannot significantly affect the entry of SGIV, but the upregulated miR-124 could significantly promote the SGIV-induced cytopathic effects (CPEs), the viral titer, and the expressions of viral genes. The target genes of miR-124 were JNK3/p38α mitogen-activated protein kinase (MAPK). Overexpression of miR-124 could dramatically inhibit the activation of NF-κB/activating protein-1 (AP-1), the transcription of proinflammatory factors, caspase-9/3, and the cell apoptosis. And opposite results happen when the expression of miR-124 was inhibited. The results suggest that E. coioides miR-124 could promote viral replication and negatively regulate host immune response by targeting JNK3/p38α MAPK, which furthers our understanding of virus and host immune interactions.

Characterization and function analysis of Epinephelus coioides Hsp40 response to Vibrio alginolyticus and SGIV infection.

Heat shock protein 40 (Hsp40), a member of Heat shock proteins (Hsps) family, plays a crucial role in regulation of cell proliferation, survival and apoptosis in mammals. In this study, Hsp40, EcHsp40, was identified from Epinephelus coioides, an economically important marine-cultured fish in China and Southeast Asian counties. The full length of EcHsp40 was 2236 bp in length containing a 1026 bp open reading frame (ORF) encoding 341 amino acids, with a molecular mass of 37.88 kDa and a theoretical pI of 9.09. EcHsp40 has two conserved domains DnaJ and DnaJ_C. EcHsp40 mRNA was detected in all tissues examined, and the expression was significantly up-regulated response to challenged with Vibrio alginolyticus or Singapore grouper iridovirus (SGIV), one of the important pathogens of marine fish. EcHsp40 was distributed in both the cytoplasm and nucleus, over-expression of EcHsp40 can inhibit the activity of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), significantly promote SGIV-induced apoptosis, intracellular caspase-3 activity and viral replication, suggesting that the EcHsp40 may play an important role in pathogenic stimulation.

Feeding containing the aerial part of Scutellaria baicalensis promotes the growth and nutritive value of rabbit fish Siganus fuscescens.

The root of Scutellaria baicalensis (Scutellaria Radix) has been used as herbal medicine for years, while its stem and leaf (aerial part) are considered as waste. The water extract from the aerial part of S. baicalensis (named as SBA) being included in the feeding of Siganus fuscescens (grey rabbit fish) has been shown to replace antibiotics in aquaculture with excellent outcome. To strengthen the usage of SBA in fish feeding, the total fish output and its nutritive value were determined here. Feeding the fishes with different doses of SBA for a month, the body length and weight were significantly increased after intake of standard feed containing 1% SBA. In parallel, the expressions of alkaline phosphatase and growth-related factors in bone, liver, and muscle of 1% SBA-fed fishes were markedly increased, suggesting the beneficial effects of SBA. The composition of amino acid and fatty acid in fish muscle, after intaking 1% SBA-containing feed, was altered. In SBA-fed fish muscle, the amounts of threonine and methionine were increased, while the amount of leucine was decreased, as compared with control group. The amounts of fatty acids, including docosahexaenoic acid, phosphatidylcholine, and phosphatidylethanolamine, were increased in the 1% SBA-fed fish, while the amounts of triglycerides were decreased. The results indicated the growth-promoting activity of SBA in an in vivo culture of S. fuscescens, as well as to increase the nutritive values of the muscle. Thus, the re-cycle of waste products during the farming of S. baicalensis herb in serving as fish feeding should be encouraged.

The Roles of Epinephelus coioides miR-122 in SGIV Infection and Replication.

In mammals, mature miR-122 is 22 nucleotides long and can be involved in regulating a variety of physiological and biological pathways. In this study, the expression profile and effects of grouper Epinephelus coioides miR-122 response to Singapore grouper iridovirus (SGIV) infection were investigated. The sequences of mature microRNAs (miRNAs) from different organisms are highly conserved, and miR-122 from E. coioides exhibits high similarity to that from mammals and other fish. The expression of miR-122 was up-regulated during SGIV infection. Up-regulation of miR-122 could significantly enhance the cytopathic effects (CPE) induced by SGIV, the transcription levels of viral genes (MCP, VP19, LITAF and ICP18), and viral replication; reduce the expression of inflammatory factors (TNF-a, IL-6, and IL-8), and the activity of AP-1 and NF-κB, and miR-122 can bind the target gene p38α MAPK to regulate the SGIV-induced cell apoptosis and the protease activity of caspase-3. The results indicated that SGIV infection can up-regulate the expression of E. coioides miR-122, and up-regulation of miR-122 can affect the activation of inflammatory factors, the activity of AP-1 and NF-κB, and cell apoptosis to regulate viral replication and proliferation.

Molecular cloning, inducible expression with SGIV and Vibrio alginolyticus challenge, and function analysis of Epinephelus coioides PDCD4.

Programmed cell death 4 (PDCD4) in mammals, a gene closely associated with apoptosis, is involved in many biological processes, such as cell aging, differentiation, regulation of cell cycle, and inflammatory response. In this study, grouper Epinephelus coioides PDCD4, EcPDCD4-1 and EcPDCD4-2, were obtained. The open reading frame (ORF) of EcPDCD4-1 is 1413 bp encoding 470 amino acids with a molecular mass of 52.39 kDa and a theoretical pI of 5.33. The ORF of EcPDCD4-2 is 1410 bp encoding 469 amino acids with a molecular mass of 52.29 kDa and a theoretical pI of 5.29. Both EcPDCD4-1 and EcPDCD4-2 proteins contain two conserved MA3 domains, and their mRNA were detected in all eight tissues of E. coioides by quantitative real-time PCR (qRT-PCR) with the highest expression in liver. The expressions of two EcPDCD4s were significantly up-regulated after Singapore grouper iridovirus (SGIV) or Vibrio alginolyticus infection. In addition, over-expression of EcPDCD4-1 or EcPDCD4-2 can inhibit the activity of the nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), and regulate SGIV-induced apoptosis. The results demonstrated that EcPDCD4s might play important roles in E. coioides tissues during pathogen-caused inflammation.

Molecular characterization, expression and function analysis of Epinephelus coioides MKK4 response to SGIV and Vibrio alginolyticus infection.

Mitogen-activated protein kinase 4 (MKK4), a member of the MAP kinase family, play important roles in response to many environmental and cellular stresses in mammals. In this study, three MKK4 subtypes, EcMKK4-1, EcMKK4-2 and EcMKK4-3, were obtained from grouper Epinephelus coioides. The open reading frame (ORF) of EcMKK4s are obtained and the EcMKK4s proteins contain highly conserved domains: a S_TKc domain, a canonical diphosphorylation group and two conserved MKKK ATP binding motifs, Asp-Phe-Gly (DFG) and Ala-Pro-Glu (APE). EcMKK4s could be found both in the cytoplasmic and nuclear. The EcMKK4s mRNA were detected in all E. coioides tissues examined with the different expression levels, and the expression were up-regulated during SGIV (Singapore grouper iridescent virus) or Vibrio alginolyticus infection. EcMKK4 could significantly reduce the activation of AP-1 reporter gene. The results suggested that EcMKK4s might play important roles in pathogen-caused inflammation.

Characterization of a macrophagic-like cell line derived from rabbit fish (Siganus fuscescens): An illustration of anti-inflammatory responses of the herbal extract of Scutellaria baicalensis.

A new cell line was isolated and characterized from the head kidney of Siganus fuscescens (rabbit fish). The new macrophagic-like cell line was named as rabbit fish macrophage (RFM), and which could be sub-cultured for over 50 cycles since the development. RFM cell line was tested for growth in different temperatures and serum concentrations: the best growing condition was optimized at 20% serum under 28 °C. In cultured RFM cells, sequencing of 18S rRNA, as well as immunostaining of cytokeratin and CD 68, confirmed the identity as macrophagic cell of S. fuscescens. Cultured RFM cells were exposed to challenge of inflammation, as triggered by LPS, showing highly sensitive responses to inflammation, including release of nitric oxide, expression of cytokine, and activation of phagocytosis. The water extract of aerial part of Scutellaria baicalensis, named as SBA, has been shown anti-inflammatory property in S. fuscescens fish. In order to extend the application of SBA in aquaculture, the extract and its effective flavonoids, i.e. baicalin and scutellarin, were applied in LPS-treated RFM cells. Application of SBA extract, baicalin or scutellarin, inhibited the expressions of LPS-induced inflammatory cytokines, i.e. IL-1ß, TNF-α, as well as the signaling of transcription factor NF-κB. The results support the established RFM cell line could be an ideal in vitro model in drug screening relating to inflammation. Additionally, the notion of SBA herbal extract in fish aquaculture is supported by its efficacy against inflammation.