Establishment of a hydrodynamic delivery system in ducks.

Chronic hepatitis B virus (HBV) poses a significant global health challenge as it can lead to acute or chronic liver disease and hepatocellular carcinoma (HCC). To establish a safety experimental model, a homolog of HBV-duck HBV (DHBV) is often used for HBV research. Hydrodynamic-based gene delivery (HGD) is an efficient method to introduce exogenous genes into the liver, making it suitable for basic research. In this study, a duck HGD system was first constructed by injecting the reporter plasmid pLIVE-SEAP via the ankle vein. The highest expression of SEAP occurred when ducks were injected with 5 µg/mL plasmid pLIVE-SEAP in 10% bodyweight volume of physiological saline for 6 s. To verify the distribution and expression of exogenous genes in multiple tissues, the relative level of foreign gene DNA and ß-galactosidase staining of LacZ were evaluated, which showed the plasmids and their products were located mainly in the liver. Additionally, ß-galactosidase staining and fluorescence imaging indicated the delivered exogenous genes could be expressed in a short time. Further, the application of the duck HGD model on DHBV treatment was investigated by transferring representative anti-HBV genes IFNα and IFNγ into DHBV-infected ducks. Delivery of plasmids expressing IFNα and IFNγ inhibited DHBV infection and we established a novel efficient HGD method in ducks, which could be useful for drug screening of new genes, mRNAs and proteins for anti-HBV treatment.

CTRP1 prevents high fat diet-induced obesity and improves glucose homeostasis in obese and STZ-induced diabetic mice.

BACKGROUND: C1q/tumor necrosis factor-related protein 1 (CTRP1) is an adipokine secreted by adipose tissue, related to chondrocyte proliferation, inflammation, and glucose homeostasis. However, the therapeutic effects on metabolic disorders and the underlying mechanism were unclear. Here, we investigated the functions and mechanisms of CTRP1 in treating obesity and diabetes. METHODS: The plasmid containing human CTRP1 was delivered to mice by hydrodynamic injection, which sustained expression of CTRP1 in the liver and high protein level in the blood. High-fat diet (HFD) fed mice and STZ-induced diabetes model were used to study the effects of CTRP1 on obesity, glucose homeostasis, insulin resistance, and hepatic lipid accumulation. The lipid accumulation in liver and adipose tissue, glucose tolerance, insulin sensitivity, food intake, and energy expenditure were detected by H&E staining, Oil-Red O staining, glucose tolerance test, insulin tolerance test, and metabolic cage, respectively. The metabolic-related genes and signal pathways were determined using qPCR and western blotting. RESULTS: With high blood circulation, CTRP1 prevented obesity, hyperglycemia, insulin resistance, and fatty liver in HFD-fed mice. CTRP1 also improved glucose metabolism and insulin resistance in obese and STZ-induced diabetic mice. The metabolic cage study revealed that CTRP1 reduced food intake and enhanced energy expenditure. The mechanistic study demonstrated that CTRP1 upregulated the protein level of leptin in blood, thermogenic gene expression in brown adipose tissue, and the gene expression responsible for lipolysis and glycolysis in white adipose tissue (WAT). CTRP1 also downregulated the expression of inflammatory genes in WAT. Overexpression of CTRP1 activated AMPK and PI3K/Akt signaling pathways and inhibited ERK signaling pathway. CONCLUSION: These results demonstrate that CTRP1 could improve glucose homeostasis and prevent HFD-induced obesity and fatty liver through upregulating the energy expenditure and reducing food intake, suggesting CTRP1 may serve as a promising target for treating metabolic diseases.

Grass carp (Ctenopharyngodon idellus) SHP2 suppresses IFN I expression via decreasing the phosphorylation of GSK3ß in a non-contact manner.

As a tyrosine phosphatase, Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) serves as an inhibitor in PI3K-Akt pathway. In mammals, SHP2 can phosphorylate GSK3ß at Y216 site to control the expression of IFN. So far, the multiple functions of SHP2 have been reported in mammals. However, little is known about fish SHP2. In this study, we cloned and identified a grass carp (Ctenopharyngodon idellus) SHP2 gene (CiSHP2, MT373151). SHP2 is conserved among different vertebrates by amino acid sequences alignment and the phylogenetic tree analysis. CiSHP2 shared the closest homology with Danio rerio SHP2. Simultaneously, SHP2 was also tested in grass carp tissues and CIK (C. idellus kidney) cells. We found that it responded to poly I:C stimulation. CiSHP2 was located in the cytoplasm just as the same as those of mammals. Interestingly, it inhibited the phosphorylation level of GSK3ß in a non-contact manner. Meanwhile CiGSK3ß interacted with and directly phosphorylated CiTBK1. In addition, we found that CiSHP2 also reduced the phosphorylation level of CiTBK1 by CiGSK3ß, and then it depressed the expression of IFN I via GSK3ß-TBK1 axis. These results suggested that CiSHP2 was involved in CiGSK3ß and CiTBK1 activity but not regulated their transcriptional level. At the same time, we also found that CiSHP2 also influenced the activity of CiIRF3. Therefore, fish SHP2 inhibited IFN I expression through blocking GSK3ß-TBK1 signal axis.

Grass carp (Ctenopharyngodon idella) IRAK1 and STAT3 up-regulate synergistically the transcription of IL-10.

In vertebrates, IL-10 is an anti-inflammatory factor that serves as a key inhibitory role in a wide range of immune responses. IRAK1 (IL-1 receptor-associated kinase 1), a key molecule in the inflammatory signal of IL-1R/TLR, plays an important pivotal role in regulating the autoimmunity of body. STAT3 (Signal transducer and activator of transcription 3) activated by IRAK1 participates in inflammation, tumorigenesis, metabolic disorders and immune response. Under the stimulation of LPS, IRAK1 enters the nucleus to form a dimer with STAT3 and regulates the expression of IL-10. However, the relationship between fish IRAK1 and STAT3 has not been reported. To explain the anti-inflammation in fish, we amplified and identified the complete open reading frame of grass carp IRAK1 (CiIRAK1) and STAT3 (CiSTAT3) based on the existing sequences. The expression of CiIRAK1 and CiSTAT3 were up-regulated significantly under the stimulation of LPS. This result suggests that both CiIRAK1 and CiSTAT3 may be involved in LPS-induced TLR4 pathway. The subcellular localization experiment revealed that CiIRAK1 is distributed in cytoplasm and enters nucleus after LPS stimulation. CiSTAT3 is distributed in both cytoplasm and nucleus with or without LPS stimulation. Immunoprecipitation assay revealed that CiIRAK1 interacted with CiSTAT3 under LPS stimulation. However in absence of LPS stimulation, CiIRAK1 and CiSTAT3 cannot interact with each other. Subsequently, immunofluorescence colocalization experiment further proved the interaction of CiIRAK1 and CiSTAT3 in nucleus under LPS stimulation. The dual luciferase reporter assays indicated that the binding of CiIRAK1 and CiSTAT3 synergistically enhanced the activity of CiIL-10 promoter.

IRF9 promotes apoptosis and innate immunity by inhibiting SIRT1-p53 axis in fish.

As a NAD+-dependent deacetylase, SIRT1 is widely involved in apoptosis and cellular inflammation via multiple pathways such as p53, NF-кB and STAT. More and more studies have shown that p53 is the first non-histone deacetylation target of SIRT1. SIRT1-p53 axis thus plays an important role in mammalian cells. IRF9 is an important member of interferon regulator factor family and performs an important role in innate immunity against foreign virus invasion. More importantly, human IRF9 can suppress the SIRT1-p53 axis. However, the functions and relationship between IRF9 and SIRT1-p53 axis are rarely studied in fish. To this end, we made a preliminary research on the functions of grass carp (Ctenopharyngodon idella) IRF9, SIRT1 and p53 in apoptosis and innate immunity. Firstly, we cloned and identified the ORF of SIRT1 (named CiSIRT1, MN125614) from C. idella and demonstrated that CiIRF9 promoted apoptosis, while CiSIRT1 inhibited apoptosis by flow cytometry and TUNEL experiments. Next, we found the interaction between CiSIRT1 and Cip53 in vivo by co-immunoprecipitation experiments. Moreover, the colocalization analysis also showed CiSIRT1 and Cip53 were mainly distributed in nucleus. Thirdly, we got a conclusion that CiIRF9 can repress the expression of CiSIRT1, implying that CiIRF9 regulates CiSIRT1-p53 axis. Finally, CiSIRT1 mRNA level was significantly up-regulated and the expression reached the highest level at 24 h post poly (I:C) stimulation in CIK cells. So, CiSIRT1 may exert an important function in innate immunity. Furthermore, we found CiSIRT1 down-regulated the expression of CiIFN1. In summary, CiIRF9 promotes apoptosis and innate immunity by inhibiting SIRT1-p53 axis. These findings will provide a new theoretical basis for the research on teleost innate immunity.

Research on the signaling pathways related to the intervention of traditional Chinese medicine in Parkinson's disease:A literature review.

ETHNOPHARMACOLOGICAL RELEVANCE: Parkinson's disease (PD) is the most common progressive neurodegenerative disorder affecting more than 10 million people worldwide and is characterized by the progressive loss of Daergic (DA) neurons in the substantia nigra pars compacta. It has been reported that signaling pathways play a crucial role in the pathogenesis of PD, while the active ingredients of traditional Chinese medicine (TCM) have been found to possess a protective effect against PD. TCM has demonstrated significant potential in mitigating oxidative stress (OS), neuroinflammation, and apoptosis of DA neurons via the regulation of signaling pathways associated with PD. AIM OF THE REVIEW: This study discussed and analyzed the signaling pathways involved in the occurrence and development of PD and the mechanism of active ingredients of TCM regulating PD via signaling pathways, with the aim of providing a basis for the development and clinical application of therapeutic strategies for TCM in PD. MATERIALS AND METHODS: With "Parkinson's disease", "Idiopathic Parkinson's Disease", "Lewy Body Parkinson's Disease", "Parkinson's Disease, Idiopathic", "Parkinson Disease, Idiopathic", "Parkinson's disorders", "Parkinsonism syndrome", "Traditional Chinese medicine", "Chinese herbal medicine", "active ingredients", "medicinal plants" as the main keywords, PubMed, Web of Science and other online search engines were used for literature retrieval. RESULTS: PD exhibits a close association with various signaling pathways, including but not limited to MAPKs, NF-κB, PI3K/Akt, Nrf2/ARE, Wnt/ß-catenin, TLR/TRIF, NLRP3, Notch. The therapeutic potential of TCM lies in its ability to regulate these signaling pathways. In addition, the active ingredients of TCM have shown significant effects in improving OS, neuroinflammation, and DA neuron apoptosis in PD. CONCLUSION: The active ingredients of TCM have unique advantages in regulating PD-related signaling pathways. It is suggested to combine network pharmacology and bioinformatics to study the specific targets of TCM. This not only provides a new way for the prevention and treatment of PD with the active ingredients of TCM, but also provides a scientific basis for the selection and development of TCM preparations.

Efficacy of probiotic supplements on Parkinson's disease: A systematic review and meta-analysis.

OBJECTIVE: This study aimed to evaluate the clinical efficacy and safety of probiotics supplementation in the treatment of Parkinson's disease (PD). METHODS: We searched China National Knowledge Infrastructure (CNKI), Weipu (VIP) database, Wanfang Database, Sinomed (CBM), PubMed, Embase, Cochrane library and Web of Science databases for eligible studies from inception to January 4th, 2024. Randomized controlled trials (RCTS) comparing the effects of probiotic supplements and placebo in patients with PD. Meta-analysis was conducted with the software Review Manager 5.4. The quality assessment was performed according to Cochrane risk of bias tool. RESULTS: A total of 11 RCTs with 756 PD patients were included in this study. We found that probiotics could increase the number of complete bowel movements (CBMs) per week and improved the scores of Patient Assessment of Constipation Quality of Life Questionnaire (PAC-QOL) (SMD = 0.73, 95 % CI: 0.54 to 0.92, P < 0.00001, I2 = 45 %; SMD = - 0.79, 95 % CI: - 1.19 to - 0.39, P < 0.001, I2 = 55 %, respectively) compared with the placebo group. However, there was no significant difference between the two groups in improving fecal traits and defecation efforts in PD patients (SMD = 0.87, 95 % CI: 0.01 to 1.74, P = 0.05, I2 = 94 %; SMD = 1.24, 95 % CI: - 1.58 to 4.06, P > 0.05, I2 = 98 %, respectively). In terms of PD composite scale scores: after treatment, there was no significant difference in Movement Disorder Society-Unified-Parkinson Disease Rating Scale Ⅲ score (MDS-UPDRSⅢ) between the probiotic group and the placebo group (SMD = - 0.09, 95 % CI: - 0.35 to 0.16, P > 0.05, I2 = 0 %). CONCLUSIONS: In conclusion, based on the overall results of the available RCTs studies, our results suggested the potential value of probiotics in improving constipation symptoms in PD patients. Therefore, probiotics may be one of the adjuvant therapy for PD-related constipation patients. The findings of this study provide more proof supporting the effectiveness of probiotics, encouraging probiotics to be utilized alone or in combination with other therapies in clinical practice for PD patients. However, more well-designed RCTs with large sample sizes are required.

Relationship between long-term shift work; night work; sleep deficiency and stroke: A systematic review and meta-analysis.

BACKGROUND: The association between shift/night work and the risk of stroke is not supported by strong evidence. OBJECTIVE: This study aimed to obtain evidence of a potential relationship between shift/night shift work and the risk of stroke. METHODS: We searched PubMed, Embase, the Cochrane Library and Web of science databases for eligible studies from inception to January 19, 2024. We followed the statement in the Preferred Reporting Items for Systematic Evaluations and Meta-Analysis (PRISMA). STATA 14.0 software was used for meta-analysis. RESULTS: A total of five studies involving 700,742 subjects were included in this meta-analysis. We found that shift/night workers had a 1.08 times higher risk of stroke than non-shift/night workers (RR: 1.08; 95 % CI: 1.05-1.10; P < 0.001). CONCLUSION: Shift/night work may be a risk factor for stroke. More objective prospective studies are needed to further support this result.

Integrated non-targeted metabolomics and network pharmacology to reveal the mechanisms of berberine in the long-term treatment of PTZ-induced epilepsy.

AIMS: The increasing resistance to anti-seizure medications (ASMs) and the ambiguous mechanisms of epilepsy highlight the pressing demand for the discovery of pioneering lead compounds. Berberine (BBR) has received significant attention in recent years within the field of chronic metabolic disorders. However, the reports on the treatment of epilepsy with BBR are not systematic and the mechanism remains unclear. MAIN METHODS: In this study, the seizure behaviors of mice were recorded following subcutaneous injection of pentetrazol (PTZ). Non-targeted metabolomics was used to analyze the serum metabolites based on ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Meanwhile, multivariate statistical methods were used for metabolite identification and pathway analysis. Furthermore, network pharmacology, molecular docking, and quantitative real-time PCR assay were used for the target identification. KEY FINDINGS: BBR had anti-seizure effects on PTZ-induced seizure mice after long-term treatment. Tryptophan metabolism and phenylalanine metabolism were involved in regulating the therapeutic effects of BBR. SIGNIFICANCE: This study reveals the potential mechanism of BBR for epilepsy treatment based on non-targeted metabolomics and network pharmacology, which provides evidence for uncovering the pathogenesis of epilepsy, suggesting that BBR is a potential lead compound for anti-epileptic treatment.

Validation of Moorong Self-Efficacy Scale among Chinese stroke survivors.

PURPOSE: To examine the psychometric properties of a Chinese version of the Moorong Self-Efficacy Scale (MSES-C) among stroke survivors. METHODS: A cross-sectional descriptive study was conducted with 160 stroke survivors recruited from the three neurology departments in China. Reliability, concurrent validity, and construct validity of the scale were determined. RESULTS: The MSES-C whole scale showed good internal consistency with a Cronbach's α of 0.953. There was a moderate to a strong positive correlation between the MSES-C and Chinese version of the General Self-Efficacy Scale (r = 0.695, p < 0.000), a strong positive correlation between the MSES-C and Chinese version of the stroke specific self-efficacy scale (r = 0.801, p < 0.000), positive correlations between the MSES-C and Chinese versions of the Modified Barthel Index (r = 0.695, p < 0.000), and a negative correlation between the MSES-C and National Institutes of Health Stroke Scale (r = -0.511, p < 0.000). Known-group validity was also supported. CONCLUSIONS: The MSES-C is a reliable and valid instrument for assessing self-efficacy in Chinese stroke survivors.Implications for RehabilitationThe Chinese version of the Moorong Self-Efficacy Scale demonstrated good internal consistency and showed satisfactory concurrent and construct validity among stroke survivors.The Moorong Self-Efficacy Scale can be used to assess stroke recovery among the Chinese population in clinical and research settings.

Grass carp (Ctenopharyngodon idellus) Cdc25a down-regulates IFN 1 expression by reducing TBK1 phosphorylation.

In vertebrates, TANK Binding Kinase 1 (TBK1) plays an important role in innate immunity, mainly because it can mediate production of interferon to resist the invasion of pathogens. In mammals, cell division cycle-25a (Cdc25a) is a member of the Cdc25 family of cell division cycle proteins. It is a phosphatase that plays an important role in cell cycle regulation by dephosphorylating its substrate proteins. Currently, many phosphatases are reported to play a role in innate immunity. This is because the phosphatases can shut down or reduce immune signaling pathways by down-regulating phosphorylation signals. However, there are no reports on fish Cdc25a in innate immunity. In this paper, we conducted a preliminary study on the involvement of grass carp Cdc25a in innate immunity. First, we cloned the full-length cDNA of grass carp Cdc25a (CiCdc25a), and found that it shares the highest genetic relationship with that of Anabarilius grahami through phylogenetic tree comparison. In grass carp tissues and CIK cells, the expression of CiCdc25a mRNA was up-regulated under poly (I:C) stimulation. Therefore, CiCdc25a can respond to poly (I:C). The subcellular localization results showed that CiCdc25a is distributed both in the cytoplasm and nucleus. We also found that CiCdc25a can down-regulate the expression of IFN 1 with or without poly (I:C) stimulation. In other words, the down-regulation of IFN1 by CiCdc25a is independent of poly (I:C) stimulation. Further functional studies have shown that the inhibition of IFN1 expression by CiCdc25a may be related to decrease of TBK1 activity. We also confirmed that the phosphorylation of TBK1 at Ser172 is essential for production of IFN 1. In short, CiCdc25a can interact with TBK1 and subsequently inhibits the phosphorylation of TBK1, thereby weakens TBK1 activity. These results indicated that grass carp Cdc25a down-regulates IFN 1 expression by reducing TBK1 phosphorylation.

The tyrosine kinase SRC of grass carp (Ctenopharyngodon idellus) up-regulates the expression of IFN I by activating TANK binding kinase 1.

In response to viral infections, various pattern recognition receptors (PRRs) are activated for the production of type I interferon (IFN I). As a center of these receptor responses, TANK binding kinase-1 (TBK1) activates interferon regulatory factor 3 (IRF3). SRC is a member of Src family kinases (SFK) which participates in TBK1-mediated IFN I signaling pathway. In mammals, the immunological function of SRC is depended on its interaction with TBK1. To date, SRC has not been studied in fish. In this paper, we cloned the ORF of grass carp (Ctenopharyngodon idellus) SRC (CiSRC). CiSRC has a closer relationship with Sinocyclocheilus rhinocerous SRC (SrSRC). The expression level of CiSRC was significantly up-regulated following poly (I:C) stimulation in grass carp tissues and cells. Subcellular localization results showed that CiSRC is located both in the cytoplasm and nucleus, while CiTBK1 is only located in the cytoplasm of CIK cells. When GFP-CiSRC and FLAG-CiTBK1 were co-transfected into CIK cells, we found that they were co-localized in the cytoplasm. GST-pulldown and Co-immunoprecipitation analysis revealed that CiSRC and CiSRC tyrosine kinase domain deletion mutant (SRC-ΔTyrkc) can interact with CiTBK1, respectively. CiSRC promotes the phosphorylation of CiTBK1. Furthermore, the phosphorylation of TBK1 is more strongly under poly (I:C) stimulation. We also demonstrated that SRC can up-regulate IFN I expression. These results above unraveled that CiSRC initiates innate immune response by binding to and then up-regulating the phosphorylation of TBK1.

Grass carp (Ctenopharyngodon idella) interferon regulatory factor 8 down-regulates interferon1 expression via interaction with interferon regulatory factor 2 in vitro.

Interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein (ICSBP), is a negative regulatory factor of interferon (IFN) and plays an important role in cell differentiation and innate immunity in mammals. In recent years, some irf8 homologous genes have been cloned and confirmed to take part in innate immune response in fish, but the mechanism still remains unclear. In this paper, a grass carp (Ctenopharyngodon idella) irf8 gene (Ciirf8) was cloned and characterized. The deduced protein (CiIRF8) possesses a highly conserved N-terminal DNA binding domain but a less well-conserved C-terminal IRF association domain (IAD). Ciirf8 was widely expressed in all tested tissues of grass carp and up-regulated following poly(I:C) stimulation. Ciirf8 expression was also up-regulated in CIK cells upon treatment with poly(I:C). To explore the molecular mechanism of how fish IRF8 regulates ifn1 expression, the similarities and differences of grass carp IRF8 and IRF2 were compared and contrasted. Subcellular localization analysis showed that CiIRF8 is located both in the cytoplasm and nucleus; however, CiIRF2 is only located in the nucleus. The nuclear-cytoplasmic translocation of CiIRF8 was observed in CIK cells under stimulation with poly(I:C). The interaction of CiIRF8 and CiIRF2 was further confirmed by a co-immunoprecipitation assay in the nucleus. Dual-luciferase reporter assays showed that the promoter activity of Ciifn1 was significantly inhibited by co-transfection with CiIRF2 and CiIRF8. The transcription inhibition of Ciifn1 was alleviated by competitive binding of CiIRF2 and CiIRF8 to CiIRF1. In conclusion, CiIRF8 down-regulates Ciifn1 expression via interaction with CiIRF2 in cells.

Grass carp (Ctenopharyngodon idella) TNK1 modulates JAK-STAT signaling through phosphorylating STAT1.

TNK1 (thirty-eight-negative kinase 1) belongs to the ACK (Activated Cdc42 Kinases) family of intracellular non-receptor tyrosine kinases that usually acts as an important regulator in cytokine receptor-mediated intracellular signal transduction pathways. JAK-STAT signal pathway acts as a key point in cellular proliferation, differentiation and immunomodulatory. Mammalian TNK1 is involved in antiviral immunity and activation of growth factors. However, TNK1 has rarely been studied in fish. To evaluate the role of fish TNK1 in JAK-STAT pathway, we cloned the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) TNK1 (CiTNK1). CiTNK1 protein consists of N-terminal Tyrkc (tyrosine kinase) domain, C-terminal SH3 (Src homology 3) domain and Pro-rich domain. Phylogenetic analysis showed that CiTNK1 has a closer relationship with Danio rerio TNK1. The expression and phosphorylation of CiTNK1 in grass carp tissues and cells was increased under poly(I:C) stimulation. Subcellular localization and co-immunoprecipitation indicated that CiTNK1 is targeted in the cytoplasm and interacts with grass carp STAT1 (CiSTAT1). Co-transfection of CiTNK1 and CiSTAT1 into cells facilitated the expression of IFN I. This is because that the presence of CiTNK1 enhanced the phosphorylation of CiSTAT1 and causes activation of CiSTAT1. Our results revealed that TNK1 can potentiate the phosphorylation of STAT1 and then regulates JAK-STAT pathway to trigger IFN I expression in fish.

Grass Carp (Ctenopharyngodon idellus) NIMA-Related Kinase 6 Blocks dsRNA-Induced IFN I Response by Targeting IRF3.

Accumulating evidence indicates that mammalian NIMA (never in mitosis, gene A)-related kinase 6 (NEK6) plays potential roles during the course of tumorigenesis, but little is known about NEK6 in lower vertebrates. Herein, we reported a mammalian ortholog of NEK6 in grass carp (Ctenopharyngodon idellus) (CiNEK6). Multiple alignment of amino acid sequences and phylogenetic analysis showed that CiNEK6 shares a high level of sequence similarity with its counterparts in birds. CiNEK6 was ubiquitously expressed in all tested tissues, and its expression level was increased under treatment with GCRV (dsRNA virus) or poly I:C (dsRNA analog). Q-PCR and dual-luciferase assays suggested that CiNEK6 overexpression suppressed IFN I activity in CIK cells treated with poly I:C. Knockdown of CiNEK6 resulted in a higher level of IFN I expression in CIK cells treated with poly I:C compared to those which received PBS. Interestingly, analysis of subcellular localization demonstrated that CiNEK6 protein scattered throughout the cytoplasm is gradually congregated together at the edges of karyotheca upon stimulation with poly I:C. Co-IP and co-localization assays suggested that CiNEK6 interacts with CiIRF3 after poly I:C challenge. In poly I:C-treated cells, the phosphorylation of CiIRF3 was increased by CiNEK6 knockdown, but was suppressed by CiNEK6 overexpression, suggesting that CiNEK6 decreases IFN I expression through inhibiting CiIRF3 activity. Cell viability assay, crystal violet staining, and detection of Vp5 also showed that CiNEK6 plays an inhibitory role in IRF3-mediated antiviral responses.

Grass carp (Ctenopharyngodon idellus) TRAF6 up-regulates IFN1 expression by activating IRF5.

In mammals, interferon regulatory factor 5 (IRF5) can be activated by tumor necrosis factor receptor-associated factor 6 (TRAF6). Upon activation, IRF5 translocates into the nucleus, where it binds to IFN promoter and up-regulates IFN expression. However, there are few reports on the molecular mechanism by which TRAF6 up-regulates IFN expression in fish. In this study, we explored how Grass carp (Ctenopharyngodon idellus) TRAF6 initiated innate immunity by activating IRF5. We found that CiTRAF6, CiIRF5 and CiIFN1 were all significantly up-regulated in LPS-stimulated CIK cells and the expression of CiTRAF6 was earlier than the expressions of CiIRF5 and CiIFN1. These findings suggested that CiIFN1 expression might be induced by CiTRAF6 in fish. CiIFN1 expression, CiIFN1 promoter activity and CO cells viability were all significantly up-regulated in the overexpression experiments, but they were significantly down-regulated in the gene silencing experiments. This indicated that CiTRAF6, along with CiIRF5, regulated CiIFN1 expression. The localization analysis found that both CiTRAF6 and CiIRF5 located in the cytoplasm. Following LPS stimulation, CiIRF5 was observed to translocate to the nucleus. GST-pull down and co-IP experiments revealed that CiTRAF6 interacted with CiIRF5. The colocalization analysis also showed that CiTRAF6 bound with CiIRF5 in the cytoplasm. Overexpression of CiTRAF6 increased the endogenous CiIRF5, promoted its ubiquitination and nuclear translocation. In conclusion, CiTRAF6 bound to CiIRF5 in the cytoplasm, and then activated CiIRF5, resulting in up-regulating the expression of CiIFN1.